BE1009826A3 - Plasmid vaccine against pseudorabies virus - Google Patents

Abstract

Vaccine comprising at least one plasmid coding for the gIII glycoprotein of PRV virus or for a protein having the same antigenicity as the gIII glycoprotein of PRV virus and a pharmaceutically acceptable excipient for same. The invention also relates to the pEVhis14gIII plasmid used in vaccine manufacture.<IMAGE>

Description

       

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  Plasmid vaccine against pseudorabic virus
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 The present invention relates to a plasmid vaccine against the pseudorabic virus, also known as Aujeszky's disease virus (ADV), swine herpes virus 1 (PHV-1) or herpes virus l Suid ( SHV-1).



  Aujeszky's disease is a viral disease to which most mammals are susceptible. However, humans do not seem to be susceptible to this disease.



  The agent causing this disease is a double-stranded DNA enveloped virus from the Herpesviridae family, a subfamily of alphaherpesvirinae, namely, the pseudorabic virus (PRV).



  The PRV virus genome is formed by a double-stranded DNA molecule of approximately 150 kb and consists of a single long region (UL) and a single short region (Us) which is flanked by two reverse repeat sequences, one terminal (TR) and the other internal (IR) (BENPORAT, T. et al, 1979, Virology 95, p. 285-294).



  The Aujeszky's disease virus genome contains at least 70 genes.



  The main characteristics and biological properties of the best characterized PRV genes are listed in Table 1 below.

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    Table 1: Properties of certain PRV genes, their characteristics and biological properties (extract from Mettenleiter T., Acta Veterinaria Hungarica, 42 (2-3), p. 153-177 (1994)).
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 <tb>
 <tb>



  Segment <SEP> from <SEP> Designation <SEP> Designation <SEP> Gene <SEP> Protein <SEP> Size <SEP> Essential <SEP> Function <SEP> 1 <SEP> Activity <SEP> Virulence
 <tb> Genome <SEP> Gene <SEP> (HSV) <SEP> (PRV) <SEP> (kDa) <SEP> (replication <SEP> in
 <tb> vitro)
 <tb> UL <SEP> L <SEP> gB <SEP> gII <SEP> 913 <SEP> aa <SEP> 110-68-55 <SEP> + <SEP> penetration, <SEP> fusion <SEP> +
 <tb> cell
 <tb> gC <SEP> gin479 <SEP> aa <SEP> 92 <SEP> adsorption, <SEP> +
 <tb> release
 <tb> TK <SEP> TK <SEP> 320 <SEP> aa <SEP> 35 <SEP> - <SEP> thymidine <SEP> kinase <SEP> +
 <tb> gH <SEP> gH <SEP> 686 <SEP> aa <SEP> 84 <SEP> + <SEP> penetration, <SEP> fusion <SEP> n. <SEP> a.
 <tb> cell
 <tb> Us <SEP> prot.

    <SEP> kinase <SEP> PK <SEP> 336 <SEP> aa <SEP> 41 <SEP> - <SEP> protein <SEP> kinase <SEP> +
 <tb> G <SEP> gX <SEP> 498 <SEP> aa <SEP> 99 <SEP> - <SEP> unknown <SEP> gD <SEP> gp50 <SEP> 402 <SEP> aa <SEP> 60 <SEP> + <SEP> penetration <SEP> n.a.
 <tb> gI <SEP> aa <SEP> 63 <SEP> - <SEP> unknown <SEP> +
 <tb> gE <SEP> gI <SEP> 577 <SEP> aa <SEP> 110 <SEP> - <SEP> release, <SEP> +
 <tb> transmission <SEP> from
 <tb> cell <SEP> to <SEP> cell
 <tb> TEG <SEP> IIK <SEP> 106 <SEP> aa <SEP> 11 <SEP> - <SEP> protein <SEP> from <SEP> unknown
 <tb> seed coat
 <tb>
 

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Transmission of the virus usually occurs by oral, respiratory or direct contact between an infected animal and a healthy animal.



   The disease has become a concern for pig farms where it manifests itself in several forms: 1. Adults develop few clinical signs, but become permanent sources of infection. However, the PRV virus can cause abortion in pregnant sows.



  2. Piglets suffer severe damage to the central nervous system.



   Piglets have a high sensitivity to birth which will gradually decrease. Up to the age of 10 days, affected piglets, not benefiting from passive immunity from the mother, succumb within a few hours. Older, they are prone to muscle tremors, contractions, but the evolution is rather mild.



   In other species the disease can be fatal and the duration of incubation is variable and ranges from 15 hours to 12 days.



   There are currently several methods of vaccination against Ausjeszky's disease.



   One of the classic methods is the injection of live viruses, which must be attenuated to prevent the disease from spreading.



   Thus, for the vaccination of pigs, PRV viruses of the Bartha strain are used which include mutations in the genome coding for the glycoproteins gI, gp63 and gin as well as for a viral capsid protein whose gene is located on the fragment BamHI 4. (Mettenleiter T., Acta Veterinaria Hungarica, 42 (2-3), pp. 153-177 (1994)).
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    <s We also use the vaccines sold under the names OMNIVAC-PRV (FERMENTA ANIMAL HEALTH Co., Kansas City, MO, USA) and
OMNIMARK-PRV (FERMENTA ANIMAL HEALTH Co., Kansas City, MO, USA) which include genetically engineered PRV viruses from the Bucharest strain. These strains contain deletions in the genes coding respectively for thymidine kinase or for thymidine kinase and gill. Of course, there are still other vaccines against Aujeszky's disease that work on the same principle.



   This vaccination method gives the vaccinated subject good protection which is due to the intracellular action of the live virus. Indeed, live viruses enter cells where viral antigens will be

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 synthesized. Next, peptides derived from these viral antigens are presented on the surface of infected cells in association with the major class 1 histocompatibility complex (MHC I). A cytotoxic response can thus be provoked in addition to the humoral response, which induces in the vaccinated subject better protection against the virus.



   Although viruses are attenuated by mutations, there is a risk that viruses will become pathogenic again by spontaneous mutations or by recombinations with wild-type viruses.



   Vaccinations with live viral agents can also lead, under certain conditions, to the proliferation of live viruses. This proliferation of live viruses, even attenuated, constitutes a risk for more susceptible subjects, such as newborns or pregnant women.



   Another technique developed more recently consists in the use of a non-pathogenic living vector carrying selected genes of the PRV virus.



   M. ELOIT et al (JT van Oirschot (ed.), Vaccination and control of Aujeszky's Disease, p. 61-66, ECSC, EEC, EAEC, Brussels and Luxembourg, 1989) have developed a vaccine based on adenovirus type 5 (AD5) recombinant which gives rise to the expression of the PRV virus gp50 gene.



   WL MENGEUNG et al (Arch. Of Virology, V 134, n 3-4, p. 259-269,1994) published results of trials with a vaccinia virus (NYVAC) containing the PRV genes coding for the gp50 glycoproteins , gII and gill. However, the effectiveness of this type of vaccine is limited.



   On the other hand, ML VAN DER LEEK et al (The Veterinary Record (1994) 134, p. 13-18) have produced encouraging results with vaccination of pigs against the PRV virus by scarification or by intramuscular injection of a recombinant virus. swine pox and PRV virus (rSPV-AD). This recombinant was obtained by insertion of the PRV genes coding for the gp50 and gp63 attached to the promoter P 7.5 of the vaccinia virus in the thymidine kinase gene of the SPV virus.



   Of course, there are still other vaccines against Aujeszky's disease that work on the same principle.



   A new approach to induce an immunological response has been described recently by ULMER et al, 1993, Sci. 259: 1745). Mice were immunized by intramuscular injection of a plasmid comprising the

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 influenza virus nucleoprotein gene under the control of a mammalian promoter. This immunization by injection of the plasmid apparently led to a transfection of muscle cells, followed by an in situ expression of the protein, resulting in a specific immunological response against the influenza virus, of cellular and humoral type, and therefore protection against attack by this virus.



   It seems, from published experiments, that parts of viral proteins produced inside transfected cells are taken up by the major class 1 histocompatibility complex and presented on the surface of the cell.



   ROBINSON et al, described in 1993, in Vaccine 11,957-960, immunization of hens against the influenza virus by intravenous, intraperitoneal and subcutaneous injections of plasmid. From 28% to 100% of the animals thus immunized resist a "challenge" with a lethal dose of the virus. It should be noted that the effectiveness strongly depends on the route and the system used for the injection and on a possible pretreatment of the injection site (DANKO et al, Vaccine 12,1499-1502 (1994)).



   GRAHAM J. M. COX et al have described a method of vaccinating cattle and mice against the BHV-1 virus by injection of plasmid DNA, in J. Virol. 1994 p. 5685-5689. It has been shown that intramuscular injection of cattle and mice (into the quadriceps) of plasmid DNA containing the gI, gm or gIV glycoprotein gene of BHV-1 elicits an immune response in the vaccinated animal. The immune response is highly dependent on the amount of DNA injected, and the glycoprotein. Thus, the gIV protein gene elicited a higher response than that of the gI and gin proteins.



   None of the plasmid vaccines described to date are effective against viral diseases infecting pigs and other species such as Aujeszky's disease caused by the PRV virus.



   The aim of the present invention is to provide a plasmid vaccine against the PRV virus responsible for Aujeszky's disease.



   This object is achieved by a vaccine comprising at least one plasmid coding for the glycoprotein gin of the PRV virus or for a protein having the same antigenicity as the glycoprotein gin of the PRV virus and a pharmaceutically acceptable excipient for the latter.

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   One of the advantages of this method lies in the fact that this method is inexpensive.



   Indeed, the plasmid can be produced, according to well-controlled techniques, in bacteria such as E. coli.



   Plasmid extraction is well known and a high yield can be obtained.



   The gene introduced into the plasmid does not have to code for the whole gill protein, it just has to code for a part or a homolog of the PRV gill protein which has the same antigenicity as the gill c protein. i.e. the same effect on the immune system as the gin protein.



  Indeed, only part of the gin protein is identified by the animal's immunological system, the other parts of the protein, although certainly playing a role in the life of the virus, are not essential in the recognition of the protein by the infected organism.



   Another advantage is that vaccinated animals can easily be distinguished from animals infected with the PRV virus. In fact, vaccinated animals only develop antibodies against the gin protein while animals infected with the PRV virus also develop antibodies against other proteins of the virus.



   In addition, the method is safe, since a proliferation of live viruses is not to be feared. As only a specific part of the genome of the virus is injected, there is no infection by viruses and therefore consequently there can be no proliferation of viruses.



   Since muscle cells have a long life and do not circulate in the animal's body, local and continuous expression of the antigen at low concentrations can stimulate the long-term immunological response.



   Vaccination with the vaccine according to the present invention can be used as a diagnostic tool because it induces the formation of monospecific antibodies. These antibodies can be used for the detection of the p antigen. ex. during ELISA or other tests.



   A surprising effect of the invention lies in the effectiveness of the immunization against the PRV virus. Although the importance of the gin glycoprotein in immunization is well known, injection of the purified gill protein does not appear to have a pronounced immunizing effect.



   Z. H. BISEEBUTSU et al describe, in the patent application

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 Japanese JP 05/246888, a vaccine against the PRV virus, based on purified glycoprotein gill and an oil-based adjuvant. This approach is not used on a commercial scale.



   A. MATSUDA TSUCHIDA et al show in an article published in J.



  Vet. med. Sci. 54 (3): 447-452,1992, that a mixture of purified gll, gill and gIV glycoproteins injected together with a conventional oil-based adjuvant gives better protection to mice against a challenge by virulent PRV viruses than the glycoproteins injected individually.



   It should be noted that vaccines using purified proteins are, in general, very expensive because the purification steps are long and complicated.



   According to a first advantageous embodiment, the plasmid is the plasmid pEVhisl4gill.



     The plasmid pEVhisl4gill has the advantage of comprising a gene conferring resistance to ampicillin, so that the transformed bacteria, having incorporated the plasmid, are easily selectable by adding ampicillin into their growth medium.



   Of course, it is possible to envisage using other plasmids. It suffices that the plasmid comprises a gene coding for a protein having the same antigenicity as the glycoprotein gill of the PRV virus, inserted into the plasmid so that it is expressed in the vaccinated organism. A marker such as a gene conferring resistance to an antibiotic makes it possible to select the bacteria transformed by the plasmid.



   To increase the efficacy of the vaccine, the plasmid contains, in addition to the gene coding for the PRV virus glycoprotein gin or for a protein having the same antigenicity as the PRV virus glycoprotein gill, one or more genes coding for cytokines.



   Certain cytokines are known to exert an adjuvant activity on vaccines. Introducing them into the plasmid so that they can be expressed in cells will increase the effectiveness of the vaccine.



   According to another advantageous embodiment, the vaccine also comprises a pharmaceutically acceptable excipient in which the plasmid is incorporated. The term "pharmaceutically acceptable excipient" mentioned in this document refers to either liquid media or

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 solid media capable of being used as excipient (vehicle) to introduce the plasmid into the animal to be vaccinated.



   Examples include liquid media, water, saline, phosphate-saline buffer, solutions containing adjuvants, detergents, stabilizers and transfection promoting substances, liposome suspensions, virosomes and emulsions.



   Let us cite as an example of solid media, the microbeads coated with plasmid, intended to be projected by bombardment ("gene gun") in the tissues of the animal and the microparticles containing DNA, usable by parenteral and oral route. .



   DNA vaccination can possibly be preceded by a pretreatment of the vaccination site (eg use of a local anesthetic) in order to improve its effectiveness.



   The present invention also provides a plasmid vaccine against the PRV virus comprising a nucleic acid sequence coding for the gin protein of the PRV virus or for a protein having the same antigenicity as the glycoprotein gill of the PRV virus or a DNA construct comprising a expression cassette including: a) a DNA sequence coding for a polypeptide containing at least one antigenic determinant of the glycoprotein gill or an immunogenic fragment thereof, and b) control sequences operatively linked to said coding sequences where said coding sequence can be transcribed and translated in a cell and where said control sequences are homologous or heterologous to said coding sequence.



   Advantageously, the plasmid vaccine contains one or more genes coding for cytokines.



   According to yet another aspect of the present invention, it is proposed to use the plasmid pEVhisl4gill in the manufacture of a vaccine.



   It is also proposed to use the plasmid pEVhisl4gill in the manufacture of a vaccine against the PRV virus.



   The invention is described in more detail, by way of illustration, in the examples which follow.



  Example 1: Obtaining a vaccine.



   The vaccine was obtained in three stages, comprising the construction of a plasmid (pEVhis14gin) containing the gene for the virus’s glycoprotein gin

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   PRV, the production of this plasmid in transformed bacteria and the formulation of the vaccine comprising the plasmid and a pharmaceutically acceptable excipient.



  Step A: Construction of a plasmid (pEVhisl4gin) containing the gin glycoprotein gene from the PRV virus.
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  The plasmid pEVhis14gill DNA was obtained from the Institute for Animal Science and Health, (ID-DLO, Lelystad, NL). It includes the PRV virus glycoprotein gin gene, under the control of the HCMV promoter, and the ampicillin resistance marker gene; it is used as a DNA vaccine (DNA +). The plasmid map is given in Figure 1.



  The plasmid was deposited under the provisions of the Budapest Treaty on November 16, 1995 in the "Belgian Coordinated Collections of Microorganisms-Laboratorium voor Moleculaire Biologie-Plasmiden collection" (LMBP), Universiteit Gent, KL Ledeganckstraat 35 Gent, Belgium B-9000 under accession number LMBP3377 Step B: Production of the plasmid in transformed bacteria.



  Preparation of E cells. coli treated with RbCI.



   Before being transformed, the cells of E. coli, strain DH 5a (Gibco) were subjected to an RbCl treatment to increase the efficiency of the transformation. The procedure which was followed, with the exception that we used an E. coli DH 5a strain, is described in the newsletter "The NEB transcript", vol. 6 (1) p7, May 1994, edited by NEW ENGLAND BIOLABS, Inc., Beverly, MA01915.



  Transformation of E cells. coli treated with RbCI.



  100 lil of cells treated with RbCl and 1 ut of DNA solution containing 250
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 ng of plasmid pEVhis14gill were incubated for 10 min on ice (at 0 ° C.) and then for 5 min at 37 ° C. The mixture was then transferred to 2 ml of RB medium (1% bactopeptone (w / v), extract of 0.5% yeast (w / v), 1% NaCl (w / v) and incubated for 30 min to 2 hours at 37 ° C with shaking.



   100 and 500 g of the cell culture were spread on a Petri dish containing an RB medium + 100 g / ml ampicillin + 2% w / v agar.



  Mini preparation of plasmid DNA pEVhis14gIII.



   The colonies obtained above were first used to make mini-preparations in order to verify the production and the structure of the plasmid pEVhisl4gill. Individual colonies were cultivated

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 overnight in 2 ml of RB medium + ampicillin 100 g / ml.



   The cultures were then treated as described in "Molecular cloning, a laboratory manual", J. Shambrook, EF Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press (1989) under the chapter "Small-scale preparations of plasmid DNA", µ 1.21-1. 28 except with the exception that for steps 1-4, the centrifugations were carried out at room temperature and that the composition of the solution m was modified so as to contain 3M sodium acetate, at pH 4.8.



   The pellets were dried under vacuum and taken up in 100 to 200 l of water (filtered on a Milli-Q device, Millipore, USA) without treatment with RNAse.



   Analysis by digestion using various restriction enzymes, followed by agarose gel electrophoresis made it possible to verify the compliance of the plasmid (see also "Molecular cloning, a laboratory manual", J. Shambrook, EF Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press (1989), µ6).



  Maxi-preparation of plasmid DNA pEVhisl4gïn
In order to obtain plasmid DNA in an amount sufficient for vaccinations, the plasmid DNA was obtained in greater quantity according to one of the following two procedures.



   A suspension of E. coli transformed with the plasmid pEVhis14gm is incubated for one hour in 2 ml of RB medium and then distributed in 1 to 4 bottles of 400 ml of RB medium containing ampicillin at the rate of 100 g / ml. The cultures were incubated overnight at 37 ° C with shaking at about 150-200 rpm.



   The cultures were centrifuged in 500 ml Nalgene tubes for 7 minutes at 8670 g. All centrifugations were carried out in a Beckman model J2-21 centrifuge.



   The supernatant was removed and the pellet was resuspended in 10 ml of solution 1 (1% glucose (w / v); 25 mM tris-HCl; pH = 8.0; 10 mM EDTA; 1% lysozyme (p / v)) and transferred to a 40 ml Nalgene tube.



  The tube was left for 5 minutes at room temperature. Then 10 ml of solution 2 (0.2 M NaOH; 1% SDS (w / v)) was added, the tubes were stirred and left for 5 minutes at room temperature. 10 ml of solution 3 (3M sodium acetate; pH = 4.8) were added and the flasks were shaken and then centrifuged for 30 minutes at 48,400 g at 0 C. 25 ml of the supernatant were transferred to a tube 50 ml Falcon

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 covered with six layers of gauze. If necessary, the volume can be adjusted with TE (10 mM tris-HCl; pH = 8.0; 1 mM EDTA). Then 15 ml of isopropanol was added at room temperature, stirred and the mixture transferred to a 40 ml Nalgene tube.

   After centrifugation for 15 minutes at 48,400 g at 0 C, the supernatant was removed. After having drained the liquid well and dissolved the pellet in 5 ml of TE, the suspensions were incubated for 30 minutes at 37 ° C. with shaking in the presence of RNase A at a final concentration of 0.1 mg / ml. Then we added 10 juices
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 proteinase K in solution (10 mg / ml) and incubated for 30 minutes at 37 ° C minimum with shaking.



  The QIAGEN TIP 500 columns (QIAGEN, INC., CA, USA) were balanced with 10 ml of QBT buffer (750 mM NaCI; 50 mM MOPS; 15% ethanol (v / v); 0.15% Triton X-100 (v / v); pH = 7.0) by flow under simple gravity. The samples were placed on the columns, and the columns were washed 6 times with 10 ml of QC buffer (1000 mM NaCl; 50 mM MOPS; 15% ethanol (v / v); pH = 7.0). After elution with 20 ml of QF 'buffer (1250 mM NaCl; 50 mM MOPS; 15% ethanol (v / v); pH = 8.2), the solution was recovered in 40 ml Nalgene tubes. 14 ml of isopropanol were added at room temperature.



  After stirring, the mixture was centrifuged for 15 minutes at 48,400 g at 0 C. The supernatant was removed and the pellet was dried under vacuum and taken up in 500 μl of Milli-Q water. After three extractions of the supernatant with 500 III of phenol / chloroform / isoamyl alcohol (25/24/1, v / v / v) and one
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 extraction with one volume of ether, the DNA was precipitated with 50 bed of 3M sodium acetate and 0.7 ml of isopropanol. After centrifugation for 10 minutes at 0 C at 18,320 g, the pellet was washed with 1 ml of 70% ethanol (v / v). The supernatant was removed after a 10 minute centrifugation at 18,320 g at 0 C and the pellet comprising the plasmid DNA was dried under vacuum and dissolved in 500 l of Milli-Q water.



   Another method for producing plasmid DNA in large quantities using PZ523 columns (5 Prime- "3 Prime, INC., Boulder, CO., USA), is repeated below.



   The 400 ml cultures were centrifuged for 10 minutes at 8670 g in a 500 ml Nalgene well (Beckman J2-21). 10 ml of solution 1 (1% glucose (w / v), 25 mM tris-HCl pH = 8; 10 mM EDTA: 1% lysozyme (w / v) (SIGMA)) were added and 10 ml of this mixture in

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 another 40 ml Nalgene tube. After incubation for 5 minutes at room temperature, 10 ml of solution 2 (0.2 M NaOH; freshly prepared SDS 1% (w / v)) was added. After gentle shaking, the mixture was incubated for five minutes on ice.

   After adding 10 ml of solution 3 (3 M sodium acetate; pH = 4.8) cold and centrifuging the tube for 20 minutes at 0 C at 48,400 g, the supernatant, about 25 ml, was transferred to a tube 50 ml falcon covered with six layers of gauze. After adding 15 ml of isopropanol, the 40 ml of sample thus obtained were transferred to another 40 ml Nalgene tube and centrifuged for 20 minutes at 0 ° C. at 48,400 g. The isopropanol supernatant was removed, the pellet obtained was washed with 1 ml of 70% ethanol (v / v) and the liquid is well drained.

   The pellet was resuspended in 5 ml of TE to which 50 μl of RNase A (10 mg / ml solution) was added and incubated for 30 minutes at 37 ° C. with shaking. 10 µl proteinase K (10 mg / ml solution) was added and the mixture was incubated for at least 30 minutes at 37 ° C with shaking. For the two consecutive phenot / chioroform / isoamyl alcohol extractions (25/24/1, v / v / v), 5 ml of this phenol solution were added to a Greiner screw-on tube, then the sample. After gentle stirring for 20 to 30 seconds to mix, the solution was centrifuged for 5 minutes at 3,920 g in a "swinging bucket" type rotor.

   After passing the solution through a Nalgene tube, 1 ml of 7.5 M ammonium acetate and 10 ml of absolute ethanol were added. After centrifugation for 20 minutes at 0 C at 48,400 g (Beckman J2-21), the pellet was washed with 1 ml of 70% ethanol, then dried under vacuum and resuspended in 1.8 ml of solution 4 (Tris 10 mM; EDTA 1 mM; NaCI 1 M).



   After removing the upper plug and then the lower plug from the column, it was placed on a collection tube and then centrifuged for 1 minute at 980 g. The collection tube that collected the balancing buffer was discarded. The column was placed on another collection tube and the dissolved sample (1.8 ml) was deposited on top of the resin. The column was centrifuged for 12 minutes at 980 g in a swinging bucket type rotor. The collected plasmid solution was divided into two Eppendorf tubes to which 600 ± 1 isopropanol was added.



  After centrifugation for 15 minutes at 18,320 g (SIGMA 2K15 centrifuge), the pellet was washed with 300 μl of 70% ethanol (v / v) and dried under

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 empty. The pellet comprising the plasmid DNA is redissolved in 500 μl of Milli-Q water (Millipore, USA) and kept cold until vaccination.



   As regards the preparation of the DNA plasmid, approximately 11 mg were prepared according to the method using the PZ523 columns and approximately 16 mg using the Qiagen columns. These two preparations were mixed and used for the examples described.



  Step C: Formulation of the vaccine comprising the plasmid and a pharmaceutically acceptable excipient.



   The pellet of plasmid DNA being redissolved in water, the DNA concentration was determined by deposition on agarose gel and revelation with ethidium bromide (see "Molecular cloning, a laboratory manual", J. Shambrook, EF Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press (1989), µ 6 and appendix E and Winnacker EL "From genes to clones", VCH (1987), µ 2.1. 2.2.). The DNA concentration was adjusted so as to be between 0.3 and 1 zu of DNA per f'1 of water.



  Example 2: Use of the plasmid vaccine in mice to stimulate the induction of a plasmid antibody response and a cytotoxic T cell response.



   The experiment carried out in mice to prove the effectiveness of the plasmid vaccine requires the construction of a control plasmid derived from the plasmid pEVhis 14gill, prior to the immunization of the animals and to the analysis of the immune response.



  Step 1: Construction of a plasmid derived from the plasmid pEVhisl4gin by deletion of the coding sequence of the gin gene.



   A plasmid derived from the plasmid pEVhisl4gm by deletion of the coding sequence of the gin glycoprotein gene was used as
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 negative control (pEVhisl4gm ', DNA-). This deleted plasmid was obtained in the following manner: the plasmid pEVhisl4gill was digested with the restriction enzymes Asp 718 and EcoRV. The DNA was then processed using T4 DNA polymerase to obtain blunt-ended fragments.



  The DNA thus obtained was ligated using T4 DNA ligase (see "Molecular cloning, a laboratory manual", J. Shambrook, E. F. Fritsch and T.



  Maniatis, Cold Spring Harbor Laboratory Press (1989), µ 1.53-1. 73).



   With regard to the preparation of the vaccine containing the deleted plasmid, the same steps were followed as those described for the plasmid

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   DNA +, with the exception that during the production of the plasmid pEVhis14gIIr by maxi-preparation, the columns PZ523 were used only.



  Step 2: Mouse immunizations
5 groups of 6 to 10 female mice of the inbred Balb / c strain, aged 16 to 18 weeks at the first injection, were used.



   Mice must be inbred to measure cytotoxic T cell responses (CTL) because the compatibility of the major histocompatibility complex (MHC) between cytotoxic T cells and target cells, - 3T3-swiss albino cells (fibroblasts) (haplotype H-2D) -, must be guaranteed. Target cells were grown in DME medium containing 10% (v / v) fetal calf serum.



   The plasmid DNA of the plasmid pEVhisl4gm, containing the PRV virus gin glycoprotein gene, was used as positive DNA, (DNA +) while the equivalent plasmid, without the gm gene, (AND-), was used. as a negative control.



   100% g of DNA were injected into each mouse intramuscularly, in the upper left and right hindquarters, in two portions containing 50-150 p. 1 of aqueous solution, according to the following immunization protocol.



   Group 1, comprising 10 mice marked with a color code, was vaccinated four times, at week 0 (DNA '+), week 3 (DNA2 +), week 5 (DNA3 +) and week 10 ( DNA4 +) with DNA +. Serum, sADNi '*', was withdrawn 2 days before the last injection, serum sADN4 + was withdrawn 6 days after the first withdrawal, i.e. 5 days after the last DNA injection.



   At week 11, the spleen cells were removed and restimulated in vitro. CTL tests were performed 4 days later.



   Group 2, also comprising 10 mice marked with a color code, was vaccinated three times, at week 0 (iDNA +), at week 3 (DNA2 +) and at week 5 (gDNA) with serum DNA , sADN2 +, was collected 2 days before the last injection and sADN3 + serum was collected at week 9 i.e. 4 weeks after the last ADNo injection.



   At week 9, the spleen cells were restimulated in vitro. CTL tests were performed 6 days later.

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  Group 3 comprising 8 mice marked with a color code was vaccinated only twice, at week 0 (DNA 1 and at week 3 (DNA2 +). Serum, sADN2 +, was taken 2 weeks after the last injection and at week 9.



   At week 9, the spleen cells were restimulated in vitro. CTL tests were performed 6 days later.



   Group 4, or control group, comprising 10 mice marked with a color code, was vaccinated three times with DNA-, at week 0 with 200 g of DNA- (DNA, at week 2 with 100 tg (DNA *) and at the
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 week 7 with 100 g of DNA '(DNA'). Serum, sADN, was collected 2 days before the last injection, and sADNo serum was collected at week 8 i.e. 1 week after the last gDNA injection.



   On week 8, the spleen cells were removed and restimulated in vitro. CTL tests were performed 4 days later.



   Group 5 (positive control group), comprising 6 mice, was vaccinated three times with live virus, strain NIA3 M207 (obtained from the Institute for Animal Science and Health, ID-DLO, NL) at a dose of 107 PFU (Forming Unit Plate) by mouse and by injection into the toes, at week 0, at week 16 and at week 17.



   Serum was collected 2 days after the second injection. A mixture of serum from 5 animals was used for the analyzes.



   At week 18, the spleen cells were removed and restimulated in vitro. CTL tests were performed 4 days later.



  Step 3: Analysis of the humoral and cellular immune response (CTL test).



   Depending on the case, the animals were euthanized and the spleen was removed under aseptic conditions between 7 days and up to six weeks after the last DNA injection.



  Part A - In vitro culture and restimulation of the effectors
The vaccinated and control mice were euthanized by cervical dislocation and rinsed with alcohol (70% v / v). The rats of these animals were placed in a petri dish containing PBS (Gibco) and were crushed in these dishes using nylon gauze and a curved plastic tube. The elimination of the aggregates and of the connective tissue surrounding the spleen was done by filtration (nylon gauze) of the ground material obtained.



  After centrifugation at 220 g for 4 minutes, the pellet was recovered

  <Desc / Clms Page number 16>

 
 EMI16.1
 and the erythrocytes were lysed by adding 4 ml / spleen of sterile ACK solution (0.15 M NH4Cl, 1 mM KHC03, 0.1 mM NaEDTA, pH = 7, 2-7, 4).



   Then, two washes were carried out with sterile effector medium [composed of DME medium (Dulbecco's modified Eagle, Gibco), supplemented by 10% (v / v) of fetal calf serum (Gibco), 1% (v / v) 200 mM L-glutamine (Gibco), 1% (v / v) of the penicillinestreptomycin antibiotic solution (10,000 U / ml penicillin and 10,000 lig / ml streptomycin (Gibco), 10 mM HEPES buffer ( pH = 7.4) (Sigma), 2 x 10-5 M 2mercaptoethanol (Gibco), 2 mM sodium pyruvate (Merck)]. The cells were resuspended at 5 x 10 6 cells per ml in this sterile environment.

   The spleen cells are distributed for their cultivation in vitro in 25 cm 2 culture flasks (Falcon) at the rate of 25 x 10 6 cells / flask. Part of these cells received an in vitro restimulation by adding one of the viral strains mentioned above with a multiplicity of infection (MOI) equal to 2 and the others served as non-restimulated controls. These bottles were placed vertically in an incubator for 4 to 7 days (at 37 ° C., 3% (v / v) of COo and a humidity greater than 90% of saturation) as described above.



  Part B-The CTL test.



   The cells of the histocompatible line (fibroblasts) 3T3-swiss albino (haplotype H-2D) are used as targets.



   The CTL test comprises several stages: a) The target cells have been infected or not with a strain of the virus
Aujeszky (NIA3 M207) at an MOI equal to 10, the infected cells being designated CV + and the non-infected cells CV. Ninety minutes later, the labeling of the target cells in suspension began (see below-Eu labeling of the target cells (3T3) in suspension. B) As for the effectors cultured in vitro 4 to 7 days (as described above) before, they were taken up in culture flasks, washed 2 x with effector medium and counted to be resuspended at the rate of 5 x 10 6 cells / ml. Six hours after the start of the infection of the target cells, these were brought into contact with the effectors (including Tc lymphocytes).

   In a 96-well round bottom plate, 5,000 target cells in 50 gel of effector medium (Eu-labeled and infected or not) were deposited on

  <Desc / Clms Page number 17>

 
 EMI17.1
 respectively 500,000, 250,000, 125,000, 62,500, 31,250, 15,625 effectors in! 00 1 (i.e. effector / target ratios ranging from 100/1 to 3/1). Repetitions (3 or 4 times) are performed for each condition. The plate was centrifuged at 50 g for 4 minutes and brought to 37 C for 4 hours. The evaluation of the quantity of target cells lysed by Tc lymphocytes was carried out by a sample of supernatant after a further centrifugation for 4 minutes at 50 g.



  This was deposited in a 96-well plate with a flat bottom in 200
 EMI17.2
 h j. of a fluorescence enhancing solution (DELFIA
Enhancement solution, Pharmacia, Sweden) in the case of EU labeling. Fluorimeter counting (delayed-time fluorimeter, 1234
DELFIA Recherche, Wallac) was carried out 12 hours later, taking care to put the plates containing the mixture in the dark and at room temperature.



   The quantification of specific lysis (in%) was estimated using the following formula: Experimental release-background noise ------------------- X 100 = Specific lysis (%) Maximum release-background noise Part C-Eu marking of target cells in suspension.



   This type of labeling is applicable both for cells growing in suspension and for adherent cells.



   Culture flasks at maximum confluence were used for the different labeling of the 3T3 target cells.



   The culture flask containing the adherent 3T3 target cells was stripped of its growth medium and washed 1 x with PBS (Gibco). 5 ml of trypsin-EDTA (Gibco) at 37 ° C. were placed in the flask on the cell mat. After one minute, the cells were taken down with small tapping on the bottle. After complete detachment, 7 ml of sterile effector medium [described above were added.

   The cells were washed 1 x with a solution composed of HEPES buffer [50 mM HEPES ((hydroxy-2-ethyl) -4-piperazinyl-1,2-ethanesulfonic acid) (pH = 7.4), (Sigma); 93 mM NACI (Merck); 5 mM KCI (Merck); 2 mM
 EMI17.3
 MgC12. 6H20 (Merck)] and resuspended at the rate of 6 x 10 6 cells / ml in said solution. The counting of living cells was carried out with Trypan Blue (0.4% solution (w / v), Sigma).

  <Desc / Clms Page number 18>

 



   One ml of this suspension was made up with: - 750 gel of the HEPES buffer solution (pH 7.4), - 200 jut of the Eu-DTPA solution (1.52 ml of the standard Eu solution ( 1000 jug / ml in 1% (v / v) nitric acid) (Aldrich); 8 ml of the HEPES buffer solution (pH 7.4); 0.5 ml of DTPA diethylene triaminepentaacetic acid (Merck) at 3.93 g in 100 ml of the solution
 EMI18.1
 HEPES buffer) and after 2 minutes, with 100 μl of dextran sulphate (50 mg dextran sulphate, M. W. = 500 KDa, Pharmacia in 10 ml of the solution of HEPES buffer).



   Thirty minutes were required for labeling at room temperature. During this labeling, the tubes were shaken gently every 10 minutes. After which, 7 ml of repair buffer [0.588 g of CaCl2. 2:20 (Merck); 1.8 g of glucose (Merck) in 1 liter of the HEPES buffer solution pH 7, 4]; 3 ml of effector medium (see above) and 12 μl of DNAase at 17,000 units / ml (Boehringer Mannheim) were added. An 8-minute break was observed. Then, washing with the effector medium was followed by a cushion of Ficoll-Paque (Ficoll and sodium diatnzoate, Pharmacia LKB).

   To do this, the cells were resuspended in 5 ml of effector medium in a 50 ml tube (Falcon), and 5 ml of Ficoll-Paque were deposited at the bottom.



   After fifteen minutes of centrifugation at 800 g and 20 C, the upper part of the solution in the tube up to the interface included was recovered.



   The cells were rid of all traces of Ficoll-Paque by washing with the effector medium. After counting, the cells were resuspended at a concentration of 105 cells / ml.



   For the evaluation of the labeling, 5,000 target cells were deposited in a 96-well round-bottom plate in the presence of 100 l of effector medium to determine the amount of background noise from the Eu.



   The same amount of cells was deposited in 100 d of Triton X-100 (1% v / v, Merck) for maximum release of Eu. After a 4-minute centrifugation at 50 g, 20 µl of supernatant was removed and deposited in 200 ftl of fluorescence enhancer solution (see above) and the 96-well flat-bottom plate was run on a fluorometer ( 1234 DELFIA Recherche, Wallac) after an incubation of 1 hour in the dark so as to reach a stable reading.

  <Desc / Clms Page number 19>

 



  Part D-CTL test results.



  Table 2: Comparison of the specific lysis of splenocytes before and after passage over a Ficoll-Paque gradient.
 EMI19.1
 
 <tb>
 <tb>



  Lyse <SEP> specific <SEP> (in <SEP>%)
 <tb> ¯ <SEP> deviation <SEP> standard <SEP> (in <SEP>%)
 <tb> Groups / in <SEP> vitro / targets <SEP> ¯ <SEP> deviation <SEP> standard <SEP> (in <SEP>%)
 <tb> Report <SEP> E / C
 <tb> 100/1 <SEP> 50/1 <SEP> 25/1 <SEP> 12/1 <SEP> 6/1 <SEP> 3/1 <SEP> n
 <tb> Group <SEP> 5
 <tb> SB ++ / ME <SEP> 2 / CV + <SEP> 37.6 <SEP> 22.9 <SEP> 16.1 <SEP> 8, <SEP> 4 <SEP> 3, <SEP> 8-0, <SEP> 5 <SEP> 4
 <tb> Before <SEP> Ficoll <SEP> + <SEP> 10.0 <SEP> ¯ <SEP> 5, <SEP> 8 <SEP> ¯ <SEP> 4, <SEP> 7 <SEP> 2, <SEP> 2 <SEP> 1, <SEP> 2 <SEP> ¯ <SEP> 0, <SEP> 2
 <tb> SB ++ / ME <SEP> 2 / CV + <SEP> 42.2 <SEP> 35.5 <SEP> 20.3 <SEP> 17, <SEP> 0 <SEP> 9.1 <SEP> 11.4 <SEP> 4
 <tb> After <SEP> Ficoll <SEP> 16, <SEP> 4 <SEP> l <SEP> 12, <SEP> 2 <SEP> 6, <SEP> 4 <SEP> I6, <SEP> 9 <SEP> ¯ <SEP> 3.3 <SEP> ¯ <SEP> 4, <SEP> 5
 <tb> Group <SEP> 1
 <tb> DNA4 + / V- / CV- <SEP> 7.5 <SEP> 6.1 <SEP> 2,

  8 <SEP> 1,2 <SEP> -0.7 <SEP> -0.9 <SEP> 4
 <tb> Before <SEP> Ficoll <SEP> ¯ <SEP> 1, <SEP> 6 <SEP> 1.1 <SEP> 0, <SEP> 4 <SEP> ¯ <SEP> 0, <SEP> 3 <SEP> ¯ <SEP> 0, <SEP> 1 <SEP> 0, <SEP> 2
 <tb> DNA <SEP> + / V- / CV- <SEP> 9, <SEP> 2 <SEP> 9.1 <SEP> 5.5 <SEP> 2.7 <SEP> 1.9 <SEP> 1,2 <SEP> 4
 <tb> After <SEP> Ficoll <SEP> 1, <SEP> 6 <SEP> 1, <SEP> 5 <SEP> 0, <SEP> 6 <SEP> ¯ <SEP> 0, <SEP> 5 <SEP> 0, <SEP> 2 <SEP> 0, <SEP> 2
 <tb> DNA4 + / V- / CV + <SEP> 11.9 <SEP> 9.3 <SEP> 1, <SEP> 8-2, <SEP> 1-2, <SEP> 7-3, <SEP> 6 <SEP> 4
 <tb> Before <SEP> Ficoll <SEP> + <SEP> 4, <SEP> 0 <SEP> I3, <SEP> 5 <SEP> ¯ <SEP> 0, <SEP> 6 <SEP> ¯ <SEP> 0, <SEP> 6 <SEP> 0, <SEP> 8 <SEP> + <SEP> 1.0
 <tb> DNA4 + / V- / CV + <SEP> 31.8 <SEP> 49.4 <SEP> 45, <SEP> 6 <SEP> 20.6 <SEP> 19.8 <SEP> 10.7 <SEP> 4
 <tb> After <SEP> Ficoll <SEP> ¯ <SEP> 14.2 <SEP> + <SEP> 20.3 <SEP> 23, <SEP> 8 <SEP> 9, <SEP> 0 <SEP> ¯ <SEP> 9,

    <SEP> 0 <SEP> 4.7
 <tb> DNA4 + / ME <SEP> 2 / CV- <SEP> -0.3 <SEP> -0.7 <SEP> -1.0 <SEP> -0.6 <SEP> -1.3 <SEP> -1, <SEP> 2 <SEP> 4
 <tb> Before <SEP> Ficoll <SEP> ¯ <SEP> 0.1 <SEP> ¯ <SEP> 0.1 <SEP> ¯ <SEP> 0.2 <SEP> ¯ <SEP> 0.1 <SEP> ¯ <SEP> 0.2 <SEP> ¯ <SEP> 0, <SEP> 2
 <tb> DNA4 + / ME <SEP> 2 / CV- <SEP> -2.0 <SEP> -1.6 <SEP> -1.2 <SEP> -2.7 <SEP> -1.1 <SEP> -1, <SEP> 3 <SEP> 4
 <tb> After <SEP> Ficoll <SEP> ¯ <SEP> 0, <SEP> 3 <SEP> 0, <SEP> 2 <SEP> 0, <SEP> 2 <SEP> 0, <SEP> 4 <SEP> 0, <SEP> 1 <SEP> 0, <SEP> 2
 <tb> DNA.

    <SEP> + / ME <SEP> 2 / CV + <SEP> -5.3 <SEP> -6.2 <SEP> -5.4 <SEP> -5.1 <SEP> -4.1 <SEP> -3, <SEP> 3 <SEP> 4
 <tb> Before <SEP> Ficoll <SEP> + <SEP> 1.6 <SEP> 1, <SEP> 6 <SEP> 1, <SEP> 4 <SEP> 1, <SEP> 5 <SEP> ¯ <SEP> 1, <SEP> 1 <SEP> ¯ <SEP> 0, <SEP> 9
 <tb> DNA4 + / ME <SEP> 2 / CV + <SEP> 15.4 <SEP> 12.8 <SEP> 19.1 <SEP> 15.3 <SEP> 7.6 <SEP> 22.9 <SEP> 4
 <tb> After <SEP> Ficoll <SEP> ¯ <SEP> 6, <SEP> 6 <SEP> ¯ <SEP> 5, <SEP> 8 <SEP> 8, <SEP> 6 <SEP> ¯ <SEP> 7, <SEP> 2 <SEP> ¯ <SEP> 2, <SEP> 9 <SEP> +
 <tb> 13.3
 <tb> Group <SEP> 4
 <tb> DNA4 + / ME <SEP> 2 / CV + <SEP> -5.5 <SEP> -5.2 <SEP> -3.9 <SEP> -3.3 <SEP> -3.9 <SEP> -3, <SEP> 7 <SEP> 4
 <tb> Before <SEP> Ficoll <SEP> ¯ <SEP> 1.7 <SEP> ¯ <SEP> 1.5 <SEP> ¯ <SEP> 1,2 <SEP> 1.1 <SEP> 1, <SEP> 2 <SEP> + <SEP> 1.3
 <tb> DNA3- / ME <SEP> 2 / CV + <SEP> -10.3 <SEP> -5.8 <SEP> -7.8 <SEP> -2.4 <SEP> -3, <SEP> 5 <SEP> 6,

  5 <SEP> 4
 <tb> After <SEP> Ficoll <SEP> + <SEP> 4.2 <SEP> + <SEP> 2.1 <SEP> ¯ <SEP> 3.4 <SEP> ¯ <SEP> 1, <SEP> 1 <SEP> ¯ <SEP> 1.3 <SEP> ¯ <SEP> 2, <SEP> 8
 <tb>
 

  <Desc / Clms Page number 20>

 
Explanatory notes concerning in vitro restimulation treatments and target preparation.



  V: spleen cells cultured in vitro in the absence of MOI 2 virus: spleen cells cultured in vitro and restimulated by addition of live virus (NIA3 M207) with a multiplicity of infection (MOI) equal to 2 .



  CV: non-infected target cells, labeled with Europium CV +: target cells infected by the NIA3 M207 virus at an MOI equal to 10, and labeled with Europium
SB + +: group 5, positive control vaccinated with live virus DNA: group 1, animals injected 4 times with DNA + DNA3-: group 4, animals injected 3 times with DNA- n: number of repetitions
The lysis of uninfected target cells was between 0 and 9% and was not affected by the passage over the Ficoll-Paque gradient. In contrast, the positive control (group 5), both before and after Ficoll treatment, presented a significantly positive cell lysis rate.

   The lysis percentages of the infected targets were increased by passage over Ficoll-Paque for animals immunized with DNA.



   The CTL group 1 test, injected 4 times with DNA + (DNA4 +) showed lytic activity.



   The CTL group 2 test, injected 3 times with DNA (DNA) did not show lytic activity.



   The group 3 CTL test, injected twice with DNA + (DNA2 +) did not show lytic activity.



   The group 4 CTL test, injected 3 times with DNA '(DNA *) did not show lytic activity.



   Of course other frequencies and other intervals are possible, as well as other dosages and routes of immunization.



  Part E-Analysis of the humoral immunological response.



   The serum was collected twice during the experiment, the last collection being made just before the sacrifice of the animals to obtain the spleen cells for a CTL test, and the antibody responses were measured by : a PRV virus seroneutralization test (SN) an enzyme immunoassay (ELISA) for the measurement in the serum of

  <Desc / Clms Page number 21>

 mice with antibodies to an extract of all PRV glycoproteins.



   The PRV virus seroneutralization test was carried out according to the procedure which is reproduced below.



   PD5 cells (SOLVAY-DUPHAR, NL) and Bartha K61 strain viruses (SOLVAY-DUPHAR, NL) were used. The experiment was carried out in 96 microwell plates with a flat bottom from Greiner (France).



   The medium in which the SN test was carried out had the following composition: 340 ml minimum Eagle essential medium (Flow), 100 ml 2.5% (w / v) lactalbumin hydrolyzate; 5-10 ml of 5.6% NaHCO3 (w / v) and 50 ml of fetal calf serum (Gibco).



   The test serum was diluted in series of 2 in 2 in the medium, the dilutions ranging from 1: 2 to 1: 4096 (50 1'1 of serum + 50 1'1 of medium each time), in the 96 flat bottom wells (Greiner). Each sample was tested in duplicate.



   The virus was diluted to 100 TCID50 (tissue culture infectious dose at 50%) in 0.05 ml of medium and 50 μl of this diluted virus solution was added to each well. The serum / virus mixture was incubated for 24 hours at 37 ° C. 50 μl of the suspension of PD5 cells having a concentration of 4 × 10 -6 cells / ml were added to each sample of serum / virus. then incubated at 37 ° C for 5 days.



   The results were observed under a microscope and the titers were calculated by taking the inverse of the dilution which corresponds to 50% of the limiting dilution.



   The virus was checked by incubating a diluted virus sample
 EMI21.1
 for 24 hours at 4 C and another virus sample for 24 hours at 37 C. The two virus suspensions were diluted (v / v) with the SN test medium (see above) 1: 10; 1: 100 and 1: 1000. Then, 0.05 ml of each dilution of the virus suspensions were added per well using 8 wells for each dilution. Then 0.05 ml of medium and 0.05 ml of cell suspension were added. The results were interpreted under the microscope after an incubation of 5 days at 37 C.



   The humoral responses measured by the SN test, after two, three and four DNA injections, showed zero or weakly positive values.



   It should be noted that after immunization with live virus in large doses,

  <Desc / Clms Page number 22>

 measurable but relatively low titers were observed in the seroneutralization test. These observations are confirmed by the literature.



   The procedure for the ELISA test is described by M. ELOIT et al in ARCH. virol. (1992), 123, 135-143.



   In addition to the serum from groups of animals treated as described under "immunization of mice", 2 additional sera were included in the analysis, a positive serum (serum +) and a negative serum (serum-). The serum comes from non-injected mice (OFI mice, 3 weeks old). A mixture of serum from 10 mice was carried out. Serum + is a mixture of serum from 10 OFI mice which received 109 TCID (tissue culture infectious dosis) of recombinant adenovirus at 3 weeks expressing the gD gene intramuscularly and collected 3 weeks later.



   Table 3 and Table 4, shown below, show the optical density (OD) as a function of the dilutions of the serum. The samples were first tested at a dilution of 1/10 (Table 3-test 1) in order to identify the positive samples c. i.e. samples for which the OD was greater than the optical density of the negative control serum (OD x 100 = 509). The positive samples thus identified were tested a second time at variable dilutions (Table 4).



   In the serum of animals injected with plasmid DNA without the gill gene (DNA * -pEVhisl4gII-), no antibody was detected against the gill glycoprotein. It has been shown that after 4 injections of DNA plasmids spaced 2 weeks or more apart, the mice showed a good humoral response against the virus. Seven out of 9 animals tested showed anti-gin antibodies after four injections.



   Even after three injections of DNA +, the serum of 9 animals out of 10 showed measurable anti-gin antibody titers. Similarly, after 2 injections of DNA +, the serum of 7 animals out of 8 showed positive responses in ELISA.

  <Desc / Clms Page number 23>

 
 EMI23.1
 



  Table 3: EUSA test: Optical density (OD) as a function of the dilutions of the serum-Identification of positive samples.
 EMI23.2
 
 <tb>
 <tb>



  OD <SEP> (x <SEP> 100)
 <tb> n <SEP> from <SEP> mouse <SEP> dilution <SEP> 1/10
 <tb> Witness <SEP> positive <SEP> (NIA3 <SEP> M207) <SEP> mixture <SEP> 2000
 <tb> sADN2-1 + 2 + 3 <SEP> 552
 <tb> 4 + 5 + 6 <SEP> 545
 <tb> 7 + 8 + 9 + 10 <SEP> 395
 <tb> sADN3- <SEP> 1 + 2 + 3 <SEP> 587
 <tb> 4 + 5 + 6 <SEP> 679
 <tb> 7 + 8 + 9 + 10 <SEP> 697
 <tb> DNA <SEP> + <SEP> 1 <SEP> 2000
 <tb> 2 <SEP> 2000
 <tb> 3 <SEP> 2000
 <tb> 4 <SEP> 511
 <tb> 5 <SEP> 1263
 <tb> 6 <SEP> 2000
 <tb> 7 <SEP> 2000
 <tb> 8 <SEP> 2000
 <tb>
 

  <Desc / Clms Page number 24>

 
 EMI24.1
 
 <tb>
 <tb> 9 <SEP> 2000
 <tb> 10 <SEP> 2000
 <tb> DNA + <SEP> 2 <SEP> 2000
 <tb> 3 <SEP> 2000
 <tb> 4 <SEP> 874
 <tb> 5 <SEP> 759
 <tb> 6 <SEP> 2000
 <tb> 7 <SEP> 2000
 <tb> 8 <SEP> 2000
 <tb> 9 <SEP> 2000
 <tb> 10 <SEP> 2000
 <tb> serum-mixture <SEP> 509
 <tb> serum <SEP> + <SEP> mixture <SEP> 2000
 <tb>
 explanatory notes: sADN2-:

   serum from animals injected 2 times with DNA- sADN3-: serum from animals injected 3 times with DNA- sADN3 +: serum from animals injected 3 times with DNA + sADN4 +: serum from animals injected 4 times with DNA + control positive (NIA3 M207): serum from animals injected with live virus NIA3 M207 serum-: serum from non-injected animals

  <Desc / Clms Page number 25>

 serum +: serum from animals injected with adenovirus expressing the gD gene.
 EMI25.1
 



  Table 4: EUSA optical density (OD) test as a function of the dilutions of test serum 2
 EMI25.2
 
 <tb>
 <tb> group <SEP> mouse <SEP>? <SEP> says. <SEP> 1/100 <SEP> 1/300 <SEP> 1/900 <SEP> 1/2700
 <tb> witness <SEP> positive <SEP> medium <SEP> 2000 <SEP> 2000 <SEP> 2000 <SEP> 2000
 <tb> (M207)
 <tb> DNA <SEP> + <SEP> 3 <SEP> 1087 <SEP> 599 <SEP> 287 <SEP> 171
 <tb> 4 <SEP> 1378 <SEP> 622 <SEP> 303 <SEP> 192
 <tb> 5 <SEP> 1506 <SEP> 866 <SEP> 405 <SEP> 261
 <tb> 6 <SEP> 673 <SEP> 261 <SEP> 152 <SEP> 136
 <tb> 7 <SEP> 1676 <SEP> 913 <SEP> 353 <SEP> 205
 <tb> 8 <SEP> 1731 <SEP> 837 <SEP> 355 <SEP> 229
 <tb> 9 <SEP> 1710 <SEP> 778 <SEP> 274 <SEP> 167
 <tb> 1 <SEP> Negative <SEP> to <SEP> dil <SEP> 1/10
 <tb> 2 <SEP> and <SEP> 10 <SEP> no <SEP> tested
 <tb> sADN3 + <SEP> + <SEP> 1 <SEP> 2000 <SEP> 1029 <SEP> 329 <SEP> 142
 <tb> 2 <SEP> 2000 <SEP> 2000 <SEP> 879 <SEP> 296
 <tb> 3 <SEP> 2000 <SEP>

  2000 <SEP> 2000 <SEP> 2000
 <tb> 5 <SEP> 197 <SEP> 123 <SEP> 89 <SEP> 83
 <tb> 6 <SEP> 2000 <SEP> 1704 <SEP> 528 <SEP> 244
 <tb>
 

  <Desc / Clms Page number 26>

 
 EMI26.1
 
 <tb>
 <tb> 7 <SEP> 2000 <SEP> 2000 <SEP> 1084 <SEP> 435
 <tb> 8 <SEP> 2000 <SEP> 809 <SEP> 188 <SEP> 122
 <tb> 9 <SEP> 2000 <SEP> 1501 <SEP> 285 <SEP> 169
 <tb> 10 <SEP> 2000 <SEP> 2000 <SEP> 722 <SEP> 263
 <tb> 4 <SEP> Negative <SEP> to <SEP> dil <SEP> 1/10
 <tb> DNA + <SEP> 2 <SEP> 2000 <SEP> 2000 <SEP> 684 <SEP> 262
 <tb> 3 <SEP> 2000 <SEP> 2000 <SEP> 2000 <SEP> 2000
 <tb> 4 <SEP> 263 <SEP> 138 <SEP> 98 <SEP> 103
 <tb> 5 <SEP> 143 <SEP> 110 <SEP> 96 <SEP> 104
 <tb> 6 <SEP> 2000 <SEP> 1641 <SEP> 566 <SEP> 256
 <tb> 7 <SEP> 2000 <SEP> 2000 <SEP> 1122 <SEP> 419
 <tb> 8 <SEP> 2000 <SEP> 1097 <SEP> 350 <SEP> 143
 <tb> 9 <SEP> 2000 <SEP> 1739 <SEP> 462 <SEP> 211
 <tb> 10 <SEP> 2000 <SEP> 2000 <SEP> 549 <SEP> 249
 <tb> 1 <SEP>

  no <SEP> tested
 <tb> serum <SEP> - <SEP> mixture <SEP> 155 <SEP> 96 <SEP> 83 <SEP> 92
 <tb> serum <SEP> + <SEP> mixture <SEP> 827 <SEP> 305 <SEP> 146 <SEP> 106
 <tb>
 

  <Desc / Clms Page number 27>

 explanatory notes: sADN2-: serum from animals injected 2 times with DNA- sADN3-: serum from animals injected 3 times with DNA- sADN3 +: serum from animals injected 3 times with DNA + sADN4 +: serum from animals injected 4 times with DNA + positive control (NIA3 M207): serum from animals injected with live virus NIA3 M207 serum-: serum from animals not injected serum +: serum from animals injected with adenovirus expressing the gD gene.



  Example 3:
Induction of protection in mice against challenge inoculation with virulent viruses.



  Part A-Immunization protocol.



   Five groups of ten mice (Charles River, Germany) females of 16 weeks of age of the inbred strain Balb c were used.



   For the group GO, the negative control, only a PBS buffer (Gibco) was used.



   Group G 1 (the positive control) was vaccinated using an attenuated virus (strain NIA3 M207) intraperitoneally.



   107 PFUs (plate forming units) were used for each mouse. This is a very high dose compared to the doses used for vaccination of pigs with the attenuated vaccine strains traditionally used at the dose of 105.5 PFU.



   The other three groups received plasmid DNA obtained according to the method described in Example 1.



   Plasmid DNA vaccination was carried out by intramuscular injection of 100 g in 2 times 100 g of water / mouse in the left and right hindquarters for several consecutive days.



   Group C was vaccinated twice, Friday and Monday respectively.



   Group B received four consecutive injections from Wednesday to Monday.



   Group A received 6 doses distributed from Monday to the following Monday.



   The animals were housed in cages, separated by group and the individuals were individually marked with blue felt.

  <Desc / Clms Page number 28>

 



   The serum taken from each animal was coded so that each animal could be followed individually with regard to the antibody dosage and the protection conferred by vaccinations.



   Part B-Analysis of immune responses.



   The development of humoral responses was monitored according to the ELISA protocol presented in Example 2.



   The serum virus neutralization test was carried out according to the method described in Example 2.



   Serum samples were taken at the start of the immunization period, i.e. 3 days after the last vaccine injection, at the end of the immunization period, i.e. one month after the last injection of vaccine and just before the inoculation of the test with the virulent virus which took place 9 days later. Finally, one month after the challenge, serum was collected again.



  Part C-Test inoculation.



   About 37 days after the last vaccination, all the mice were exposed to infection with a live virulent virus of the NIA3 strain at the rate of 7,000 PFU in 200 IL1 per animal injected intraperitoneally.



  The dosage is very high because the lethal dose for 50% of the animals is approximately 100 times lower (LD50 = 70 PFU).



   The animals are observed and the deaths are recorded during the 15 days following the challenge as indicated in Table 5.



   The results show the evolution of the survival rate expressed as a percentage as a function of the days following the challenge.



   All animals in the negative control group died after the challenge.



  Table 5: Monitoring of the survival rate of mice after a challenge inoculation with virulent viruses.

  <Desc / Clms Page number 29>

 
 EMI29.1
 
 <tb>
 <tb>



  Groups <SEP> from
 <tb> mouse <SEP> (Ten <SEP> Rate <SEP> from <SEP> survival <SEP> (in <SEP>%) <SEP> in <SEP> function <SEP> of <SEP> days <SEP> after <SEP> the test
 <tb> by <SEP> group)
 <tb> 4 <SEP> days <SEP> 5 <SEP> days <SEP> 6 <SEP> days <SEP> 7 <SEP> days <SEP> 10 <SEP> days <SEP> 15 <SEP> days
 <tb> GO <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0
 <tb> GI <SEP> 100 <SEP> 80 <SEP> 80 <SEP> 80 <SEP> 80 <SEP> 80
 <tb> DNA <SEP> 50 <SEP> 20 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0
 <tb> 2 <SEP> x <SEP> 100 <SEP> gg
 <tb> DNA <SEP> 80 <SEP> 40 <SEP> 30 <SEP> 30 <SEP> 30 <SEP> 30
 <tb> 4 <SEP> x <SEP> 100 <SEP> g
 <tb> DNA + <SEP> 90 <SEP> 60 <SEP> 60 <SEP> 60 <SEP> 60 <SEP> 60
 <tb> 6 <SEP> x <SEP> 100 <SEP> j, <SEP> tg
 <tb>
 
 EMI29.2
 The results show that protection took place even at the lowest vaccination dose, i.e. 2 x 100 gg.

   Indeed, the death of these animals was delayed compared to the negative control group (GO).



  At a dosage of 4 x 100 mg, 30% of the animals survived, while at 6 x 100 jug, 60% of the animals survived. The results show that the protection induced by this new plasmid vaccination method is effective, especially when compared to the 80% survival rate observed in group G 1 (the positive control), i.e. the group of animals vaccinated with the very high dose attenuated virus (107 PFU).



  It should be noted that the vaccination method used in this example can still be optimized. According to the results of the cytotoxicity test against the PRV virus, vaccination by plasmid injection for several consecutive days did not induce a CTL response while the injection of the same quantity of DNA at intervals of 3 weeks or more, as used in Example 2 induced a pronounced CTL response.



  In addition, the induced humoral response, measured by the anti-gin antibody response in the ELISA test, was lower following plasmid injections for several consecutive days than the humoral response induced by injections of the same amount of DNA into 3 week intervals, described in Example 2.



  Example 4: Induction of protection in mice against challenge inoculation with virulent viruses.



  For these experiments, the immunization protocol of Example 3 was

  <Desc / Clms Page number 30>

 followed, the only difference being that injections of plasmid DNA were given every three weeks and that the mice were 19 weeks old.



   For the GO group (negative control), only PBS buffer (Gibco) was used.



   Group G 1 (positive control) was vaccinated using the attenuated virus (strain NIA3 M207) in the necks with a dose of 107 PFU per mouse. Four injections of plasmid DNA were carried out intramuscularly in the two hindquarters of groups of mice each comprising 10 animals, marked with a colored code.



   Serum was collected either the same day or three days before the new immunization.



   Three weeks after the last plasmid injection and six weeks after the immunizations of the control groups, all the mice were exposed to the virulent NIA3 virus at a rate of 7,000 PFU / animal, injected intraperitoneally. Deaths are recorded during the 15 days following the test and are shown in Table 6. The results show the survival rates expressed as a percentage as a function of the days after the test.



  Table 6: Monitoring of the survival rate of mice after a challenge inoculation with virulent viruses.
 EMI30.1
 
 <tb>
 <tb>



  Groups <SEP> from <SEP> Rate <SEP> from <SEP> survival <SEP> (in <SEP>%) <SEP> in <SEP> function <SEP> of <SEP> days <SEP> after <SEP> the test
 <tb> mouse <SEP> (Ten
 <tb> by <SEP> group)
 <tb> 4 <SEP> days <SEP> 5 <SEP> days <SEP> 6 <SEP> days <SEP> 7 <SEP> days <SEP> 10 <SEP> days <SEP> 15 <SEP> days
 <tb> GO <SEP> 40 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0
 <tb> Gel <SEP> 100 <SEP> 90 <SEP> 90 <SEP> 80 <SEP> 80 <SEP> 80
 <tb> DNA + <SEP> 80 <SEP> 70 <SEP> 60 <SEP> 50 <SEP> 40 <SEP> 40
 <tb> 4 <SEP> x <SEP> 100 <SEP> p. <SEP> g
 <tb>
 All GO group animals (negative control) died after
 EMI30.2
 the ordeal. An 80% survival rate is observed in the Gl, c group. -to do animals vaccinated by the attenuated virus at a very high dose.



  At a dosage of 4 times 100 g of plasmid DNA, 40% of the animals survived. The immunization method by which animals are vaccinated with plasmid DNA (4 injections) every three weeks

  <Desc / Clms Page number 31>

 was more effective compared to daily injections (better survival rate of 40% instead of 30% and relative delay in the death of animals).



  Example 5: Induction of a humoral response in pigs.



   Three groups of pigs of 3 animals, 5 weeks old were used. The animals come from a PRV-free farm and from the same litter. They are individually marked with an ear ring.



  A group of 3 animals (&num; 1,2 and 3) which have not been vaccinated is used as a negative control.



   For the other 2 groups, three intramuscular injections of the plasmid pEVhis14gm were carried out at the ages of 5.7 and 9 weeks.



  Three animals (&num; 4,5 and 6) received a dose of 75 gg of plasmid, while 3 other pigs (U 7, 8 and 9) received 560 g of plasmid with each injection. The DNA dose was diluted in 4 ml of PBS buffer (Gibco, USA) and administered in 4 portions of 1 ml at 4 places of inoculation: on both sides of the neck and in the center of the left hindquarters and law. The injection was made using a syringe fitted with a 40 mm long Terumo needle with a 0.9 mm opening.



   Serum was collected during each injection and 2 weeks after the last injection. Analysis of the humoral responses (antibodies against the gm protein) before and during the immunization was carried out by a seroneutralization test (sensitive test mediated by complement. See Bitsch and Eskilsen, curr. Top. Vet. Med. Anim. Sci. 12,41-49, 1982) and by an IPMA test (Immuno-Peroxidase-Monolayer-Assay).



  The IPMA test was carried out according to the procedure which is shown below. On each well 96-well plates (Corning, USA) covered
 EMI31.1
 500 TCID50 of PRV virus of strain 89V87 ((Nauwynck H., Pensaert M., Am. J. Vet. Research, 53 (4) 489) were added to a carpet of confluent SK-6 cells (ATCC, USA) (1992)) in MEM medium (Gibco, USA) As soon as a cythopathic effect appears, the plates are thermofixed: after washing with PBS buffer, the plates are dried at 37 ° C. until the liquid evaporates. Then they are incubated at 80 ° C for 1 hour.



  The test serum, diluted 2 in 2 in series in PBS was distributed in the wells and the plate was incubated for 1 hour at 37 C.



  The serum was removed and the plates were washed twice with PBS, followed by

  <Desc / Clms Page number 32>

 one hour incubation in the presence of anti-porcine antibodies labeled with peroxidase (Nordic, Holland) and diluted 100 times in PBS. After 1 hour, the plates were incubated in the presence of 3-amino-9-ethylcarbazole substrate (2 mg AEC in 10 ml of sodium acetate buffer (0.05 M at pH 5) and 75 l of 30% H 2 O 2).



  The appearance of a red coloration is observed under an optical microscope. The reaction was stopped after 15 minutes by 3 washes with tap water.



  The IPMA titers are calculated by taking the inverse of the highest dilution which generates a red coloration of the foci of viral antigen on the infected cells.



  The results of the seroneutralization test and the IPMA test are shown in Table 7.



  Table 7:
Antibody titers against the PRV virus determined by the seroneutralization test and by the IPMA test
 EMI32.1
 
 <tb>
 <tb> Titles <SEP> in <SEP> antibody <SEP> in <SEP> function <SEP> from <SEP> age
 <tb> age
 <tb>? <SEP> from <SEP> Dose <SEP> from <SEP> 5 <SEP> weeks <SEP> 7 <SEP> weeks <SEP> 9 <SEP> weeks <SEP> 11 <SEP> weeks
 <tb> pork <SEP> plasmid
 <tb> pEVhis14gm
 <tb> administered
 <tb> 1 <SEP> 'Og < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5 < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5
 <tb> 2 <SEP> gas <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5
 <tb> 3 <SEP> 0 <SEP> g <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5
 <tb> 4 <SEP> (75 <SEP> g)

    <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> 4 <SEP> < <SEP> 5 <SEP> 6 <SEP> 128
 <tb> 5 <SEP> (75 <SEP> g) <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5
 <tb> 6 <SEP> (75 <SEP> g) <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> 4 <SEP> 128
 <tb> 7 <SEP> (560 <SEP> g) <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP>, 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5
 <tb> 8 <SEP> (560 <SEP> g) <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> < <SEP> 5
 <tb> 9 <SEP> (560 <SEP> g) <SEP> < <SEP> 2 <SEP> < <SEP> 5 <SEP> < <SEP> 2 <SEP> <5 <SEP> < <SEP> 2 <SEP> <5 <SEP> 3 <SEP> 128
 <tb>
 Explanatory notes SN:

   serum neutralizing antibody titer EPMA: antibody titer determined by the IPMA method

  <Desc / Clms Page number 33>

 
The results show that none of the unvaccinated animals developed antibodies against the PRV virus. The results indicate that even at the lowest vaccination dose, that is to say 3 × 75 p. g, a humoral response was induced in 2 out of 3 pigs. Only 1 animal out of 3 reacted to a dosage of 560 mg. The responses are weak and the differences between the 2 immune groups are not significant.



  Example 6: Induction of protection against a test with a virulent virus in pigs.



   At least 2 groups of pigs, at least 3 animals aged 5 weeks will be used. The animals come from a PRV-free farm and preferably come from the same litter within a group. They are individually marked with an ear ring.



  A first group, which will not be vaccinated, will be used as a negative control.



   The other animals will be injected at least once by intramuscular injections (see details of the method in Example 5) of at least 50 to 2000 jug of DNA + plasmid (plasmid pEVhisl4gill) repeated at 2 weeks or more apart .



   The virulent virus test will be carried out according to the method described in Vaccine 12 (7), p. 661-665 (1994).



   The effectiveness of DNA + vaccination will be demonstrated by observing a decrease in clinical signs (temperature and weight) and less proliferation of the virus used for the test load in immunized animals compared to to the negative control group.


    

Claims (9)

 CLAIMS 1-Vaccine comprising a plasmid containing a gene coding for the PRV virus glycoprotein gin or for a protein having the same antigenicity as the PRV virus glycoprotein gin and a pharmaceutically acceptable excipient for the latter.  2-Vaccine according to claim 1, characterized in that the plasmid also contains a "human Cytomegalovirus promoter".  3-Vaccine according to claim 1, characterized in that the plasmid used is the plasmid pEVhisl4gID.  4-Vaccine according to claims 1 to 3 characterized in that the plasmid also contains at least one gene or a portion thereof coding for at least one cytokine or a fragment of a cytokine.  5-Vaccine according to any one of claims 1 to 4 characterized in that the pharmaceutically acceptable excipient comprises tiny gold beads coated with the vaccine which are introduced into the tissue of the animal to be vaccinated.  6-Use of a plasmid for a vaccine against the PRV virus, said plasmid comprising a nucleic acid sequence coding for the gin protein of the PRV virus or for a protein having the same antigenicity as the gin glycoprotein of the PRV virus or a construct DNA comprising an expression cassette including: a) a DNA sequence coding for a polypeptide containing at least one antigenic determinant of the glycoprotein gin or an immunogenic fragment thereof, and b) control sequences operatively linked to said coding sequences where said coding sequence can be transcribed and translated in a cell and where said control sequences are homologous or heterologous to said coding sequence.  7-A plasmid vaccine against the PRV virus according to claim 6 characterized in that it also contains at least one gene or a portion thereof coding for at least one cytokine or a fragment of a cytokine.  <Desc / Clms Page number 35>    EMI35.1   8 - Plasmid pEVhisl4gID used in the manufacture of a vaccine. 9-pEVhisl4gm plasmid used in the manufacture of a vaccine against the PRV virus.