Polynucleotide formula against porcine reproductive and respiratory pathologies

The present invention relates to a vaccine formula allowing in particular the vaccination of pigs against reproductive and respiratory pathologies. It also relates to a corresponding method of vaccination.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
 Not applicable.
 BACKGROUND OF THE INVENTION
 1. Field of the Invention
 The present invention relates to a vaccine formula allowing in particular
 the vaccination of pigs against reproductive and respiratory pathologies.
 It also relates to a corresponding method of vaccination.
 2. Description of Related Art including Information Disclosed under 37
 C.F.R. .sctn. 1.97 AND 37 C.F.R. .sctn. 1.98
 During the past decades, the methods for the production of pigs have
 changed fundamentally. The intensive breeding in an enclosed space has
 become generalized with, as a corollary, the dramatic development of
 respiratory pathologies.
 The range of symptoms of porcine respiratory pathology is in general
 grouped under the complex name of pig respiratory disease and involves a
 wide variety of pathogenic agents comprising viruses as well as bacteria
 and mycoplasmas.
 The principal agents involved in the respiratory disorders are
 Actinobacillus pleuropneumoniae, the infertility and respiratory syndrome
 virus (PRRS) also called mysterious disease virus, the Aujeszky's disease
 virus (PRV) and the swine flu virus.
 Other viruses cause reproductive disorders leading to abortions,
 mummifications of the fetus and infertility. The principal viruses are
 PRRS, the parvovirus and the conventional hog cholera virus (HCV).
 Secondarily, the swine flu virus PRV and A. pleuropneumoniae can also
 cause such disorders. Deaths may occur with A. pleuropneumoniae, HCV and
 PRV.
 In addition, interactions between microorganisms are very important in the
 porcine respiratory complex. Indeed, most of the bacterial pathogens are
 habitual hosts of the nasopharangeal zones and of the tonsils in young
 animals. These pathogens, which are derived from the sows, are often
 inhaled by the young pigs during their first few hours of life, before the
 cholostral immunity has become effective. The organisms living in the
 upper respiratory tract may invade the lower tract when the respiratory
 defense mechanisms of the host are damaged by a precursor agent such as A.
 pleuropneumoniae or by viruses. The pulmonary invasion may be very rapid,
 in particular in the case of precursor pathogens such as A.
 pleuropneumoniae which produce potent cytotoxins capable of damaging the
 cilia of the respiratory epithelial cells and the alveolar macrophages.
 Major viral infections, such as influenza, and respiratory coronavirus and
 Aujeszky's virus infections, may play a role in the pathogenicity of the
 respiratory complex, besides bacteria with respiratory tropism and
 mycoplasmas.
 Finally, some agents have both a respiratory and a reproductive effect.
 Interactions may also occur from the point of view of the pathology of
 reproduction.
 It therefore appears to be necessary to try to develop an effective
 prevention against the principal pathogenic agents involved in porcine
 reproductive and respiratory pathologies.
 The associations developed so far were prepared from inactivated vaccines
 or live vaccines and, optionally, mixtures of such vaccines. Their
 development poses problems of compatibility between valencies and of
 stability. It is indeed necessary to ensure both the compatibility between
 the different vaccine valencies, whether from the point of view of the
 different antigens used from the point of view of the formulations
 themselves, especially in the case where both inactivated vaccines and
 live vaccines are combined. The problem of the conservation of such
 combined vaccines and also of their safety especially in the presence of
 an adjuvant also exists. These vaccines are in general quite expensive.
 Patent applications WO-A-90 11092, WO-A-93 19183, WO-A-94 21797 and WO-A-95
 20660 have made use of the recently developed technique of polynucleotide
 vaccines. It is known that these vaccines use a plasmid capable of
 expressing, in the host cells, the antigen inserted into the plasmid. All
 the routes of administration have been proposed (intraperitoneal,
 intravenous, intramuscular, transcutaneous, intradermal, mucosal and the
 like). Various vaccination means can also be used, such as DNA deposited
 at the surface of gold particles and projected so as to penetrate into the
 animals' skin (Tang et al., Nature, 356, 152-154, 1992) and liquid jet
 injectors which make it possible to transfect at the same time the skin,
 the muscle, the fatty tissues and the mammary tissues (Furth et al.,
 Analytical Biochemistry, 205, 365-368, 1992). (See also U.S. Pat. Nos.
 5,846,946, 5,620,896, 5,643,578, 5,580,589, 5,589,466, 5,693,622, and
 5,703,055; Science, 259:1745-49, 1993; Robinson et al., seminars in
 IMMUNOLOGY, 9:271-83, 1997; Luke et al., J. Infect. Dis. 175(1):91-97,
 1997; Norman et al., Vaccine, 15(8):801-803, 1997; Bourne et al., The
 Journal of Infectious Disease, 173:800-7, 1996; and, note that generally a
 plasmid for a vaccine or immunological composition can comprise DNA
 encoding an antigen operatively linked to regulatory sequences which
 control expression or expression and secretion of the antigen from a host
 cell, e.g., a mammalian cell; for instance, from upstream to downstream,
 DNA for a promoter, DNA for a eukaryotic leader peptide for secretion, DNA
 for the antigen, and DNA encoding a terminator.)
 The polynucleotide vaccines may also use both naked DNAs and DNAs
 formulated, for example, inside cationic lipid liposomes.
 M-F Le Potier et al., (Second International Symposium on the Eradication of
 Aujeszky's Disease (pseudorabies) Virus Aug. 6th to 8 th 1995 Copenhagen,
 Denmark) and M. Monteil et al., (Les Journees d'Animation Scientifique du
 Departement de Pathologie Animale [Scientific meeting organized by the
 department of animal pathology], INRA-ENV, Ecole Nationale Veterinaire,
 LYON, Dec. 13-14, 1994) have tried to vaccinate pigs against the
 Aujeszky's disease virus with the aid of a plasmid allowing the expression
 of the gD gene under the control of a strong promoter, the type 2
 adenovirus major late promoter. In spite of a good antibody response
 level, no protection could be detected. Now, satisfactory results in the
 area of protection have been recorded after inoculation of pigs with a
 recombinant adenovirus into which the gD gene and the same promoter have
 been inserted, proving that the gD glcyoprotein could be sufficient for
 inducing protection in pigs.
 The prior art gives no protective result in pigs by the polynucleotide
 vaccination method.
 BRIEF SUMMARY OF THE INVENTION
 The invention proposes to provide a multivalent vaccine formula which makes
 it possible to ensure vaccination of pigs against a number of pathogenic
 agents involved in particular in respiratory pathology and/or in
 reproductive pathology.
 Another objective of the invention is to provide such a vaccine formula
 combining different valencies while exhibiting all the criteria required
 for mutual compatibility and stability of the valencies.
 Another objective of the invention is to provide such a vaccine formula
 which makes it possible to combine different valencies in the same
 vehicle.
 Another objective of the invention is to provide such a vaccine formula
 which is easy and inexpensive to use.
 Yet another objective of the invention is to provide such a vaccine formula
 and a method for vaccinating pigs which makes it possible to obtain
 protection, including multivalent protection, with a high level of
 efficiency and of long duration, as well as good safety and an absence of
 residues.

DETAILED DESCRIPTION OF THE INVENTION
 The subject of the present invention is therefore a vaccine formula in
 particular against porcine reproductive and/or respiratory pathology,
 comprising at least 3 polynucleotide vaccine valencies each comprising a
 plasmid integrating, so as to express it in vivo in the host cells, a gene
 with one porcine pathogen valency, these valencies being selected from
 those of the group consisting of Aujeszky's disease virus (PRV or
 pseudorabies virus), swine flu virus (swine influenza virus, SIV), pig
 mysterious disease virus (PRRS virus), parvovirosis virus (PPV virus),
 conventional hog cholera virus (HCV virus) and bacterium responsible for
 actinobacillosis (A. pleuropneumoniae), the plasmids comprising, for each
 valency, one or more of the genes selected from the group consisting of gB
 and gD for the Aujeszky's disease virus, HA, NP and N for the swine flu
 virus, ORF5 (E), ORF3, ORF6 (M) for the PRRS virus, VP2 for the
 parvovirosis virus, E1, E2 for the conventional hog cholera virus and
 apxI, apxII and apxIII for A. pleuropneumoniae.
 Valency in the present invention is understood to mean at least one antigen
 providing protection against the virus for the pathogen considered, it
 being possible for the valency to contain, as subvalency, one or more
 modified natural genes from one or more strains of the pathogen
 considered.
 Pathogenic agent gene is understood to mean not only the complete gene but
 also the various nucleotide sequences, including fragments which retain
 the capacity to induce a protective response. The notion of a gene covers
 the nucleotide sequences equivalent to those described precisely in the
 examples, that is to say the sequences which are different but which
 encode the same protein. It also covers the nucleotide sequences of other
 strains of the pathogen considered, which provide cross-protection or a
 protection specific for a strain or for a strain group. It also covers the
 nucleotide sequences which have been modified in order to facilitate the
 in vivo expression by the host animal but encoding the same protein.
 Preferably, the vaccine formula according to the invention will comprise
 the Aujeszky and porcine flu valencies to which other valencies,
 preferably selected from the PRRS and A. pleuropneumoniae
 (actinobacillosis) valencies, can be added. Other valencies selected from
 the parvovirosis and conventional hog cholera valencies can be optionally
 added to them.
 It goes without saying that all the combinations of valencies are possible.
 However, within the framework of the invention, the Aujeszky and porcine
 flu, followed by PRRS and A. pleuropneumoniae, valencies are considered to
 be preferred.
 From the point of view in of a vaccination directed more specifically
 against the porcine respiratory pathology the valencies will be preferably
 selected from Aujeszky, porcine flu, PRRS and actinobacillosis.
 From the point of view of a vaccination directed specifically against the
 reproductive pathology, the valencies will be preferably selected from
 PRRS, parvovirosis, conventional hog cholera and Aujeszky.
 As regards the Aujeszky valency, either of the gB and gD genes may be used.
 Preferably, both genes are used, these being in this case mounted in
 different plasmids or in one and the same plasmid.
 As regards the porcine flu valency, the HA and NP genes are preferably
 used. Either of these two genes or both genes simultaneously can be used,
 mounted in different plasmids or in one and the same plasmid. Preferably,
 the HA sequences from more than one influenza virus strain, in particular
 from the different strains found in the field, will be combined in the
 same vaccine. On the other hand, NP provides cross-protection and the
 sequence from a single virus strain will therefore be satisfactory.
 As regards the PRSS valency, the E and ORF3 or alternatively M genes are
 preferably used. These genes can be used alone or in combination; in the
 case of a combination, the genes can be mounted into separate plasmids or
 into plasmids combining 2 or 3 of these genes. Genes derived from at least
 two strains, especially from a European strain and an American strain,
 will be advantageously combined in the same vaccine.
 As regards the conventional hog cholera valency, either of the E1 and E2
 genes or also E1 and E2 genes combined, in two different plasmids or
 optionally in one and the same plasmid, can be used.
 As regards the actinobacillosis valency, one of the three genes mentioned
 above or a combination of 2 or 3 of these genes, mounted in different
 plasmids or mixed plasmids, may be used in order to provide protection
 against the different stereotypes of A. pleuropneumoniae. For the apxI, II
 and III antigens, it may be envisaged that the coding sequences be
 modified in order to obtain the detoxified antigens, in particular as in
 the examples.
 The vaccine formula according to the invention can be provided in the form
 of a dose volume generally of between 0.1 and 10 ml, and in particular
 between 1 and 5 ml especially for vaccinations by the intramuscular route.
 The dose will be generally between 10 ng and 1 mg, preferably between 100
 ng and 50 .mu.g and preferably between 1 .mu.g and 250 .mu.g per plasmid
 type.
 Use will preferably be made of naked plasmids simply placed in the
 vaccination vehicle which will be in general physiological saline (0.9%
 NaCl), ultrapure water, TE buffer and the like. All the polynucleotide
 vaccine forms described in the prior art can of course be used.
 Each plasmid comprises a promoter capable of ensuring the expression of the
 gene inserted, under its control, into the host cells. This will be in
 general a strong eukaryotic promoter and in particular a cytomegalovirus
 early CMV-IE promoter of human or murine origin, or optionally of another
 origin such as rats, pigs and guinea pigs.
 More generally, the promoter may be either of viral origin or of cellular
 origin. As viral promoter, there may be mentioned the SV40 virus early or
 late promoter or the Rous sarcoma virus LTR promoter. It may also be a
 promoter from the virus from which the gene is derived, for example the
 gene's own promoter.
 As cellular promoter, there may be mentioned the promoter of a cytoskeleton
 gene, for example the desmin promoter (Bolment et al., Journal of
 Submicroscopic Cytology and Pathology, 1990, 22, 117-122; and Zhenlin et
 al., Gene, 1989, 78, 243-254), or alternatively the actin promoter.
 When several genes are present in the same plasmid, these may be presented
 in the same transcription unit or in two different units.
 The combination of the different vaccine valencies according to the
 invention may be preferably achieved by mixing the polynucleotide plasmids
 expressing the antigen(s) of each valency, but it is also possible to
 envisage causing antigens of several valencies to be expressed by the same
 plasmid.
 The subject of the invention is also monovalent vaccine formulae comprising
 one or more plasmids encoding one or more genes from one of the viruses
 selected from the group consisting of PRV, PRRS, PPV, HCV and A.
 pleuropneumoniae, the genes being those described above. Besides their
 monovalent character, these formulae may possess the characteristics
 stated above as regards the choice of the genes, their combinations, the
 composition of the plasmids, the dose volumes, the doses and the like.
 The monovalent vaccine formulae may be used (i) for the preparation of a
 polyvalent vaccine formula as described above, (ii) individually against
 the actual pathology, (iii) combined with a vaccine of another type (live
 or inactivated whole, recombinant, subunit) against another pathology, or
 (iv) as booster for a vaccine as described below.
 The subject of the present invention is in fact also the use of one or more
 plasmids according to the invention for the manufacture of a vaccine
 intended to vaccinate pigs first vaccinated by means of a first
 conventional vaccine of the type in the prior art, namely, in particular,
 selected from the group consisting of a live whole vaccine, an inactivated
 whole vaccine, a subunit vaccine, a recombinant vaccine, this first
 vaccine (monovalent or multivalent) having (that is to say containing or
 capable of expressing) the antigen(s) encoded by the plasmids or
 antigen(s) providing cross-protection. Remarkably, the polynucleotide
 vaccine has a potent booster effect which results in an amplification of
 the immune response and the acquisition of a long-lasting immunity.
 In general, the first-vaccination vaccines can be selected from commercial
 vaccines available from various veterinary vaccine producers.
 The subject of the invention is also a vaccination kit grouping together a
 first-vaccination vaccine as described above and a vaccine formula
 according to the invention for the booster. It also relates to a vaccine
 formula according to the invention accompanied by a leaflet indicating the
 use of this formula as a booster for a first vaccination as described
 above.
 The subject of the present invention is also a method for vaccinating pigs
 against the porcine reproductive pathology and/or respiratory pathology,
 comprising the administration of an effective dose of a vaccine formula as
 described above. This vaccination method comprises the administration of
 one or more doses of the vaccine formula, it being possible for these
 doses to be administered in succession over a short period of time and/or
 in succession at widely spaced intervals.
 The vaccine formulae according to the invention can be administered in the
 context of this method of vaccination, by the different routes of
 administration proposed in the prior art for polynucleotide vaccination
 and by means of known techniques of administration. The vaccination can in
 particular be used by the intradermal route with the aid of a liquid jet,
 preferably multiple jet, injector and in particular an injector using an
 injection head provided with several holes or nozzles, in particular
 comprising from 5 or 6 holes or nozzles, such as the Pigjet apparatus
 manufactured and distributed by the company Endoscoptic, Laons, France.
 The dose volume for such an apparatus will be reduced preferably to between
 0.1 and 0.9 ml, in particular between 0.2 and 0.6 ml and advantageously
 between 0.4 and 0.5 ml, it being possible for the volume to be applied in
 one or several, preferably 2, applications.
 The subject of the invention is also the method of vaccination consisting
 in making a first vaccination as described above and a booster with a
 vaccine formula according to the invention. In a preferred embodiment of
 the process according to the invention, there is administered in a first
 instance, to the animal, an effective dose of the vaccine of the
 conventional, especially inactivated, live, attenuated or recombinant,
 type, or alternatively a subunit vaccine, so as to provide a first
 vaccination, and, after a period preferably of 2 to 6 weeks, the
 polyvalent or monovalent vaccine according to the invention is
 administered.
 The invention also relates to the method of preparing the vaccine formulae,
 namely the preparation of the valencies and mixtures thereof, as evident
 from this description.
 The invention will now be described in greater detail with the aid of the
 embodiments of the invention taken with reference to the accompanying
 drawings.
 EXAMPLES
 Example 1
 Culture of the Viruses
 The viruses are cultured on the appropriate cellular system until a
 cytopathic effect is obtained. The cellular systems to be used for each
 virus are well known to persons skilled in the art. Briefly, the cells
 sensitive to the virus used, which are cultured in Eagle's minimum
 essential medium (MEM medium) or another appropriate medium, are
 inoculated with the viral strain studied using a multiplicity of infection
 of 1. The infected cells are then incubated at 37.degree. C. for the time
 necessary for the appearance of a complete cytopathic effect (on average
 36 hours).
 Example 2
 Culture of the Bacteria and Extraction of the Bacterial DNA
 The A. pleuropneumoniae strains were cultured as described by A. Rycroft et
 al. (J. Gen. Microbiol., 1991, 137, 561-568). The high-molecular weight
 DNA (chromosomal DNA) was prepared according to the standard techniques
 described by J. Sambrook et al. (Molecular Cloning: A Laboratory Manual,
 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
 1989).
 Example 3
 Extraction of the Viral Genomic DNAs:
 After culturing, the supernatant and the lysed cells are harvested and the
 entire viral suspension is centrifuged at 1000 g for 10 minutes at
 +4.degree. C. so as to remove the cellular debris. The viral particles are
 then harvested by ultracentrifugation at 400,000 g for 1 hour at
 +4.degree. C. The pellet is taken up in a minimum volume of buffer (10 mM
 Tris, 1 mM EDTA; pH 8.0). This concentrated viral suspension is treated
 with proteinase K (100 .mu.g/ml final) in the presence of sodium dodecyl
 sulphate (SDS) (0.5% final) for 2 hours at 37.degree. C. The viral DNA is
 then extracted with a phenol/chloroform mixture and then precipitated with
 2 volumes of absolute ethanol. After leaving overnight at -20.degree. C.,
 the DNA is centrifuged at 10,000 g for 15 minutes at +4.degree. C. The DNA
 pellet is dried and then taken up in a minimum volume of sterile ultrapure
 water. It can then be digested with restriction enzymes.
 Example 4
 Isolation of the Viral Genomic RNAs
 The RNA viruses were purified according to techniques well known to persons
 skilled in the art. The genomic viral RNA of each virus was then isolated
 using the "guanidium thiocyanate/phenol-chloroform" extraction technique
 described by P. Chromczynski and N. Sacchi (Anal. Biochem., 1987, 162,
 156-159).
 Example 5
 Molecular Biology Techniques
 All the constructions of plasmids were carried out using the standard
 molecular biology techniques described by J. Sambrook et al. (Molecular
 Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory,
 Cold Spring Harbor, N.Y., 1989). All the restriction fragments used for
 the present invention were isolated using the "Geneclean" kit (BIO 101
 Inc. La Jolla, Calif.).
 Example 6
 RT-PCR Technique
 Specific oligonucleotides (comprising restriction sites at their 5' ends to
 facilitate the cloning of the amplified fragments) were synthesized such
 that they completely cover the coding regions of the genes which are to be
 amplified (see specific examples). The reverse transcription (RT) reaction
 and the polymerase chain reaction (PCR) were carried out according to
 standard techniques (J. Sambrook et al., Molecular Cloning: A Laboratory
 Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor,
 N.Y., 1989). Each RT-PCR reaction was performed with a pair of specific
 amplimers and taking, as template, the viral genomic RNA extracted. The
 complementary DNA amplified was extracted with phenol/chloroform/isoamyl
 alcohol (25:24:1) before being digested with restriction enzymes.
 Example 7
 Plasmid pVR1012
 The plasmid pVR1012 (FIG. No. 1) was obtained from Vical Inc., San Diego,
 Calif., USA. Its construction has been described in J. Hartikka et al.
 (Human Gene Therapy, 1996, 7, 1205-1217).
 Example 8
 Construction of the Plasmid pAB090 (PRV gB Gene)
 The plasmid pPR2.15 (M. Riviere et al., J. Virol., 1992, 66, 3424-3434) was
 digested with ApaI and NaeI in order to release a 2665 bp ApaI-NaeI
 fragment (fragment A) containing the gene encoding Aujeszky's disease
 virus (NIA3 strain) gB glycoprotein (FIG. No. 2 and SEQ ID No. 1).
 By hybridizing the following 2 oligonucleotides:
 AB166 (33 mer) (SEQ ID No. 3)
 5' GATGCCCGCTGGTGGCGGTCTTTGGCGCGGGCC 3'
 AB167 (33 mer) (SEQ ID No. 4)
 5' ACGTCTACGGGCGACCACCGCCAGAAACCGCGC 3'
 a 33 bp fragment containing the sequence of the gD gene, from the initial
 ATG codon up to the ApaI site, was reconstructed, with the creation of a
 PstI site in 5' (fragment B).
 By hybridizing the following 2 oligonucleotides:
 AB168 (45 mer) (SEQ ID No. 5)
 5' GGCACTACCAGCGCCTCGAGAGCGAGGACCCCGACGCCCTGTAGG 3'
 AB169 (49 mer) (SEQ ID No. 6)
 5' GATCCCTACAGGGCGTCGGGGTCCTCGCTCTCGAGGCGCTGGTAGTGCC 3'
 a 45 bp fragment containing the sequence of the gD gene, from the NaeI site
 to the TAG stop codon was reconstructed, with the creation of a BamHI site
 in 3' (fragment C).
 The fragments A, B and C were ligated together into the vector pVR1012
 (Example 7), previously digested with PstI and BamHI, to give the plasmid
 pAB090 (7603 bp) (FIG. No. 3).
 Example 9
 Construction of the Plasmid pPBO98 (PRV gD Gene)
 The plasmid pPR29 (M. Riviere et al., J. Virol., 1992, 66, 3424-3434) was
 digested with SalI and BglII in order to liberate a 711 bp SalI-BglII
 fragment (fragment A) containing the 3' part of the gene encoding the
 Aujeszky's disease virus (NIA3 strain) gD glycoprotein (FIG. No. 4 and SEQ
 ID No. 6).
 The plasmid pPR29 was digested with Eco47III and SalI in order to liberate
 a 498 bp Eco47III-SalI fragment containing the 5' part of the gene
 encoding the Aujeszky's disease virus (NIA3 strain) gD glycoprotein
 (fragment B).
 By hybridizing the following 2 oligonucleotides:
 PB101 (15 mer) (SEQ ID No. 9)
 5' GATGCTGCTCGCAGC 3'
 PB102 (19 mer) (SEQ ID No. 10)
 5' GCTGCGAGCAGCATCTGCA 3'
 a 15 bp fragment containing the 5' sequence of the gD gene, from the
 initial ATG codon up to the Eco47III site was reconstructed, with the
 creation of a PstI site in 5' (fragment C).
 After purification, the fragments A, B and C were ligated together into the
 vector pVR1012 (Example 7), previously digested with PstI and BglII, to
 give the plasmid pPB098 (6076 bp) (FIG. No. 5).
 Example 10
 Construction of the Plasmid pBP143 (porcine flu HA gene, H1N1 strain)
 An RT-PCR reaction according to the technique described in Example 6 was
 carried out in the porcine flu virus (SIV, H1N1 "SW" strain) genomic RNA,
 prepared according to the technique described in Example 4, and with the
 following oligonucleotides:
 PB107 (32 mer) (SEQ ID No. 11)
 5' GTTCTGCAGCACCCGGGAGCAAAAGCAGGGGA 3'
 PB108 (33 mer) (SEQ ID No. 12)
 5' ATTGCGGCCGCTAGTAGAAACAAGGGTGTTTTT 3'
 so as to precisely isolate the gene encoding the HA protein from SIV H1N1
 (FIG. No. 6 and SEQ ID No. 11) in the form of a 1803 bp PCR fragment.
 After purification, this fragment was ligated with the vector PCRII-direct
 (Invitrogen Reference K2000-01), to give the vector pPB137 (5755 bp). The
 vector pPB137 was digested with EcoRV and NotI in order to liberate a 1820
 bp EcoRV-NotI fragment containing the HA gene. This fragment was then
 ligated into the vector pVR1012 (Example 7), previously digested with
 EcoRV and NotI, to give the plasmid pPB143 (6726 bp) (FIG. No. 7).
 Example 11
 Construction of the Plasmid pPB142 (porcine flu NP gene, H1N1 strain)
 An RT-PCR reaction according to the technique described in Example 6 was
 carried out with the porcine flu virus (SIV H1N1 "SW" strain) genomic RNA,
 prepared according to the technique described in Example 4, and with the
 following oligonucleotides:
 PB097 (36 mer) (SEQ ID No. 15)
 5' CCGGTCGACCGGGATAATCACTCACTGAGTGACATC 3'
 PB098 (33 mer) (SEQ ID No. 16)
 5' TTGCGGCCGCTGTAGAAACAAGGGTATTTTTCT 3'
 so as to precisely isolate the gene encoding the NP protein from SIV H1N1
 (FIG. No. 8 and SEQ ID No. 14) in the form of an SalI-NotI fragment. After
 purification, the 1566 bp RT-PCR product was ligated with the vector
 PCRII-direct (Invitrogen Reference K2000-01), to give the vector pPB127
 (5519 bp).
 The vector pPB127 was digested with SalI and NotI in order to liberate a
 1560 bp SalI-NotI fragment containing the NP gene. This fragment was then
 ligated into the vector pVR1012 (Example 7), previously digested with SalI
 and NotI, to give the plasmid pPB142 (6451 bp) (FIG. No. 9).
 Example 12
 Construction of the Plasmid pPB144 (porcine flu HA gene, H3N2 strain)
 An RT-PCR reaction according to the technique described in Example 6 was
 carried out with the porcine flu virus (strain SIV H3N2 Cotes du Nord
 1987) genomic RNA, prepared according to the technique described in
 Example 4, and with the following oligonucleotides:
 PB095 (31 mer) (SEQ ID No. 19)
 5' GTTCTGCAGGCAGGGGATAATTCTATCAACC 3'
 PB096 (36 mer) (SEQ ID No. 20)
 5' TTGCGGCCGCAAGGGTGTTTTTAATTACTAATATAC 3'
 so as to precisely isolate the gene encoding the HA protein from SIV H3N2
 (FIG. No. 10 and SEQ ID No. 17) in the form of a PstI-NotI fragment. After
 purification, the 1765 bp RT-PCR product was ligated with the vector
 PCRII-direct (Invitrogen Reference K2000-01) to give the vector pPB120
 (5716 bp).
 The vector pPB120 was digested with NotI in order to liberate a 1797 bp
 NotI-NotI fragment containing the HA gene. This fragment was then ligated
 into the vector pVR1012 (Example 7), previously digested with NotI, to
 give the plasmid pPB144 (6712 bp) containing the H3N2 HA gene in the
 correct orientation relative to the promoter (FIG. No. 11).
 Example 13
 Construction of the Plasmid pPB132 (porcine flu NP gene, H3N2 strain)
 An RT-PCR reaction according to the technique described in Example 6 was
 carried out with the porcine flu virus (strain SIV H3 N2 Cotes du Nord
 1987) genomic RNA, prepared according to the technique described in
 Example 4, and with the following oligonucleotides:
 PB097 (36 mer) (SEQ ID No. 15)
 5' CCGGTCGACCGGGATAATCACTCACTGAGTGACATC 3'
 PB098 (33 mer) (SEQ ID No. 16)
 5' TTGCGGCCGCTGTAGAAACAAGGGTATTTTTCT 3'
 so as to precisely isolate the gene encoding the NP protein from SIV H3N2
 (FIG. No. 12 and SEQ ID No. 18) in the form of a SalI-NotI fragment. After
 purification, the 1564 bp RT-PCR product was ligated with the vector
 PCRII-direct (Invitrogen Reference K2000-01) in order to give the vector
 pPB123 (5485 bp).
 The vector pPB123 was digested with SalI and NotI in order to liberate a
 SalI-NotI fragment of 1558 bp containing the NP gene. This fragment was
 then ligated into the vector pVR1012 (Example 7), previously digested with
 SalI and NotI, to give the plasmid pPB132 (6449 bp) (FIG. No. 13).
 Example 14
 Construction of the Plasmid pAB025 (PRRSV ORF5 gene, Lelystad strain)
 An RT-PCR reaction according to the technique described in Example 6 was
 carried out with the PRRSV virus (Lelystad strain) genomic RNA (J.
 Meulenberg et al., Virology, 1993, 19, 62-72), prepared according to the
 technique described in Example 4, and with the following oligonucleotides:
 AB055 (34 mer) (SEQ ID No. 25)
 5' ACGCGTCGACAATATGAGATGTTCTCACAAATTG 3'
 AB056 (33 mer) (SEQ ID No. 26)
 5' CGCGGATCCCGTCTAGGCCTCCCATTGCTCAGC 3'
 so as to precisely isolate the "ORF5" gene encoding the envelope
 glycoprotein E (gp25) from the PRRS virus, Lelystad strain. After
 purification, the 630 bp RT-PCR product was digested with SalI and BamHI
 in order to isolate a 617 bp SalI-BamHI fragment. This fragment was
 ligated with the vector pVR1012 (Example 7), previously digested with SalI
 and BamHI, to give the plasmid pAB025 (5486 bp) (FIG. No. 14).
 Example 15
 Construction of the Plasmid pAB001 (PRRSV ORF5 gene, USA strain)
 An RT-PCR reaction according to the technique described in Example 6 was
 carried out with the PRRSV virus (ATCC VR2332 strain) genomic RNA (M.
 Murtaugh et al., Arch Virol., 1995, 140, 1451-1460), prepared according to
 the technique described in Example 4, and with the following
 oligonucleotides:
 AB001 (30 mer) (SEQ ID No. 27)
 5' AACTGCAGATGTTGGAGAAATGCTTGACCG 3'
 AB002 (30 mer) (SEQ ID No. 22)
 5' CGGGATCCCTAAGGACGACCCCATTGTTCC 3'
 so as to precisely isolate the gene encoding the envelope glycoprotein
 E("gp25") from the PRRS virus, ATCC-VR2332 strain. After purification, the
 620 bp RT-PCR product was digested with PstI and BamHI in order to isolate
 a 606 bp PstI-BamHI fragment. This fragment was ligated with the vector
 pVR1012 (Example 7), previously digested with PstI and BamHI, to give the
 plasmid pAB001 (5463 bp) (FIG. No. 15).
 Example 16
 Construction of the Plasmid pAB091 (PPRSV ORF3 gene, Lelystad strain)
 An RT-PCR reaction according to the technique described in Example 6 was
 carried out with the PRRSV virus (Lelystad strain) genomic RNA (J.
 Meulenberg et al., 1993), prepared according to the technique described in
 Example 4, and with the following oligonucleotides:
 AB170 (32 mer) (SEQ ID No. 29)
 5' AAACTGCAGCAATGGCTCATCAGTGTGCACGC 3'
 AB171 (30 mer) (SEQ ID No. 30)
 5' CGCGGATCCTTATCGTGATGTACTGGGGAG 3'
 so as to precisely isolate the "ORF3" gene encoding the envelope
 glycoprotein "gp45" from the PRRS virus, Lelystad strain. After
 purification, the 818 bp RT-PCR product was digested with PstI and BamHI
 in order to isolate an 802 bp PstI-BamHI fragment. This fragment was
 ligated with the vector pVR1012 (Example 7), previously digested with PstI
 and BamHI, to give the plasmid pAB091 (5660 bp) (FIG. No. 16).
 Example 17
 Construction of the Plasmid pAB092 (PPRSV ORF3 gene, USA strain)
 An RT-PCR reaction according to the technique described in Example 6 was
 carried out with the PRRSV virus (ATCC-VR2332 strain) genomic RNA (M.
 Murtaugh et al., 1995), prepared according to the technique described in
 Example 4, and with the following oligonucleotides:
 AN172 (32 mer) (SEQ ID No. 31)
 5' AAACTGCAGCAATGGTTAATAGCTGTACATTC 3'
 AB173 (32 mer) (SEQ ID No. 32)
 5' CGCGGATCCCTATCGCCGTACGGCACTGAGGG 3'
 so as to precisely isolate the "ORF3" gene encoding the envelope
 glycoprotein "gp45" from the PRRS virus, ATCC-VR2332 strain. After
 purification, the 785 bp RT-PCR product was digested with PstI and BamHI
 in order to isolate a 769 bp Pst-BamHI fragment. This fragment was ligated
 with the vector pVR1012 (Example 7), previously digested with PstI and
 BamHI, to give the plasmid pAB092 (5627 bp) (FIG. No. 17).
 Example 18
 Construction of the Plasmid pAB004 (porcine parvovirus VP2 gene)
 An RT-PCR reaction according to the technique described in Example 6 was
 carried out with the porcine parvovirus (NADL2 strain) genomic RNA (J.
 Vasudevacharya et al., Virology, 1990, 178, 611-616), prepared according
 to the technique described in Example 4, and with the following
 oligonucleotides:
 AB007 (33 mer) SEQ ID No. 33)
 5' AAAACTGCAGAATGAGTGAAAATGTGGAACAAC 3'
 AB010 (33 mer) (SEQ ID No. 34)
 5' CGCGGATCCCTAGTATAATTTTCTTGGTATAAG 3'
 so as to amplify a 1757 bp fragment containing the gene encoding the
 porcine parvovirus VP2 protein. After purification, the RT-PCR product was
 digested with PstI and BamHI to give a 1740 bp PstI-BamHI fragment. This
 fragment was ligated with the vector pVR1012 (Example 7), previously
 digested with PstI and BamHI, to give the plasmid pAB004 (6601 bp) (FIG.
 No. 18).
 Example 19
 Construction of the Plasmid pAB069 (hog chlolera HCV E1 gene)
 An RT-PCR reaction according to the technique described in Example 6 was
 carried out with the hog cholera virus (HCV) (Alfort strain) genomic RNA
 (G. Meyers et al., Virology, 1989, 171, 18-27), prepared according to the
 technique described in Example 4, and with the following oligonucleotides:
 AB126 (36 mer) (SEQ ID No. 35)
 5' ACGCGTCGACATGAAACTAGAAAAAGCCCTGTTGGC 3'
 AB127 (34 mer) (SEQ ID No. 36)
 5' CGCGGATCCTCATAGCCGCCCTTGTGCCCCGGTC 3'
 so as to isolate the sequence encoding the E1 protein from the HCV virus in
 the form of a 1363 bp RT-PCR fragement. After purification, this fragment
 was digested with SalI and BamHI to give a 1349 bp SalI-BamHI fragment.
 This fragment was ligated with the vector pVR1012 (Example 7), previously
 digested with SalI and BamHI, to give the plasmid pABO69 (6218 bp) (FIG.
 No. 19).
 Example 20
 Construction of the Plasmid pAB061 (hog cholera HCV E2 gene)
 An RT-PCR reaction according to the technique described in Example 6 was
 carried out with the hog cholera virus (HCV) (Alfort strain) genomic RNA
 (G. Meyers et al., 1989), prepared according to the technique described in
 Example 4, and with the following oligonucleotides:
 AB118 (36 mer) (SEQ ID No. 37)
 5' ACGCGTCGACATGTCAACTACTGCGTTTCTCATTTG 3'
 AB119 (33 mer) (SEQ ID No. 38)
 5' CGCGGATCCTCACTGTAGACCAGCAGCGAGCTG 3'
 so as to isolate the sequence encoding the E2 protein from the HCV virus in
 the form of a 1246 bp RT-PCR fragment. After purification, this fragment
 was digested with SalI and BamHI to give a 1232 bp SalI-BamHI fragment.
 This fragment was ligated with the vector pVR1012 (Example 7), previously
 digested with SalI and BamHI, to give the plasmid pAB061 (6101 bp) (FIG.
 No. 20).
 Example 21
 Construction of the Plasmid pBP162 (deleted Actinobacillus pleuropneumoniae
 apxI gene)
 The A. pleuropneumoniae apxI gene was cloned so as to delete the
 glycine-rich amino acid region (involved in the binding of the calcium
 ion) which is between amino acids 719 and 846.
 A PCR reaction was carried out with the A. pleuropneumoniae (serotype 1)
 genomic DNA (J. Frey et al., Infect. Immun., 1991, 59, 3026-3032),
 prepared according to the technique described in Examples 2 and 3, and
 with the following oligonucleotides:
 PB174 (32 mer) (SEQ ID No. 39)
 5' TTGTCGACGTAAATAGCTAAGGAGACAACATG 3'
 PB189 (29 mer) (SEQ ID No. 40)
 5' TTGAATTCTTCTTCAACAGAATGTAATTC 3'
 so as to amplify the 5' part of the apxI gene encoding the A.
 pleuropneumoniae haemolysin I protein, in the form of a SalI-EcoRI
 fragment. After purification, the 2193 bp PCR product was digested with
 SalI and EcoRI in order to isolate a 2183 bp SalI-EcoRI fragment (fragment
 A).
 A PCR reaction was carried out with the A. pleuropneumoniae (serotype 1)
 genonic DNA (J. Frey et al., 1991) and with the following
 oligonucleotides:
 BP190 (32 mer) (SEQ ID No. 41)
 5' TTGAATTCTATCGCTACAGTAAGGAGTACGG 3'
 PB175 (31 mer) (SEQ ID No. 42)
 5' TTGGATCCGCTATTTATCATCTAAAAATAAC 3'
 so as to amplify the 3' part of the apxI gene encoding the A.
 pleuropneumoniae haemolysin I protein, in the form of an EcoRI-BamHI
 fragment. After purification, the 576 bp PCR product was digested with
 EcoRI and BamHI in order to isolate a 566 bp EcoRI-BamHI fragment
 (fragment B). The fragments A and B were ligated together with the vector
 pVR1012 (Example 7), previously digested with SalI and BamHI, to give the
 plasmid pPB162 (7619 bp) (FIG. No. 21).
 Example 22
 Construction of the Plasmid pPB163 (deleted A. pleourpneumoniae apxII gene)
 The A. pleuropneumoniae apxII gene was cloned so as to delete the
 glycine-rich amino acid region (involved in the binding of the calcium
 ion) which is between amino acids 716 and 813.
 A PCR reaction was carried out with the A. pleuropneumoniae (serotype 9)
 genomic DNA (M. Smits et al., Infection and Immunity, 1991, 59,
 4497-4504), prepared according to the technique described in Examples 2
 and 3, and with the following oligonucleotides:
 PB176 (31 mer) (SEQ ID No. 43)
 5' TTGTCGACGATCAATTATATAAAGGAGACTC 3'
 PB191 (30 mer) (SEQ ID No. 44)
 5' TTGAATTCCTCTTCAACTGATTTGAGTGAG 3'
 so as to amplify the 5' part of the apxII gene encoding the A.
 pleuropneumoniae haemolysin II protein, in the form of an SalI-EcoRI
 fragment. After purification, the 2190 bp PCR product was digested with
 SalI and EcoRI in order to isolate a 2180 bp SalI-EcoRI fragment (fragment
 A).
 A PCR reaction was carried out with the A. pleuropneumoniae (serotype 9)
 genomic DNA (M. Smits et al., 1991) and with the following
 oligonucleotides:
 PB192 (29 mer) (SEQ ID No. 45)
 5' TTGAATTCGTAAATCTTAAAGACCTCACC 3'
 PB177 (30 mer) (SEQ ID No. 46)
 5' TTGGATCCACCATAGGATTGCTATGATTTG 3'
 so as to amplify the 3' part of the apxII gene encoding the A.
 pleuropneumoniae haemolysin II protein, in the form of an EcoRI-BamHI
 fragment. After purification, the 473 bp PCR product was digested with
 EcoRI and BamHI in order to isolate a 463 bp EcoRI-BamHI fragment
 (fragment B).
 The fragments A and B were ligated together with the vector pVR1012
 (Example 7), previously digested with SalI and BamHI, to give the plasmid
 pPB163 (7513 bp) (FIG. No. 22).
 Example 23
 Construction of the Plasmids pPB174', pPB189 and pPB190 (deleted A.
 pleuropneumoniae apxIII gene)
 First Example of Deletion in AxIII (plasmid pPB174'):
 The Actinobacillus pleuropneumoniae apxIII gene was cloned so as to delete
 the glycine-rich amino acid region (involved in the binding of the calcium
 ion) which is between amino acids 733 and 860.
 A PCR reaction was carried out with the A. pleuropneumoniae (serotype 8)
 genomic DNA (M. Smits, 1992, Genbank sequence accession No.=X68815),
 prepared according to the technique described in Examples 2 and 3, and
 with the following oligonucleotides:
 PB278 (30 mer) (SEQ ID No. 47)
 5' TTTGTCGACATGAGTACTTGGTCAAGCATG 3'
 PB279 (28 mer) (SEQ ID No. 48)
 5' TTTATCGATTCTTCTACTGAATGTAATTC 3'
 so as to amplify the 5' part of the apxIII gene (encoding the
 Actinobacillus pleuropneumoniae haemolysin III protein) in the form of an
 SalI and ClaI fragment. After purification, the 2216 bp PCR product was
 digested with SalI and ClaI in order to isolate a 2205 bp SalI-ClaI
 fragment (fragment A).
 A PCR reaction was carried out with the A. pleuropneumoniae (serotype 8)
 genomic DNA (M. Smits, 1992, Genbank sequence accession No.=X68815) and
 with the following oligonucleotides:
 PB280 (33 mer) (SEQ ID No. 49)
 5' TTTATCGATTTATGTTTATCGTTCCACTTCAGG 3'
 PB307 (32 mer) (SEQ ID No. 50)
 5' TTGGATCCTTAAGCTGCTCTAGCTAGGTTACC 3'
 so as to amplify the 3' part of the apxIII gene (encoding the A.
 pleuropneumoniae haemolysin III protein) in the form of a ClaI-BamHI
 fragment. After purification, the 596 bp PCR product was digested with
 ClaI and BamHI in order to isolate a 583 bp ClaI-BamHI fragment (fragment
 B).
 The fragments A and B were ligated together with the vector pVR1012
 (Example 7), previously digested with SalI and BamHI, to give the plasmid
 pPB174' (7658 bp) (FIG. No. 23).
 Second Example of Deletion in ApxIII (plasmid pPB189):
 The A. pleuropneumoniae apxIII gene was cloned so as to delete the
 glycine-rich amino acid region (involved in the binding of the calcium
 ion) which is between amino acids 705 and 886.
 A PCR reaction was carried out with the A. pleuropneumoniae (serotype 8)
 genomic DNA (M. Smits, 1992, Genbank sequence accession No.=X68815),
 prepared according to the technique described in Examples 2 and 3, and
 with the following oligonucleotides:
 PB278 (30 mer) (SEQ ID No. 47)
 5' TTTGTCGACATGAGTACTTGGTCAAGCATG 3'
 PB303 (32 mer) (SEQ ID No. 51)
 5' TTTATCGATTTCTTCACGTTTACCAACAGCAG 3'
 so as to amplify the 5' part of the apxIII gene (encoding the A.
 pleuropneumoniae haemolysin III protein) in the form of a SalI-ClaI
 fragment. After purification, the 2133 bp PCR product was digested with
 SalI and ClaI in order to isolate a 2122 bp SalI-ClaI fragment (fragment
 A).
 A PCR reaction was carried out with the A. pleuropneumoniae (serotype 8)
 genomic DNA (M. Smits, 1992, Genbank sequence accession No.=X68815) and
 with the following oligonucleotides:
 PB306 (31 mer) (SEQ ID No. 52)
 5' TTTATCGATTCTGATTTTTCCTTCGATCGTC 3'
 PB307 (32 mer) (SEQ ID No. 50)
 5' TTGGATCCTTAAGCTGCTCTAGCTAGGTTACC 3'
 so as to amplify the 3' part of the apxIII gene (encoding the A.
 pleuropneumoniae haemolysin III protein) in the form of a ClaI-BamHI
 fragment. After purification, the 518 bp PCR product was digested with
 ClaI and BamHI in order to isolate a 506 bp ClaI-BamHI fragment (fragment
 B).
 The fragments A and B were ligated together with the vector pVR1012
 (Example 7), previously digested with SaII and BamHI, to give the plasmid
 pPB189 (7496 bp) (FIG. No. 24).
 Third Example of Deletion in ApxIII (plasmid pPB190):
 The A. pleuropneumoniae apxIII gene was cloned so as to delete the
 glycine-rich amino acid region (involved in the binding of the calcium
 ion) which is between amino acids 718 and 876.
 A PCR reaction was carried out with the A. pleuropneumoniae (serotype 8)
 genomic DNA (M. Smits, 1992, Genbank sequence accession No.=X68815),
 prepared according to the technique described in Examples 2 and 3, and
 with the following oligonucleotides:
 PB278 (30 mer) (SEQ ID No. 47)
 5' TTTGTCGACATGAGTACTTGGTCAAGCATG 3'
 PB304 (33 mer) (SEQ ID No. 53)
 5' TTTATCGATACCTGATTGCGTTAATTCATAATC 3'
 so as to amplify the 5' part of the apxIII gene (encoding the A.
 pleuropneumoniae haemolysin III protein) in the form of a SalI-ClaI
 fragment. After purification, the 2172 bp PCR product was digested with
 SalI and ClaI in order to isolate a 2161 bp SalI-ClaI fragment (fragment
 A).
 A PCR reaction was carried out with the A. pleuropneumoniae (serotype 8)
 genomic DNA (M. Smits, 1992, Genbank sequence accession No.=X68815) and
 with the following oligonucleotides:
 PB305 (31 mer) (SEQ ID No. 54)
 5' TTTATCGATAAATCTAGTGATTTAGATAAAC 3'
 PB307 (32 mer) (SEQ ID No. 50)
 5' TTGGATCCTTAAGCTGCTCTAGCTAGGTTACC 3'
 so as to amplify the 3' part of the apxIII gene (encoding the A.
 pleuropneumoniae haemolysin III protein) in the form of a ClaI-BamHI
 fragment. After purification, the 548 bp PCR product was digested with
 ClaI and BamHI in order to isolate a 536 bp ClaI-BamHI fragment (fragment
 B).
 The fragments A and B were ligated together with the vector pVR1012
 (Example 7), previously digested with SalI and BamHI, to give the plasmid
 pPB190 (7565 bp) (FIG. No. 25).
 Example 24
 Preparation and Purification of the Plasmids
 For the preparation of the plasmids intended for the vaccination of
 animals, any technique may be used which makes it possible to obtain a
 suspension of purified plasmids predominantly in the supercoiled form.
 These techniques are well known to persons skilled in the art. There may
 be mentioned in particular the alkaline lysis technique followed by two
 successive ultracentrifugations on a caesium chloride gradient in the
 presence of ethidium bromide as described in J. Sambrook et al. (Molecular
 Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory,
 Cold Spring Harbor, N.Y., 1989). Reference may also be made to patent
 applications PCT WO 85/21250 and PCT WO 96/02658 which describe methods
 for producing, on an industrial scale, plasmids which can be used for
 vaccination. For the purposes of the manufacture of vaccines (see Example
 17), the purified plasmids are resuspended so as to obtain solutions at a
 high concentration (&gt;2 mg/ml) which are compatible with storage. To do
 this the plasmids are resuspended either in ultrapure water or in TE
 buffer (10 mM Tris-HCl; 1 mM EDTA, pH 8.0).
 Example 25
 Manufacture of the Associated Vaccines
 The various plasmids necessary for the manufacture of an associated vaccine
 are mixed starting with their concentrated solutions (Example 16). The
 mixtures are prepared such that the final concentration of each plasmid
 corresponds to the effective dose of each plasmid. The solutions which can
 be used to adjust the final concentration of the vaccine may be either a
 0.9% NaCl solution, or PBS buffer.
 Specific formulations such as liposomes, cationic lipids, may also be used
 for the manufacture of the vaccines.
 Example 26
 Vaccination of Pigs
 The pigs are vaccinated with doses of 100 .mu.g, 250 .mu.g or 500 .mu.g per
 plasmid.
 The injections can be performed with a needle by the intramuscular route.
 In this case, the vaccinal doses are administered in a volume of 2 ml.
 The injections can be performed by the intradermal route using a liquid jet
 injection apparatus (with no needle) delivering a dose of 0.2 ml at 5
 points (0.04 ml per point of injection) (for example "PIGJET" apparatus).
 In this case, the vaccinal doses are administered in 0.2 or 0.4 ml
 volumes, which corresponds to one or two administrations respectively.
 When two successive administrations are performed by means of the PIGJET
 apparatus, these administrations are spaced out so that the two injection
 zones are separated from each other by a distance of about 1 to 2
 centimetres.