AU711915B2 - Plasmid vaccine against pseudorabies virus - Google Patents

Description

Code: 295-57857 Ref. AHP-97020 PCT PLASMID VACCINE AGAINST PSEUDORABIES VIRUS The present invention concerns a plasmid vaccine against the pseudorabies virus, also known under the Aujeszky's disease virus (ADV), the porcine herpes 1 virus (PHV-1) or the Suid herpes-i virus (SHV-1).

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

The causative agent of this disease is a coated virus with two-stranded DNA of the family of the Herpesviridae, subfamily of the alpha-herpesvirinae [sic; herpesviridae], that is the pseudorabies virus (PRV).

The genome of the PRV virus is formed by an approximately 150-kb two-strand DNA molecule and it consists of a long unique region (UL) and a short unique region (Us) which is flanked by two reverse repeated sequences, a terminal sequence (TR) and an internal sequence (IR) BEN-PORAT, T. et al., 1979, Virology, Vol. 95, pp. 285-294) The genome of the virus of Aujeszky's disease contains at least 70 genes.

The principal characteristics and biological properties of the genes of PRV which have been best characterized are listed in Table I below.

Table I. Properties of certain genes of PRy, their characteristics and their biological properties (except from T.

Mettenleiter, Acta Veterinaria Hungarica, 42 pp. 153-177 (1994)) Segment Designation Designation Prot6ine Taille (kDa) Essentielle Fonction /Activit6 Virulence duG~nome Gene (HSV) Gene PRV) ,<(r~plication in Lf~ J~ vitro) UL gB gll 913 aa 110-68-55 p~n~tration, fusion gC glll 479 aa 92- adsorption, relargage___ TK 320 aa 35 -thymnidine kinas /0 gH gH 686 aa 84 +p~n~tration, fusion n.a.

cellulaire prot. kinase 336 aa.

I f -t 4 498 aa 1 99 prot6ine kinas inconnu I -t t I t 4 402 aa 1 60 I~~rtn I2' gp6 350 aa gE jgI 577 aa 110 inconnu relargage, transmision de cele Acelul +nc TEG 106 aa tt~m] -nt Key: 1 2 3 4 6 Segment of the genome Gene designation (HSV) Gene designation (PRV) Protein Size (kDa) Essential (replication in vitro) 7 Function/activity 8 Penetration, cellular fusion 9 Adsorption, release Thymidine kinase 11 Protein kinase 12 Unknown 13 Penetration 14 Release, transmission from cell to cell Integument protein Usually viruses are transmitted by the oral, or respiratory route, or by direct contact between an infected animal and a healthy animal.

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

2. The piglets suffer a severe attack on the central nervous system. The piglets present a high sensitivity at the birth, followed by .a decrease in sensitivity. Until the age of 10 days, piglets which have been affected, as they do not benefit from any passive immunity from the mother, die within a few hours. Older piglets are subjected to muscle tremors, contractions, but the outcome is rather benign.

In other species, the disease can be fatal, and the duration of incubation is variable, between 15 h and 12 days.

At this time there are several methods of vaccination against Aujeszky's disease.

One of the standard methods consists in injecting live viruses, which must be attenuated to prevent the disease from declaring itself.

Thus, for the vaccination of the pigs, PRV viruses from the Bartha strain are used, which comprise mutations in the genome coding for the glycoproteins gI, gp63 and gIII and for a protein of the viral capsid whose gene is in the BamHI-4 fragment Mettenleiter, Acta Veterinaria Hungarica, Vol. 42 12-3), pp. 153-177 (1994)).

The vaccines sold under the names of OMNIVAC®-PRV (FERMENTA ANIMAL HEALTH Co., Kansas City, MO, and OMNIMARKI-PRV (FERMENTA ANIMAL HEALTH Co., Kansas City, MO, are also used, which consist of PRV viruses from the Bucharest strain, which have been genetically manipulated. These strains comprise deletions in the genes coding respectively for thymidine kinase or thymidine kinase and gIII. Naturally, other vaccines against Aujeszky's disease exist, which work on the same principle.

This vaccination method imparts a good protection to the vaccinated subject, which is due to the intracellular action of the live virus. Indeed, the live viruses penetrate the cells where the viral antigens are synthesized. Then, the peptides derived from these viral antigens are presented at the surface of the infected cells in association with the major histocompatibility complex of class I (MHC A cytotoxic response can thus be triggered, in addition to the humoral response, resulting in a better protection against the virus in the vaccinated subject.

Although the viruses are attenuated by mutations, there is a risk of the viruses recovering their pathogenicity as a result of spontaneous mutations or recombinations with wild viruses.

The vaccinations with live viral agents can also cause, under certain conditions, a proliferation of live viruses. This proliferation of live viruses, even though they are attenuated, constitutes a risk for more susceptible subjects, such as newborns and pregnant subjects.

Another more recently developed technique consists in using a nonpathogenic live vector which carries selected genes from the PRV virus.

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

W.L. MENGELING et al. (Arch. of Virology, Vol. 134, No. 3-4, pp. 259-269, 1994) published results of tests with a vaccinia virus (NYVAC) containing the genes of PRV coding for the glycoproteins gp50, gII and gIII. However, the efficacy of this type of virus is limited.

In contrast, M.L. VAN DER LEEK et al. (The Veterinary Record (1994), Vol. 134, pp. 13-18) have reached encouraging results with a vaccination of pigs against the PRV virus by scarification or intramuscular injection of the recombinant of the virus of porcine variola and of the PRV virus (rSVP-AD). This recombinant was obtained by insertion of the PRV genes coding for the and gp63 attached to the P 7.5 promoter of the vaccinia virus in the gene of the thymidine kinase of the SPV virus.

Naturally, other vaccines exist against Aujeszky's disease, which work according to the same principle.

A new approach to induce an immunological response was recently described by ULMER et al., 1993, Sci., Vol. 259, p. 1745). Mice were immunized by intramuscular injection of a plasmid comprising the gene of the nucleoprotein of the influenza virus under the control of a mammalian promoter. This immunization by injection of the plasmid apparently has led to a transfection of the muscle cells, followed by an in situ expression of the protein, leading to a specific immunological response against the influenza virus, which response is of the cellular and humoral type and, consequently, it is protected against an attack by this virus.

It appears, according to the published experiments, that parts of viral proteins produced inside the transfected cell are taken on by the major histocompatibility complex of class I and presented at the surface of the cell.

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

GRAHAM J.M. COX et al. have described a method for the vaccination of cattle and mice against the BHV-1 virus by injection of plasmidic DNA, in J. Virol., 1994, pp. 5685-5689. It has been possible to show that the intramuscular injection of cattle and mice (in the quadriceps) of plasmidic DNA containing the gene of the glycoproteins gI, gIII and gIV of BHV-1 triggered an immune response in the vaccinated animal. The immune response strongly depends on the quantity of DNA injected, and on the glycoprotein. Thus, the gene of the protein gIV triggered a response which was superior to that of the proteins gI and gIII.

None of the plasmidic vaccines described to this day is effective against viral diseases which infect pigs and other species, such as PRV-caused Aujeszky's disease.

The purpose of the present invention is to propose a plasmidic vaccine against the PRV virus, which is responsible for Aujeszky's disease.

This purpose is achieved by a vaccine comprising at least one plasmid coding for the glycoprotein gIII of the PRV virus or for a protein presenting the same antigenicity as the glycoprotein gIII of the PRV virus and a pharmaceutically acceptable excipient for it.

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

Indeed, the plasmid can be produced, using techniques which have been well mastered, in bacteria such as E. coli.

The extraction of plasmid is well known, and a high yield can be obtained.

It is not necessary for the gene introduced into the plasmid to code for the entire gIII protein, rather it is sufficient for it to code for a part or a homolog of the gIII protein of PRV, which has the same antigenicity as the gIII protein, that is the same effect on the immunological system as the protein gill.

Indeed, only a part of the protein gIIl's are identified by the immunological system of the animal; the other parts of the protein, although they certainly play 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 it is easy to distinguish the vaccinated animals from animals infected by the PRV virus.

Indeed, the vaccinated animals develop only antibodies against the protein gIII, whereas the animals infected by the PRV virus also develop antibodies against other proteins of the virus.

In addition, the method is reliable; because no proliferation of live viruses need be feared, there is no infection by viruses, and consequently, there can be no proliferation of viruses.

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

The 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 antigen, for example, in ELISA tests or other tests.

A surprising effect of the invention resides in the efficacy of the immunization against the PRV virus. Although the importance of the glycoprotein gIII in the immunization is well known, the injection of the purified protein gIII does not seem to have any pronounced immunization effect.

Z.H. BISEIBUTSU et al. describe, in Japanese Patent Application No. JP 05/246888, a vaccine against the PRV virus, based on the 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., Vol. 54 pp. 447-452, 1992, that a mixture of glycoproteins gII, gIII and gIV, purified and injected together with a conventional oil-based adjuvant, imparts a better protection to mice against a challenge with virulent PRV viruses than the glycoproteins injected individually.

It remains to be noted that vaccines which use 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 which imparts a resistance to ampicillin, so that the transformed bacteria, having incorporated the plasmid, can easily be selected for by adding ampicillin to their growth medium.

Naturally, one can consider using other plasmids. It is sufficient for the plasmid to contain a gene, which codes for a protein having the same antigenicity as the glycoprotein gIII of the PRV virus, inserted in the plasmid so that it is expressed in the vaccinated organism. A marker such as a gene which imparts resistance to an antibiotic allows the selection of bacteria transformed by the plasmid.

To increase the efficacy of the vaccine, the plasmid can contain, besides the gene coding for the glycoprotein gIII of the PRV virus or for a protein presenting the same antigenicity as the glycoprotein gIII of the PRV virus, one or more genes coding for these cytokines.

Certain cytokines are known to exert an adjuvant activity on the vaccines. The fact of introducing them into the plasmid so that they can be expressed in the cells will increase the efficacy 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 which can be used as an excipient (vehicle) to introduce the plasmid into the animal to be vaccinated.

Let us cite, as examples of liquid media, water, physiological serum, the phosphate-salt buffer, solutions containing the adjuvants, detergents, stabilizers and substances which promote the transfection, suspensions of liposomes, of virosomes, and emulsions.

Let us cite, as examples of solid media, gold microbeads covered with plasmid, intended to be projected by bombardment ("gene gun") into the tissue of the animals and the microparticles containing the DNA, which can be used for administration by the parenteral or oral route.

The vaccination with DNA can optionally be preceded by a pretreatment of the vaccination site (for example, use of a local anesthetic) so as to improve its efficacy.

The present invention also proposes a plasmidic vaccine against the PRV virus which comprises a nucleic acid sequence coding for the protein gill of the PRV virus or for a protein presenting the same antigenicity as the glycoprotein gill of the PRV virus or a DNA construct comprising an expression cassette including: a) a DNA sequence coding for a polypeptide containing at least one antigenic determinant of the glycoprotein gill or an immunogenic factor of the latter, and b) controlled sequences which are operatively connected to said coding sequence where said coding sequence can be transcribed and translated in a cell and where said control sequences are homologous or heterologous with respect to said coding sequence.

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

According to another aspect of the present invention, the proposal is made to use the plasmid pEVhisl4gIII in the manufacture of a vaccine.

It is also propose to use the plasmid pEVhisl4gIII in the manufacture of a vaccine against the PRV virus.

The invention is described in detail, as an illustration, in the following examples.

Example 1: Obtention of a vaccine The vaccine was obtained in three steps, comprising the construction of a plasmid (pEVhisl4gIII) containing the gene of the glycoprotein gill of the PRV virus, the production of this plasmid in transformed bacteria and the formulation of the vaccine comprising the plasmid and the pharmaceutically acceptable excipient.

Step A: Construction of a plasmid (pEVhisl4gIII) containing the gene of the glycoprotein gIII of the PRV virus The DNA of the plasmid pEVhisl4gIII was obtained from the Institute for Animal and Science and Health (ID DLO, Lelystad, NL). It comprises the gene of the glycoprotein gIII of the PRV virus, under the control of the HCMV promoter and the marker gene of resistance to ampicillin; it is used as a DNA (DNA vaccine.

The map of the plasmid is given in Figure 1.

The plasmid was deposited according to the Budapest Treaty on November 16, 1995, in the collection called "Belgian Coordinated Collections of Microorganisms--Laboratorium voor Moleculaire Biologie--Plasmiden collectie [Laboratory for Molecular Biology--Plasmid collection]" (LMBP), University of Ghent, K.L. Ledeganckstraat 35 Ghent, Belgium B 9000 under the accession number LMBP3377.

Step B: Production of the plasmid in transformed bacteria Preparation of E. coli cells treated with RbCl Before being transformed, the E. coli cells, strain DH (Gibco), were subjected to a treatment with RbCl to increase the effectiveness of the transformation. The procedure which was used, with the exception that we used a strain of E. coli DH is described in the information bulletin The NEB Transcript, Vol. 6 p. 7, May, 1994, edited by NEW ENGLAND BIOLABS, Inc., Beverly, MA, 01915.

Transformation of E. coli cells treated with RbCl 100 iL of cells treated with RbCl and 1 pL of DNA solution containing 250 ng of plasmid pEVhisl4gIII were incubated for min on ice (at 0°C) and then for 5 min at 37 0 C. The mixture was then transferred into 2 mL of RB buffer (bactopeptone 1% (wt/vol), 0.5% yeast extract (wt/vol), 1% NaCl (wt/vol)) and incubated between 30 min and 2 h at 37 0 C with stirring. The abbreviation (wt/vol) represents a percentage expressed in weight by volume.

100 and 500 pL of the cell culture were spread in a Petri dish containing RB medium ampicillin 100 pg/mL agar 2% (wt/vol).

Minipreparation of pEVhisl4gIII plasmidic DNA The colonies obtained above were first used to prepare minipreparations for verifying the production and the structure of the plasmid pEVhisl4gIII. Individual colonies were cultured overnight in 2 mL of RB medium ampicillin 100 pg/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) in the chapter, Small scale preparations of plasmid DNA, 1.21-1.28, with the exceptions of steps 1, 4, the centrifugations were carried out at ambient temperature, and the fact that the composition of the solution III was modified so as to contain 3M sodium acetate, at pH 4.8.

The pellets were dried under a vacuum and redissolved in 100-200 pL of water (filtered using a Milli-Q apparatus, Millipore, without treatment with RNAse.

The analysis by digestion using various restriction enzymes, followed by electrophoresis on agarose gel has allowed the verification of the conformity of the plasmid (see also Molecular Cloning, a Laboratory Manual, J. Shambrook, E.F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press (1989), 6).

Maxipreparation of pEVhisl4gIII plasmidic DNA To obtain plasmidic DNA in a sufficient quantity for the vaccinations, the plasmidic DNA was produced in a larger quantity according to the following two protocols.

A suspension of E. coli transformed by the plasmid pEVhisl4gIII was incubated for 1 h in 2 mL of RB medium and then distributed in 1-4 flasks of 400 mL of RB medium containing ampicillin at the concentration of 100 pg/mL. The cultures were incubated for one night at 37 0 C with stirring at approximately 150-200 rpm.

The cultures were centrifuged in 500-mL Nalgene tubes for 7 min at 8670 G. Centrifugations were carried out in a Beckman centrifuge, model J2-21.

The supernatant was eliminated and the pellet resuspended in mL of solution 1 (glucose 1% (wt/vol); Tris-HCL 25mM; pH EDTA 10mM; lysozyme 1% (wt/vol)), and transferred into a mL Nalgene tube. The tube was left for 5 min at ambient temperature. Then 10 mL of solution 2 (NaOH 0.2M; SDS (sodium dodecyl sulfate) 1% (wt/vol)) were added, the tubes were stirred and left for 5 min at ambient temperature. 10 mL of solution 3 (sodium acetate 3M; pH 4.8) were added, and the flasks were stirred and then centrifuged for 30 min at 48,400 g at 0°C. 25 mL of the supernatant were transferred into a 50-mL Falcon tube covered with six layers of gauze. If necessary, the volume can be adjusted with TE (Tris-HCL 10mM; pH 8.0; EDTA ImM). Then, 15 mL of isopropanol at ambient temperature were added, followed by stirring, and transfer of the mixture into a 40-mL Nalgene tube.

After centrifugation for 15 min at 48,400 G at 0 C, the supernatant was eliminated. After draining the liquid well, and 1 I after dissolution of the pellet in 5 mL of TE, the suspensions were incubated for 30 min at 37°C with stirring in the presence of RNase A at a final concentration of 0.1 mg/mL. Then, 10 uL of proteinase K in solution (10 mg/mL) were added, followed by incubation for 30 min at minimum 37 0 C, and with stirring.

The columns TIP 500 QIAGEN (QIAGEN, INC., CA, were equilibrated with 10 mL of QBT buffer (NaCl 750mM; MOPS ethanol 15% (vol/vol); Triton X-100, 0.15% (vol/vol); pH by simple gravity flow. The abbreviation MOPS represents 3-(N-morpholino)propanesulfonic acid. The samples were loaded onto the columns, and the columns were washed 6 times with mL of QC buffer (NaCl 1000mM; MOPS 50mM; ethanol (vol/vol); pH After elution with 20 mL of QF buffer (NaCI 1250mM; MOPS 50mM; ethanol 15% (vol/vol); pH the solution was recovered in 40-mL Nalgene tubes. 14 mL of isopropanol were added at ambient temperature. After stirring, the mixture was centrifuged for 15 min at 48,400 G at 0°C. The supernatant was eliminated, and the pellet was dried under a vacuum and dissolved in 500 pL of Milli-Q water. After three extractions of the supernatant with 500 pL of phenol/chloroform/isoamyl alcohol (25/24/1, vol/vol/vol) and an extraction with one volume of ether, the DNA was precipitated with 50 pL of 3M sodium acetate and 0.7 mL of isopropanol. After centrifugation for 10 min at 18,320 G at 0 C, the pellet was washed with 1 mL of 70% ethanol (vol/vol). The supernatant was eliminated after a centrifugation at 18,320 G at 0 0 C, and the pellet comprising the plasmidic DNA was dried under a vacuum and dissolved in 500 pL of Milli-Q water. The abbreviation (vol/vol) represents the percentage of volume by volume.

Another method for producing plasmidic DNA in large quantity using PZ523 columns (5 Prime 3 Prime, INC., Boulder, CO, U.S.) is used below.

The 400-mL cultures were centrifuged for 10 min at 8670 g in a 500-mL Nalgene beaker (Beckman J2-21). 10 mL of solution 1 (glucose 1% (wt/vol); Tris-HCL 25mM, pH 8, EDTA 10mM; lysozyme 1% (wt/vol) (SIGMA)) were added, and 10 mL of this mixture were transferred into another 40-mL Nalgene tube. After incubation for min at ambient temperature, 10 mL of a solution 2 (NaOH 0.2M; SDS 1% (wt/vol)) which was freshly prepared were added. After slight stirring, the mixture was incubated for 5 min on ice.

After the addition of 10 mL of cold solution 3 (sodium acetate 3M; pH and centrifugation of the tube for 20 min at 0 C and 48,400 G, the supernatant, approximately 25 mL, was transferred into a 50-mL Falcon tube, and covered with-six layers of gauze. After the addition of 15 mL of isopropanol, the 40 mL of sample so obtained were transferred into another 40-mL Nalgene tube and centrifuged for 20 min at 0°C and 48,400 G. The supernatant of isopropanol was eliminated, the pellet obtained was washed with 1 mL of 70% (vol/vol) ethanol, and the liquid was well drained. The pellet was resuspended in 5 mL of TE to which pL of RNase A (10 mg/mL solution) were added, and incubated for 30 min at 37 0 C with stirring. 10 pL of proteinase K (10 mg/mL solution) were added, and the mixture was incubated for at least min at 37 0 C with stirring. For the two successive extractions with phenol/chloroform/isoamyl alcohol (25/24/1, vol/vol/vol), mL of this solution of phenol were added in a Greiner tube with threading screw cap, followed by the sample. After slight stirring for 20-30 sec to mix, the solution was centrifuged for min at 3920 G in a "swinging bucket" rotor. After having transferred the solution into a Nalgene tube, 1 mL of ammonium acetate and 10 mL of absolute ethanol were added. After centrifugation for 20 min at 0°C and 48,400 G (Beckman J2-21), the pellet was washed with 1 mL of 70% ethanol and then dried under a vacuum and resuspended in 1.8 mL of solution 4.(Tris EDTA ImM; NaCl 1M).

After having removed the top stopper, and then the lower stopper of the column, the column was placed on a collection tube and then centrifuged for 1 min at 980 G. The collection tube which collected the equilibration buffer was discarded. The column was placed on another collection tube and the dissolved sample (1.8 mL) was loaded the top of the resin. The column was centrifuged for 12 min at 980 G in a rotor of the "swinging bucket" type. The plasmidic solution collected was divided into two Eppendorf tubes to which 600 pL of isopropanol were added.

After centrifugation for 15 min at 18,320 G (SIGMA 2K15 centrifuge), the pellet was washed with 300 pL of 70% ethanol (vol/vol) and dried under a vacuum. The pellet comprising the plasmidic DNA was redissolved in 500 pL of Milli-Q water (Millipore, and stored cold until the vaccination.

As far as the preparation of the DNA plasmid is concerned, 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 described examples.

Step C: Formulation of the vaccine comprising the plasmid and a pharmaceutically acceptable excipient The plasmidic DNA pellet having been resuspended in water, the DNA concentration was determined by loading agar gel and development with ethidium bromide (see Molecular Cloning, a Laboratory Manual, J. Shambrook, E.F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press (1989), 6 and Appendix E, and Winnacker, E. "From Genes to Clones," VCH (1987), The DNA concentration was adjusted to 0.3-1 pg of DNA per IL of water.

Example 2: Use of the plasmidic vaccine in mice to stimulate the induction of an antibody response and a cytotoxic T-cell response The experience performed on mice to prove the efficacy of the plasmidic vaccine requires the construction of a control plasmid derived from the plasmid pEVhisl4gIII, prior to the immunization of the animals and the analysis of the immune response.

Step 1: Construction of a plasmid derived from the plasmid pEVhisl4gIII by the deletion of the sequence coding for the gene gill A plasmid derived from the plasmid pEVhisl4gIII by deletion of the sequence coding for the gene of the glycoprotein gill was used as a negative control (pEVhisl4gIII-, DNA) This deleted plasmid was obtained as follows: the plasmid pEVhisl4gIII was digested using the restriction enzymes Asp 718 and EcoRV. The DNA was then treated with the T4 DNA polymerase to obtain fragment with blunt ends. The DNA so obtained was ligated with the T4 DNA ligase (see "Molecular Cloning, a Laboratory Manual," J.

Shambrook, E. F. Fritsch, and T. Maniatis, Cold Spring.Harbor Laboratory Press (1989), 1.53, 1.73).

With regard to the preparation of the vaccine against the deleted plasmid, the same steps were used as those described for the DNA plasmid, except that during the production of the plasmid pEVhisl4gIII by the maxipreparation, only the PZ523 columns were used.

Step 2: Immunization of mice Five groups of 6-10 female mice from the consanguineous strain Balb/c with ages from 16 to 18 weeks at the first injection were used.

The mice must be consanguineous to measure the responses of such toxic T cells (CTL), because the major histocompatibility complex (MHC) between the cytotoxic T cells and the target cells--3T3 Swiss albino cells (fibroblasts) (haplotype H-2D)must be guaranteed. The target cells were cultivated in a 10% DME medium (vol/vol) in fetal calf serum.

The plasmidic DNA of the plasmid pEVhisl4gIII, containing the gene of the glycoprotein gIII of the PRV virus, was used as positive DNA (DNA whereas the equivalent plasmid, without the gene gIII was used as the negative control.

100 pg of DNA were intramuscularly injected in each mouse, in the left and right upper hind quarters in two portions containing 50-150 pL of aqueous solution, according to the following immunization protocol.

Group I comprising 10 mice labeled by a color code was vaccinated four times, in week 0 (DNA/) in week 3 (DNA 2 in week 5 (DNA3 and in week 10 (DNA4+) with DNA. Serum sDNA3+ was removed 2 days before the last injection; serum sDNA 4 was removed 6 days after the first collection, that is, 5 days after the last DNA4+ injection.

In week 11, spleen cells were collected and restimulated in vitro. The CTL tests were carried out 4 days later.

Group 2, also consisting of 10 mice labeled with a color code, was vaccinated three times, in week 0 in week 3

(DNA

2 and in week 5 (DNA 3 with DNA+. Serum sDNA2 was collected 2 days before the last injection and sDNA 3 serum was collected in week 9, that is, 4 weeks after the last DNA 3 injection.

During week 9, the spleen cells were restimulated in vitro.

The CTL test were carried out 6 days later.

Group 3, consisting of 8 mice labeled by a color code, was vaccinated only two times--in week 0 (DNAi+); and week 3 (DNA 2 Serum sDNA2+ was removed 2 weeks after the last injection and in week 9.

During week 9, the spleen cells were restimulated in vitro.

The CTL tests were carried out 6 days later.

Group 4, or the control group, consisting of 10 mice labeled with a color code, was vaccinated three times with DNA~--in week 0 with 200 pg of DNA" (DNA 1 in week 2 with 100 pg (DNA 2 and in week 7 with 100 pg of DNA" (DNA3-). Serum sDNA 2 was collected 2 days before the last injection and serum sDNA 3 s was collected in week 8, that is, 1 week after the last DNA3s injection.

During week 8, the spleen cells were collected and restimulated in vitro. The CTL tests were carried out 4 days later.

Group 5 (positive control group), consisting of 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 (Plaque Forming Units) per mouse and per injection in the instep, in week 0, in week 16, and in week 17.

Serum was collected 2 days after the last injection. A mixture of serum originating from 5 animals was used for the analyses.

During week 18, the spleen cells were removed and restimulated in vitro. The CTL tests were carried out 4 days later. Step 3: Analysis of the humoral and cellular immune response (CTL test) Depending on the case, the animals were euthanized; the spleen was removed under aseptic conditions between 7 days and up to six weeks after the last DNA injection.

Part A In vitro culturing and restimulation of the effectors The vaccinated mice and the control mice were euthanized by cervical dislocation and rinsed with alcohol (70% vol/vol). The spleens of these animals were deposited in a Petri dish containing PBS (Gibco) and they were crushed in these dishes by means of a piece of nylon gauze and a curved plastic tube. The elimination of the aggregates and of the conjunctive tissue surrounding the spleen was done by filtration (through the nylon gauze) of the crushed material obtained. After a centrifugation at 220 G for 4 min, the pellet was recovered and the erythrocytes were lysed by the addition of 4 mL/spleen of sterile ACK solution (0.15M NH 4 C1, 1mM KHCO 3 0.1mM NaEDTA, pH 7.2-7.4).

Two washings were then performed with sterile effector medium (compound of DME medium (Dulbecco's modified Eagle, Gibco), completed with 10% (vol/vol) of fetal calf serum (Gibco), 1% (vol/vol) of 200mM L-glutamine (Gibco), and 1% (vol/vol) of the penicillin-streptomycin antibiotic solution (10,000 U/mL of penicillin and 10,000 pg/mL of streptomycin (Gibco), along with of HEPES buffer (pH 7.4) (Sigma), 2 x 10-5M of 2mercaptoethanol (Gibco), and 2mM of sodium pyruvate (Merck)). The cells were resuspended at a concentration 5 x 10 6 cells per mL in this sterile medium. The spleen cells were distributed for culturing in vitro in 25-cm 2 culture flasks (Falcon) at the rate of 25 x 106 cells/flask. A part of these cells was restimulated in vitro by the addition of the viral strains cited above with an infection multiplicity (MOI) equal to 2; the others were used as nonrestimulated controls. These flasks were vertically deposited in an incubator for 4-7 days (at 37 0 C, 3% (vol/vol) of CO2, and a humidity of more than 90% of saturation level), as described above.

Part B The CTL test The cells of the histocompatible line (fibroblasts) 3T3- Swiss albino (haplotype H-2D) were used as targets.

The CTL test comprises several steps: a) The target cells were either infected or not infected by a strain of the Aujeszky virus (NIA3 M207) at an MOI equal to with the infected cells being denoted as CV and the noninfected cells denoted as CV~. Ninety minutes later, the labeling of the target cells in suspension starts (see below--Labeling of the suspended target cells (3T3) with b) With regard to the effectors cultured in vitro for 4-7 days (as described above) earlier, they were dissolved in culture flasks, washed 2 times with the effector medium and counted to be resuspended at a concentration of 5 x 106 cells/mL. Six hours after the start of the infection of the target cells, they were brought in contact with the effectors (which include Tc lymphocytes). In a plate with 96 wells with round bottoms, 5000 target cells in 50 pL of effector medium (labeled with Eu, and either infected or not infected) were deposited on, respectively, 500,000, 250,000, 125,000, 62,500, 31,250, and 15,625 effectors in 100 pL (that is, at effector/target ratios from 100/1 to 3/1).

Repetitions (3 or 4 times) were carried out for each condition.

The plate was centrifuged at ±50 G for 4 min and kept at 37 0 C for 4 h. The evaluation of the quantity of target cells lysed by the Tc lymphocytes was made by collecting the supernatant after a new centrifugation for 4 min at ±50 G. The supernatant was placed into a plate with 96 wells with flat bottoms in 200 pL of a fluorescence amplifying solution (DELFIA® Enhancement solution, Pharmacia, Sweden) in the case of labeling with Eu. The fluorimeter count (delayed-time fluorimeter, 1234 DELFIA Recherche, Wallac) was carried out ±12 h, later taking care to place the plates containing the mixture in darkness and at ambient temperature.

The quantification of the specific lysis (in was estimated by means of the following formula: (Experimental release background noise)/(Maximum release background noise) x 100 specific lysis Part C Labeling of the suspended target cells with Eu This type of labeling is applied both for cells that develop in suspension and for adhering cells.

Culture flasks with maximum confluence were used for the different labelings of the 3T3 target cells.

The growth medium was removed from the culture flask containing the adhering 3T3 target cells; the flask was washed one time with PBS (Gibco). 5 mL of trypsin-EDTA (Gibco) at 37°C were deposited in the flask on the cell lawn. After 1 min, the cells were detached by small abrupt knocks against the flask.

Once the detachment was completed, 7 mL of sterile effector solution (described above) were added. The cells were washed one time with a solution consisting of HEPES buffer (50mM HEPES hydroxyethyl)-4-piperazinyl-l,2-ethanesulfonic) acid (pH 7.4), (Sigma); 93mM NaCl (Merck); 5mM KC1 (Merck); 2mM MgC1 2 6H20 (Merck)) and resuspended at a concentration of 6 x 106 cells/min in said solution. The live cell count was performed with Trypan Blue (wt/vol) solution, Sigma).

1 mL of this solution was completed with: 750 pL of the HEPES buffer solution (pH 7.4), 200 pL of the solution of Eu-DTPA (1.52 mL of the standard solution of Eu (1000 pg/mL in 1% (vol/vol) of nitric acid) (Aldrich); 8 mL of the solution of the HEPES buffer (pH and mL of DTPA diethylenetriaminepentaacetic acid (Merck) at 3.93 g in 100 mL of the HEPES buffer solution); after 2 min.it was completed with 100 uL of dextran sulfate (50 mg of dextran sulfate, M.W. 500 kd, Pharmacia in 10 mL of the HEPES buffer solution).

Thirty minutes were required for the labeling at ambient temperature. During this labeling, the tubes were slightly stirred every 10 min. Afterwards, 7 mL of repair buffer (0.588 g of CaCl 2 *2H 2 0 (Merck), with 1.8 g of glucose (Merck) in 1 L of HEPES buffer solution, pH 3 mL of effector medium (see above), and 12 pL of DNAase at 17,000 units/mL (Boehringer Mannheim) were added. A pause of 8 min was observed. A washing with the effector medium was carried out, followed by a Ficoll- Paque gauze (Ficoll and sodium diatrizoate, Pharmacia LKB). For this purpose, 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. 15 min of centrifugation at 800 G and 20 0 C, the top part of the solution in the tube, up to and including the interface, was recovered.

Any traces of Ficoll-Paque were removed from the cell 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, 5000 target cells were deposited in a plate with 96 wells with round bottoms in the presence of 100 pL of effector medium to determine the quantity of the background noise of the Eu.

26 The same quantity of cells was deposited in 100 pL of Triton X-100 v/v, Merck), for the maximum release of Eu. After a centrifugation of 4 min at 50 G, 20 pL of supernatant were collected and deposited in 200 pL of a fluorescence amplifying solution (see above); the plate with 96 wells with flat bottoms was passed through the fluorimeter (1234 DELFIA Recherche, Wallac) after a 1-h incubation in darkness to obtain a stable reading.

Part D Results of the CTL tests Table II. Comparison of the specific lysis of the splenocytes before and after passage through a Ficoll-Paque gradient

C

0 Groupes/in vitro/cibles Lyse sp6cifique (en t viation standard (en Ra port E/C 100/1 50/1 /1 12/1 6/1 3/1 n Groupe SB+/MOI 2/CV+ 37,6 22,9 16,1 8,4 3,8 -0,5 4 Avant Ficoll 10,0 ±5,8 4,7 ,2 1 0,2 SB+/MOI 2/CV+ 42,2 35,5 20,3 17,0 9,1 11,4 4 Apr~s Ficoll 16,4 12,2 ±6,4 ±6,9 ±3,3 Groupe I

ADN

4 V-/CV- 7,5 6,1 2,8 1,2 -0,7 -0,9 4 Avant Ficoll 1,6 1,1 0 4 0,3 Oil 0,2

ADN

4 9,2 9,1 5,5 2,7 1,9 1,2 4 Apr~s Ficoll 1,5 0,6 ±0,5 0, 2

ADN

4 11,9 9,3 1,8 -2,1 -2,7 -3,6 4 Avant Ficoll 4,0 3,5 06 0,6 0,8

ADN

4 +N/CV+ 31,8 49,4 45,6 20,6 19,8 10,7 4 Apr~s Ficoll 14,2 20,3 ±238 9,0 9,0 4,7

ADN

4 f/MOI 2/CV- -0,3 -0,7 -1,0 -0,6 -1,3 -1,2 4 Avant Ficoll 0 1 O,01 2 ±0,2 0 ,2_

ADN

4 +/MOI 2/CV- -2,0 -1,6 -1,2 -2,7 -1,1 -1,3 4 Apr~sFicoll ±0,3 ±0,2 ±0,4 ±0,1 0,2

ADN

4 +/MOI 2/CV+ -5,3 -6,2 -5,4 -5,1 -4,1 -3,3 4 Avant Ficoll ±1,6 ±1,6 ±1,4 1, ±1,1 ±0,9

ADN

4 +/MOI 2/CV+ 15,4 12,8 19,1 15,3 7,6 22,9 4 Ars Ficoll ±6,6 58 ±8,6 ±7,2 ±2,9 13,3 Groupe 4 ADN3-/MOI 2/CV+ -5,5 -5,2 -3,9 -3,3 -3,9 -3,7 4 Avant Ficoll 1,7 1,5 1,2 ±1,1 ±1,2 1,3

ADN

3 -/MOI 2/CV+ -10,3 -5,8 -7,8 -2,4 -3,5 6,5 4 Aprts Ficoll ±4,2 2,1 3,4 1,1 ±1,3 2,8 0

LU

Q-

Key: 1 Groups/in vitro/targets 2 Specific lysis (in ±standard deviation (in 3 E/C ratio 4 Group SB++/MOI 2/CV+ Before Ficoll 6 SB++/MOI 2/CV+ After Ficoll, 7 Group 1 8 DNA 4

+/V/CV-

Before Ficoll 9 DNA 4 /Vr/CT- After Ficoll

DNA

4 +/V7/CV+ Before Ficoll 11 DNA4+/V7/CV+ After Ficoll 12 DNA 4 +/MOI 2/CVF Before Ficoll 13 DNA 4 +/MOI 2/CV- After Ficoll 14 DNA 4 +/MOI 2/CV+ Before Ficoll

DNA

4 +/MOI 2/CV+ After Ficoll 16 Group 4 17 DNA 3 7/MOI 2/CV+ Before Ficoll 18 DNA/MOI 2/CV+ After Ficoll Explanatory notes concerning the in vitro restimulation treatments and the preparation of the targets: spleen cells cultured in vitro without virus MOI 2: spleen cells cultured in vitro and restimulated by the addition of live virus (NIA3 M207) with an infection multiplicity (MOI) equal to 2.

CV-: noninfected target cells, labeled with Europium CV+: target cells infected with the virus NIA3 M207 at a MOI equal to 10, and labeled with Europium group 5, positive control vaccinated with live virus

DNA

4 group 1, animals injected 4 times with DNA+ DNA3-: group 4, animals injected 3 times with DNAn: number of repetitions The lysis of the noninfected target cells occurred between 0 and and it was not affected by the passage through the Ficoll-Paque gradient. In contrast, the positive control (group both before and after the Ficoll treatment, presented a significantly positive cell-lysis rate. The lysis percentage of infected cells was increased by the passage through Ficoll-Paque for the animals immunized with DNA.

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

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

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

The CTL test of group 4, injected 3 times with DNA (DNA3-) did not show any lytic activity.

Naturally, other frequencies and other intervals can be -considered, as well as other dosages and routes of immunization.

Part E Analysis of the humoral immunological response The serum was collected two times during the experiment, with the last collection occurring just before the sacrifice of the animals to obtain the spleen cells for the CTL test, and the antibody responses were measured by: a test of seroneutralization of the PRV virus (SN) an immunoenzymatic test (ELISA) for measuring, in the mouse serum, antibodies directed against an extract of all the PRV glycoproteins.

The seroneutralization test of the PRV virus was carried out according to the protocol indicated below.

PDs cells (SOLVAY-DUPHAR, NL) and viruses of the Bartha K61 strain (SOLVAY-DUPHAR, NL) were used. The experiment was carried out in plates with 96 microwells with flat bottoms from Greiner (France).

The medium in which the SN test was carried out had the following composition: 340 mL of essential Eagle minimal medium (flow), 100 mL of hydrolysate of lactalbumin 2.5% (wt/vol), 5-10 mL of NaHCO 3 at 5.6% (wt/vol), and 50 mL of fetal calf serum (Gibco).

The serum to be tested was subjected to in a 2-by-2 series dilution in the medium, with the dilutions ranging from 1:2 to 1:4096 (50 pL of serum 50 pL of medium, each time), in the plate with 96 wells with flat bottom (Greiner). Each sample was tested in duplicate.

The virus was diluted to 100 TCID0o (tissue culture infectious dose at 50%) in 0.05 mL of medium, with 50 uL of this diluted virus solution being added to each well. The serum/virus mixture was incubated for 24 h at 37 0 C. 50 pL of the PDs cell suspension, at a concentration of 4 x 10 5 cell/mL, were added to each serum/virus sample. The plates were then incubated at 37°C for 5 days.

The results were observed under microscope, then the titers were calculated by taking the inverse of the dilution that corresponds to 50% of the limit dilution.

The virus was controlled by incubating a diluted virus sample for 24 h at 4 0 C and another virus sample for 24 h at 37 0

C.

The two virus suspensions were diluted (vol/vol) with the SN test medium (see above) at 1:10, 1:100, and 1:1000. Subsequently, 0.05 mL of each dilution of the virus suspensions was 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 interpretation of the results under the microscope took place after an incubation of days at 37 0

C.

The humoral responses measured using the SN test--after two, three, and four injections of DNA--showed values of zero or Weakly positive values.

It should be noted that after immunization with live virus at high doses, measurable but relatively slow titers were observed in the seroneutralization test. These observations were confirmed by the literature.

The protocol of the ELISA test is described by M. ELOIT et al. in ARCH. virol. (1992), Vol. 123, pp. 135-143.

In addition to the serum originating from the treated animal groups as described under "Immunization of the mice," 2 additional sera were included in the analysis--a positive serum (serum and a negative serum (serum The serum .originates from noninjected mice (OF1 mice, 3 weeks old A mixture of serum from 10 mice was prepared. The serum is a mixture of serum originating from 10 OF1 mice having received, after 3 weeks, 109 TCID (tissue culture infectious dose) of recombinant adenovirus expressing the gene gD by intramuscular injection, with collection/sampling 3 weeks later.

Tables III and IV, 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 III--test 1) to identify the positive examples, that is, the samples for which the OD was greater than the optical density of the serum of the negative control (OD x 100 509). The positive samples so identified were tested a second time at variable dilutions (Table IV).

In the serum of the animals that had received an injection of plasmidic DNA without the gene gIII (DNA- pEVhisl4gIII-), no antibodies directed against the glycoprotein gIII were found. It has been shown that after 4 injections of DNA' plasmids, at intervals of 2 weeks or longer, the mice showed a good humoral response against the virus. Seven animals out of 9 tested showed anti-gIII antibodies after four injections.

Even after three injections of DNA+, the sera of 9 animals out of 10 showed measurable titers of anti-gIII antibodies.

Similarly, after 2 injections of DNA', the sera of 7 animals out of 8 showed positive responses in the ELISA test.

33 Table III. ELISA test: Optical density (OD) as a function of the dilutions of the serum--identification of the positive examples OD (x 100) de souris dilution 1/10 3T6moin positif (NIA3 M207) 2m~Iange 2000 2 1+2+3 552 4+5+6 545 7+8±9+10 395 C(4 sADN3 1+2+3 587 4+5+6 679 7+8+9+10 697 7 sADN 3 1 2000 2 2000 3 2000 4 511 1263 6 2000 LU7 2000 2000 9 2000 2000

AD

4 ~2 2000 3 2000 4 874 759 6 2000 7 2000 8 2000 9 2000 2000 serum m~lange 509 s6rum CO m6lange 2000 Explanatory notes: sDNA2-: sDNA3 sDNA 3 sDNA 4 Positive serum serum serum originating from animals injected 2 times with DNAserum originating from animals injected 3 times with DNAserum originating from animals injected'3 times with DNA+ serum originating from animals injected 4 times with DNA+ control (NIA3 M207): serum originating from animals injected with live virus NIA3 M207 serum originating from noninjected animals serum originating from animals injected with adenovirus expressing the gene gD Key: 1 2 3 4 6 7 8 9 11 Mouse No.

OD (x 100) 1/10 dilution Positive control Mixture sDNA2- SDNA3-

SDAN

3 sDNA4 serum mixture serum Table IV. ELISA test--optical density (OD) as a function of the dilutions of the serum test 2 groupe souris N' dii. 1/100 1/300 1/900 1/2700 0 t6moin positif myne 2000 2000 2000 2000 5(M207) sADN 2 3 1087 599 287 171 4 1378 622 303 192 1506 866 405 261 6 673 261 152 136 7 1676 913 353 205 8 1731 837 355 229 .9 .1710 778 274 167 1 N~gatif A dil 1/10 ~2et 10 ~non test6 9sADN+ 1 2000 1029 329 142 2 2000 2000 879 296 3 2000 2000 2000 2000 197 1 123 1 83 6 200 1704 528 244 7 2000 2000 1084 435 8 2000 809 188 122 9 2000 1501 285 169 2000 2000 722 263 sADN 4 2 2000 2000 684 262 3 2000 2000 2000 2000 4 263 138 98 103 143 110 96 104 6 2000 1641 566 256 7 2000 2000 1122 419 8 2000 1097 350 143 9 2000 1739 462 211 20001 2000 549 249 1 non teste s6rum @b6lange 155 96 83 92 s~rum+ P1 6lange 827 305 146 106 Explanatory notes: sDNA2 sDNA3 sDNA3: sDNA+: Positive serum serum serum originating from animals injected 2 times with DNAserum originating from animals injected 3 times with DNAserum originating from animals injected'3 times with DNA+ serum originating from animals injected 4 times with DNA control (NIA3 M207): serum originating from animals injected with live virus NIA3 M207 serum originating from noninjected animals serum originating from animals injected with the adenovirus expressing the gene gD Key: 1 2 3 4 6 7 8 9 11 13 14 Group Mouse No.

Positive control Mean sDNA 2 Negative at 1/10 2 and Not tested sDNA3 Negative at 1/10 sDNA 4 Mixture serum serum (M207) dilution dilution Example 3: Induction of a protection in mice against a challenge inoculation with virulent viruses Part A Immunization protocol Five groups of ten female mice (Charles River, Germany) aged 16 weeks and of the consanguineous strain Balb c were used.

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

The group G1 (the positive control) was vaccinated using an attenuated virus (strain NIA3 M207) by the intraperitoneal route.

107 PFU (plaque forming units) were used for each mouse.

This is a very high dose compared to the doses used for the vaccination of pigs with the attenuated vaccination strains traditionally used at the dose of 105.5 PFU.

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

The vaccination with plasmidic DNA was carried out by the intramuscular injection of 100 pg in 2 x 100 pL of water/mouse in the left and right hindquarters for several consecutive days.

The group C was vaccinated two times, on Friday and Monday, respectively.

The group B received four consecutive injections, distributed from Wednesday to Monday.

The group A received six doses distributed from Monday to the next Monday.

The animals were housed in cages, separated by groups, and the individuals were labeled with a blue felt tip pen.

The serum collected from each animal was coded so as to be able to track each animal individually, as far as the antibody dosage and the protection conferred by the vaccinations are concerned.

Part B Analysis of the immune responses The development of the humoral responses was controlled according to the ELISA protocol presented in Example 2.

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

Serum samples were collected at the beginning of the immunization period, that is 3 days after the last injection of vaccine, at the end of the immunization period, that is one month after the last injection of the vaccine, and just before the test inoculation with virulent virus that took place 9 days later.

Finally, one month after the challenge, the serum was again sampled.

Part C Challenge inoculation Approximately 37 days after the last vaccination, all the mice were exposed to infection with a live virulent virus of the NIA3 strain, in the amount of 7000 PFU in 200 mL per animal, injected peritoneally. The dosage is very high because the lethal dose for the animals is approximately 100 times lower 70 PFU).

The animals were observed, and the deaths were recorded during the 15 days after the challenge, as indicated in Table V.

The results show the variations of the survival rate expressed in percentages as a function of the days after the challenge.

All the animals of the negative control group were dead after the challenge.

Table V. Monitoring of the survival rate of mice after a challenge inoculation with virulent viruses Groupes de sounis (Dix par groupe) Ory~ d s en fonction desior*~~ I I 4 jotn 1 ours 1 6 jours I7 jours 1 10 )ours 1 15 jours GO 0 0 0 0 0 0 GI 100 80 80 80 80 SADN+ 50 20 0 0 0 0 2 x 100 mg AD+ 80 40 30 30 30 U4 x 100 mg___ C, ADN+ 90 60 60 60 60 6 xI100mg Key: 1 Groups of mice (ten per group) 2 Survival rate (in as a function of the days after the challenge 3 Days 4 5 days 6 days 6 7 days 7 10 days 8 15 days 9 DNA 2 x 100 mg

DNA

4 x 100 mg 11 DNA+ 6 x 100 mg The results show that a protection is given, even at the lowest vaccination dose, that is, 2 x 100 pg. Indeed, the death of these animals was delayed in comparison to the negative control group (GO).

At a dosage of 4 x 100 pg, 30% of the animals survived, whereas at a dosage of 6 x 100 pg, 60% of the animals survived.

The results show that the protection induced by this new plasmidic vaccination method is effective, especially if one compares it to the survival rate of 80% observed in the G1 group (positive control), that is, the group of animals vaccinated with the attenuated virus at a very high dose (107 PFU).

It remains to be noted that the vaccination method used in this example can be further optimized. According to the results of the cytotoxicity test against the PRV virus, the vaccination by plasmidic injection for several consecutive days did not induce any CTL response, whereas the injection of the same quantity of DNA at intervals of 3 weeks or longer, as used in Example 2, induced a pronounced CTL response. In addition, the induced humoral response, measured by the response of the antigIII antibodies in the ELISA test, was weaker due to the plasmidic injections over several consecutive days than the humoral response induced by injections of the same quantity of DNA at 3-week intervals, described in Example 2.

Example 4: Induction of a protection in mice against a challenge inoculation with virulent viruses For these experiments, the immunization protocol of Example 3 was used, with the only difference being that plasmidic DNA injections were carried out every three weeks, and the age of the mice was 19 weeks.

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

The group G1 (positive control) was vaccinated using the attenuated virus (strain NIA3 M207) in the instep with a dose of 107 PFU per mouse. Four injections of plasmidic DNA were administered by the intramuscular route in the two hindquarters of the mice groups, each consisting of ten animals, labeled with a color code.

Serum was collected either on the same day, or within three days prior to the next 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, using a dose of 7000 PFU/animal, and peritoneal injections. The deaths were recorded during 15 days after the challenge; they are listed in Table VI.

The results show the survival rates expressed in percentages as a function of the days after the challenge.

Table VI. Monitoring of the survival rate of the mice after a challenge inoculation with virulent viruses Groupes de Aux de survie (en en fonction des jours aprs 1'6preuve souris (Dix par groupe) SO 4 fours 5 iurs 6 jours 7 lours I1ours I1jours GO 40 0 0 0 0 0 G1 100 90 90 80 80 )ADN+ 80 70 60 50 40 4 x 100tg Key: 1 Groups of mice (ten per group) 2 Survival rate (in as a function of the days after the challenge 3 4 days 4 5 days 6 days 6 7 days 7 10 days 8 15 days 9 DNA 4 x 100 pg All the animals of the GO group (negative control) were dead after the challenge. A survival rate of 80% was observed in the group Gl, that is, the animals vaccinated with the attenuated virus at a very high dose.

of the animals survived a dosage of 4 x 100 mg of plasmidic DNA. The immunization method used for the vaccination of the animals with plasmidic DNA (four injections) every three weeks was shown to be more effective in comparison to daily injections (better survival rate of 40% instead 30%, and a relative delay before the death of the animals).

Example 5: Induction of a humoral response in pigs Three groups of pigs, each consisting of 3 animals aged weeks, were used. The animals originated from a PRV-free farm and they were part of the same litter. They were individually labeled with an earring.

One group of 3 animals (Nos. 1, 2, and 3) that were not vaccinated was used as the negative control.

M

For the 2 other groups, three intramuscular injections of the plasmid pEVhisl4gIII were administered at the ages of 5, 7, and 9 weeks.

Three animals (Nos. 4, 5, and 6) received a dose of 75 pg of plasmid, whereas 3 other pigs (Nos. 7, 8, and 9) received 560 pg of plasmid at each injection. The dose of DNA was diluted in 4 mL of PBS buffer (Gibco, and administered in 4 portions of 1 mL at 4 inoculation sites: on the 2 sides of the neck and in the center of the left and right hindquarters. The injection was carried out using a syringe with a Terumo needle having a length of 40mM and an opening of 0.9mM.

The serum was collected at the time of each injection and 2 weeks after the last injection. The analysis of the humoral responses (antibody against the protein gIII) 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. Animi. Sci., 12, 41, 49, 1982) and by an IPMA test (Immuno Peroxidase Monolayer Assay).

The IPMA test was carried out according to the protocol listed below. To each well of 96-well plates (Corning, U.S.) covered with a confluent lawn of SK 6 cells (ATCC, 500 of PRV virus of strain 89V87 (Nauwynck Pensaert Am.

J. Vet. Research, 53 489 (1992)) were added in MEM medium (Gibco, As soon as a cythopathic effect was observed, the plates were thermofixed: after a washing with a PBS buffer, the plates were dried at 37 0 C until evaporation of the liquid. They were then incubated for 1 h at 80 0

C.

The serum to be tested, subjected to 2-by-2 series dilution in PBS, was distributed into the wells, then the plates were incubated for 1 h at 37 0

C.

The serum was removed, then the plates were subjected to 2 washings with PBS, followed by a 1-h incubation in the presence of anti-porcine antibodies labeled with peroxidase (Nordic, Holland) and diluted 100 times in PBS. After 1 h, the plates were incubated in the presence of 3-amino-9-ethylcarbazole substrate (2 mg of AEC in 10 mL of sodium acetate buffer (0.05M at pH and 75 pL of H 2 0 2 at The appearance of a red coloration was observed under an optical microscope. The reaction was interrupted after 15 min with 3 washings of tap water. The IPMA titers were calculated using the inverse of the strongest dilution that produces a red coloration of foci of viral antigen on the infected cells.

The results of the seroneutralization test and of the IPMA test are listed in Table VII.

47 Table VII. Titers of antibodies against the PRV virus, determined by the seroneutralization test and by the IPMA test Titres en anticorPs en fonction de Page ~ge__ NO Dose de 5 semaines 7 semaines 9 du plasmide porc pEVhis 1 4gII1 Sadministre __SN 1wMA SN rIPM semaines I1I semaines 2

IPMA

5

SN

2

IPMIA

n M, 1<2 5 I~ 2~llb~I 0 Me 5 <2 t 5 5 Om~ 0 Ma_ _II i-r mg) <5 1 (75 mg) 1j<2 1 <5 1 2 1~ <2 (75 mg) <5 I- I I I I I 9 (560 mg) (560 mg) (560 mg) 2 2 2 5 5 5 2 2 2 5 2 2 2 5 5 5 5 2 2 6 2 4 <21 <2 3 128 128 128 I I- I I I I Key: 1 2 3 4 6 7 8 Antibody titers as a function of age Age Pig No.

Dose of plasmid pEVhisl4gIII administered 5 weeks 7 weeks 9 weeks 11 weeks Explanatory notes: SN: titer of seroneutralizing antibodies IPMA: titer of antibodies determined by the IPMA method The results show that none of the nonvaccinated animals developed antibodies against the PRV virus. The results indicate that even at the lowest vaccination dose, that is, 3 x 75 pg, a humoral response was induced in 2 pigs out of 3. Only 1 animal out of 3 reacted at a dosage of 560 pg. The responses were weak, and the differences between the two immunized groups were not significant.

Example 6: Induction of a protection against a challenge with a virulent virus in pigs The protocol of Example 5 was repeated exactly.

Three groups of 3 pigs aged 5 weeks were used. The animals originated from a PRV-free farm and they belonged to the same litter. They were individually labeled by an earring.

A group of 3 animals that were not vaccinated was used as negative control.

For the 2 other groups, three intramuscular injections of the plasmid pEVhisl4gIII were carried out at the ages of 5, 7, and 9 weeks, according to the technique described in Example Three animals (pig Nos. 4, 5, and 6) received a dose of 75 ig of plasmid, whereas the three other animals (pig Nos. 7, 8, and 9) received a dose of 560 pg of plasmid.

The challenge with virulent virus was carried out according to the method described in Vaccine, 1994, 12 pp. 661-665, and it is described below.

27 weeks later, that is, 18 weeks after the last injection of plasmid, all the animals were transferred into an isolation unit for exposure to virulent PRV virus of strain 75V19 (Andries, Pensaert, M. Vandeputte, Am. J. Vet. Res., 1978, Vol.

39, pp. 1282-1285) The temperature of the isolation unit was kept at 18'C, and the ventilation at 0.2 m/sec.

105.0 TCID 5 o of virus PRV (strain 75V19) suspended in 5 mL of phosphate buffer were administered to all the animals, 2 mL of this suspension were administered orally and 1.5 mL of this suspension were instilled into each nostril.

All the animals were observed daily during the two weeks following the challenge. The temperature of the animal bodies was recorded, aswell as the animal weights. The relative weight gain (RDWG) was calculated according to the formula indicated below, for the comparison of the performances of the three groups.

RDWG from the day of the challenge to day x: weight at day x weight on the day of the challenge RDWG weight on the day of the challenge The nasal secretions of all the animals were collected with a swab every two days for 14 days after the challenge. The weight of the nasal secretions collected was recorded. The collected nasal secretions was suspended in a phosphate buffer. The decimal [1/10] dilutions of these suspensions were inoculated into monolayer cell cultures, where the cells originated from a

L

continuous cell line of porcine testicles The presence of a cytolytic effect for these cell cultures was then checked for five days. The lethal dose 50 [LD 5 0] was calculated using the method of Reed and Muench (Amer.-J. Hyg., 1938, Vol. 27, pp. 493- 497) The viral titers were expressed in TCID 5 0 per gram of nasal secretion.

The results of the serological examinations are combined in Table VIII.

Table VIII. Titers of antibodies against the PRV virus determined by the seroneutralization test (SN) and the IPMA test N, Dosc de dec Plainidc Ixirc pEilki 14gH11 ~)Titres en anticorps en fonction de l'Sgc 7 seinaines 9 sernaines II semaines 27 semaines 27 s~iii~jncs i~nes scinaines (Ire vaccination) 7 sernaines (2e vaccination) (2e vt~cinalion) (3e vaccination) (pev) Sor 1 or p~ I I sernaines 27 sernaines (6prcuve)

SN

Col Sel SjN Co* Se' IPMA SN Co* Se*

SN

Co* SC* IPMA SN Co* Sc' <2 <2 <2 <5 <2 <2 <2 <2 Q2 <2 <2 <2 <2 <5 <5 I I <2 <2 <2 <5

<S

<5 <2 4 <2 <2 <2 <2 <2 6 <2 <2 <2 4 128 <5 128 <2 <2 <2 <2 <2 3 i8 <2 <2 6 24 <2 4 24 <2 8 (jours apr.2s I~ru lNpreuve) Co' Se' Co Sc <2 <2 96 38 <2 <2 64 8 <2 <2 18 8 3 8 8 3- <2 <2 18 8 4* 32 34 38 <2 2 18 8 4 48 12 8 <2 24 12 8 r1o) 29ftr raines (14 jours apr s Mpretive)

SN

Co' Se, 96 384 64 e-384 128 384 2384 084 128 1-384 2!384 084 -4 1 I I I 7 560 pg 1<2 <2 8 $60 jig 1<2 <2 9 560 pG 1<2 <2 Co* conventionnet 1 7 Se* sensible <2 <2 <2 <5

<S

<5 <2 <2 <2 <2 <2 <2 <2 3 <5 <5 128 Key: 1 2 3 4 6 7 8 9 11 Pig No.

Dose of plasmid pEVhisl4gIII administered Antibody titers as a function of the age 5 weeks (first vaccination) 7 weeks (second vaccination) 9 weeks (third vaccination) 11 weeks 27 weeks (challenge) 27 weeks 5 days (5 days after the challenge) 29 weeks (14 days after the challenge) Co* conventional Sc* sensitive The analysis of the humoral responses was carried out by a sensitive seroneutralization test (test sensitive to the mediation by the complement according to the technique described by Bitsch and Eskilsen, Curr. Top. Vet. Anim. Sci., 1982, Vol.

12, pp. 41-49) and by a conventional seroneutralization test (according to the technique described by Andries, Pensaert, M. Vandeputte, Am. J. Vet. Res., 1978, Vol. 39, pp. 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, titers of seroneutralizing antibodies between 3 and 24 were found, respectively, for three of the animals (Pig Nos. 4, 6, and 9) and for four of the animals (Pig Nos. 4, 6, 8, and 9).

14 days after the challenge, the titers of seroneutralizing antibodies were observed to be between 64 and 128 for the animals that belonged to the negative control group, between 128 and >384 for the animals that received a dose of 75 pg of plasmid, and between 128 and 192 for the animals that received a dose of 560 pg of plasmid.

All the animals were listless and anorexic. They sneezed from the third day after the challenge to the eighth or ninth day after the challenge. Vomiting and nervous disorders were observed in two animals (Pig Nos. 3 and All the animals, with the exception of one (pig No. had a fever (>40 0 C) from the third day to the seventh day after the challenge. The mean maximum temperature was 41.3 0 C for the negative control group and 41 0 C for the two other groups.

The results concerning the changes in the animal weight are collected in Table IX.

Table IX. Changes in the weights of the animals (j Groupe N° de po A G7 A G14 RDWG 7 RDWG 14 1 -5,4 2,8 -0,861 0,223 2 -5,6 3,4 -0,845 0,256 3 -5 -10,7 -1,26 -1,352 4 -0,4 9,7 -0,092 1,110 -5,4 -8,1 -1,467 -1,099 6 -3,3 4,3 -0,630 0,411 7 0,8 7,7 0,178 0,857 8 -1,1 7 -0,185 0,587 9 -3,5 7,6 -0,661 0,718 poids 7 jours aprs 1'6preuve poids le jour de I'epreuve poids 14 jours apres 1'6preuve poids le jour de 1'epreuve RDWG 7 jours apres 1'preuve RDWG 14 jours apres I'epreuve G7 AG14 RDWG7 RDWG14 Key: 1 Group 2 Pig No.

3 Negative control 4 75 pg Vaccinated 560 pg Vaccinated 6 A G7 weight 7 days after the challenge weight on the day of the challenge A G14 weight 14 days after the challenge weight on the day of the challenge RDWG7 RDWG 7 days after the challenge RDWG14 RDWG 14 days after the challenge All the animals, except for one (pig No. lost weight during the first week after the challenge.

Fourteen days after the challenge, only two animals (pig Nos. 3 and 5) had not regained weight to return to their prechallenge weight. Moreover, they continued to lose weight. The three animals having received a dose of 560 pg of plasmid, and one animal (pig No. 4) having received a dose of 75 pg of plasmid, showed a compensatory weight gain between the seventh and fourteenth day after the challenge, so that they had returned to their initial growth curve between the eleventh and fourteenth day after the challenge. The two groups of vaccinated animals presented a significantly positive average weight gain, whereas the control group, nonvaccinated animals, presented a negative mean weight gain value weight loss).

The results of the virus titrations in the nasal secretions are collected in Table X.

G

Table X. Secretion of virus after the challenge IC pe N' de S~rdion de virus expdmrri en porc (log I OTCID 50) en fonction du nomrbre dejouns apr~s 0 M~preuve V/jours apr~s N'preuve Contr6le 1 <1,5 3,2 5,5 5,5 2,0 <1,5 1,7 1, n~gatif 2 <5 1,5 1, 5 5,0 7,5 3,0 2,3 2,0 3 <1,5 2,7 6,7 7,3 4,5 4,5 4,3 [ig 4 <1,5 2,0 6,5 7,3 1,7 <1,5 <1,5 vaccin6 5 <1,5 4,7 6,3 3,7 2,0 <1,5 <1,5 <51,5 6 <1,5 1,5 16,7 7,5 <1,55<1,55<1,5 11,5 560 jig 7 <1,5 2,7 6,3 8,0 4,5 <51,5 <1,5 vaccin6 8 <1,5 51,5 7,3 7,0 <1,5 1,5 <1,5 9 <1,5 5,3 6,3 6,3 <1,5 51,5 <51,5 <51,5 Key: 1 2 3 Group Pig No.

Secretion of virus expressed in (loglO TCID 5 0) as a function of the number of days after the challenge 4 Days after the challenge Negative control 6 75 pg vaccinated 7 560 pg vaccinated The first viruses were isolated from nasal secretions two days after the challenge, for two of the three animals in each group. The viral titers reached a maximum level between the fourth and sixth day after the challenge, with the viral titers being between 10 55 and 108.0 TCID 50 The viral secretion stopped between the sixth and eighth day after the challenge for the vaccinated animals, whereas it continued until the twelfth day after the challenge for the animals of the negative control group.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

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Claims (11)

1. Vaccine comprising a plasmid containing a gene coding for the glycoprotein gIIl of the PRV virus, or for the protein presenting the same antigenicity as the glycoprotein gill of the PRV virus, and a pharmaceutically acceptable excipient for said vaccine.

2. Vaccine according to claim 1, wherein the plasmid also contains a promoter obtained from human cytomegalovirus.

3. Vaccine according to claim 1 wherein the plasmid used is the pEVhisl4gIII plasmid.

4. Vaccine according to claims 1 to 3 wherein the plasmid also contains at least one gene, or a portion of a gene, coding for at least one cytokine or a fragment of a cytokine.

5. Vaccine according to any one of claims 1 to 4, wherein the pharmaceutically acceptable excipient comprises minibeads made of or coated 20 with vaccine, which are introduced into the tissue of the animal to be vaccinated. t

6. Use of a plasmid for a vaccine against the PRV virus, with said plasmid comprising a nucleic acid sequence coding for the protein gill of the PRV virus or for a protein presenting the same antigenicity as the glycoprotein gill of the PRV virus or a DNA construct comprising an expression cassette including: a) a DNA coding sequence for a polypeptide containing at least one antigenic determinant of the glycoprotein gill or an immunogenic fragment thereof; and b) control sequences that are operatively connected to said coding sequences where said coding sequence can be transcribed and translated in a C \WINWORD\STACY\SPEC1M03594 DOC 57 cell and where said controlled sequences are homologous or heterologous with respect to said coding sequence.

7. A plasmidic vaccine against the PRV virus according to claim 6, wherein it also contains at least a gene, or a portion of a gene, coding for at least one cytokine or a fragment of a cytokine.

8. Plasmid pEVhisl4 gIII used in the manufacture of a vaccine.

9. Plasmid pEVhis14 gIII used in the manufacture of a vaccine against the PRV virus.

A method of vaccinating an animal against PRV virus comprising administering to said animal an effective amount of vaccine according to any one of claims 1 to

11. A vaccine substantially as hereinbefore described with reference to any one of the examples. 20 DATED: 23 March 1999 PHILLIPS ORMONDE FITZPATRICK Attorneys for: AMERICAN HOME PRODUCTS CORPORATION °l *o C:\WINWORD\STACY\SPECI\503594.DOC