EP0817799A1

EP0817799A1 - Plasmid vaccine for pseudorabies virus

Info

Publication number: EP0817799A1
Application number: EP96943118A
Authority: EP
Inventors: Christine Swysen; Christophe Depierreux
Original assignee: Solvay SA; Dimminaco AG
Current assignee: Dimminaco AG
Priority date: 1995-12-21
Filing date: 1996-12-06
Publication date: 1998-01-14
Also published as: CA2216308A1; AU711915B2; AU1194997A; WO1997023502A1

Abstract

A vaccine including at least one plasmid coding for pseudorabies virus (PRV) glycoprotein gIII, or for a protein having the same antigenicity as PRV glycoprotein gIII, and a pharmaceutically acceptable carrier, is disclosed. The pEVhis14gIII plasmid used for making a vaccine is also disclosed.

Description

Plasmid vaccine against pseudorabic virus

The present invention relates to a plasmid vaccine against the pseudorabic virus, also known under the name of Aujeszky's disease virus (ADV), of swine virus of herpes 1 (PHV-1) or of virus heφès-1 Suid ( SHV-1) Aujeszky's disease is a viral disease to which most mammals are sensitive However, humans do not seem to be susceptible to this disease

The agent causing this disease is a double stranded DNA enveloped virus of the family Herpesviridae, subfamily of alpha-herpesvirinae, namely, 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 (UjJ and a single short region (U _s ) which is flanked by two reverse repeat sequences, one terminal (TR) and the other internal (IR) (BEN-PORAT, T et al, 1979, Virology 95, p 285-294)

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.

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)) κ5 m U) o

I to

1 n5

H

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 undergo severe damage to the central nervous system Piglets have a high sensitivity to birth which will gradually decrease Until 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 between 15 hours and 12 days

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

One of the conventional methods consists in injecting live viruses, which must be attenuated to prevent the disease from declaring itself Thus, PRV viruses of the strain are used for vaccination of pigs

Bartha which include mutations in the genome coding for the glycoproteins gl, gp63 and glll as well as for a viral capsid protein whose gene is located on the BamHI 4 fragment (Mettenleiter T, Acta Veterinaria Hungarica, 42 (2 - 3) , p 153-177 (1994)) The vaccines sold under the names OMNIVAC ^® -PRV are also used

(FERMENTA ANIMAL HEALTH Co, Kansas City, MO, USA) and OMMMARK ^® -PRV (FERMENTA ANIMAL HEALTH Co, Kansas City, MO, USA) which include genetically engineered PRV viruses of the Bucharest strain These strains contain deletions in the genes coding respectively for thymidine kinase or for thymidine kinase and glll Of course, there are still other vaccines against Aujeszky's disease which work on the same principle

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

Then, peptides derived from these viral antigens are present on the surface infected cells in association with the major histocompatibility class I 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 (J.T. van Oirschot (ed.), Vaccination and control of Aujeszky's Disease, p. 61-66, ECSC, EEC, EAEC, Brussels and Luxembourg,

1989) developed a vaccine based on the recombinant adenovirus type 5 (AD5) which gives rise to the expression of the gp50 gene of the PRV virus

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

On the other hand, M L. VAN DER LEEK et al (The Veterinary Record (1994) 134, p. 13-18) have led to encouraging results with vaccination of pigs against the PRV virus by scarification or by intramuscular injection of a recombinant swine pox virus 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 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 the cellular and humoral type, and consequently in a protection against an 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 I histocompatibility complex and presented on the surface of the cell.

ROBINSON et al, describe in 1993, in Vaccine 1 1, 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 to a "challenge" by a lethal dose of the virus II 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 JM COX et al have described a method of vaccination of 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 the intramuscular injection of bovines and mice (into the quadriceps) of plasmid DNA containing the gene for gl, gll1 or gIV glycoproteins of BHV-1 elicited an immune response in the vaccinated animal The immune response is highly dependent on the amount of DNA injected, and on the glycoprotein. Thus, the gIV protein gene elicited a higher response than that of the gl and glll 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 glll of the PRV virus or for a protein having the same antigenicity as the glycoprotein glll of the PRV virus and a pharmaceutically acceptable excipient for the latter.

One of the advantages of this method is that it 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 got.

The gene introduced into the plasmid does not need to code for the whole glll protein, it is sufficient that it codes for a part or a homologue of the PRV glll protein which has the same antigenicity as the glll c protein. ie the same effect on the immunological system as the glll protein Indeed, only part of the glll 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, is 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, the vaccinated animals develop only antibodies against the glll protein while the animals infected with the PRV virus also develop antibodies against other proteins of the virus. In addition, the method is safe, because a proliferation of live viruses is not to be feared Since only a well-defined part of the virus genome is injected, there is no infection by viruses and therefore consequently there can be no proliferation of viruses.

Because muscle cells have a long lifespan and do not circulate in the animal's body, local and continuous expression of the antigen at low concentrations can stimulate the long-term immune 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 resides in the effectiveness of the immunization against the PRV virus. Although the importance of the glycoprotein glll in immunization is well known, the injection of the purified glll protein does not seem to have any effect. pronounced effect of immunization. Z H. BISEIBUTSU et al describe, in the Japanese patent application

JP 05/246888, a vaccine against the PRV virus, based on purified glycoprotein glll 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 glycoproteins gll, glll and purified gIV injected together with a conventional oil-based adjuvant, confers a better protection in mice against a challenge by virulent PRV viruses than the glycoproteins injected individually.

It remains to 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 pEVhisl4gIII.

The plasmid pEVhisl4gIII has the advantage of comprising a gene conferring resistance to ampicillin, so that the transformed bacteria, having incorporated the plasmid, are easily selected by adding ampicillin in 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 gll1 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 effectiveness of the vaccine, the plasmid may contain, in addition to the gene coding for the glycoprotein glll of the PRV virus or for a protein having the same antigenicity as the glycoprotein glll of the PRV virus, 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 either to liquid media or to solid media capable of being used as excipient (vehicle) for introducing the plasmid into the animal to be vaccinated. liquids, water, saline, phosphate-saline buffer, solutions containing adjuvants, detergents, stabilizers and transfection promoting substances, liposome, virosome suspensions and emulsions.

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

DNA vaccination can possibly be preceded by 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 glll protein of the PRV virus or for a protein having the same antigenicity as the glycoprotein glll 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 glll or an immunogenic fragment thereof, and b) control sequences operatively linked to said coding sequences where said sequence coding 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 pEVhisHglII in the manufacture of a vaccine

It is also proposed to use the plasmid pEVhisHglII 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 (pEVhisHglII) containing the gene for the glycoprotein gll1 of the PRV virus, the production of this plasmid in transformed bacteria and the formulation of the vaccine comprising the plasmid and an excipient pharmaceutically acceptable Step A: Construction of a plasmid (pEVhisHglII) containing the PRV virus glycoprotein glll gene The plasmid DNA pEVhisHglII was obtained from the Institute for Animal Science and Health, (ID DLO, Lelystad, NL) . It includes the PRV virus glycoprotein glll 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 in accordance with the Budapest Treaty on November 16, 1995 in the collection named "Belgian Coordinated Collections of Microorganisms - Laboratorium voor Moléculaire Biologie - Plasmiden collectie" (LMBP), Universiteit Gent, KL Ledeganckstraat 35 Gent, Belgium B - 9000 sous accession number LMBP3377

Step B Production of the plasmid in transformed bacteria Preparation of E. cells. coli treated with RbCl.

Before being transformed, the cells of E coli, strain DH 5 * (Gibco) were subjected to a treatment with RbCl to increase the efficiency of the transformation. The procedure which was followed, with the exception that we used an E coli DH 5α strain, is described in the information bulletin "The NEB transcript", vol 6 (1) p7, May 1994, published by NEW ENGLAND BIOLABS, Ine, Beverly, MA01915 Transformation of E cells . coli treated with RbCl. 100 μl of cells treated with RbCl and 1 μl of DNA solution containing

250 ng of plasmid pEVhisHglII were incubated for 10 minutes on ice (at 0 ° C) and then for 5 minutes at 37 ° C The mixture was then transferred into 2 ml of RB medium (1% bactopeptone (w / v ), yeast extract 0 5% (w / v), NaCl 1% (w / v)) and incubated between 30 minutes and 2 hours at 37 ° C with shaking The abbreviation% (w / v) represents a percentage expressed by weight per volume

100 and 500 μl of the cell culture were spread on a Petri dish containing RB medium + ampicillin 100 μg / ml + 2% agar (w / v) Mini preparation of plasmid DNA pEVhisHglII. The colonies obtained above were first used to carry out mini preparations in order to verify the production and the structure of the plasmid pEVhisHglII Individual colonies were cultured overnight in 2 ml of RB medium + ampicillin 100 μg / ml

The cultures were then treated as described in "Molecular cloning, a laboratory manual", J Shambrook, E.F Fritsch and T Maniatis, Cold

Spring Harbor Laboratory Press (1989) under the chapter "Small scale preparations of plasmid DNA", § 1 21 1 28 with the exception that for steps 1 4, the centrifugations were carried out at room temperature and that the composition of the solution III has been modified to contain 3M sodium acetate, pH 4.8

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

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), §6) Maxi-preparation of plasmid DNA pEVhisHglII.

In order to obtain plasmid DNA in an amount sufficient for vaccinations, the plasmid DNA was produced in greater quantity according to one of the following two procedures. A suspension of E. coli transformed by the plasmid pEVhisl4gIII 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 a rate of 100 μg / ml The cultures were incubated overnight at 37 ° C. with shaking at approximately 150- 200 revolutions per minute The cultures were centrifuged in 500 ml Nalgene tubes for 7 minutes at 8,670 g All the 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, SDS (sodium dodecyl sulfate) 1% (w / v )) were added, the tubes were shaken 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 50 ml Falcon tube covered with six layers of gauze If necessary, the volume can be adjusted with TE (10 mM tris-HCl, pH = 8 , 0, EDTA 1 mM) Then, 15 ml of isopropanol was added at room temperature, stirred and the mixture was transferred to a 40 ml Nalgene tube After centrifugation for 1 5 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 10 μl of proteinase K in solution (10 mg / ml) was added and incubated for 30 minutes at 37 ° C minimum with shaking

The QIAGEN TIP 500 columns (QIAGEN, INC, CA, USA) have been equilibrated with 10 ml of QBT buffer (NaCl 750 mM, MOPS 50 mM, ethanol 15% (v / v), Triton X- 100 0.15% (v / v), pH = 7.0) by flow under simple gravity The abbreviation MOPS represents sulfonic acid 3- (N-morpholino) propane The samples were deposited 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 ¹ 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 μl of phenol / chloroform / isoamyl alcohol (25/24/1, v / v / v) and extraction with a volume of ether, DNA has was precipitated with 50 μl 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 10 minutes centrifugation at 18,320 g at 0 ° C and the pellet comprising the plasmid DNA was dried under vacuum and dissolved in 500 μl of Milli-Q L 'water. abbreviation% (v / v) represents the percentage of volume by volume Another method for producing plasmid DNA in large quantities using columns PZ523 (5 Prime -> 3 Prime, INC, Boulder, CO, USA), is resumed 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), tris-HCl 25) was added mM pH = 8, EDTA 10 mM; lysozyme 1% (w / v) (SIGMA)) and 10 ml of this mixture were transferred to another 40 ml Nalgene tube. After incubation for 5 minutes at room temperature, we added 10 ml of solution 2 (0.2 M NaOH, 1% SDS (w / v) freshly prepared After gentle shaking, the mixture was incubated for five minutes on ice After adding 10 ml of solution 3 (acetate 3 M sodium, pH = 4.8) cold and centrifuge the tube for 20 minutes at 0 ° C at 48,400 g, the supernatant, about 25 ml, was transferred to a 50 ml Falcon tube 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. 48,400 g The isopropanol supernatant was removed, the pellet obtained was washed with 1 ml of 70% ethanol (v / v) and the liquid was 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 of proteinase K (solution at 10 mg / ml) were added and the mixture was incubated for at least 30 minutes at 37 ° C. with shaking For the two consecutive phenol / chloroform / 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 light stirring for 20 to 30 seconds to mix, the solution was centrifuged for 5 minutes at 3,920 g in a rotor. "swinging bucket" type 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 e and resuspended in 1.8 ml of solution 4 (10 mM Tris, 1 mM EDTA, 1 M NaCl) After removing the upper plug and then the lower plug from the column, this was placed on a tube. collection and then centrifuged for 1 minute at 980 g The collection tube which collected the balancing buffer was eliminated. 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" rotor. The plasmid solution collected was divided into two Eppendorf tubes to which 600 μl of isopropanol were 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 vacuum The pellet comprising the plasmid DNA is redissolved in 500 μl of Milli-Q water (Millipore, USA) and kept cold until vaccination

With regard to 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 development with ethidium bromide (see "Molecular cloning, a laboratory manual", J

Shambrook, EF Fritsch and T Maniatis, Cold Spring Harbor Laboratory Press (1989), § 6 and appendix E and Winnacker EL "From genes to clones", VCH (1987), § 2 1.2.2) The DNA concentration has been adjusted to be between 0 3 and 1 μg of DNA per μl of water

Example 2 Use of the Plasmid Vaccine in Mice to Stimulate the Induction of an Antibody Response and a Cytotoxic T Cell Response The experiment carried out in mice to prove the efficacy of the plasmid vaccine requires the construction of a control plasmid derived from the plasmid pEVhisl4gIII, prior to the immunization of animals and the analysis of the immune response

Step 1 Construction of a plasmid derived from the plasmid pEVhisHglII by deletion of the coding sequence of the glll gene A plasmid derived from the plasmid pEVhisHglII by deletion of the coding sequence of the glycoprotein glll gene was used as a negative control (pEVhisl4gIH ", DNA ") This deleted plasmid was obtained in the following way, the plasmid pEVhisHglII 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, EF Fritsch and T Maniatis, Cold Spring Harbor Laboratory Press (1989), § 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 DNA ⁺ plasmid, with the exception that during the production of the plasmid pEVhisl4gIII by maximum 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 The target cells were cultured in a DME medium containing 10% (v / v) fetal calf serum The plasmid DNA of the plasmid pEVhisHglII, containing the gene for the glycoprotein glll of the PRV virus , was used as positive DNA, (DNA ⁺ ) while the equivalent plasmid, without the glll gene, (DNA "), was used as a negative control

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

Group I, comprising 10 mice marked with a colored code, was vaccinated four times, at week 0 (DNA] ⁺ ) at week 3 (DNA2 ⁺ ), at week 5 (DNA3 ⁺ ) and at week 10 ( DNA4 ⁺ ) with DNA ⁺ Serum, SADN3 ^{4 "} , was collected 2 days before the last injection, serum sADN4 ⁺ was collected 6 days after the first collection i.e. 5 days after the last injection DNA ^{4 "}

Week 1 1, 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 (DNA] ⁺ ), at week 3 (DNA2 ⁺ ) and at week 5 (DNA3 ⁺ ) with DNA ⁺ Serum, sADN2 ⁺ , was taken 2 days before the last injection and serum sADN3 ⁺ a was removed at week 9, i.e. 4 weeks after the last DNA3 ⁺ injection. At week 9, the spleen cells were restimulated in vitro.

CTL were performed 6 days later

Group 3, comprising 8 mice marked with a color code, was vaccinated only twice, at week 0 (DNA j ⁺ ) and at week 3 (DNA2 ^{4 "} ) Serum, sADN2 ⁺ , was collected 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 μg of DNA" (DNA] "), at week 2 with 100 μg (DNA2 ") and at week 7 with 100 μg of DNA" (DNA3 ") Serum, SADN2", was taken 2 days before the last injection and SADN3 "serum was taken at week 8 ie 1 week after the last DNA3 injection ^"

On week 8, 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 N1A3 M207 (obtained from the Institute for Animal Science and Health, ID-DLO, NL) at a dose of 10 ⁷ PFU (Plate Forming Unit) per mouse and by injection into the necks , 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, 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 - Culture and in vitro 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 PB S (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 (through the nylon gauze) of the ground material obtained. After centrifugation at 220 g for 4 minutes, the pellet was recovered and the erythrocytes were lysed by adding 4 ml / spleen of sterile ACK solution (0.15 M NH ₄ C1, 1 mM KHCO3, 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 penicillin-streptomycin antibiotic solution (penicillin at 10,000 U / ml and streptomycin at 10,000 μg / ml (Gibco), 10 mM buffer HEPES (pH = 7.4) (Sigma), 2 x 10 ^"5 M 2-mercaptoethanol (Gibco), 2 mM sodium pyruvate (Merck)]. The cells were resuspended at 5 x 10 ⁶ cells per ml in this sterile medium. The spleen cells are distributed for their cultivation in vitro in 25 cm 3 culture flasks (Falcon) at the rate of 25 x 10 "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. were placed vertically in an incubator for 4 to 7 days (at 37 ° C. 3% (v / v) of CO2 and a humidity higher 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 steps a) The target cells were infected or not infected with a strain of the Aujeszky virus (NIA3 M207) at an MOI equal to 10, the infected cells being designated CV ⁺ and the uninfected cells CV "Ninety minutes later, the labeling of the target cells in suspension began (see below - Eu labeling of the cells targets (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 returned suspended at a rate of 5 x

10 ^ cells / ml Six hours after the start of the infection of the target cells, these were brought into contact with the effectors (including T _c lymphocytes) In a 96-well plate with a round bottom, 5,000 target cells in 50 μl of effector medium (marked with Eu and infected or not) were deposited on respectively 500,000, 250,000, 125,000, 62,500, 31,250, 15,625 effectors in 100 μl (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 set at 37 ° C for 4 hours L evaluation of the quantity of target cells lysed by Te lymphocytes was carried out by a sample of supernatant after a further centrifugation of 4 minutes at ± 50 g This was placed in a 96-well plate with a flat bottom in 200 μl a fluorescence enhancing solution (DELFIA ^® Enhancement solutio n, Pharmacia, Sweden) in the case of marking with Eu The counting with the fluorimeter (delayed-time fluorimeter, 1234 DELFIA Recherche, Wallac) was carried out ± 12 hours later having taken care to put the plates containing the mixture in the dark and at room temperature

The quantification of the 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 suspended target cells 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 freed from its growth medium and washed 1 x with PB S (Gibco). 5 ml of trypsin-EDTA (Gibco) at 37 ° C. were deposited in the flask on the cell mat. After one minute, the cells were removed by short tapping on the flask. The complete detachment achieved, 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) acid - 4-piperazinyl-1,2-ethanesulfonic) (pH = 7.4), (Sigma); 93 mM NaCl (Merck); 5 mM KC1 (Merck), 2 mM MgCl2-6H2θ (Merck)] and resuspended at the rate of 6 x 10 ^ cells / ml in said solution. The counting of living cells was carried out with Blue Trypan (0.4% solution (w / v), Sigma). One ml of this suspension was completed with:

- 750 μl of the HEPES buffer solution (pH 7.4),

- 200 μl of the Eu-DTPA solution (1.52 ml of the standard solution of Eu (1000 μg / ml in 1% (v / v) of nitric acid) (Aldrich); 8 ml of the solution of HEPES buffer (pH 7.4); 0.5 ml of DTPA diethylene triaminepentaacetic acid (Merck) at 3.93 g in 100 ml of the solution of HEPES buffer) and after 2 minutes, per 100 μl of dextran sulfate (50 mg dextran sulfate, M W. = 500 KDa, Pharmacia in 10 ml of the HEPES buffer solution).

Thirty minutes were necessary 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 2H2O (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 Ficoll-Paque cushion (Ficoll and sodium diatrizoate, 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 in 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 a 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 placed in a 96-well round-bottom plate in the presence of 100 μl of effector medium to determine the amount of background noise from the Eu.

The same quantity of cells was deposited in 100 μl of Triton X-100 (1% v / v, Merck) for the maximum release of Eu. After a 4-minute centrifugation at 50 g, 20 μl of supernatant were removed and deposited in 200 μl of a fluorescence enhancer solution (see above) and the 96-well plate with flat bottom was passed through a fluorimeter ( 1234 DELFIA Recherche, Wallac) after an incubation of 1 hour in the dark so as to reach a stable reading. Part D - CTL test results.

Table 2 Comparison of the specific lysis of splenocytes before and after assa e on a Ficoll-Paque gradient

Explanatory notes concerning in vitro restimulation treatments and the preparation of V "target spleen cells cultured in vitro in the absence of virus MOI 2 spleen cells cultured in vitro and restimulated by adding 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 with the NIA3 M207 virus at an MOI equal to

10, and marked with Europium

SB ++ group 5, positive control vaccinated with live virus

DNA ₄ ⁺ group 1, animals injected 4 times with DNA ^{4 "} DNA ₃ " group 4, animals injected 3 times with DNA "number of repetitions Lysis of uninfected target cells was between 0 and 9% and was not affected by the transition to the Ficoll-Paque gradient. On the other hand, the positive control (group 5), both before and after treatment Ficoll presented a significantly positive cell lysis rate. infected targets were increased by passage over Ficoll-Paque for animals immunized with DNA.

The CTL test group 1, injected 4 times with DNA ^{4 "(ADN4} ⁺⁾ ^showed lytic activity.

The CTL test Group 2, injected 3 times with DNA ^{4 "(DNA3} ^4") did not show any lytic activity.

The CTL test group 3 injected 2 times with DNA ^{4 "(DNA2} ^4") did not show any lytic activity.

The group 4 CTL test, injected 3 times with DNA "(DNA3") 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 seroneutralization test of the PRV virus (SN)

- an immunoenzymatic test (ELISA) for the measurement in the serum of mice of the antibodies directed against an extract of all the PRV glycoproteins.

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

PD5 cells (SOLVAY-DUPHAPv, NL) and strain viruses Bartha K61 (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 Eagle minimum essential medium (Flow), 100 ml lactalbumin hydrolyzate 2.5% (w / v); 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 μl of serum + 50 μl of medium each time), in the 96-well plate with flat bottom (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 were added to each well The serum mixture / virus was incubated for 24 hours at 37 ° C 50 μl of the suspension of PD5 cells having a concentration of 4 × 10 ^ cells / ml were added to each serum / virus sample The plates were 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 virus sample diluted 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 test medium SN (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 was 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 high doses, 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 of non-injected mice (OF1 mice, 3 weeks old) A mixture serum from 10 mice was carried out Serum + is a mixture of serum from 10 OF 1 mice having received at 3 weeks 10 ^ TCID (tissue culture infectious dosis) of recombinant adenovirus expressing the gD gene intramuscularly and collected 3 weeks later Table 3 and Table 4, reproduced 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 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 to which plasmid DNA was injected without the glll gene (DNA "- pEVhis 14gIII"), no antibody was found against the glycoprotein glll II was shown only after 4 injections of plasmids ^{4 "} DNA spaced 2 weeks or more apart, the mice showed a good humoral response against the virus. Seven out of 9 animals tested showed anti-glll antibodies after four injections

Even after three injections of ^{4 "} DNA, the serum of 9 animals out of 10 showed measurable anti-glll antibody titers. Similarly, after 2 injections of DNA ^4" , the serum of 7 animals out of 8 showed responses positive in ELISA

Table 3 ELISA optical density (OD) test as a function of the dilutions of the

explanatory notes

SADN2 "serum from animals injected twice with DNA ^" SADN3 "serum from animals injected 3 times with DNA" SADN3 ^{4 "} serum from animals injected 3 times with DNA ^4" SADN4 ^{4 "} serum from animals injected 4 times with DNA ^{4 "} 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 Table 4: ELISA test - optical density (OD) as a function of dilutions of test serum 2

10 2000 2000 549 249

1 not tested

serum - mixture 155 96 83 92

serum + mixture 827 305 146 106

explanatory notes.

SADN2 "serum from animals injected twice with DNA"

SADN3 "serum from amino injected 3 times with DNA"

SADN3 ^{4 "} serum from animals injected 3 times with DNA ^4"

SADN4 ^{4 "} serum from animals injected 4 times with DNA ^4" positive control (NI A3 M207) serum from animals injected with live virus NIA3 M207 serum - serum from non-injected animals 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 group G0, the negative control, only a PBS buffer (Gibco) was used

Group G 1 (the positive control) was vaccinated using an attenuated virus (strain N1A3 M207) intraperitoneally lθ7 PFU (plaque-forming units) were used for each mouse This is a very high dose by comparison with the doses used for the vaccination of pigs with the attenuated vaccine strains traditionally used at the dose of 10 ^ 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 μg in 2 times 100 μl of water / mouse in the hindquarters left and right 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

The serum taken from each animal was coded so as to be able to follow each animal individually with regard to the antibody assay and the protection conferred by the vaccinations. Part B - Analysis of the immune responses

The development of humoral responses was controlled according to the ELISA protocol presented in Example 2 The test for neutralization of serum virus 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 injection of vaccine and just before the test inoculation with the virulent virus which took place 9 days later Finally, one month after the challenge, serum was taken 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 ml 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.

The results show that protection took place even at the lowest vaccination dose, i.e. 2 x 100 μg. Indeed, the death of these animals was delayed compared to the negative control group (GO).

At a dosage of 4 x 100 μg, 30% of the animals survived, while at 6 x 100 μg, 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 the Gl group (the positive control), i.e. the group of animals vaccinated with the attenuated virus at very high dose (lθ7 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-glll antibody response in the ELISA test, was weaker 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 followed, with the only difference that injections of plasmid DNA were performed every three weeks and that the mice were 19 weeks old.

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

The Gl group (positive control) was vaccinated using the attenuated virus (strain NI A3 M207) in the necks with a dose of 10? 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 injection of plasmid and six weeks after the immunizations of the control groups, all the mice were exposed to the virulent virus NIA3 at the rate of 7000 PFU / animal, injected intraperitoneally Deaths are recorded during the 15 days following the test and are listed in Table 6 The results show the survival rates expressed as a percentage as a function of the days following the test Table 6 Monitoring of the survival rate of mice after an inoculation of test with virulent viruses

All animals in group G0 (negative control) died after the test An 80% survival rate was observed in the group Gl, i.e. animals vaccinated with the attenuated virus at very high dose

At a dosage of 4 times 100 mg of plasmid DNA, 40% of the animals survived The immunization method by which the animals are vaccinated with plasmid DNA (4 injections) every three weeks has been shown to be more effective compared to daily injections (better survival rate of 40% instead of 30% and relative delay in animal death) 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 breeding free from GRP and from the same litter Us are individually marked with an ear ring

A group of 3 animals (# 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 pEVhisl4gIII were carried out at the age of 5, 7 and 9 weeks

Three animals (# 4, 5 and 6) received a dose of 75 μg of plasmid, while 3 other pigs (# 7, 8 and 9) received 560 μg of plasmid on each injection The dose of DNA 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 and right hindquarters The injection was performed on the using a syringe fitted with a Terumo needle 40 mm long with an opening of 0 9 mm

Serum was collected during each injection and 2 weeks after the last injection. Analysis of the humoral responses (antibodies against the glll protein) before and during the immunization was carried out by a seroneutralization test (test sensitive to mediation by the complement, see Bitsch and Eskilsen, curr Top Vet Med Anim Sci. 12, 41 49, 1982) and by an EPMA test (Immuno Peroxidase Monolayer Assay) The IPMA test was carried out according to the procedure which is repeated below Each then 96-well plates (Corning, USA) covered with a mat of confluent SK 6 cells (ATCC, USA) were added 500 TCID50 of PRV virus of strain 89V87 ((Nauwynck H, Pensaert M, Am J Vet Research , 53 (4) 489 (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 liquid evaporation 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 of an hour's 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 substrate 3 amino 9 ethylcarbazole (2 mg AEC in 10 ml of sodium acetate buffer (0 05 M at pH 5) and 75 μl of H 2 O 2 at 30%) The appearance of a red coloration is observed under an optical microscope The reaction was stopped after 15 minutes by 3 city water washes IPMA titers are calculated by taking the inverse of the highest dilution which causes a red coloration of the viral antigen foci on the infected cells

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

Table 7 Titers of antibodies against the PRV virus determined by the seroneutralization test and by the IPMA test

Explanatory notes

SN: titer in seroneutralizing antibodies

10 IPMA: antibody titer determined by the IPMA method

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, ie 3 x 75 μg, a humoral response was induced in 2 out of 3 pigs. Only one animal out of 3 reacted to a dosage of 560 μg

15 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 The protocol of example 5 is reproduced exactly. Three groups of 3 pigs aged 5 weeks were used. The animals come from a farm free from GRP. and of the same scope They are individually marked by a ring at the ear

A group of 3 animals which were not vaccinated, was used as a negative control

For the other 2 groups, three intramuscular injections of the plasmid pEVhisHglII were carried out at the age of 5, 7 and 9 weeks according to the technique described in Example 5. Three animals (pigs Nos. 4, 5 and 6) were received a dose of 75 μg of plasmid, while the other three animals (pig number 7, 8 and 9) received a dose of 560 μg of plasmid

The virulent virus test was carried out according to the method described in Vaccine 1994, 12 (7), p 661 665 and is described below.

27 weeks later, ie 18 weeks after the last plasmid injection, all the animals are transferred to an isolated unit for exposure to the virulent PRV strain 75 VI 9 virus (Andries K, Pensaert MB, Vandeputte J , Am J Vet Res., 1978, 39, p. 1282 1285). The temperature of the isolated unit is maintained at 18 ° C and the ventilation at

0.2 m / s

10 ⁵ * ⁰ TCID50 of PRV virus (strain 75V 19) suspended in 5 ml of phosphate buffer are administered to all animals 2 ml of this suspension is given orally and 1.5 ml of this suspension is instilled in each nostril

All the animals were observed daily during the two weeks following the test. The body temperature of the animals was noted, as well as their weight. The relative weight gain (RDWG) was calculated according to the formula below, in order to compare the performances of the three RDWG groups from the day of the test until day x day weight x weight on day of the test

RDWG = weight on the day of the test The nasal secretions of all the animals were taken with a swab every other day for 14 days after the test The weight of the nasal secretions taken was noted The nasal secretions taken were suspended in phosphate buffer. Decimal dilutions of these suspensions were inoculated into monolayer cell cultures, these cells coming from a continuous pig testicle (ST) cell line. Then, the presence of a cytological effect for these cell cultures was checked for five days. The lethal dose 50 was calculated according to the method of Reed and Muench (Amer. J. Hyg., 1938, 27, 493 497). The virus titers were expressed in TCID50 per gram of nasal secretion.

The results of the serological examinations are collated in Table 8

The analysis of the humoral responses was carried out by a sensitive serum neutralization test (test sensitive to complement mediation according to the technique described by Bitsch and Eskilsen, Curr Top Vet Anim Sci, 1982, 12, p 41 49) and by a conventional seroneutralization test (according to the technique described by Andries K, Pensaert MB, Vandeputte J, Am J Vet Res, 1978, 39, p 1282 1285)

These results show that a serological response was found for only one of the six animals vaccinated at the time of the last vaccination. Two and eighteen weeks later, seroneutralizing antibody titers between 3 and 24 were observed respectively for three of the animals (N ° of pigs 4, 6 and 9) and for four of the animals (No. of pigs 4, 6, 8 and 9)

14 days after the test, the seroneutralizing antibody titers were observed between 64 and 128 for the animals belonging to the negative control group, between 128 and> 384 for the animals having received a dose of 75 μg of plasmid, and between 128 and 192 for animals having received a dose of 560 μg of plasmid

All the animals were dazed and anorexic They sneezed from the third day after the test until the eighth or ninth day after the test Vomiting and nervous disorders were observed for two animals (No. of pigs 3 and 5)

All animals, except one (pig number 8), had a fever (> 40 ° C) from the third day until the seventh day after the test. The maximum average temperature was 41.3 ° C for the negative control group and 41 ° C for the other two groups. The results concerning the changes in weight per animal are collated in Table 9.

Table 9. Weight change of animals

Δ G7 = weight 7 days after the test - weight on the day of the test

Δ G14 = weight 14 days after the test - weight on the day of the test RDWG7 = RDWG 7 days after the test RDWG 14 = RDWG 14 days after the test

All animals except one (pig number 8) lost weight during the first week after the test.

Fourteen days after the test, only two animals (pig numbers 3 and 5) had not regained the weight they had before the test. They also continued to lose weight. The three animals having received a dose of 560 μg of plasmid and one animal (pig number 4) having received a dose of 75 μg of plasmid, showed a compensatory weight gain between the seventh and the fourteenth day after the test , so that they had resumed their initial growth curve between the eleventh and the fourteenth day after the test The two groups of vaccinated animals show an average value of weight gain significantly positive, while the control group, unvaccinated animals, shows an average value of negative weight gain (= weight loss)

The results of the virus titrations in the nasal secretions are collated in Table 10 Table 10: Secretion of viruses after testing

The first viruses were isolated from the nasal secretions, two days after the test, for two of the three animals in each group. The titles virus reached a peak between the fourth and sixth day after the test, virus titers ranging between 10 ^, 10 ^ 5 _e t _^'υ TCID 50. The secretion of virus was stopped between the sixth and the eighth day after the test for the vaccinated animals, while it continued until the twelfth day after the test for the animals of the negative control group.

INDICATIONS FOR A DEPOSITED MICRO-ORGANISM

(rule

A. The indications relate to the microorganism referred to in the description on page _9_, lines 2 ~ 5

B. IDENTIFICATION OF THE DEPOSIT Other deposits are the subject of an additional sheet [|

Name of depository institution

BELGIAN C∞RDINATED COLLECTIONS OF tŒCROORGANISMS (BCCM)

Address of the depositary institution (including postal code and country)

Laboratorium voor Moléculaire Biologie - Plasmidencollectie (LMBP)

Universiteit Gent

K.L. Ledeganckstraat, 35

B-9000 GENT (Belgium)

Date of filing order no.

NOVEMBER 16, 1995 LMBP3377

An additional sheet is attached ι - ι

C. ADDITIONAL INFORMATION (if applicable) for the continuation of this information I - I

pEV hisl4gHI

The accessibility of the micro-organism can only be achieved by providing a sample to an expert designated by the applicant.

D. DESIGNATED STATES FOR WHICH THE INDICATIONS ARE GIVEN

(if Here indications are not given * pow all designated Stalls)

E. INDICATIONS PROVIDED SEPARATELY (if applicable)

The indications listed below will be supplied later to the International Bureau fψeaftei the general pledge of the indications p e \. "deposit idiocy")

Reserved for the International Bureau that the I This sheet reached the International Bureau on

Authorized official

PCI '/ RO / IM form (July 1992

Claims

R E V E N D 1 C A T I O N S

1 - Vaccine comprising a plasmid containing a gene coding for the glycoprotein glll of the PRV virus or for a protein having the same antigenicity as the glycoprotein glll of the PRV virus 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 pEVhisHglII

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 glll protein of the PRV virus or for a protein having the same antigenicity as the glycoprotein glll 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 glycoprotein glll 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 8 - Plasmid pEVhisHglII used in the manufacture of a vaccine.

9 - Plasmid pEVhisHglII used in the manufacture of a vaccine against the PRV virus.