ITTO940713A1 - VACCINE FOR THE PREVENTION OF REPRODUCTIVE AND RESPIRATORY PORCINE SYNDROME. - Google Patents

Abstract

A new virus capable of reproducing reproductive changes associated with porcine reproductive and respiratory syndrome (PRRS) and in pigs, respiratory disorders has been isolated. This virus can be used in the formulation of a vaccine that can protect sows against PRRS. A vaccine that contains a suitable amount of viral antigen, inactivated, as well as a suitable adjuvant is described. The vaccine has proven effective in test experiments with PRRS viruses other than the Spanish strain. This vaccine is used in veterinary medicine.

Description

DESCRIPTION

The present invention relates to a vaccine capable of preventing porcine reproductive and respiratory syndrome, in particular to an inactivated vaccine containing the causative virus of said disease in inactivated form.

The disease known as porcine reproductive and respiratory syndrome (PRRS) attacks pregnant sows in which it can lead to anorexia, miscarriages, fetal mortality, mummified fetuses, weak piglets that die after a few hours or days of life, post-partum respiratory problems and problems (Loula T., "Clinical Presentation of Mystery Pig Disease in the breeding herd and suckling piglets", Proceedings of the Mystery Swine Disease Committee Meeting, 6 October 1990, Denver, Colorado, Liverstock Conservation Insti tu te, Madison WI, USA ). Some cases have been described in which infected sows have blue spots on their ears, for which reason the disease has also been known as "blue abortion, blue pig ear disease" ["Veterinary Record", 130, n. 3, (January 18, 1992)]. Other names given to ma (MPD) (MSD), "Mysterious Reproductive Syndrome '' (MRS)," Pig Infectivity and Respiratory Syndrome "(SIARS) or" Pig Respiratory and Epidemic Abortion Syndrome "(PEARS).

The first epizootic manifestations of this disease appeared in the United States and Canada in 1987. In Europe, the first manifestation was detected in Germany in 1990, where it spread to Holland and Belgium in the late 1990s and early 1990s. of 1991. In Spain, the first cases of the disease were identified in mid-January 1991 when important respiratory changes were observed in a consignment of 300 piglets imported from Germany [Plana et al., "Med. Vet.", 8 , no. 11 (1991)]. Shortly thereafter, in two breeding groups located 500 meters from the group in which the first problems appeared, a disease was identified characterized by an abnormally high number of abortions during the last stages of gestation, as well as a mortality of 70% in the piglets. Analyzing the clinical signs observed, and bearing in mind that (i) these groups had undergone an intensive vaccination program against porcine parvovirus, Aujeszky's disease and swine flu and that (ii) laboratory tests were carried out -laprseza-di- other abortive diseases, the clinical presence of PRRS was suspected. In samples from these farms, the causative agent of the disease was isolated, as described below in greater detail.

Numerous agents have been correlated with this infectious process, including: encephalomyocarditis virus, swine flu, classical swine fever, African swine fever, mucosal disease, Aujeszky's disease, brucellosis, leptospirosis, Q fever, parvovirus and chlamydial, although some authors have related this to mycotoxin [Loula T., "Clinical Presentation of Mystery Pig Disease in the breeding herd and suckling pìglets", Proceedings of the Mystery Swine Disease Committee Meeting, supra, Mengeling, W.L. and Lager K.M., "Mystery Pig Disease: Evidence and consider tionz for its etiology", in Proceedings of the Mystery Swine Disease Committee Meeting, supra; Dea et al., "Virus isolation from farms in Quebec experiencing severe outbreaks of respiratory and reproductive probìems ", Proceedings of the Mystery Swine Disease Committee Meeting, supra; Van Alstine W.," Past diagnostic approaches and findings and potential useful diagnostic strategies ", in Proceedings of the Mystery Swine Disease Committee Meeting-supra-Loula-T.-Agri- Practice-12 (1): 23-33

(1991); Woolen N. et al.,. "J. Am. Vet. Assoc.", 197: 600-601 (1990) ..

The PCT patent application published under publication number WO 92/21375, in the name of Stitching Centraal Diergeneeskundig_ Instituut (CDI), describes the isolation of a virus named "Lelystacl · 'Agent" (LA) or "Lelystad Virus" ( LV) which is identified as the causative agent of the disease known at that time as MSD. Isolated LV when intranasally inoculated into pregnant sows produces loss of appetite and also refusal to ingest during days 4 to 10-12 after inoculation, reproductive disturbances, and a blue discoloration on the ears of some of the infected sows on days 9- 10 after inoculation. However, it was also observed that in two of the eight experimentally infected sows the disease does not reproduce (see table 6 of said PCT request), since in one of the sows the number of piglets born alive and surviving the first week is large (6 out of 9, sow 1305) and another sow has two dead fetuses while another nine already survive the first week (sow 1065). The LV virus isolated from CDI belongs to the genus Arteriviride, has a genome formed by a polyadenylated RNA molecule with a length from 14.5 to 15.5 Kb (determined in neutral agarose gel) which replicates by means of a series of subgenomic RNAs at the end 3 '. The above PCT request illustrates the nucleotide sequence of the LV genome, the eight possible open reading bases (ORFs), the amino acid sequence deduced from the identified ORFs and the putative sites of N-glycosylation. Subsequently, other PRRS causative viruses were isolated on German, French and Spanish farms. The virus isolated in Tubingen, Germany (TV) ["Virology", 193.

329.339 (1993)] has 99.3% homology with LV at the level of nucleotides contained in ORF from-2 to 7 (there are 24 different base pairs (bp) out of a total of 3216 bp included in this region). The deduced TV amino acid sequence has 99.2% homology with LV (there are only 10 different amino acids in the deduced amino acid sequences encoded by ORF 2 to 5, as there are no differences in the sequences deduced from ORF 6 and 7) .

On the other hand, the homology found between LV and TV against the virus isolated in our laboratory (Spanish or SV strain) is less. So far, only the nucleotide and amino acid sequences corresponding to ORFs 3 to 7 have been compared, with the following results:

L. there is 95.5% homology at the nucleotide level of SV in front of LV: ò TV (out of a total of "25999 '· d bp there are 144 which are different); and

2 . there is 94.9% homology to the amnmino acid level of SV opposite to LV or TV (out of a total of 955 amino acids, a total of 47 amino acids are observed as different).

These variations in amino acids may be related to the increased pathogenicity of a. strain in comparison to another, for the reason that the virus isolated in our laboratories (Spanish strain) is more pathogenic than other known PRRS viruses. For example, the virus isolated in France (French strain), as illustrated in example 8 of this description, where it can be seen that the percentage of piglets born alive from a sow infected with the French strain is 75%, while the percentage is 9.5% when sows are infected with the Spanish strain (Tables 12 and 14). The percentage is 61% when sows are infected with LV (table 6 of PCT application No. WD 92/21375), as 53 piglets were born alive out of a total of 95 piglets.

The disease causes severe losses in the pig farming industry as it can cause, in acute spreads, a mortality of 70% of pigs in a litter. One means of solving the problem created would be to carry out a suitable vaccination program that allows the prevention of the onset of the disease. To this end, vaccines capable of effective prevention of PRRS would be required.

The PCT patent application published under number WO 93/06211 in the name of Collins J.E. and Benfield D.A., refers to a vaccine against MSD containing an infectious agent isolated from lungs of pigs infected with MSD. However, this PCT request does not describe the characterization or identification of the infectious agent, nor has it been filed with any Authorized Depositary Institution. For these reasons, the realization of the knowledge derived from this PCT request presents serious reproducibility problems since the product described in the PCT request cannot be verified nor can the results obtained be compared with those obtained by the PCT applicants. Similarly, this PCT request does not clearly express the antigen or the adjuvant used in the formulation of the vaccine which, as will be verified later, has a very important function, nor are examples described that demonstrate the potency and efficacy of said vaccines.

The PCT request published under the number -WO 93/07898 refers, among other things, to the identification of the causative agent of PRRS and to vaccines derived from it. The virus isolated exhibits LV-like characteristics but, unlike the virus isolated in our laboratory, it is unable to grow on ST cells (pig testes) to detectable levels. Apparently, the vaccine was effective, but the level of protection was only effective in approximately 52% of the vaccinated animals, which can be considered a relatively low level of protection, especially considering the high viral titer employed in the vaccine. vaccine dose. Furthermore, this PCT request does not provide any information on the organization of the virus genome or on the proteins encoded for which reason the comparison between this virus and the virus obtained in our laboratories cannot be carried out or can be carried out but by exerting an excessive research effort. . Therefore, there is a need for vaccines that can prevent PRRS. In order to solve this problem, the

effectively protect sows against infectious disease. The antigenic phase of this vaccine is composed of an isolate; . Spanish inactivated The invention also provides combinations of the isolated PRRS viral antigen (Spanish strain) together with different porcine pathogens in order to provide bi- or multivalent vaccines.

Figures 1 to 5 illustrate the cDNA sequences corresponding to the ORFs 3 to 7, respectively, of the PRRS virus (Spanish strain) as well as the sequence deduced from the amino acids encoded by each ORF;

Figures 6 to 10 illustrate the homology and the differences <'> existing between LV and the virus isolated in our laboratories, at the level of the amino acid sequences deduced from ORFs 3 to 7. Amino acids are expressed according to a one-letter code. The top line of every two lines corresponds to the LV amino acid sequence, while the bottom line corresponds to the virus isolated in our laboratories (Spanish strain). Homologous amino acids are represented by two vertical lines, while when there are substitutions of some amino acids with others, these are represented by two vertical points in cases of conservative substitution, i.e. the substitution of one amino acid with another functionally equivalent. vertical and dot lines between two amino acids represent the existence of non-conservative substitutions.

1. Sample

1.1. Animals chosen for virus isolation Mummified fetuses, stillborn pigs and living but weak pigs aged from 1 to 10 days, offspring of country sows with clinical problems due to PRRS are chosen.

1.2 Preparation of the samples

Samples of the lung, spleen, liver, kidney, brain and heart of piglets are obtained by necropsy. In one particular case, samples are prepared from the lungs of a piglet stillborn from a sow suspected of having PRRS - With this lung, a homogenate is prepared by means of DMEM (GIBCO) (10 g of lung in 90 ml of DMEM) supplemented with an antibiotic solution (PEG) consisting of 1000 Ul / ml of penicillin, 1 mg / ml of streptomicin and 0.5 mg / ml of gentamicin. The resulting suspension is left to rest for one hour at 4 ° C, is frozen and thawed twice, centrifuged and the supernatant obtained is stored at -70 ° C to be used in the infection of alveolar macrophages of pork lung. In 'another' case, lung-samples are prepared from a pig born alive but which dies within a few hours and from birth, by means of a similar procedure.

moreover, blood is extracted from animals by perforating the vena cava in order to obtain (i) blood plasma, which is stored at -60 ° C and used for virus isolation and (ii) serum, which is used to carry out titration of the antibody.

2. Pork lung alveolar microfages

2 .1 Obtaining

Pigs seronegative to Aujeszky's disease, porcine parvovirus, foot and mouth disease, classical swine fever, swine flu (types H1N1 and H3N2) and transmissible gastroenteritis are used. The age of the pigs used varies between 7 and 8 weeks. The animals are anesthetized with sodium phenobarbital before lung extraction, which is performed by first ligating the trachea below the epiglottis and then dissecting above the ligature. Once the lung has been extracted, it is washed externally - with physiological saline solution, and then solutions of PBS and PEG of antibiotics are introduced by subsequent washes. The cells obtained from these washes are subjected to centrifugation for 10 minutes at 300 g and resuspended in DMEM [medium (DMEM medium supplemented with non-essential amino acids (GIBCO), 1% sodium pyruvate 1 oM and 1 % glutamine 2 mM)], 10% fetal bovine serum (FCS) and PEG solution of antibiotics at 1 ° / °°. Counting of the cells is carried out 'in Newbauer chambers.

2.2. Sterility control

The alveolar macrophages of the lung of pigs are verified to be free from contamination with bacteria, fungi and other viruses, by means of suitable tests. Similarly, the general state of the cells is checked by optical microscopy.

The absence of mycoplasma contamination is also verified by cytochemical detection with DAPI (4, 6-diamidine-2-phenes 1-indole) which selectively binds to ONA and forms DNA-DAPI complexes with high fluorescence and high specificity.

3. Virus isolation

3.1. Virus isolation and viral growth on pig lung alveolar macrophages

A culture of alveolar macrophages of pig lung · prepared previously, in the midst of DMES / E-FCS at 10%, is infected with a homogenate of a sample suspected of containing the causative agent of; PRRS. The sample is composed, in one case, of a lung isolate from a stillborn pig from a sow showing clinical signs of PRRS, while in the other cases a lung isolate from a pig born alive but which died after a few hours, offspring from a sow with PRRS symptoms, as well as a blood plasma isolate. The homogenate is kept in contact with the macrophage culture at 37 ° C for one hour. Subsequently, the inoculum is removed and fresh DMEM is added with a solution of 2% of FCS and 1 ° / °° of PEG, buffering the culture with C02 and leaving it to incubate at 37 ° C for several days during which the cytopathic effect (CPE) produced by the virus on macrophages is observed under a microscope: at 3-4 days after infection (dpi) the CPE is 70-80% (giant and deformed cells appear). The culture is frozen at -80 ° C.

Simultaneously, a PRR5-free pig lung alveolar macrophage culture is prepared for use as a negative control.

Subcultures are prepared with the virus isolated-and-it-is-observed-that-there-is-100%-of-CPE-from

second dpi. The virus is frozen at -80 ° C for its subsequent identification. It is characterized.

Similarly, the virus is isolated from blood plasma and other samples from stillborn pigs or living but weak pigs born from experimentally infected sows.

One of the isolated viruses, in particular the virus called PRRS-CY-218-JPD-P56-91, is isolated from the lung of a stillborn pig. This is capable of experimentally reproducing the disease, has the characteristics mentioned in Section A and is registered in the European Collection of Animai Celi Coltures (ECACC), Salisbury (United Kingdom), on 29 June 1993, with accession number V93070108. .

3.2. Viral growth in other cell systems Infections with the virus isolate (Spanish strain) have been carried out in alveolar macrophages of pig lungs and in co-culture of ST cells [continuous cell lines of porcine testes (ATCC CRL 1745 SI)], as a first step to adapt the virus to SI cells. After several passages in series (5-6) on the ST cell and co-cultures of macrophages and cultures of ST-only, the infectious titers of the virus are of the order of IO6 TCID50 / ml when co-cultures of macrophage-cell, ST , are-infected-and-of-the-order

of IO4.5 TCIDc0 / ml for the virus obtained in single ST cells. (TCID50, 50% of the colturaid, infectious dose. Tissue].

In addition, alveolar macrophages from pig lung. they can also be immortalized by fusing them into ST cells by hybridization.

Alternatively, alveolar macrophages from pig lung can be immortalized by fusing them with and or. 2 the L-14 cell line (porcine peripheral blood B cells) ECACC No. 91012317, or with Jag-1 cell line (porcine trophoblast cell line) provided by Dr. Jag Ramsoondar

The casting processes are carried out following the conventional techniques of use in this field.

Alternatively, viruses can be cultured on ST cells or any other porcine cell line in which genes encoding membrane receptors of the pig lung alveolar macrophage for PRRS virus have been introduced.

Viruses produced with these cell systems are susceptible to use in the formulation of both living vaccines and inactivated vaccines.

4. Identification and characterization of the virus 4.1. Naming and storage of the virus

The virus isolated from a stillborn pig lung, named .. PRRS-CY-218J.PD-P5-6-91 -is, has been deposited at the European Collection of Animai Cell Cultures (ECACC), Salisbury (United Kingdom), June 29, 1993 under accession number V93070108 In the present description, the virus is possibly described without distinction as a Spanish virus (SV) or a Spanish strain.

4.2. Characteristics of the isolated virus

This virus (PRRS-CY-218- JPD-P5-6-91.) Has the following characteristics:

a) the production of a light CPE on a continuous line of ST cells (ATCC CRL 1746 ST) (fetal porcine testis) with an average titre of IO4, 5 TciD5Q / ml and on alveolar macrophages of pig lung with average titers of IO5 , 5 TCID50 / ml;

b) when this infects pig lung alveolar macrophage and ST cell co-cultures, mean titers of IO6.3 TCID50 / ml are obtained [which titres represent a logarithmic unit (1 log10) higher than when only alveolar macrophages are infected pork lung];

c) cytoplasmic replication;

d), ecytoplasmic blood circulation production

e) lipid envelope;

f) dimensions 40-50 nm;

g) no haemadsorption or haemagglutination is observed with chickens, guinea pigs, pigs or group 0 human red blood cells;

h) loss of infectivity at acid pH (pH <5);

i) production of microscopic (interstitial pneumonia) and macroscopic lesions in pigs aged two months;

j) production of adverse reproductive effects in pregnant sows with fetal mortality, mummified fetuses and live but weak piglets;

k) cross reaction with Lelystad reference serum (IPMA = monolayer immunoassay of peroxidase);

l) cross-reaction with sera from animals with clinical field infections (IPMA);

m) serum from sows infected with this virus cross-reacts with IV;

n) RNA polyadenylated genome approximately 15000 bp long (neutral agarose gel);

o) replication by means of a subgenomic RNA series present at the 3 'end;

p) sequence-of-nucleotides-having-8-hours

q) in a purified suspension subjected to electrophoresis in polyacrylamide gel and subsequent transfer by immunoblot, 4 majority bands corresponding to proteins with apparent molecular weights of 15,000, 23,000, 54,000 and 66,000 daltons, which are not detected in negative controls (uninfected macrophages); r) when the 0RFs from 3 to 7 of this virus are compared with those of LV and / or TV, 95.5% homology is observed at the nucleotide level (there are 114 different bp out of a total of 2599 b: ' .p, and 94.9% homology at the amino acid level (51 different amino acids out of a total of 955); and

the 'virus belongs to the Arterivirus genus.

4.3. Techniques used for virus identification

4.3.1. Experimental disease reproduction in pregnant sows

systematic serology against the viruses of Aujeszky's disease, foot-and-mouth disease, parvovirosiporcin, classical swine fever, swine flu (type H1N1 and H3N2) and transmissible gastroenteritis. In addition, the 'test * · of · is performed. titration of the antibody against the PRRS causative virus. Between days 77 and 90 of gestation, the animals are infected, with an isolate obtained on alveolar macrophages of pig lung in one case, intravenously (IV) and intranasal (IN), while in another case, only through the via intranasal (example 2.1). The absorption of food, the rectal temperature and the clinical condition of the animals are monitored throughout the experiment. In order to exclude the aforementioned agents, blood samples are taken from all sows prior to infection. These appear to be seronegative for all agents. Similarly, after the experimental infection, all the sows are still seronegative, all said viruses and seropositive against PRRS (verified with the reference LV). The results obtained are shown in table 1.

TABLE 1

Tests performed:

Legend

AD Aujeszky's disease

FMD = foot and mouth disease

PP - porcine parvo

CSF - classical swine fever

SF Swine flu (H1N1, H2N3)

TG Transmissible gastroenteritis

PRRS = porcine reproductive and respiratory syndrome HAI = peroxidase-linked hemagglutination assay

NPLA = neutralization assay linked to peroxidxis ELISA = immunosorbent assay linked to enzyme

SN = seroneutralization

IPM monolayer immunoassay of peroxidase

(1 Vanni "er et al.," Rec. Mèd. Vet. "155

158 (1979)

(2) - Charley B., Doctoral thesis, Alfort (1976) (3) Trepsta et al., "Vet. Microbiol.", 9, 113-120 (1984)

(4) = Elliot M., "J. Rech. Poro.", 20141-146

(5) Lucam F., "Diagnostic sero-immunologique des viroses himains et animals", F. Bricout; L; Joubert: J.M. Huraux (1977)

(6) = Jemènez et al., "J. Virol.", 60, 131-139 (1986) (7) = Wensvoort et al., "Vet. QUARTERLY", 13, n. 3,

(July 1991)

(-) = negative

(+) positive

NA = not performed

4.3.2. Experimental disease reproduction in piglets

The goal of this experiment is to verify whether the virus causing reproductive alterations in sows is able to produce respiratory-and-injury symptoms in pigs aged two months

macroscopic and microscopic at the level of the lungs. To this end, several piglets are infected with the virus (Spanish strain), via the IN route, which are killed several days after infection (example 2.2).

The most important resulting data show that this virus produces multiple foci of consolidation at the macroscopic level as well as interstitial pneumonia at the microscopic level '(table 4 From a health point of view, no relevant clinical signs are observed.

4.3.3. Sensitivity to the chloroform test This test is performed to determine whether the isolated virus has a lipid envelope. To this end, the method of Feldman H. and Wang S. described in "A Manual of Fundamental Virological Techniques", Prentice-Hall Inc., New Jersey, 146-148 (1978) is used. The results obtained show that the untreated virus has a titer of 10 <5,6> TCID50 / ml, while after treatment with chloroform the titer is lower than 10 <1>, <3> TCID50 / ml so it can be established that the isolated virus has a lipid envelope.

4.3.4. Viral genome sequencing

Five steps are carried out:

i) purification of the virus

ii) purification of the viral RNA

iii) synthesis of cDNA

iv) cloning and characterization of cDNA clones v) sequencing and comparison of sequences with those of LV

i) Virus purification

Viruses replicated in alveolar macrophages are clarified and concentrated by filtration using MILLIPORE filters. Next, the virus is subjected to 10% to 50% metrizamide gradient centrifugation (SIGMA). Once the centrifugation is completed, a band is obtained which is concentrated by centrifugation. With the purified virus, polyacrylamide gel electrophoresis is performed and an immunoblot is developed with a specific serum, illustrating proteins whose apparent molecular weights are 15, 23, 54 and 66 K dalton. ii) Purification of viral RNA

A technique is used to select and purify the RNA based on the fact that the RNA contains a poly (A) sequence at the 3 'end. For this purpose, a commercial kit (Pharmacia) is used which only allows the binding of the poly (A) chain of the RNA to a matrix of cellulose-oligo (dT) and its subsequent elution.

iii) Synthesis-of-cDNA

a commercial kit is used (Boehringer, Mannheim), following the producer's destructions: In short, the viral genomic RNA is incubated 'in the presence of an oligo (dT), dATP, dCTP, dGTP, dTTP and reverse transcriptase'.

iv) Cloning and characterization of cDNA clones The cDNA is cloned into a vector derived from pUC18 and a series of clones containing the complete sequence of nucleotides corresponding to ORF 3 to 7 are obtained.

v) Sequencing and comparison of the sequence with those of LV

v.a.) Sequencing

The region corresponding to ORFs 3 to 7 of the virus isolated in our laboratories is completely sequenced. Figures 1 to 5 illustrate the cDNA sequences obtained, as well as the sequences deduced from the amino acids encoded by each ORF: The total length of the sequenced region is approximately 2599 nucleotides (nt).

As can be seen, ORF 3 is approximately 798 nt in length and encodes a protein of 266 amino acid residues.

ORF 4 has a length of approximately 552 nt encoding-a protein-of-183 amino acid residues. The initiation of this ORF 4 is located in the ATG codon located approximately 540 bp from the ATG codon of the initial ORF 3. ORF 3 and ORF 4 have in common a sequence of about 246 nt.

ORF 5 is approximately 606 nt in length and encodes a protein of 200 amino acid residues. The initial codon of this ORF 5 practically overlaps the terminal codon of the ORF 4 (these have in common the TG nucleotides, the ATG codon at the beginning of the ORF 5 and the terminal codon TGA of the ORF 4). The initial ATG codon of ORF 5 is located approximately 1092 nt from the initial ATG codon of ORF 3.

ORF 6 is approximately 522 nt in length and encodes a protein of 173 amino acid residues. This initial ATG codon of ORF 6 is arranged 8 nt upstream from the beginning of the termination TAG codon of ORF 5 (approximately 1682 nt approximately from the initial ATG codon of ORF 3).

ORF 7 is approximately 387 nt in length and encodes a protein of 129 amino acid residues. This initial ATG codon of ORF 7 is arranged 5 nt upstream from the beginning of the TAA codon of termination-of-ORF-6.-to-approximately-2193-nt

approximately from the initial ATG codon -of-ORF

The proteins encoded by ORF 3 to 6 are membrane proteins, while the protein encoded by ORF 7 is a nucleocapsid protein.

v.b. ) Comparison with LV

By comparing the cDN A sequences of the LV ORFs 3 to 7 with those of the virus isolated in our laboratories, it is observed that:

i) at the nucleotic level, out of 2599 nucleotides compared 114 are different, which represents approximately 95.5% - of homology,

ii) at the level of amino acids, out of 955 amino acids compared, there are 47 different ones, representing approximately 94.9% homology,

iii) of the 47 different amino acids there are 35 considered as non-conservative replacements of which there are:

- 12 in the product of the gene of ORF 3 (figure 6); - 9 in the gene product of ORF 4 (Figure 7); - 10 in the gene product of ORF 5 (Figure 8); although it should be pointed out that the LV di-ORF-5 gene product contains one-amino acid more than the Spanish virus ORF 5 product, specifically the amino acid 35 (Asn) of the LV gene product of ORF 5 does not it is present in the product expressed by the Spanish virus;

- 4 in the product of the gene fi ORF 6 (figure, 9); while

- the product of the gene of ORF 7 does not contain any non-conservative substitution (figure 10) iv) partial homology of each of the products expressed by the different ORFs of the Spanish virus and LV is 93.6% for the products of ORF 3 and ORF 5, 94.0% for the product of ORF 4, 95.6% for the product of ORF 6, and 99.2% for the product of ORF 7.

As mentioned above, changes in amino acids can be linked to the greater pathogenicity of one strain compared to another, since the virus isolated in our laboratories (Spanish strain) is more pathogenic than the other known PRRS viruses such as, for example, the virus. used in sol ato-in - France (example 8) and LV (table 6 of PCI application no. WO 92/21375.

5. Vaccines

The invention provides a vaccine capable of treating porcine reproductive and respiratory syndrome (PRRS). The vaccine has been shown to be effective in preventing reproductive disturbances in sows such as stillborn pigs, mummified pigs or live but weak piglets, return to service and similar problems produced by the PRRS virus. Similarly, the vaccine has been found to induce cellular immunity in vaccinated animals.

The vaccine contains a suitable amount of PRRS viral antigen, Spanish strain, inactivated, as well as an adjuvant and a condom.

Tests performed with these vaccines have demonstrated the efficacy of the vaccine, as evidenced by Examples 7 and 8. Furthermore, the vaccine has been shown to be effective in avoiding return to service which appears in infected sows. In practice, sows vaccinated with the vaccines resulting from the present invention and infected with the PRRS causative virus mate and become pregnant at the first post-partum ovulation and weaning of piglets.

5.1. Components

5.1.a) Antigenic phase

As an active component, the vaccine contains inactivated PRRS viral antigen, Spanish strain, at a concentration greater than or equal to 10 <5.5> TCID50 per vaccine dose. The inactivation can be performed by chemical means which include treatment with beta-propiolactone or other conventional inactivating agents, such as ethylenimine or formaldehyde, or by physical means.

5.1.b.) Adjuvants

Although it is possible to use adjuvants of the aluminum hydroxide type, Quii A or their mixtures, as well as oily adjuvants, for the formulation of the vaccine, it has proved possible to verify that the best results are obtained when an oily adjuvant is used (example 4). In particular, it has been found that an oily adjuvant consisting of a mixture of Marcol 52, Simulsol 5100 and Montanide 888 provides excellent results.

Marcol 52 is a low density mineral oil produced Simulsol 5100 is a polyethoxyoleate ether marketed by SEPIC; and Montanide 888 is a high purity anhydrous mannitol ether octadecenoate marketed by SEPIC.

It has been possible to verify that the adjuvant plays an essential part in the efficacy of the vaccine. Thus, an immunity test, (example - was performed using sows vaccinated with two different vaccines, one sow with the oily adjuvant indicated above (Ref. 1) and the other, sow using the adjuvant munokyn <R> (Ref f2) Although both vaccines produce seroconversion, it occurs in an experimental infection trial that sows vaccinated with the Ref. 1 vaccine are protected against experimental PRRS virus infection, while the other sows, those vaccinated with the Ref. 2, are not protected despite the fact that at the time of the immunity test they have antibodies against the virus. This establishes the fact that a suitable adjuvant can have a very important part in relation to the modulation and stimulation of the immune response, mainly at the level of cellular immunity. Furthermore, this aspect was confirmed by the immunity test carried out using the Spanish strain of the PRRS virus, since ofe vaccinated and revaccinated with the Rif vaccine. 1 do not show a serological response at the time of infection, but, nevertheless, are protected (example 7, table 8).

T.

vaccine Ref. 2 (with Munokynin) can cause cellular immunity: To this end it would be-necessary to add substances that enhance the cell response (CRP), i.e. substances that enhance the subpopulations of helper T cells XTh, and Th2) , such as IL-./ 1 (interleukin-1), IL-2, IL-4, IL-5, IL-6 ;, IL-12, g-IFN (gamma-interferon), cell necrosis factor and similar substances . Evidently, it would also be possible to add these PRC substances to oil adjuvant vaccines, in which case their cell immunity effect would be enhanced.

It is also possible to use other types of adjuvants which modulate and immunostimulate the cell response, such as MDP (muramyl-dipeptide), ISCOM (Immune stimulating complex) or liposomes.

5.1. c) Condoms

Any of the condoms normally used in the formulation of vaccines can be used. One of these is Thimerosaì [sodium salt of (2-carboxy-phenylthio) -ethyl-mercury] (ALDRICH).

5.2. Method for preparing the vaccine

Vaccines resulting from the present invention can be obtained by mixing an antigenic phase containing inactivated viral antigen and another phase. as an adjuvant, which may or may not be oily depending on the adjuvant chosen. Optionally, CRP substances could be added to any one of the two phases. When the adjuvant is oily, an emulsion is formed which, in a preferable particular case (when the adjuvant is a mixture of Marco! 52, Simulsol 5100 and Montanide 888), is a double water / oil / water emulsion.

5.3. Vaccine control tests

In addition to conventional tests, the vaccine must pass, prior to its administration, for (i) purity (against foreign bacteria, fungi, mycoplasma and viruses), (ii) identification, (iii) safety, (iv) potency and (v ) physico-chemical controls, several field trials (example 5.b) were carried out in relation to the safety and efficacy of the vaccine in a total of five farms, of which 508 sows were vaccinated and revaccinated with one of the resulting vaccines from the present invention, while the remaining 472 sows have not been vaccinated and are kept as control sows, making it possible to observe how the vaccine is safe and at the same time effective, since in some of the farms the natural PRRS disease has been found in animals not vaccinated after the vaccination process

5.4. Posology and instructions for administration of the vaccine '

It has been possible to verify that a dose of 2 ml of oily vaccine with a concentration of inactivated viral ahtigene equal to or greater than 105.5 'TCID50, administered via a deep intramuscular route, is able to protect a very high percentage of animals. vaccinated against PRRS.

The following vaccination schedule is recommended:

* First vaccination: all breeding animals (sows and boars) are vaccinated and revaccinated after 21 days. Thereafter, one dose is given during each lactation (sows) and every 6 months (boars).

* Posterior vaccination

- animals intended for breeding: the first vaccination should be at 6 months of age »the revaccination after 21 days.

- Sows: it is advisable to vaccinate during

the lactation period, if possible 15

days before mating.

Boars: vaccinated twice a year (every 6-months

Alternatively, if no .CPR substance has been included in the vaccine, these substances can be injected at a site other than the injection site but simultaneously.

6. Polyvalent vaccines

By means of an additional objective embodied by the present invention, combinations are provided

of different porcine pathogens containing in addition

to inactivated viral PRRS antigen (Spanish strain) one or more of the pathogens listed below, in order to allow the preparation of bi-, or multivalent vaccines.

In this way, vaccines can be prepared

bi- or multivalent containing viral PRRS antigen

inactivated and one or more of the following pathogens:

Actinobacillus pleuropneumoniae, Haemophilus parasuis.

Porcine Parvovirus. _ Leptospira, Escherichia coli. Erysipelothrix rhusiopathiae, Pasteurella multocida, Bordetella bronchiseptica. Porcine respiratory coronavirus.

Rotavirus, or against the causative pathogens of

Aujeszky's disease, swine flu or transmissible gastroenteritis

EXAMPLES

Example 1. Virus isolation

1.A. Sample preparation

Dapomonedunmaanonaomooprgne

of a sow with classic PRRS symptoms [the sow was free of antibodies. check Aujeszky's disease, porcine parvovirus, foot and mouth disease, classical swine fever, swine flu (types H1N1 and H3N2) and gas troenteritis transmissible] ... comes. prepared a 10% suspension in culture medium. DMEM, supplemented with an antibiotic solution (PEG) consisting of 1000 IU / ml penicillin, 1 mg / ml streptomycin 5 0.5 mg / ml gentamicin, in a lung: DMEM ratio of 1:10 (weight / volume). The suspension produced is homogenized and left to rest for one hour at room temperature (20-22 * C). The homogenate is frozen and thawed twice, centrifuged and the supernatant obtained is stored at -70 ° C to be used in the infection of alveolar macrophages of pork lung.

Similarly, lung samples are prepared from a pig that was born alive but died within hours. In addition, blood is extracted and mixed with and without anticoagulant used for the isolation of the virus (from blood plasma) and to obtain serum, respectively

1.8. Obtainment of alveolar macrophages from pig lung

Alveolar macrophages are obtained from the lungs of pigs seronegative to AuJ.eszky's disease, parvovi rosi porcina foot and mouth disease, classical swine fever, swine flu (types H1N 1 and H 3N 2) and transmissible gastroenteritis. The age of the pigs used varies between 7 and 8 weeks. Before lung extraction, the animals are anesthetized with sodium phenobarbital and then killed. Immediately, the lungs together with the trachea are extracted after ligament below the epiglottis. The extracted lung is washed externally with physiological saline solution, and in subsequent washes 50 ml of PBS supplemented with 27 ”of antibiotic PEG solution are introduced until a total of 500 ml of PBS is introduced. The cells obtained from these washes are centrifuged for 10 minutes at 300 g. This washing step by centrifugation is repeated two more times. The cells obtained are washed with PBS and PEG solution of antibiotics and resuspended in DMEM medium [DMEM supplemented with non-essential amino acids (GIBC0), 1% sodium pyruvate 1 mM and 1% glutamine 2 mM], 10% serum fetal bovine (FCS) and PEG solution of antibiotics at 1 ° / °°Cell counting is performed in Newbauer's chambers and for this purpose 1/10 dilutions of the macrophage suspension are prepared by adding 0.4 ml of DMEM and 0.5 ml of trypan blue solution at 0.1; mi of suspension of macrophages. A variable number of cells between 1 and 1.2 x 10 is obtained

1.C. Virus isolation

A 25 cm surface culture flask containing a culture of previously prepared pig lung alveolar macrophages (3 x 106 cells / ml) in DMEM medium and 10% FCS is infected with 1 ml of the homogenate of a sample derived from the lung of a stillborn pig (example 1.A). The homogenate is left in contact with the macrophage culture at 37 ° C for one hour, buffered with C02 at pH 7.0-7.4 and incubated at 37 ° C for several days, during which the CPE produced by the virus on macrophages is observed. At 3-4 dpi, the CPE is observed to be 70-80%, which is why the crops are frozen at -80 ° C.

Simultaneously, a culture of alveolar macrophages from uninfected pig lung is prepared.

used as a negative control.

Sub-cultures are carried out with the isolated virus in which it is observed that the CPE starting from the second dpi is 100%. The virus is frozen at -80 ° C for its subsequent identification and characterization. After the fourth .pass in. macrophages, the corresponding titrations are carried out in 96-well microplates, obtaining the average titer of 105.6 TCID50 / ml according to the method of Reed e.Muench ["Am; J. Hug." ,.

27, 493-497 (1938)]

An isolated virus sample (Spanish strain) named PRRS-CY- 218- JPD- P5-6-9I, isolated from a stillborn pig lung, is capable of experimentally reproducing the disease, and is deposited at ECACC on 29 June 1993, with the corresponding accession number V93070108.

In a similar way, the virus is isolated from live and stillbirth pigs, progeny of experimentally infected sows.

Example 2. Virus identification and characterization

Example 2.1 Experimental reproduction of the disease in pregnant sows

Twelve sows are used, "German Landrace x Large White cross", originating from farms with systematic serological control against Aujeszky's disease viruses, foot and mouth disease, porcine parvovirus, classical swine fever, swine flu (types H1N1 and H3N2), and gastornteritis transmissible. In addition, the antibody evaluation test against the PRRS causative virus is performed. "

The sows are moved to the safety stalls of the research center a week before infection and placed in separate pens. Between days 77 and 90 of gestation, '' sows identified with numbers 53, 76, 3, 62, 91, 93 and 19 are infected with 5 ml intravenously (IV) and with 5 ml intranasally (IN) of PRRS virus, Spanish strain, isolated on alveolar macrophages of pig lung identified as PRRS-CY-218_JPD-P5-6-91, from a fourth pass on macrophages filtered through a 200 nm filter and with a titer of 10 <5, 6> TCID50 / ml. The remaining five sows (# 14, 40, 13, 30 3 85) are inoculated between days 65 and 85 of gestation with 5 ml via IN (only) of the virus.

During the experiment, the food intake, rectal temperature and clinical status of the animals are checked daily, as well as reproductive abnormalities (premature deliveries, retarded deliveries, live but weak pigs, stillborn pigs, mummified pigs and healthy pigs. ),

In order to proceed with the exclusion of the aforementioned pathogens, blood samples are taken from the sows pre and post-infection

The animals were found to be seronegative for pathogens before and after infection and seropositive against PRRS after infection (see table 1, section 4.3.1). The reproductive results are shown in Tables 2 and 3. As shown in Table 2, of the 93 piglets born, 15 are mummified, 35 stillbirths, 22 were born alive but die on the third day and 21 survive 7 days.

Some sows show loss of appetite for 2-4 days, on days 6 and 8 after infection, while other sows show loss of appetite on the second post-infection day. Hyperthermia is not observed in any case. In 4 sows (n. 8, 62, 92 and 93) the farrowing is premature from 1 to 6 days, while in the other three sows the farrowing is delayed by one or two days.

Piglets born alive, 1 or 2 per litter have edema in the eyes. Weak piglets exhibit in / coordination, hindquarter paresis, brittle hair and myoclonia. In the necropsy carried out on some of the stillborn and weak pigs.

the presence of abundant clear liquid is observed in the thoracic cavity. Healthy piglets born to infected mothers killed at 8-12 days of age exhibit gray foci of consolidation. On microscopic examination, the most relevant change is a slight multifocal interstitial pneumonia - with enlargement of the septa-alveolar - due to the infiltration of mononuclear cells. These lesions appear in all animals analyzed in the experiment.

As shown in table 3, of the 65 piglets born, 36 were stillborn, 26 were born alive but weak, dying on the second day of life, and 3 survive for the first week of life. One sow gives birth 12 days prematurely (# 40) while the others give birth 1 or 2 days prematurely. The clinical signs of the piglets born: weak are similar to duels obtained by IN-IV. Pneumonia is also observed. Infected serophae do not show inappetence or hyperthermia.

The most notable difference between the two infection systems is that mummified pigs are observed via IN IV and that 1 to 2 of the piglets born alive in each litter exhibit edema around the eyes.

In view of the results obtained, it can be concluded by defining that the infection model

experimental in sows, at about 80 days of

gestation via both routes of infection (IN and

IV) with the virus isolated (Spanish strain) from animals

in pregnant sows and causes a high proportion of

mummified fetuses, stillborn piglets and live piglets, 'but weak, very similar to the' proportion

observed in the spread of natural infection

acute. It is advisable to artificially infect

via the IN way for the reason that this is the way

natural of infection in the field and therefore the way more

appropriate to evaluate the effectiveness of the vaccine.

Using this experiment, it was as well

It is possible to define an infection model

experimental in pregnant sows that allows the

verification of the effectiveness of the vaccine.

A: sow

3: time of infection (gestation days)

C: delivery (days of gestation)

0: total piglets

E: mummified pigs

F: weak piglets died within 48 hours

G: stillborn pigs

H: pigs apparently in good health

h1: deaths between days 2 and 7

h2: live after a week

I: interstitial pneumonia

NT: not tested.

Example 2.2 Experimental reproduction of the disease in piglets

This experiment was designed in order to verify that the isolated virus (Spanish strain) that produces reproductive alterations in sows, is capable of producing clinical signs as well as macroscopic and microscopic lesions in the lungs of pigs aged 2 months. To this end, 10 pigs are infected via IN with 5 ml of virus, Spanish strain, with a titer of 10 <5'6> TCID50 / ml and 6 other pigs are left as controls (all of these derive from two litters). Animals are killed on days 3, 7, 8, 9 and 11 after infection. The results obtained are shown in table 4.

TABLE 4

No: number of pigs

T / C: infected (I) or control (C)

PPE: days post infection

I.P .: interstitial pneumonia

v / l .: virus isolation

2-t-: mild interstitial pneumonia

3+: intermediate interstitial pneumonia

4+: severe interstitial pneumonia

NT: not tested

NL: no injury

During the days of the experiments}, it is not observed

although there is weight loss in infected animals compared to non-infected animals: Under microscopic observation, the most important aspect is the presence of multiple foci of consolidation in the lungs, congested lymph nodes in the jaw and 'some haemorrhages.' intestinal. At the microscopic level, interstitial pneumonia is observed.

The virus is isolated from the solutions obtained from the lung washes of infected animals of a fresh macrophage culture, - but the virus is not isolated from the control animals, in which no seroconversion or macroscopic or microscopic lesions are observed in the lungs .

By carrying out cell counts from the washes of lungs of infected animals, 30% of dead cells (macrophages) are observed, which can be an important factor in secondary infections due to the destruction of a key immunological defense element (macrophages).

The absence of respiratory signs may be due to the fact that the experimental infections were carried out in stables with continuous disinfection treatments and, therefore, the bacterial concentration is

conditions in the field.

Example 2.3 Sensitivity to the test with chloroform The method of Feldman H. and Wang-S is used. (section 4.3.3.) which leads to the knowledge that the isolated virus has a lipid envelope since, there is a 4 log10 titer drop from the controls compared to those treated with chloroform. Example 2.4. Genome sequencing viral

i) Virus purification

The virus, replicated on alveolar macrophages of pig lung, is purified by filtration and centrifugation in a metrizamide gradient from 10% to 50% (SIGMA), leading to a band that is centrifuged again as mentioned in section 4.3.4. A polyacrylamide gel electrophoresis is performed with the purified virus, and an immunoblot is developed with a specific serum, illustrating proteins with apparent molecular weights of 15, 23, 54 and 66 K Dalton. ii) Purification of viral RNA

The viral RNA is purified using a commercial kit (Pharmacia) which allows the binding of the poly (A) RNA tail to a cellulose-oligo (dT) matrix and its subsequent elution.

A commercial kit is used (Boehringer Mannheim) (section 4. 3. 4. Iii).

iv) Cloning and characterization of cDNA clones The cDNA is cloned into a vector derived from pUC18 and a series of clones containing the complete nucleotide sequence corresponding to ORFs 3 to 7 is obtained.

v) Sequencing and comparison of the sequences with those of LV

The results of the cDNA sequencing of the virus isolated in our laboratories (ORF 3 to 7) as well as the comparison between this sequence and the LV sequence, are cited in section 4.3.4.V, where it can be seen that, at the level of amino acids, there is a degree of approximately 94.9% homology and a total of 47 different amino acids of which 35 correspond to non-conservative substitutions. These amino acid differences may be responsible for the different pathogenicities that exist between the various isolates of PRR5 virus.

Example 3. Formulation of a vaccine

A vaccine, capable of protecting against PRRS, is prepared in the form of an emulsion following the procedure described below.

pig is infected with a MOI (multiplicity order infection) of 0.001 and incubated at 37 'C for 24 hours, after which the culture medium is replaced with the infection medium (DMEM integrated with 2% FCS). The culture is incubated for 4 days, a. 37 <°> C until 70-80% CPE is observed. Once this period is completed, an IPMA test is performed in order to confirm identification. The virus is collected by vacuum aspiration and frozen at -80 <e> C.

The viral suspension intended for the vaccine formulation should have a minimum titer of 10 <5.5> TCID50 / ml (before its inactivation) and should not be contaminated by bacteria, fungi, Hd mycoplasma and other viruses. In the event that the titer may be lower, this should be adjusted by concentrating the antigen. For the inactivation of the viral suspension, a 2 ° / °° solution of beta-propiolactone is added and stirred at 4 <e> C for one night maintaining the pH at 7.4 by adding 0.5N NaOH. Once the inactivation period is complete, the viral suspension is kept at 37 ° C for one hour.

It is then prepared:

a) an antigenic phase of the inactivated viral antigen with the minimum concentration of 10 <5.5> TCID50 / dose and the preservative; And

b) an oily phase consisting of Marcol 52, Simulsol -1500 and Montanide 888.

The aqueous phase, kept under stirring, is slowly added to the reactor containing the oil phase kept under stirring as well. After the aqueous phase has been added completely, the stirring is continued for 10 minutes.

In a particular preferred case, vaccines have been prepared capable of preventing PRRS, comprising for doses of 2 ml:

a) 53% of an antigenic phase, containing:

i) PRRS viral antigen in DMEM culture medium, Spanish strain, inactivated with betapropriolactone at a minimum concentration of 10 <5.5> TCID50, and

ii) thimerosal 0.01%; And

b) 47% of an oily phase containing:

i) Marcol 52. 790.0 mg

ii) Simulsol 5100. 70.0 mg

iii) Montanide 888 80.0 mg.

The oil phase / aqueous phase ratio is a weight / volume ratio. This vaccine was called MSS Ref. 1. The vaccine obtained comes

of employment.

Another vaccine is prepared in a similar way using Munokynin <R> (aluminum hydroxide and Quii A, supplied by American Cyanamid) as an adjuvant while maintaining the same amount of inactivated virus. This vaccine was called MSD Ref. 2.

Example 4. Evaluation of the adjuvant

A field test is performed with a total of 128 sows of which 49 are vaccinated with a dose of the vaccine called MSD Ref. 1, 50 sows are vaccinated with a dose of the vaccine called MSD Ref.

2 (Munokynin) and the remaining 29 are not vaccinated and are kept as controls. After 22 days, the sows are revaccinated with a corresponding vaccine dose.

The following parameters are evaluated

1. Serological response by IPMA determination at the following times:

T0: vaccination and bloodletting

T22: revaccination and bloodletting

T51: phlebotomy at 50 days post-vaccination.

General reactions (appetite for food, hyperthermia, echo.).

The results obtained are illustrated in tables-5

with positive serological reaction (table 5) and the arithmetic mean; of the serological titers, reached (table 6).

TABLE S

% OF ANIMALS -CON- SEROLOGICAL REACTION

vaccinated and revaccinated in the same days with the MSD Rif vaccine. 2 (doses of 2 ml, 10 <5.5> TCID50 / dose of inactivated virus titer).

During the first five days after

remarks:

a) Local reaction

It consists of macroscopic observation of the injection site and palpation, noting the degree of inflammation in comparison with objects of known size.

b) General reaction

It consists of macroscopic observation of the animals and verifying their appetite for food. (If not, the rectal temperature is checked every 12 hours until the hyperthermia or any other unfavorable sign has disappeared.

The results obtained reflect the fact that there is a slight inflammatory reaction in some sows, prevalent at the injection site, which disappears in any case within a few days; no purulent formations are observed. Only one of the sows refuses to ingest all of the food in the first post-inoculation feeding, but food ingestion is normal in subsequent feedings so that it is not necessary to take the rectal temperature. There are no substantial differences in the response to the different vaccines tested when identified, on the basis that both vaccines can be said to be safe.

Example 5.A.2. Pregnant sows

Seven pregnant sows (German Landrace'x Large White cross) from a pig breeding farm are randomly selected: 6 primiparous sows aged about 9 months and a multiparous sow, aged 3 years and 7 months.

The vaccine called MSD Ref. 1- is subsequently used.

Sows are vaccinated by deep IM with a dose of 2 ml of vaccine containing inactivated virus at a titre of 105.5 TCID50 / ml, and 15 days later they are revaccinated with a dose of the same titre.

During the first five days after inoculation, the following observations are made:

a) Local reactions

It consists of macroscopic observations of the injection site and palpation taking note of the degree of inflammation in comparison with objects of well-known size.

b) Lack of appetite

It consists of macroscopic observation of animals and verification of loss of appetite for-food

c) Rectal temperature

Measurement of the rectal temperature at 24 hours after the glue. vaccination and 24 hours after revaccination.

The results obtained reflect the fact that there is a slight local reaction in two of the animals, which is not serious due to its small size, disappearing within a few days. Lack of appetite or hyperthermia are not observed in any case. Based on this, it can be said that the vaccine is safe.

Example 5.B. Trial in the field of safety and efficacy This experiment is carried out in the 5 farms listed below. A varying number of sows from each farm are vaccinated by deep IM with a 2ml dose of the MSD Ref. 1 vaccine containing a titer of 10 <5.5> TCID50 / dose of inactivated virus, and revaccinated 21 days later with an inactivated virus. other dose of the same title, while the other sows are not vaccinated and are used as controls.

FARM VACCINATES TOTAL CONTROL

TL = Total

I: "RAMON DEL QUINTA" farm (Banyoles)

II: Farm "CAL SABATER" (Orriols)

III: Farm "E: CANELA" (Preixana)

IV: "R. CUNILLERA" Farm (L’Albi)

V: "INVERSORS PICBER" farm (Bellpuig)

It was possible to verify that the vaccine is safe based on the observation of general and local reactions. Local reactions are observed in only 1% of animals. In relation to the production parameters observed in the aforementioned farms, no variations are observed in comparison with their clinical histories.

Regarding the serological response, some farms have a seroconversion for the vaccine while in others the response is negative. This is not indicative of a low level of protection since in experimental laboratory infection tests, seronegative animals resist experimental infection (examples 7 and 8).

In-connection-with-the-transmission-of-maternal-immunity from vaccinated animals to their offspring, there is a large fall in antibody titers at the age of the month.

In vaccinated and revaccinated sows that are serologically positive, there is a large drop in antibody titers two months after revaccination, Example 6. Verification of cell immunity Five pregnant sows (German. Landrace x Larga White cross) are used from one pig breeding farm. The animals are moved to the security stalls of the research center.

Sows are randomly selected e. they are vaccinated with the vaccine called MS Ref. 1. Another sow is vaccinated with the vaccine called MSD Ref. 2. The two remaining sows are not vaccinated.

Sows are vaccinated by deep IM with a 2 ml dose of MSD Ref. 1 or MSD Ref. 2 vaccine containing inactivated virus with titer IO5'5 TCIDC, Q, and 20 days later the sows are vaccinated with a other dose of the same title.

Subsequently, between days 7 7 and 90 of pregnancy all sows are infected via

with the titer of IO5.8 .. T C I D c Λ / m 1. At the time of infection, it is verified that all vaccinated files have antibodies against the causative virus of PRRS (positive serology). Table 7 illustrates the restorative-obtained results together: '

(A) number of sows

(B) vaccine used

(C) total number of piglets

(0) number of piglets born alive in good health (E) number of piglets born alive but weak

(F) number of piglets alive after the first week (G) number of piglets born dead

The results obtained show that sows vaccinated with the MSD Ref. 1 vaccine (oily adjuvant) resist infection better than sows vaccinated with the MSD Ref. 2 vaccine (adjuvant

adjuvant has an important function in establishing the immunity of the cell.

Example 7. Efficacy in pregnant sows

11 sows (German Landrace x Large White cross) are used from a pig farm. The animals are "moved to the security stalls of the research center.

Three sows are chosen at random (sows no.57, 63 and 74) and are vaccinated with the vaccine called MSD Rif. 1. Three sows (no. 15, 18 and 23) are vaccinated with the vaccine called MSD "Ref. 2, and the remaining 5 sows (no. 14, 40, 13, 30 3 85) are not vaccinated.

Sows are vaccinated by deep IM route with a 2 ml dose of the MSD Ref. 1 vaccine or the MSD Ref. 2 vaccine, containing inactivated virus with a titre of 105.5 TCID50 / dose, and revaccinated with another dose of the MSD Ref. same title 20 days later. Local and general reactions are observed.

The serological response in animals is verified by the IPMA test according to the following schedule:

T0: Bloodletting and vaccination

T20 bloodletting-revaccination

42 bloodletting

78 phlebotomy and experimental infection

bloodletting after experimental infection

25 bloodletting after experimental infection

50: bloodletting after experimental infection.

The infection, experimental It is carried out in the security stables of the research center. All animals are infected in the amount of 5 ml of virulent virus PRRS-CY-218-JPD-P5-6-91 with a titer of 10 <5.8> TCID50 / ml via the IN route. The serological response is noted as well as the number of pigs born live and stillborn from each sow. Pre- and post-colostrum bloodletting is done in piglets, and samples of. lung and brain are taken from stillborn pigs and pigs killed on different days for virus isolation and histological cuts.

The results obtained are reported in the following tables from 8 to 10.

TABLE 8

Serological results

TABLE 9

Results of calving

*: not pregnant

these sows died due to excessively high ambient temperatures. Caesarean section is performed at 112 days of gestation.

(A) date of infection (days of gestation) (B): calving (days of gestation)

(C) total number of piglets (caesarean section) (D): total number of piglets (born)

(E): pigs-born-with-normal-health

(F): piglets born weak

(G): live born piglets with crooked legs

(H): stillborn pigs (dead)

(I): stillborn pigs (mummified)

(J): piglets that died in the first week

TABLE 10

Piglet serology A. Vaccinated sows

3- Control sows (unvaccinated)

Legend:

*): These sows died due to an excessively high ambient temperature. The caesarean section was performed at 112 days of gestation.

1: number attributed to each animal

2: Piglets (number attributed to each pig corresponds to the order of birth)

3: pre-colostrum serology

4: post-colostrum serology

5: virus isolation

NT: -not-tested

-: negative

: positive

H: hours

NP: not pregnant

It is evident from the results obtained that there is a positive seroconversion against the PRRS causative virus. Furthermore, there is a satisfactory behavior against experimental infection, in comparison with control animals in which death is produced in the majority of fetuses.

Consequently, it can be said that this vaccination is an effective measure for the prevention of PRRS

Example 8. Vaccine efficacy against experimental infections with another PRRS causative agent (cross-protection)

This experiment was designed to verify the efficacy of the vaccine identified as MSD Ref.

1 in an experimental infection test using 2 pathogenic strains of the PRRS causative virus.

The pathogenic strains of PRRS used are:

i) Spanish strain, PRRS-CY-218-JPD-P5-6-91; And

iiceppoFrancesceSDRII-8B-provisto-da-D-E-Albina of the "Laboratoire Central de Recherches Avicole. et Porcine", Ploufragan, France

The vaccine used is the vaccine called MSD Ref. 1 of which the formula is given in example 5.

30 are used

causative sera of PRRS, to the reproductive cycle not included between 10 days before and 10 days after mating, and 10 days before and 10 days after delivery.

Four groups of animals are formed:

A: 10 sows vaccinated and revaccinated via the IM route and infected via the IN route with Spanish strain PRRS-CY-218-JPD-P5-6-91 of the virus.

B: 5 sows not subject to any vaccination and infected via the IN route with the Spanish strain PRRS-CY-218-JPD-P5-6-91.

C; 10 sows vaccinated and revaccinated via the IM route and infected via the IN route with the French SDRP II 8B strain.

0: 5 sows not subject to any vaccination and infected via the IN route with the French SDRP II 8B strain.

Experiment carried out

Twenty sows of groups A and C mentioned above are vaccinated with a dose of 2 billion of the vaccine called MSD Ref. 1 via the deep IM route, and revaccinated 21 days later with the same dose of vaccine.

Subsequently, between days 70 and 80 of gestation, all the sows are experimentally infected with:

Groups A and B. 5 ml of PRRS-CY-218-JPD-P5-6-91 virus with a titer of 105.8 TCID50 / ml via the IN route; And

Groups C and D. 5 ml of SDRP II 88 virus with a titer of 105.8 TCID50 / ml via the IN route.

Parameters evaluated

1. Serological response by IPMA assay to:

TQ: bloodletting and vaccination

T21: bloodletting and revaccination

T41: bloodletting

T1: bloodletting and infection

Tj 7: bloodletting 7 days after infection.

2. Evaluation of rectal temperature, local reaction and general reaction during the 4 days following vaccination and revaccination, or until the temperature or clinical signs, if any, disappear.

3. Evaluation of the rectal temperature, intake

following the experimental infection.

4. Detection of antibody in the serum, and isolation of the virus in serum and monocytes extracted from whole blood.

Reproductive parameters at the time of parturition, such as the number of pigs born alive, / number of stillborn or mummified pigs, and number of live but weak piglets that die within the first week.

6. Determination of the amount of virus present - in the serum by titration on macrophages based on CPE.

7. Determination of the virus in the pleural fluid and in the lungs of pigs.

The reproductive parameter results are shown in Tables 11-12 (immunity test with PRRS-CY-218-JPD-P5-6-91) and 13-14 (immunity test with SDRP II 8B).

TABLE 11 (vaccinated sows)

Immunity test with PRRS-CY-218-JPD-P5-6-91

^

Total TL

% of live pigs - 68% (75 born / 51 survive) A protection of 68% is observed when comparing pigs born alive with those that survive 7 days of life. The fact that experimental infection is much more potent than natural infection in the field, in addition to the previous results, makes it possible to predict that the prospects for protection are even better.

TABLE 12 (unvaccinated sows)

Immunity test with PRRS-CY-218-JPD-P5-6-91

TL = total

% of live pigs: 9.5% (42 births / 4 survive). The Spanish strain used for the infection has an extremely high pathogenic efficacy (90.5%) since only 4 pigs survive in the first week out of 42 pigs born.

TL = Total

% of live pigs: 82% (105 births / 86 survive) A protection of approximately 1’82% is observed.

TL: total

% of piglets alive: 75.7% (66 births / 50 survive) A mortality of approximately 24.3% is observed. The pathogenicity of the French strain is very weak (24.3%) compared with the results obtained from the test with the Spanish strain (mortality 90.5%). The results used for recovery

comparing vaccinated and non-vaccinated sows. are very significant in the case of the French strain: However and-in any case, the pathogenicity in vaccinated animals is reduced to 17.8%.

Key to tables 11-14 a) a different disease (not PRRS) (piglets are not included)

b) sick, they die before infection

c) sick, they die for another cause

(l) reference sow

(2) total number of piglets

C3) number of piglets born healthy

(4) number of piglets born weak

(5) number of live born pigs with crooked legs (6) number of stillborn pigs

(7): number of live pigs after the first week

Claims (1)

CLAIMS 1. - Vaccine capable of preventing porcine reproductive and respiratory syndrome (PRRS), characterized by the fact that it includes a quantity Spanish, inactivated, as well as a suitable adjuvant and, optionally, a preservative. 2. - Vaccine according to claim 1, characterized in that said PRRS virus, accession V9307G108. 3. Vaccine according to Claim 1, characterized in that it contains an amount of inactivated virus of at least 10 <5.5> TCID50 / dose. 4. Vaccine according to Claim 1, characterized in that said virus has been cultured on a culture of alveolar macrophages of pig lung. 5. Vaccine according to Claim 1, characterized in that said virus has been cultured on an alveolar macrophage of pig lung and co-cultured on an ST cell (ATCC CRL 1746 ST). or. - Vaccine according to claim 1, characterized in that said virus has been cultured on ST cell culture (ATCC CRL 1746 ST). 7. Vaccine according to claim 1, characterized in that said virus has been cultured on hybrid cells of alveolar macrophage of pig lung fused with ST cells by means of hybridization. 8. .- Vaccine according to claim 1 characterized in that said virus has been cultured on hybrid cells of alveolar macrophage of pig lung - fused-with-the-cell line, LL-c14 (ECACC n. 91012317 ') ; or with the JAG-1 cell-line. 9. - Vaccine according to Claim 1, characterized in that said virus has been cultured on ST cells or on another line of porcine cells in which genes coding for the receptors of the membrane of the alveolar macrophage of lung of pig for PRRS virus. 10. Vaccine according to Claim 1, characterized in that said adjuvant is an oily adjuvant. 11. - Vaccine according to Claim 10, characterized in that said oily adjuvant is constituted by a mixture of Marcol 52, Simulsol 5100 and Montanide 888. 12. - Vaccine according to claim 1, characterized in that it is an emulsion of (i) an aqueous antigenic phase containing inactivated virus and (ii) an oily phase containing the adjuvant. characterized by the fact that de tta emu lsione, is. made up of 53% by volume; of an aqueous phase containing the inactivated virus and 47% by <'> weight of an oily phase containing the adjuvant. 14. '- Vaccine according to any of the preceding claims, - characterized in that it is capable of inducing cellular immunity in the vaccinated animal. 15. - Vaccine according to claim 14, characterized in that it also contains cell response enhancing substances (CRP) which enhance the immune effect of the cells, such as IL-1, IL-2, IL-4, IL-5 , IL-6, IL-12, g-IFN, cell necrosis factor and similar substances. 16. - Vaccine according to Claim 1, characterized in that the adjuvant is an aqueous adjuvant. 17. - Vaccine according to claim 16, characterized in that it also contains cell response enhancing substances (CRP) which induce an immune effect in the cell, such as IL-1, IL-2, IL-4, IL-5 , IL-6, IL-12, g-IFN, cell necrosis factor and similar substances. 18. Vaccine according to claim 1, characterized - in that-the-adjuvant -is-an adjuvant which can modulate and immunostimulate via response. of cells, such as MDP, ISCOM or liposomes. -19. - Vaco ino according to any of the preceding claims, characterized by the fact that it is able to avoid the return to service in the vaccinated animal 20. - Vaccine capable of preventing porcine reproductive and respiratory syndrome (PRRS) characterized by the fact that it is an emulsion comprising: a) 53% of an aqueous phase containing the PRRS viral antigen in DMEM culture medium, Spanish strain, inactivated, at a minimum concentration of 105'5 TCIDS0 / dose, and b) 47% of an oily phase containing a mixture of Marcol 52, Simulsol 5100 and Montanide 888. 21. - Vaccine according to claim 20, characterized in that said viral antigen is the PRRS virus, Spanish strain, called PRRS-CY-2.18-JPD-P5-6-91, registered with ECACC, with accession number V93070108 . 22. - Vaccine according to one of claims 20 or 21, characterized by the fact that it is able to induce the immunity of the cell in the animal 23. - Vaccine according to. claim 22, characterized in that it also contains cell response enhancing substances (CRP) which enhance the immune effect of the cell, such as IL-1, IL-2, IL-4, IL-.5, IL-6, IL -12, g-IFN, cell necrosis factor and similar substances " 24. - Vaccine according to one of claims 20 to 23, characterized in that it is able to avoid the vaccinated animal returning to service. 25. - Bi- or multivalent vaccine capable of preventing; porcine reproductive and respiratory syndrome and another or other porcine infection, characterized by the fact that it contains a suitable amount of viral antigen or PRRS virus, Spanish strain, inactivated, plus one or more porcine pathogens. 26. - Vaccine according to claim 25, characterized in that said viral antigen is the PRRS virus, Spanish strain, called PRRS-CY-218-JPD-P5-6-91, registered with ECfìCC, with accession number V93070108 . 27. Vaccine according to the claim characterized in that it comprises at least one porcine pathogen selected from the group consisting of Actinobacillus pleuropneumoniae. Haemophilus parasuis. Porcine’parvovirusvt-Laptospirap Escherichia coli; Erysipelothrix rhuziopathiae. Pasteurella multocida. Bordetella bronchisèptica. Procine respiratory: coronavirus. Rotavirus. or against the causative pathogens of Aujeszky's disease, swine flu or transmissible gastroenteritis. 28. - Process for the preparation of a vaccine capable of preventing porcine reproductive and respiratory syndrome (PRRS), containing a suitable amount of inactivated Spanish virus PRRS, plus a suitable adjuvant and, optionally, a preservative, characterized by fact which includes: 1) culture of the PRRS causative virus, Spanish strain, in a suitable cell system, 2) collection of the virus from said cell system when the minimum titer of 10 <5.5> TCID50 / ml has been reached, 3) the inactivation of the virus by physical or chemical methods, e A) mixing of the inactivated virus with adjuvant and condom. 29. - Process according to Claim 28, characterized in that said PRRS virus, Spanish strain, is the virus called PRRS-CY-218-JPD-P5691 deposited-at-the-ECAC-with-the-number-of accession V93070108. 30. - Process according to claim 28, characterized in that said virus has been grown on: i) culture of alveolar macrophages from pig lung, or on ii) a co-culture of alveolar macrophages from pig lung and ST cells (ATCC CRL 1746 ST), or on iii) culture of ST cells (ATCC CRL 1746 ST), or on iv) hybrid cells of pig lung alveolar macrophages fused with ST cells, or on v) Hybrid cells of alveolar macrophage from pig lung fused with L-14 cell line (ECACC No. 91012317) or with Jag-1 cell line, or on vi) ST cells or any other porcine cell line in which genes encoding the pig lung alveolar macrophage membrane receptor for PRR5 -31 virus have been introduced. - DNA sequence of the causative virus of PRR5, Spanish strain, essentially comprising the DNA sequences illustrated in Figures 1 to 5. 32. - PRRS causative virus, Spanish strain, the characteristics of which essentially correspond to those of the virus called PRRS-CY-218-JPD-P5-6-91, deposited at 1st ECACC, with accession number V93070108. 33. Virus according to Claim 32, inactivated, capable of being used in the formulation of vaccines capable of preventing porcine reproductive and respiratory syndrome. 34. Virus according to: claim 32, inactivated, capable of being used in the formulation of bi- or multivalent vaccines capable of preventing porcine reproductive and respiratory syndrome and other eorcinic infections.