# Patent

## Title
Viral vectors and viral vaccines based on recombinant porcine adenoviruses

## Abstract

An in vivo replicative and recombined porcine adenovirus characterized in that it comprises a heterologous nucleotide sequence inserted into the porcine adenovirus in conditions enabling the latter to be replicated in vivo and to express the inserted heterologous nucleotide sequence, and in that the adenovirus genome comes from a 3 or 5 serotype (PAV-3 or PAV-5) adenovirus. Insertion occurs in a non-essential zone of the E3 region, preferably with deletion of said zone. The invention also relates to a recombined porcine vaccine comprising one such porcine adenovirus. The invention further relates to a serotype 3 or 5 porcine adenovirus vector that is replicative in vivo and is deleted in a non-essential region of the genome thereof. The invention also relates to a DNA fragment comprising all or part of the referenced SEQ ID NO.5 nucleotide sequence.

## Background
The present invention relates to serotype 3 and serotype 5 porcine adenoviruses which are recombined by insertion of heterologous nucleotide sequences, these adenoviruses being capable of replicating autonomously in vivo and of expressing these heterologous sequences. The invention also relates to the porcine vaccines obtained, to methods for immunizing pigs using them, to deleted and replicative adenoviral vectors, and to methods for obtaining these adenoviruses, vaccines and vectors.
The documents cited in the description and also the documents cited in those documents are incorporated herein by way of reference.
Adenoviruses are viruses containing a linear double-stranded DNA molecule, with inverted terminal repeat sequences at each end. Adenoviruses are part of the family of Adenoviridae, which are responsible for diseases affecting a large number of species, such as the simian, bovine, ovine, porcine, equine, murine, canine and avian species. Adenovirus infections can be characterized, depending on the species and on the types of adenovirus, by signs of encephalitis, of pneumonia, of renal lesions, of diarrhea and of hepatitis.
The adenovirus genome can vary from one species to the other [(T. Adrian et al., Arch. Virol. 1986, 91, 277–290), (J. Hamelin et al., J. Clin. Microbiol. 1988, 26, 31–33), (R. Assaf et al., Can. J. Comp. Med. 1983, 47, 460–463), (T. Kurokawa et al., J. Virol. 1978, 28, 212–218), (S.-L. Hu et al., J. Virol, 1984, 51, 880–883), (L. Zsak et al., Intervirology 1984, 22, 110–114)]. Animal adenoviruses, unlike human adenoviruses, have a very limited host spectrum and they often infect only the animals belonging to their species of origin.
The first porcine adenovirus (or PAV) was isolated in 1964 (D. Haig et al., J. Comp. Pathol. 1964, 74, 81–84). Since that date, 5 porcine adenovirus serotypes have been identified and described (Hirahara et al., J. Vet. Sci. 1990, 52, 407–409). Serotype 1 and serotype 2 adenoviruses are associated with diarrhea; serotype 4 adenoviruses are associated with symptoms of pneumonia and of encephalitis. Porcine adenoviruses have been classified in a fifth serotype due to their absence of cross-neutralization with antiserum specific for the serotype 1 to serotype 4 PAVs. The genome of PAV-5 has been analyzed and a restriction map has been established (Tuboly et al., Virus Res. 1995, 37, 49–54). This analysis showed the absence of similarity between the PAV-5 restriction maps and those of the PAVs of the other serotypes: PAV-1 and PAV-2 (Reddy et al., Arch. Virol. 1995, 140, 195–200, PAV-3 (Reddy et al., Intervirology 1993, 36, 161–168) and PAV-4 (Kleiboeker et al., Arch. Virol. 1993, 133, 357–368) Among the serotypes 3 and 5, strains exist which are naturally nonpathogenic for pigs.
PAVs have a genome which is approximately 32 to approximately 34 kbp in size. The genome of PAV-1 is approximately PAV-2 is approximately 33.3 kbp in size. The complete sequence of the PAV-3 virus has recently been established and filed in the GenBank data bank under the number AF083132 (SEQ ID NO: 6) (P. S. Reddy et al., Virol. 1998, 251, 414–426); it is 34094 base pairs (bp) in size. The genome of PAV-4 is approximately 32 kbp in size and that of PAV-5 is approximately 33.2 kbp in size. The complete genome of PAV-5, and in particular the E3 region, has not been sequenced or published.
The main application envisaged for adenoviruses is in the field of gene therapy in humans, and a very large number of constructs and systems have been proposed. Adenoviruses have also been proposed as recombinant vectors in immunization compositions for protecting humans and animals against many pathogenic viruses.
To date, two strategies for constructing recombinant adenoviral vectors have been developed (M. Eloit & M. Adam, J. Gen. Virol. 1995, 76, 1583–1589).
The first strategy consists of the insertion of expression cassettes into regions which are essential for replication of the adenovirus, hence the need to develop transcomplementation systems, in parallel, in order to be able to ensure the replication of the virus. For porcine adenoviruses, the E1 region has been proposed as an insertion site (P. S. Reddy et al., Virus Res. 1998, 58, 97–106). The recombinant vectors thus constructed are termed “nonreplicative” since they cannot replicate in the host to which they are administered.
As a variant of this first strategy, it is possible to use a virus which is nonreplicative in the target animal. The PRV gD expression cassette has in particular already been inserted into the E1A site of the serotype 5 human adenovirus, which is nonreplicative in pigs (M. Monteil et al., J. Gen. Virol. 1997, 78, 3303–3310; A. Ambriovic et al., Virol. 1997, 238, 327–335).
The second strategy consists of the insertion of expression cassettes into regions of the viral genome which are not essential for replication of the adenovirus. The difficulty in determining nonessential regions in adenoviruses and the presence of a rigid capsid make this second strategy difficult to carry out.
Specifically, one of the essential characteristics of adenoviruses is that they have a rigid capsid which strictly limits the size of the DNA molecule which may be encapsidated therein. In general, it is considered possible to encapsidate a DNA molecule corresponding to 105 to 114% of the size of the genome, which corresponds, depending on the size of the genome, to inserts of approximately 1.7 to approximately 3.9 kbp. In the case of excessive sizes, unwanted deletions and/or rearrangements in the recombinant genome may take place.
The E3 region is neither conserved in terms of size nor in terms of genetic organization from an adenovirus of one given species to an adenovirus of another species, nor between porcine adenoviruses of the various serotypes (S. Kleiboeker, Virus Res. 1994, 31, 17–25).
The E3 region of the PAV-3 virus is 1179 base pairs in size. It is a complex region which encodes at least three open reading frames (ORFs). These ORFs overlap one another, and the first ORF also partly overlaps the gene encoding the pVIII protein (P. S. Reddy et al., Virus Res. 1995, 36, 97–106), which is an essential protein.
The presence of these overlapping ORFs in the E3 region represents a major difficulty for determining therein the insertion sites and/or the deletion limits such that the recombinant adenoviruses remain replicative in pigs.
The amino acid sequence encoded by the second ORF of the E3 region shows no homology between the PAV-3 adenovirus and the HAV-2 adenovirus (which is a serotype 2 human adenovirus), nor with any other currently known adenoviral protein (P. S. Reddy et al., Virus Res. 1995, 36, 97–106). The amino acid sequences predicted from the nucleotide sequence of the first ORF of the E3 region in PAV-3 have only 33.3% identity with that of the serotype 2 canine adenovirus (CAV-2). Not only are the proteins encoded by E3 regions from various adenoviruses not homologous, but, in addition, those encoded by the ORFs of the E3 region show no homology between the PAV-3 and PAV-4 adenoviruses (S. B. Kleiboeker, Virus Res. 1994, 31, 17–25).
The E3 region is 1162 bp in size in PAV-1 (with 5 ORFs); it is 1222 bp in PAV-2 (with 5 ORFs) (P. S. Reddy et al., Virus Res. 1996, 43, 99–109), and 1879 bp in PAV-4 (with 6 ORFs). Only the fourth ORF of the E3 region of the PAV-4 virus (corresponding to a 13.2 kDa protein) shows homology with another known ORF of the E3 region, encoding the 14.7 kDa protein of the type 5 human adenovirus (known as Human Adenovirus 5 or HAV-5). The E3 region of PAV-4 shows no sequence homology with the E3 regions of the other porcine adenoviruses, and in particular with those of PAV-3 and of PAV-5.
Besides the difficulty in defining their limits for conserving the replicative nature in vivo, deletions in the E3 region are, moreover, not without risk. Deletions in the E3 region may, in fact, have consequences on the pathogenicity of the recombinant adenoviruses. Thus, it has been observed that a deletion in the E3 region of the HAV-5 virus increases the pulmonary pathogenicity of this virus in the cotton rat experimental model of infection (Ginsberg et al., Proc. Natl. Acad. Sci. USA 1989, 86, 3823–3827).
This shows that the consequences of deletions in the E3 region of an adenovirus must be considered case by case and not by simply transposing the observations made on one particular adenovirus to another. The genetic organization of the E3 region of PAV-3 is unique, as is that of the E3 region of PAV-5.
The precise location of an insertion site in the E3 region is further complicated by the fact that complex splicing zones and polyadenylation zones are present, these zones being characteristic of adenoviruses (Imperiale et al., Curr. Top. Microbial. Immunol. 1995, 199, 139–171). The splicing zones located in the E3 region are important for the maturation of many essential messenger RNAs encoded by the sequences located outside the E3 region. The E3 region is, itself, located in a part of the viral genome which displays high transcriptional activity (P. Sharp, 1984, in The Adenovirus, Ed. H. S. Ginsberg, Plenun Press, New York and London, 173–204); the insertion of a heterologous nucleotide sequence into the E3 region may have negative impacts on the biological activity of the recombinant virus. In addition, the E3 region is located downstream of the major late promoter (MLP), this being a region in which interference between the transcription of the insert and that initiated by the MLP promoter have been demonstrated (Xu et al., J. Gen. Virol. 1995, 76, 1971–1980).
The results obtained with adenoviruses originating from other species (human, bovine, ovine, etc.) cannot therefore be transposed to porcine adenoviruses. No replicative recombinant vector has thus been produced to date from a porcine adenovirus.
The Applicant Company set itself the aim of making possible the insertion of heterologous genes into the genome of the PAV-3 and PAV-5 viruses without substantially modifying their capacity for replication in vivo.
Another aim of the invention is to provide insertion regions which can be deleted without placing this capacity for replication in doubt, so as to increase the capacity for insertion into the virus.
Yet another aim of the invention is to provide recombinant PAV-3 and PAV-5 viruses which conserve a capacity for replication in vivo, allowing their use as live recombinant vaccines, including vaccines administered mucosally and/or in the presence of antibodies of maternal origin.
The Applicant Company has succeeded in modifying the E3 region of PAV-3 and PAV-5 while at the same time conserving the capacity for replication in vivo. It has also been able to demonstrate the in vivo expression of a gene inserted into this region. It therefore makes it possible for PAV-3 and PAV-5 to be used as replicative expression vectors.
A subject of the present invention is therefore a serotype 3 or 5 porcine adenovirus comprising at least one heterologous nucleotide sequence inserted into the genome of PAV-3 or of PAV-5, under conditions which allow the recombinant viruses obtained to replicate in vivo in pigs and to express the inserted sequence. These modified viruses have considerable advantages in terms of vaccination and, in particular, they are effective even in the presence of maternal antibodies and can then be effectively used mucosally.
Preferably, a heterologous sequence is inserted into a nonessential zone of the genome, in particular the E3 zone, with a deletion of this insertion zone. The expression “deletion of a zone” is intended to mean a total or partial, preferably total, deletion of the insertion zone. In other words, the heterologous sequence is inserted in place of all or part of the insertion zone, preferably in place of all of this zone.
For PAV-3, the preferred zone for insertion into E3 is a zone comprising all or part of nucleotides 706 to 1624 of the sequence published by P. S. Reddy et al., Virus Research 1995. 36-97-106, which corresponds to positions 27 794 to 28 712 of the sequence published in GenBank AN # U10433. In other words, this insertion zone and its possible deletion can comprise all or only part of the sequence 706 to 1624 and/or can extend in E3 beyond the limit 706 and/or the limit 1624 and can optionally comprise the entire E3. Preferably, this zone consists, or comprises, E3, nucleotides 706 to 1624, nucleotides 1002 to 1624, or all or part of the sequence 706 to 1002. In particular, it comprises at least the sequence 1002 to 1624.
For PAV-5, E3 is defined by the nucleotide sequence 2382 to 4042 in SEQ ID No. 5. The insertion zone and its possible deletion includes E3 and comprises all or part of nucleotides 2064 to 4083, for example all or part of nucleotides 2389 to 3861, of SEQ ID No. 5 (FIG. 13). In other words, this insertion zone and its possible deletion can comprise only part of the sequence 2389 to 3861 and/or can extend in E3 beyond the limit 2389 and/or beyond the limit 3861 and can optionally comprise the entire E3. When a deletion is made up to nucleotide 4083 inclusive, a splice acceptor site is then preferably inserted in place of nucleotide 4083. Preferably, this insertion zone consists essentially of nucleotides 2389 to 3861.
Needless to say, the invention also covers the insertion into the corresponding zones defined by the nucleotide sequence of other strains of PAV-3 and of PAV-5, the sequences of which differ from those of the reference strains according to the invention.
A subject of the invention is also a PAV-3 or PAV-5 virus which has a greater capacity for insertion in terms of size, while at the same time remaining replicative in vivo in pigs. Maintaining the property of replication in the host is desired in order to obtain optimum protection effectiveness, in particular using an immunization composition mucosally and/or in the presence of antibodies of maternal origin.
Increasing the capacity for insertion is achieved by means of a deletion, e.g. of all or part of the E3 region, the part of E3 which can be deleted being located between the gene encoding the pVIII protein and that encoding the fiber. In particular, the present invention describes deletions of 622 bp and 919 bp which can be made in the E3 region of the PAV-3 virus, while at the same time conserving the replicative nature of the recombinant viruses (see examples 6 to 10). The present invention moreover describes a deletion of 1472 bp in the E3 region of the PAV-5 virus, resulting in the same phenotypic characteristics of the recombinant virus (see examples 14 to 17).
The recombinant PAV-3 or PAV-5 adenovirus obtained according to the invention may advantageously be used to insert a maximum size of approximately 3.0 kbp for the PAV-3 virus and approximately 3.5 kbp for the PAV-5 virus and to express it in pigs.
A subject of the invention is thus also the PAV-3 and PAV-5 vectors deleted, in particular in the E3 region, while at the same time remaining replicative, in particular deleted in the abovementioned zones, i.e. deleted of all or part of the zone considered, preferably of all of this zone.
The heterologous nucleotide sequences which may be inserted are sequences encoding immunogens or immunologically active fragments, e.g. epitopes, of immunogens, and in particular genes or fragments of genes (e.g. epitopes) encoding immunogens of porcine pathogens.
In the context of the present invention, it is of course possible to insert more than one heterologous sequence into the same virus. In particular, heterologous sequences originating from the same virus or from different viruses may be inserted therein.
According to one particular method, it is possible to insert artificial sequences combining several fragments or epitopes originating from the same pathogen or from different pathogens, which are known as “polyepitope sequences”. In the latter case, the polyepitope sequence is placed under the control of a single promoter.
It is also possible to insert sequences encoding immunomodulators, in particular cytokins, preferably porcine cytokins such as porcine granulocyte-macrophage colony stimulating factor (poGM-CSF) porcine interleukin 4 (poIL-4), porcine IL-12 or porcine IL-18.
The expression of several heterologous genes inserted into the insertion locus can also be made possible by inserting a sequence known as an “IRES” (Internal Ribosome Entry Site) in particular originating from a picornavirus such as swine vesicular disease virus (SVDV; B.-F. Chen et al., J. Virology, 1993, 67, 2142–2148), encephalomyocarditis virus (EMCV; R. J. Kaufman et al., Nucleic Acids Research, 1991, 19, 4485–4490) or foot-and-mouth disease virus (FMDV; N. Luz and E. Beck, J. Virology, 1991, 65, 6486–6494), or from another origin. Those skilled in the art may refer to the content of the 3 articles cited. The expression cassette for two genes would therefore have the following minimum structure: promoter (optional)-gene 1-IRES-gene 2-polyadenylation signal. The live recombinant vaccine according to the invention may therefore comprise, inserted into the insertion locus, an expression cassette successively comprising a promoter, two or more genes separated in pairs by an IRES, and a polyadenylation signal.
The porcine pathogens are viruses, bacteria and parasites chosen in particular from those of the group consisting of Aujeszky's disease virus (PRV or pseudorabies virus), swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRS virus), parvovirosis virus (PPV virus), Hog Cholera Virus (HCV), porcine circovirus, in particular type 2 porcine circovirus (Porcine CircoVirus type 2, PCV2 or piglet systemic wasting syndrome virus), Actinobaccilus pleuropneumoniae and Mycoplasma hyopneumoniae. 
With regard to the Aujeszky valency, it is possible to use the gB gene (Robbins A. K. et al., J. Virol. 1987, 61, 2691–2701 GenBank AN # M17321), gC gene (Ishikawa K. et al., Vet. Microbiol. 1996, 49, 267–272 GenBank AN # D49435) and/or the gD gene (Rivière M. et al., J. Virol. 1992, 66, 3424–3434 and Hong W. Z. et al., GenBank AN # AF086702), in their native or secreted forms.
With regard to the swine influenza valency, it is preferred to use the HA gene (WO-A-98/03658, examples 10 and 12) (native or secreted forms), the NA gene (Nerome K. et al., J. Gen. Virol. 1995, 76, 613–624 GenBank AN # D21194) (native or secreted forms) and/or the NP gene (WO-A-98/03658, examples 11 and 13).
With regard to the PRRS valency, it is preferred to use the ORF4 gene, the ORF3 gene (native or secreted forms), the ORF5 gene (native or secreted forms), the ORF6 gene and/or the ORF7 gene from European strains (Meulenberg J. et al., Virology, 1993, 192, 62–72) and from American strains (Mardassi H. et al., Arch. Virol. 1995, 140, 1405–1418).
With regard to the hog cholera valency, it is preferred to use the EO gene, the E1 gene (native or secreted forms) and/or the E2 gene (native or secreted form) (Meyers G. et al., Virology, 1989, 171, 18–27).
With regard to the parvovirosis valency, it is preferred to use the VP2 capsid gene (Vasudevacharya J. et al., Virology, 1990, 178, 611–616).
With regard to the PCV2 valency, it is preferred to use the ORF1 and/or ORF2 genes, but preferably ORF1 and ORF2 (Meehan B. et al., J. Gen. Virol. 1998, 79, 2171–2179).
The heterologous sequences are inserted under the control of regulatory signals for transcription and in particular of a promoter, preferably introduced during the recombination. However, it is not excluded to express these heterologous sequences under the control of signals intrinsic to the adenovirus acting as the vector.
Among the useful promoters which are preferably used are strong eukaryotic promoters, such as the Rous sarcoma virus LTR and, preferably, the cytomegalovirus immediate-early (CMV-IE) promoter, in particular of murine origin (MCMV-IE) or human origin (HCMV-IE), or alternatively those of other origins, e.g. from pig, monkey, rat or guinea pig.
In a particularly advantageous manner, the CMV-IE promoters can be used with an enhancer (U.S. Pat. No. 5,168,062, U.S. Pat. No. 4,968,615, U.S. Pat. No. 4,963,481). It may be advantageous to use the fragments of these promoters, which conserve the promoter activity, preferably with the enhancer, e.g. the truncated CMV-IE promoters according to WO-A-98/00166. For further details, those skilled in the art may refer to WO-A-98/00166, and in particular example 12 therein.
The subject of the present invention is also the immunogenic or immunological preparations, and the porcine vaccines, comprising a recombinant adenovirus in accordance with the invention, in a vehicle or an excipient which is acceptable from a veterinary point of view, and optionally an adjuvant. By definition, an immunogenic or immunological preparation induces at least an immune response (cellular and/or humoral) after it has been administered to an animal. A vaccine induces a protective response.
The adjuvants are preferably chosen from acrylic or methacrylic acid polymers and copolymers of maleic anhydride and of an alkenyl derivative.
The preferred compounds are acrylic or methacrylic acid polymers which are crosslinked, in particular with polyalkenyl ethers of sugars or of polyalcohols. These compounds are known by the term “carbomer” (Pharmeuropa Vol. 8, No. 2, June 1996). Those skilled in the art may also refer to U.S. Pat. No. 2,909,462 which describes such acrylic polymers crosslinked with a polyhydroxy compound containing at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced with unsaturated aliphatic radicals containing 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 can, themselves, contain other substituents, such as methyl. The products sold under the name Carbopol® (BF Goodrich, Ohio, USA) are particularly suitable. They are crosslinked with an allyl sucrose or with allylpentaerythritol. Among these, mention may be made of Carbopol® 974P, 934P and 971P.
Among the copolymers of maleic anhydride and of an alkenyl derivative, preference is given to the EMAs® (Monsanto) which are copolymers of maleic anhydride and of ethylene, which may be linear or crosslinked, for example crosslinked with divinyl ether. Reference may be made to J. Fields et al., Nature, 186, 778–780, Jun. 4, 1960.
In terms of their structure, the acrylic or methacrylic acid polymers and the EMAs® are preferably formed from base units of the following formula:




in which:

    
    
        R1 and R2, which may be identical or different, represent H or CH3 
        X=0 or 1, preferably x=1
        Y=1 or 2, with x+y=2.
    
    


For the EMAs®, x=0 and y=2. For the carbomers, x=y=1.
Dissolution of these polymers in water gives an acidic solution which will be neutralized, preferably to physiological pH, to give the adjuvant solution into which the actual vaccine will be incorporated. The carboxylic groups of the polymer are then partly in COO form.
Preferably, a solution of adjuvant according to the invention, in particular of carbomer, is prepared in distilled water, preferably in the presence of sodium chloride, the solution obtained being at acid pH. This stock solution is diluted by adding it to the amount required (to obtain the final concentration desired), or a large proportion thereof, of water containing NaCl, preferably physiological saline (6 g/l NaCl), in one or more stages with concomitant or subsequent neutralization (pH 7.3 to 7.4), preferably with NaOH.
This solution at physiological pH will be used, without further modification, to take up the vaccine, which is in particular stored in lyophilized form.
The polymer concentration in the final immunization composition will be from 0.01% to 2% W/V, more particularly from 0.06 to 1% W/V, preferably from 0.1 to 0.6% W/V.
The immunogenic preparations and vaccines according to the invention can comprise several recombinant adenoviruses in accordance with the invention, comprising different heterologous nucleotide sequences originating from identical and/or different pathogens.
A subject of the present invention is also the methods for immunizing and for vaccinating pigs against one or more porcine pathogens, comprising the administration of effective doses of the above immunogenic preparations and vaccines. The doses will, for example, be from 1 to 107 CCID50.
The invention relates in particular to the administration of these immunogenic preparations and vaccines mucosally, e.g. orally, nasally or ocularly, and/or to young animals exhibiting maternal antibodies. The other routes conventionally used for vaccinating pigs (intramuscular in particular, intradermal, etc.) can also be used.
A subject of the present invention is also methods for obtaining recombinant PAV-3 and PAV-5 vectors, and immunogenic preparations and vaccines incorporating them.
A subject of the invention is also the use of these vectors for the manufacture of the immunogenic preparations and vaccines according to the invention, which are in particular intended for mucosal administration and/or for administration to young animals exhibiting maternal antibodies and/or for administration via the conventional routes.
A subject of the present invention is also a fragment of PAV-5 DNA which includes E3 and which, in the particular strain used here, has the nucleotide sequence referred to as SEQ ID NO. 5. The invention also relates to any fragment of this sequence, in particular any fragment which conserves the PAV-5 specificity. A subject of the invention is thus the sequence 2064 to 4083 and fragments thereof, in particular the E3 sequence of PAV-5 which, in the particular strain used here, is defined by nucleotides 2382 to 4042, and also fragments thereof, in particular all or part of the sequence from 2389 to 3861 in SEQ ID NO. 5. It goes without saying that the invention automatically covers the equivalent sequences, i.e. sequences which do not change the PAV-5 strain functionality or specificity of the sequence described or of the polypeptides encoded by this sequence. Needless to say, sequences which differ through degeneracy of the code will be included.
The invention also covers the sequences specific for PAV-5, which are equivalent in the sense that they are capable of hybridizing to the sequence above under highly stringent conditions and/or in that they have strong homology with the strains of the invention, especially homology greater than or equal to 85%, in particular to 90% and more particularly to 95%.
The invention will now be described in greater detail with the aid of the embodiments given by way of nonlimiting examples, and with reference to the drawing in which:



List of figures:
FIG. 1: Scheme of the pKS-Right ITR3, pPolyII Right ITR3 and pITRsPAV3 plasmids
FIG. 2: Scheme of the pE3PAV3, pE3dl622CMV-EGFP and pE3dl622CMV-gD plasmids
FIG. 3: Restriction map of the pE3PAV3 and pE3dl919 plasmids
FIG. 4: Scheme of the pCMV-EGFP, pCMV-gD and pgD plasmids
FIG. 5: Restriction map of the pCMV-EGFP, pgD, pE3dl919CMV-EGFP and pE3dl919CMV-gD plasmids
FIG. 6: Restriction map of the pE3dl622EGFP and pE3dl919EGFP plasmids
FIG. 7: Restriction map of the pE3dl622gD and pE3dl919CMV-gD plasmids
FIG. 8: Restriction map of the pCR-Right ITR5, pCR-Left ITR5, pPolyII-Right ITR5 and pITR-PAV5 plasmids
FIG. 9: Restriction map of the pE3PAV5 plasmid
FIG. 10: Restriction map of the pE3dln472CMV-EGFP and pE3dl1472CMV-gD plasmids
FIG. 11: Restriction map of the pE3dl1472EGFP and pE3dl1472gD plasmids
FIG. 12: Physical map of PAV-5, digested with BamHI
FIG. 13: Sequence of 5614 base pairs (E3 region and adjacent sequences of the PAV-5 virus)



SEQUENCE LISTING


    SEQ ID NO 1: Oligonucleotide ITRPAV3
    SEQ ID NO 2: Oligonucleotide T3
    SEQ ID NO 3: Oligonucleotide T7
    SEQ ID NO 4: Oligonucleotide ITRPAV5
    SEQ ID NO 5: Sequence of the E3 region of the PAV-5 virus


Examples
Example 1
Cells and Viruses
The cell culture media and the reagents were supplied by Gibco BRL. The media (Dulbecco's MEM with Glutamax-1 Cat # 61965-026) were supplemented with 1 mM sodium pyruvate (Cat # 11360-039), gentamycin (50 μg/ml, Cat # 15710-031) and 10% fetal calf serum (Cat # 10270-106 batch 40Q5774K).
PK-15 cells (ATCC No. CCL33) and ST cells (ATCC No. CRL 1756) were used for culturing the viruses.
The wild-type type 3 porcine adenovirus (=PAV-3), which is nonpathogenic in pigs, was obtained from the laboratory of Dr. Eva Nagy (University of Guelph, Pathobiology Department, 50 Stone Road Guelph, Ontario, Canada, NIG 2W1) (Reddy P. et al., Virus Res. 1996, 43, 99–109).
The wild-type type 5 porcine adenovirus (=PAV-5), which is nonpathogenic in pigs, was obtained from the laboratory of Dr. Tadashi Hirahara (Kyoto Biken Laboratories Inc., Division of Veterinary Microbiology, 24–16 Makishima-cho, Uji-shi, Kyoto, 611 Japan) (Hirahara T. et al., J. Vet. Sci. 1990, 52, 407–409). Culture and passage conditions: twice a week, the cells are trypsinized (2.5% trypsin solution (Sigma Cat # 044-5075) diluted 50-fold in Earle's balanced salt solution (Gibco-BRL Cat. # 14155-030)) and resuspended in culture medium (dilution 1:6). The cells are cultured at +37° C. in the presence of 5% CO2 before being recultured.
Example 2
Enzymes, Bacteria, Plasmids and Molecular Biology Techniques
The restriction enzymes, the Taq polymerase for the chain amplification reactions, and the other enzymes required for the various DNA modifications were supplied, respectively, by New England Biolabs Inc., Roche Diagnostics and Gibco-BRL.
All these enzymes were used according to the manufacturers' recommendations. The standard molecular biology techniques were performed as described in “Molecular Biology: A Laboratory Manual”, 2nd edition, (Sambrook et al., Cold Spring Harbor Laboratory, New York, 1989).
The BJ5183 competent bacteria (Hanahan D., J. Mol. Biol. 1983, 166, 557–580) were prepared as follows: 60 ml of a culture of these bacteria in the exponential phase were prepared according to the calcium chloride technique (Sambrook et al., 1989). They were harvested in a final volume of 2 ml of 50 mM CaCl2. 300 μl of this bacterial suspension were mixed with the various homologous-recombination partner DNAs contained in a volume of 100 μl of 100 mM Tris-HCl pH 7.4 and left in contact for 30 min on melting ice, and the bacteria were then treated with a heat shock at +45° C. for 2 minutes, left to stand on melting ice for 10 minutes and finally plated out on a selective medium.
Example 3
Extraction of PAV-3 and PAV-5 Viral DNA
The viruses are cultured and amplified in 10 cm2 Falcon flasks under the conditions of example 1. When the CPE is complete, the cells are harvested, lysed by 3 cycles of freezing/thawing and the viral suspension is then clarified by low-speed centrifugation. The PAV-3 and PAV-5 viruses are purified on a cesium chloride gradient (1.34 g/ml) and the viral band is harvested and purified and the viral DNAs are then obtained after treatment with proteinase K and extraction with phenol/chloroform (Sambrook et al., 1989). These DNAs are then used for the various cloning or polymerase chain amplification (PCA) steps.
Example 4
Construction of the “pITRs PAV3” Plasmid
The genomic DNA of the PAV-3 virus, prepared according to example 3, was digested with EcoRI and the 245-bp EcoRI D fragment was isolated after agarose gel electrophoresis. This fragment was then ligated with the pBlueScript KS plasmid (pBS-KS) (Stratagene Inc. La Jolla, Calif.) (GenBank # X52327), predigested with EcoRI and dephosphorylated, to give the plasmid referred to as “pKS-EcoDPAV3”.
A PCA reaction was carried out with the pKS-EcoDPAV3 plasmid matrix and with the following oligonucleotides:
ITRPAV3 (SEQ ID NO: 1) (50-mer):
5′CCTTAATTAAGGTAGGGATAACAGGGTAATCATCATCAATAATATACCGC 3′ and
T3 (SEQ ID NO: 2) (20-mer):
5′ ATTAACCCTCACTAAAGGGA 3′
The amplification product (367 bp) was treated with T4 DNA polymerase to make the ends blunt and was then digested with the ClaI enzyme and ligated with the pBS-KS plasmid, predigested with ClaI and EcoRV, to give the plasmid referred to as “pKS-Right ITR3” (FIG. 1).
The EcoRI D fragment was then freed from the pKS-Right ITR3 plasmid by digestion with the enzymes BamHI and KpnI, and the 353-bp BamHI-KpnI restriction fragment was ligated with the pPolyII plasmid (Lathe R. et al., Gene, 1987, 57, 193–201) (GenBank # G209113), predigested with BamHI and KpnI, to generate the plasmid referred to as “pPolyII-Right ITR3” (FIG. 1). The genomic DNA of the PAV-3 virus, prepared according to example 3, was digested with KpnI in order to isolate, after agarose gel electrophoresis, the 939-bp KpnI F terminal fragment. This fragment was ligated with the pBS-KS plasmid, predigested with KpnI and dephosphorylated, to give the plasmid referred to as “pKS-KpnFPAV3”.
A PCA reaction was then carried out with the pKS-KpnFPAV3 plasmid matrix and with the following oligonucleotides:
ITRPAV3 (SEQ ID NO: 1) (50-mer):
5 CCTTAATTAAGGTAGGGATAACAGGGTAATCATCATCAATAATATACCGC 3′
and
T7 (SEQ ID NO: 3):
5′ TAATACGACTCACTATAGGG 3′
The amplification product (1086 bp) was treated with T4 DNA polymerase to make the ends blunt and was then digested with the XhoI enzyme and ligated with the pPolyII-Right ITR3 plasmid, predigested with BglII, treated with T4 DNA polymerase and then digested with XhoI, to generate the plasmid referred to as “pITRsPAV3” (FIG. 1).
Example 5
Construction of the pPAV3 Plasmid
The pITRsPAV3 plasmid (example 4) was linearized by digestion with ClaI and then co-transformed in BJ5183 competent bacteria with the PAV-3 virus genomic DNA prepared according to example 2. This co-transformation made it possible to generate, by homologous recombination in Escherichia coli (Chartier C. et al., J. Virol. 1996, 70, 4805–4810), a plasmid containing the complete cloned genome of PAV-3. This plasmid was designated as “pPAV3”.
Example 6
Construction of the PAV-3 Shuttle Plasmids
6.1. Construction of the Plasmid with a Deletion of 622 Base Pairs (Small Deletion) in the E3 Region
The pNEB193 plasmid (New England BioLabs Cat # 305-1) was digested with PacI, treated with T4 DNA polymerase and then self-religated to generate the “pNEBPac-” plasmid.
The pPAV3 plasmid (example 5) was digested with KpnI and BamHI to isolate the 4344-bp KpnI-BamHI fragment containing the E3 region (Reddy et al., Virology, 1998, 251, 414–426). This fragment was ligated with the pNEBPac-plasmid, predigested with KpnI and BamHI, to give the “pE3PAV3” plasmid (FIG. 2). The sequence of the KpnI-BamHI fragment was checked and was found to be identical to the sequence published by Reddy P. et al. (Virus Research, 1995, 36, 97–106 and Virology, 1998, 251, 414–426) (GenBank AN # U10433 and AN # AF083132) Those skilled in the art may refer to these references.
The pEGFP-F plasmid (Chalfie M. et al., Science, 1994, 263, 802–805; Clontech Cat # 6074-1) was modified in order to completely remove the polylinker. This deletion was obtained by digestion with BamHI, followed by a treatment with T4 DNA polymerase. Self-religation of the vector thus modified generated the pCMV-EGFP plasmid. The pCMV-EGFP plasmid was then digested with the AsnI and MluI enzymes and then treated with T4 DNA polymerase, to isolate the 1.6-kbp AsnI (blunt end)-MluI (blunt end) fragment (=CMV-EGFP expression cassette). This fragment was ligated with the pE3PAV3 plasmid, predigested with BsrGI and SnaBI and treated with T4 DNA polymerase, to generate the pE3dl622CMV-EGFP plasmid (FIG. 2). The deletion created between the BsrGI and SnaBI sites of the E3 region of the PAV-3 virus is 622 base pairs in size. This deletion extends from nucleotides 1002 to 1624 of the sequence published by P. Reddy et al. (Virus Research, 1995, 36, 97–106) (GenBank AN # U10433).
The pCMV-gD plasmid (Ambriovic A. et al., Virology, 1997, 238, 327–335) was digested with SnaBI and ClaI to isolate the 1.8-kbp SnaBI-ClaI fragment (containing the 3′ portion of the CMV promoter and the PRV gD gene). This fragment was then treated with T4 DNa polymerase to make the ends blunt and was then ligated with the pE3dl622CMV-EGFP plasmid, predigested with SnaBI and HpaI, to generate the pE3dl622CMV-gD plasmid (FIG. 2).
6.2. Construction of the Plasmid with a Deletion of 919 Base Pairs (Large Deletion) in the E3 Region
The pE3PAV3 plasmid (see section 6.1. above) was digested with SgrAI and SacI to isolate the 644-bp SgrAI-SacI fragment. This fragment was treated with T4 DNA polymerase to make the ends blunt and was then ligated with the pNEBPac-plasmid (6.1. above), predigested with EcoRI, treated with T4 DNA polymerase and then digested with SacI, to generate the pSgrAI-SacI plasmid.
The pE3PAV3 plasmid was digested with SnaBI and HindIII to isolate the 2.4-kbp SnaBI-HindIII fragment. This fragment was ligated with the pSgrAI-SacI plasmid, predigested with PmeI and HindIII, to generate the pE3dl919 plasmid (FIG. 3). The deletion created between the SacI and SnaBI sites of the E3 region of the PAV-3 virus is 919 base pairs in size. This deletion extends from nucleotides 706 to 1624 of the sequence published by P. Reddy et al. (Virus Research, 1995, 36, 97–106) (GenBank AN # U10433).
The pCMV-gD plasmid (example 6.1) was digested with SnaBI and ClaI to isolate the 1.8-kbp SnaBI-ClaI fragment. This fragment was then treated with the Klenow fragment of DNA polymerase to make the ends blunt and was then ligated with the pCMV-EGFP plasmid (example 6.1), predigested with SnaBI and HpaI and then dephosphorylated, to generate the pgD plasmid (FIG. 4).
The pCMV-EGFP plasmid (example 6.1) was digested with AsnI and MluI to isolate the 1.8-kbp AsnI-MluI fragment (CMV-EGFP cassette). This fragment was treated with the Klenow fragment of DNA polymerase to make the ends blunt and was then ligated with the pE3dl919 plasmid, predigested with AscI, treated with the Klenow fragment of DNA polymerase to make the ends blunt and then dephosphorylated, to generate the pE3dl919CMV-EGFP plasmid (FIG. 5).
The pgD plasmid (above) was digested with AsnI and MluI to isolate the 2.3-kbp AsnI-MluI fragment. This fragment was treated with T4 DNA polymerase to make the ends blunt and was then ligated with the pE3dl919 plasmid, predigested with AscI and treated with the Klenow fragment of DNA polymerase to make the ends blunt and then dephosphorylated, to generate the pE3dl919CMV-gD plasmid (FIG. 5).
Example 7
Construction of the Recombinant Genomes pPAV3dl622CMV-EGFP and pPAV3dl622CMV-gD
The pE3dl622CMV-EGFP plasmid (example 6.1) was digested with KpnI and BamHI to isolate the 5.5-kbp KpnI-BamHI fragment. This fragment was co-transformed in BJ5183 competent bacteria with the pPAV3 plasmid (example 5) linearized by digestion with the SnaBI enzyme. This co-transformation led to the generation, by homologous recombination in Escherichia coli, of the pPAV3dl622CMV-EGFP plasmid.
The pE3CMVgD plasmid (example 6.1) was digested with EcoRI and PmeI to isolate the 6.0-kbp EcoRI-PmeI fragment. This fragment was co-transformed in BJ5183 competent bacteria with the pPAV3 plasmid (example 5) linearized by digestion with the SnaBI enzyme. This co-transformation led to the generation, by homologous recombination in Escherichia coli, of the pPAV3dl622CMVgD plasmid.
Example 8
Construction of the Recombinant Genomes pPAV3dl919CMV-EGFP and pPAV3dl919CMV-gD
The pE3dl919CMV-EGFP plasmid (example 6.2) was linearized with HindIII and then co-transformed in BJ5183 competent bacteria with the pPAV3 plasmid (example 5) linearized by digestion with the SnaBI enzyme. This co-transformation led to the generation, by homologous recombination in Escherichia coli, of the pPAV3dl919CMV-EGFP plasmid.
The pE3dl919CMV-gD plasmid (example 6.2) was linearized with HindIII and then co-transformed in BJ5183 competent bacteria with the pPAV3 plasmid (example 5) linearized by digestion with the SnaBI enzyme. This co-transformation led to the generation, by homologous recombination in Escherichia coli, of the pPAV3dl919CMV-gD plasmid.
Example 9
Construction of the Recombinant Genomes pPAV3dl622EGFP, pPAV3dl622gD, pPAV3dl919EGFP and pPAV3dl919gD (Insertion of the Coding Sequences without the CMV Promoter Sequences)
9.1. Construction of the pE3dl622EGFP and pE3dl919EGFP Plasmids
The pCMV-EGFP plasmid (example 6.1) was digested with NheI and MluI to isolate the 1.0-kbp NheI-MluI fragment. This fragment was treated with the Klenow fragment of DNA polymerase to make the ends blunt and then ligated with the pE3PAV3 plasmid (example 6.1), predigested with BsrGI, treated with the Klenow fragment of DNA polymerase and finally digested with SnaBI, to generate the pE3dl622EGFP plasmid (FIG. 6).
The pCMV-EGFP plasmid was digested with NheI and MluI to isolate the 1.0-kbp NheI-MluI fragment. This fragment was treated with the Klenow fragment of DNA polymerase to make the ends blunt and was then ligated with the pE3dl919 plasmid, predigested with AscI, treated with the Klenow fragment of DNA polymerase and then dephosphorylated, to generate the pE3dl919EGFP plasmid (FIG. 6).
9.2. Construction of the pE3dl622gD and pE3dl919gD Plasmids
The pgD plasmid (example 6.2) was digested with XhoI and ClaI to isolate the 1.5-kbp XhoI-ClaI fragment. This fragment was treated with the Klenow fragment of DNA polymerase to make the ends blunt and was then ligated with the pE3PAV3 plasmid (example 6.1), predigested with BsrGI, treated with the Klenow fragment of DNA polymerase and then digested with SnaBI, to generate the pE3dl622gD plasmid (FIG. 7).
The pgD plasmid (example 6.2) was digested with XhoI and ClaI to isolate the 1.5-kbp XhoI-ClaI fragment. This fragment was treated with the Klenow fragment of DNA polymerase to make the ends blunt and was then ligated with the pE3dl919 plasmid (example 6.2), predigested with AscI, treated with the Klenow fragment of DNA polymerase and finally dephosphorylated, to generate the pE3dl919gD plasmid (FIG. 7).
9.3. Construction of the pPAV73dl622EGFP, pPAV3dl622gD, pPAV3dl919EGFP and pPAV3dl919gD Plasmids
The pE3dl622EGFP plasmid (example 9.1) was digested with KpnI and BamHI and then co-transformed in BJ5183 competent bacteria with the pPAV3 plasmid (example 5), prelinearized by digestion with SnaBI. This co-transformation made it possible to generate, by homologous recombination in Escherichia coli, the pPAV3dl622EGFP plasmid.
The pE3dl622gD plasmid (example 9.2) was digested with EcoRI and PmeI and then co-transformed in BJ5183 competent bacteria with the pPAV3 plasmid (example 5), prelinearized by digestion with SnaBI. This co-transformation generated, by homologous recombination in Escherichia coli, the pPAV3dl622gD plasmid.
The pE3dl919EGFP plasmid (example 9.1) was linearized by digestion with HindIII and then co-transformed in BJ5183 competent bacteria with the pPAV3 plasmid (example 5), prelinearized by digestion with SnaBI. This co-transformation generated, by homologous recombination in Escherichia coli, the pPAV3dl919EGFP plasmid.
The pE3dl919gD plasmid (example 9.2) was linearized by digestion with HindIII and then co-transformed in BJ5183 competent bacteria with the pPAV3 plasmid (example 5), prelinearized by digestion with SnaBI. This co-transformation generated, by homologous recombination in Escherichia coli, the pPAV3dl919gD plasmid.
Example 10
Production of Recombinant PAV3 Viruses by Transfection with the Recombinant PAV3 Genomes Cloned into Escherichia coli 
PK-15 or ST cells (see example 1) are transfected in 6-well plates to a confluence density of 60–80% with 2 μg of recombinant PAV-3 genomic DNA, predigested with PacI, diluted in the presence of 4 to 12 μl of LipofectAMINE (Gibco-BRL Cat # 18324-012) depending on the cell line.
After transfection according to the conditions recommended by the supplier, the cells are left in culture for a few days until the appearance of a viral CPE. A further cell culture passage is then carried out to amplify the recombinant virus obtained.

    
    
        The transfection carried out with the pPAV3dl622CMV-EGFP plasmid (example 7) generated the recombinant virus vPAV3-1.
        The transfection carried out with the pPAV3dl622CMV-gD plasmid (example 7) generated the recombinant virus vPAV3-2.
        The transfection carried out with the pPAV3dl919CMV-EGFP plasmid (example 8) generated the recombinant virus vPAV3-3.
        The transfection carried out with the pPAV3dl919CMV-gD plasmid (example 8) generated the recombinant virus vPAV3-4.
        The transfection carried out with the pPAV3dl622EGFP plasmid (example 9.3) generated the recombinant virus vPAV3-5.
        The transfection carried out with the pPAV3dl622gD plasmid (example 9.3) generated the recombinant virus vPAV3-6.
        The transfection carried out with the pPAV3dl919EGFP plasmid (example 9.3) generated the recombinant virus vPAV3-7.
        The transfection carried out with the pPAV3dl919gD plasmid (example 9.3) generated the recombinant virus vPAV3-8.
    
    


Example 11
Construction of the pITRsPAV5 Plasmid
11.1. Cloning of the Right-Terminal Genomic Fragment
The genomic DNA of the PAV-5 virus, prepared according to example 3, was digested with EcoRI and the 0.9-kbp EcoRI I right-terminal fragment was isolated after agarose gel electrophoresis. This fragment was ligated with the pBS-KS plasmid, predigested with EcoRI and dephosphorylated, to generate the “pKS-Right ITR5” plasmid.
A PCA reaction was then carried out with the pKS-Right ITR5 plasmid matrix and with the following oligonucleotides:
ITRPAV5 (SEQ ID NO: 4) (50-mer):
5′CCTTAATTAAGGTAGGGATAACAGGGTAATCATCATCAATAATATACGGA 3′
and T3 (SEQ ID NO: 2) (20-mer)
5′ ATTAACCCTCACTAAAGGGA 3′
to produce a 1.0-kbp fragment. This fragment was ligated with the pCRII plasmid (InVitrogen Original TA Cloning Kit Cat # K2000-01) to give the “pCR-Right ITR5” plasmid (FIG. 8).
11.2. Cloning of the Left-Terminal Genomic Fragment
The genomic DNA of the PAV-5 virus, prepared according to example 3, was digested with HindIII and the 1.2-kbp HindIII left-terminal fragment was isolated after agarose gel electrophoresis. This fragment was ligated with the pBS-KS plasmid, predigested with HindIII and dephosphorylated, to generate the “pKS-Left ITR5” plasmid.
A PCA reaction was then carried out with the pKS-Left ITR5 plasmid matrix and with the following oligonucleotides:
ITRPAV5 (SEQ ID NO: 4):
5′CCTTAATTAAGGTAGGGATAACAGGGTAATCATCATCAATAATATACGGA 3′
and T7 (SEQ ID NO: 3 20-mer):
5′ TAATACGACTCACTATAGGG 3′
to produce a 1.3-kbp fragment. This fragment was ligated with the pCRII plasmid to give the “pCR-Left ITR5” plasmid (FIG. 8).
11.3. Construction of the pPolyII-Right ITR5 Plasmid
The pCR-Right ITR5 plasmid (example 11.1) was digested with BamHI and HindIII to isolate the 1.0-kbp BamHI-HindIII fragment. This fragment was then ligated with the pPolyII plasmid (see example 4), predigested with BamHI and HindIII, to generate the “pPolyII-Right ITR5” plasmid (FIG. 8).
11.4. Construction of the pITRsPAV5 Plasmid
The pCR-Left ITR5 plasmid (example 11.2) was digested with BamHI and HindIII to isolate the 1.3-kbp BamHI-HindIII fragment. This fragment was then ligated with the pPolyII-Right ITR5 plasmid, predigested with BglII and HindIII, to generate the “pITRs PAV5” plasmid (FIG. 8).
Example 12
Construction of the pPAV5 Plasmid
The pITRsPAV5 plasmid was linearized by digestion with HindIII. The fragment obtained was then co-transformed in BJ5183 competent bacteria with the genomic DNA of the PAV-5 virus prepared according to example 3. This co-transformation generated, by homologous recombination, a plasmid containing all of the genome of the PAV-5 virus. This plasmid was denoted as “pPAV5”.
Example 13
Construction of the PAV-5 Shuttle Plasmids
13.1. Construction of the E3 PAV-5 Shuttle Plasmid
The pPAV5 plasmid (example 12) was digested with SacI and MluI to isolate the 4372-bp SacI-MluI fragment containing the E3 region. This fragment was ligated with the pNEB193 plasmid (example 6.1), predigested with SmaI and AscI, to give the “pE3PAV5” plasmid (FIG. 9).
The SacI-MluI fragment cloned into the pE3PAV5 plasmid was entirely sequenced and analyzed. The analysis of this sequence and of the adjacent sequences present on the pPAV5 plasmid confirmed that the cloned fragment did indeed represent the E3 region of the PAV-5 virus. The sequence of this region (5614 base pairs) (SEQ ID NO: 5) is given in FIG. 13.
13.2. Construction of the Donor CMV-EGFP and CMV-PRV gD Plasmids with a Deletion of 1472 Base Pairs in the E3 Region.
The pCMV-EGFP plasmid (example 6.1) was digested with the AsnI and MluI enzymes and then treated with T4 DNA polymerase to isolate the 1.6-kbp AsnI (blunt end)-MluI (blunt end) fragment. This fragment was ligated with the pE3PAV5 plasmid (example 13.1), predigested with PvuII and BglI, and treated with T4 DNA polymerase, to generate the pE3dl1472CMV-EGFP plasmid (FIG. 10). The deletion introduced between the PvuII and BglI sites is 1472 bp in size. This deletion is between nucleotides 2389 and 3861 of the sequence given in FIG. 13 (SEQ ID NO: 5).
The pgD plasmid (example 6.1) was digested with the AsnI and MluI enzymes to isolate the 2.3-kbp AsnI-MluI fragment. This fragment was then treated with T4 DNA polymerase to make the ends blunt and was then ligated with the pE3PAV5 plasmid, predigested with PvuII and BglI, and treated with T4 DNA polymerase, to generate the pE3dl1472CMVgD plasmid (FIG. 10).
The pCMV-EGFP plasmid was digested with NheI and MluI to isolate the 1.0-kbp NheI-MluI fragment. This fragment was treated with the Klenow fragment of DNA polymerase to make the ends blunt and was then ligated with the pE3PAV5 plasmid, predigested with PvuII and BglI, and treated with T4 DNA polymerase, to generate the pE3dl1472EGFP plasmid (FIG. 11).
The pCMVgD plasmid (example 6.1) was digested with XhoI and ClaI to isolate the 1.5-kbp XhoI-ClaI fragment. This fragment was treated with the Klenow fragment of DNA polymerase to make the ends blunt and was then ligated with the pE3PAV5 plasmid, predigested with PvuII and BglI, and treated with T4 DNA polymerase, to generate the pE3dl1472gD plasmid (FIG. 12).
Example 14
Construction of the Recombinant Genomes pPAV5dl1472CMV-EGFP and pPAV5dl1472CMV-gD
The pE3dl1472CMV-EGFP plasmid (example 13.2) was linearized with PmeI and then co-transformed in BJ5183 competent bacteria with the pPAV5 plasmid (example 12) linearized by partial digestion with the BamHI enzyme (site of joining of the restriction subfragments BamHI A-BamHI D, FIG. 12). This co-transformation generated, by homologous recombination in Escherichia coli, the pPAV5dl1472CMV-EGFP plasmid.
The pE3dl1472CMVgD plasmid (example 13.2) was linearized with PmeI and then co-transformed in BJ5183 competent bacteria with the pPAV5 plasmid (example 12) linearized by partial digestion with the BamHI enzyme (site of joining of the restriction subfragments BamHI A-BamHI D, FIG. 12). This co-transformation generated, by homologous recombination in Escherichia coli, the pPAV5dl1472CMV-gD plasmid.
Example 15
Construction of the Recombinant Genomes pPAV5dl1472EGFP and pPAV5dl1472 gD (Insertion of the Coding Sequences without the CMV Promoter Sequences)
The pE3dl1472EGFP plasmid (example 13.2) was linearized with PmeI and then co-transformed in BJ5183 competent bacteria with the pPAV5 plasmid (example 12) linearized by partial digestion with the BamHI enzyme (site of joining of the restriction subfragments BamHI A-BamHI D, FIG. 12). This co-transformation generated, by homologous recombination in Escherichia coli, the pPAV5dl1472EGFP plasmid.
The pE3dl1472gD plasmid (example 13.2) was linearized with PmeI and then co-transformed in BJ5183 competent bacteria with the pPAV5 plasmid (example 12) linearized by partial digestion with the BamHI enzyme (site of joining of the restriction subfragments BamHI A-BamHI D, FIG. 12). This co-transformation generated, by homologous recombination in Escherichia coli, the pPAV5dl1472gD plasmid.
Example 16
Production of Recombinant PAV-5 Viruses by Transfection with the Recombinant PAV-5 Genomes Cloned into Escherichia coli 
PK-15 or ST cells (see example 1) are transfected in 6-well plates to a confluence density of 60–80% with 2 μg of recombinant PAV-5 genomic DNA, predigested with PacI, diluted in the presence of 4 to 12 μl of LipofectAMINE (Gibco-BRL Cat # 18324-012) depending on the cell lines.
After transfection according to the conditions recommended by the supplier, the cells are left in culture for a few days until the appearance of a viral CPE. A passage is then carried out to amplify the recombinant virus obtained.

    
    
        The transfection carried out with the pPAV5dl1472CMV-EGFP plasmid (example 14) generated the recombinant virus vPAV5-1.
        The transfection carried out with the pPAV5dl1472CMV-gD plasmid (example 14) generated the recombinant virus vPAV5-2.
        The transfection carried out with the pPAV5dl1472EGFP plasmid (example 15) generated the recombinant virus vPAV5-3.
        The transfection carried out with the pPAV5dl1472gD plasmid (example 15) generated the recombinant virus vPAV5-4.
    
    


Example 19
Manufacture of the Vaccines According to the Invention
The recombinant viruses obtained according to the invention are cultured and amplified in PK-15 cell cultures. The supernatants are harvested after a complete cytopathogenic effect has been observed. After clarification by low-speed centrifugation to remove the cell debris, followed by centrifugation to concentrate the virions and washing of the viral pellet, the pellet is taken up in a solution of culture medium. The viral titer of these suspensions is measured. The viral titer is then optionally adjusted by dilution so as to obtain a final viral titer of between 104 50% cell culture infective doses (CCID50) and 107 CCID50/ml.
The viral suspensions can then be frozen, or preferably lyophilized in the presence of a lyophilization substrate.
Example 20
Vaccination of Pigs
After thawing or after taking up the lyophilized doses in water for injectable preparations, and optional addition of an adjuvant, pigs are vaccinated with doses of from 104 to 107 CCID50 of each recombinant virus. The vaccines comprise one or more recombinant PAV viruses each expressing different immunogens.
The vaccines are administered either by injection with a needle (intramuscular route) in a volume of 2 ml, or mucosally (intranasally or by ocular instillation) (volumes adapted to each route).
The intradermal injections can also be carried out using a needle-free injector such as the PIGJET device (Société Endoscoptic, Laon, France).
Example 21
Protocol for Vaccination/Testing for Pseudorabies
A vaccination/testing protocol was used to evaluate the efficacy of a vaccine consisting of a suspension of the adjuvant-free recombinant vPAV3-2 virus (example 10 of the present invention).
Groups of six 8–10 week-old piglets, born from sows which had been vaccinated against pseudorabies virus (PRV) at the start of gestation, were established. All the piglets definitely had maternal anti-PRV antibodies on day D0 of the vaccination. The piglets of groups 1 and 2 were vaccinated on D0 intranasally with 1 milliliter (ml) of a suspension of the recombinant virus vPAV3-2 (PAV-3/PRV gD) having a titer of 107 CCID50/ml (0.5 ml per nostril). Group 2 was vaccinated in the same way, but also received a second administration on D21. Group 3 received on D0, intramuscularly, 1 ml of a viral suspension of PAV-3/gD with a titer of 107 CCID50/ml. Group 4 was vaccinated in the same way as Group 3, but received a second injection of viral suspension on D21. Group 5 was not vaccinated and served as a negative control group for the test.
All the groups were tested on D35 by administration of 2 ml (1 ml per nostril) of a suspension of the PRV NIA3 strain at a titer of at least 107.3 pfu/ml.
After testing, the pigs were monitored on the basis of the criteria of weight change (delta G7) (Stellmann C. et al., J. Biol. Standard, 1989, 17, 1–11) (D0 to D7 after testing) and of the level of viral excretion via the nasal mucosae, and on the basis of ELISA antibody titers and anti-PRV seroneutralizing titers.
It should be clearly understood that the invention defined by the attached claims is not limited to the specific embodiments indicated in the description above, but encompasses the variants which depart neither from the context nor from the spirit of the present invention.

## Claims

1. A recombinant in vivo replication competent porcine adenovirus (PAV) serotype 5 (PAV-5) which contains at least one heterologous nucleic acid molecule encoding at least one product in the E3 region of the PAV-5 genome, whereby the recombinant PAV replicates in vivo and expresses the product.

2. The recombinant PAV according to claim 1, wherein the PAV has a deletion in the E3 region.

3. A recombinant in vivo replication competent porcine adenovirus (PAV) serotype 5 (PAV-5) which contains at least one heterologous nucleic acid molecule encoding at least one product in the E3 region of the PAV-5 genome, wherein the heterologous nucleic acid molecule is within nucleotides 2064 to 4083 in SEQ ID NO: 5 and whereby the recombinant PAV replicates in vivo and expresses the product.

4. The recombinant PAV of claim 3, wherein the heterologous nucleic acid molecule is within nucleotides 2389 to 3861 in SEQ ID NO: 5.

5. The recombinant PAV of claim 3, wherein the heterologous nucleic acid molecule is within nucleotides 2382 to 4042 in SEQ ID NO: 5.

6. A recombinant in vivo replication competent porcine adenovirus (PAV) serotype 5 (PAV-5) which contains at least one heterologous nucleic acid molecule encoding at least one product in the E3 region of the PAV-5 genome, wherein the heterologous nucleic acid molecule is in place of nucleotides 2389–3861 in SEQ ID NO: 5 and whereby the recombinant PAV replicates in vivo and expresses the product.

7. The recombinant PAV of claim 6, wherein the heterologous nucleic acid molecule is in place of nucleotides 2064–4083 in SEQ ID NO: 5.

8. The recombinant PAV of claim 6, wherein the heterologous nucleic acid molecule is in place of nucleotides 2382–4042 in SEQ ID NO: 5.

9. The recombinant PAV according to any of claims 1, 3 or 6, wherein the heterologous nucleic acid molecule encodes an immunogen or an immunologically active fragment thereof or an epitope thereof, of a porcine pathogen; or an immunomodulator; or an immunogen or an immunologically active fragment thereof or an epitope thereof, of a porcine pathogen, and an immunomodulator.

10. The recombinant PAV according to any of claims 1, 3 or 6, containing more than one heterologous nucleic acid molecule.

11. The recombinant PAV according to any of claims 1, 3 or 6, wherein the heterologous nucleic acid molecule encodes a polyepitope.

12. The recombinant PAV according to any of claims 1, 3 or 6, containing an IRES sequence.

13. The recombinant PAV according to any of claims 1, 3 or 6, wherein the heterologous nucleic acid molecule encodes a product from a porcine pathogenic agent selected from the group consisting of viruses, bacteria and parasites.

14. The recombinant PAV according to claim 13, wherein the porcine pathogenic agent is selected from the group consisting of pseudorabies virus, swine influenza virus, porcine reproductive and respiratory syndrome virus, parvovirus virus, Hog Cholera Virus, porcine circovirus type 2, Actinobacillus pleuropneumoniae and Mycoplasma hyopneumoniae.

15. The recombinant PAV according to claim 13, wherein the porcine pathogenic agent is pseudorabies virus.

16. The recombinant PAV according to claim 13, wherein the porcine pathogenic agent is swine influenza virus.

17. The recombinant PAV according to claim 13, wherein the porcine pathogenic agent is porcine reproductive and respiratory syndrome virus.

18. The recombinant PAV according to claim 13, wherein the porcine pathogenic agent is parvovirus.

19. The recombinant PAV according to claim 13, wherein the porcine pathogenic agent is Hog Cholera Virus.

20. The recombinant PAV according to claim 13, wherein the porcine pathogenic agent is porcine circovirus type 2.

21. The recombinant PAV according to claim 13, wherein the porcine pathogenic agent is Actinobacillus pleuropneumoniae.

22. The recombinant PAV according to claim 13, wherein the porcine pathogenic agent is Mycoplasma hyopneumoniae.

23. The recombinant PAV according to claim 13, wherein the heterologous nucleic acid molecule(s) encode(s): pseudorabies virus gB, pseudorabies virus gC, pseudorabies virus gD, swine influenza HA, swine influenza NA, swine influenza NP, ORF4 of porcine reproductive and respiratory syndrome virus, ORF7 gene porcine reproductive and respiratory syndrome virus, Hog Cholera Virus E1, Hog Cholera Virus E2 gene, parvovirus VP2, porcine circovirus type 2 ORF1, or porcine circovirus type 2 ORF2.

24. The recombinant PAV according to claim 13, wherein the heterologous nucleic acid molecule(s) encode(s) an immunologically active fragment(s) or epitope(s) of: pseudorabies virus gB, pseudorabies virus gC, pseudeorabies virus gD, swine influenza HA, swine influenza NA, swine influenza NP, ORF4 of porcine reproductive and respiratory syndrome virus, ORF7 gene porcine reproductive and respiratory syndrome virus, Hog Cholera Virus E1, Hog Cholera Virus E2 gene, parvovirus VP2, porcine circovirus type 2 ORF1, or porcine circovirus type 2 ORF2.

25. The recombinant PAV according to claim 13, wherein the heterologous nucleic acid molecule(s) encode(s) porcine circovirus type 2 (PCV2) ORF1 or PCV2 ORF2 or PCV2 ORF 1 and PCV ORF2.

26. The recombinant PAV according to claim 13, wherein the heterologous nucleic acid molecule(s) encode(s) pseudorabies virus gB or pseudorabies virus gC or pseudorabies virus gD or pseudorabies virus gB and gC or pseudorabies virus gB, gC and gD.

27. The recombinant PAV according to claim 13, wherein the heterologous nucleic acid molecule(s) encode swine influenza HA or swine influenza NA or swine influenza NP or swine influenza HA and NA and NP.

28. The recombinant PAV according to claim 13, wherein the heterologous nucleic acid molecule encodes porcine parvovirus VP2.

29. The recombinant PAV according to claim 13, wherein the heterologous nucleic acid molecule encodes porcine respiratory and reproductive virus ORF6.

30. The recombinant PAV according to claim 13, wherein the heterologous nucleic acid molecule encodes Hog Cholera Virus E1 or Hog Cholera Virus E1 and E2.

31. The recombinant PAV according to any of claims 1, 3 or 6, wherein the heterologous nucleic acid molecule encodes a porcine cytokine.

32. The recombinant PAV according to claim 31, wherein the porcine cytokine is selected from the group consisting of GM-CSF, IL-4, IL-12 and IL-18.

33. A porcine vaccine comprising at least one recombinant PAV according to claim any of claims 1, 3 or 6, and a veterinarily acceptable vehicle or excipient.

34. The porcine vaccine of claim 33, comprising more than one recombinant PAV, wherein the recombinant PAVs comprise different heterologous nucleic acid molecules and a veterinarily acceptable vehicle or excipient.

35. The porcine vaccine according to claim 33, which contains an adjuvant.

36. The porcine vaccine according to claim 35, wherein the adjuvant is an acrylic or methacrylic acid polymer, or a copolymer of maleic anhydride and an alkenyl derivative.

37. The porcine vaccine of claim 36, wherein the adjuvant is a carbomer.

38. The porcine vaccine of claim 36, wherein the adjuvant is EMA.

39. An in vivo replication competent porcine adenovirus (PAV) serotype 5 (PAV-5) comprising a nucleotide sequence as depicted in SEQ ID NO: 5, which contains at least one deletion in the E3 region of the PAV-5 genome, whereby the recombinant PAV replicates in vivo.

40. The recombinant PAV of claim 39, wherein the deletion is in a region comprising nucleotides 2389 to 3861 in SEQ ID NO: 5.

41. The recombinant PAV of claim 39, wherein the deletion is of nucleotides 2389 and 3861 in SEQ ID NO: 5.

42. The recombinant PAV of claim 39, wherein the deletion is in a region comprising nucleotides 2064 to 4083 in SEQ ID NO: 5.

43. The recombinant PAV of claim 39, wherein the deletion is of nucleotides 2064 and 4083 in SEQ ID NO: 5.

44. The recombinant PAV of claim 39, wherein the deletion is in a region comprising nucleotides 2382 to 4042 in SEQ ID NO: 5.

45. The recombinant PAV of claim 39, wherein the deletion is of nucleotides 2382 and 4042 in SEQ ID NO: 5.