NL194940C - Virus strain and vaccine to prevent pig reproduction and respiration syndrome. - Google Patents

Description

1 194940

Vaccine for the prevention of pig reproduction and respiration syndrome

FIELD OF THE INVENTION

The present invention relates to a vaccine that is capable of preventing pig reproduction and respiratory syndrome. In particular, the invention relates to an inactivated vaccine containing the causative virus of the disease in inactivated form

History of the invention

The disease known as Pig Reproduction and Respiratory Syndrome (PRRS) has an effect on 10 pregnant sows in which anorexia, abortions, birth of dead piglets, mummified fetuses, weak piglets that die several hours or days after birth, respiratory problems after delivery and reproductive problems can be caused (Loula, T., "Clinical Presentation of Mystery Pig Disease in the breeding and suckling piglets", Proceedings of the Mystery Swine Disease Committee Meeting, October 6, 1990, Denver, Colorado, Livestock Conservation Institute, Madison , Wl, USA) A number of cases have been described in which infected sows showed blue dots on ears so that the disease is also known as blue abortion or blue-headed pig disease (Veterinary Record, Vol. 130, no. 3.18 January 1992). Other names given to the disease are "Mystery Pig Disease" (MPD), Mystery Swine Disease "(MSD)," Mysterious Reproductive Synd Rome ”(MRS),“ Swine Infertility and Respiratory Syndrome ”(SIARS) or“ Porcine Epidemic Abortion and Respiratory Syndrome ”(PEARS).

20 The first epidemic outbreaks of this disease appeared in the United States and Canada in 1987. In Europe, the first outbreak was detected in Germany in 1990, from which it spread to the Netherlands and Belgium at the end of 1990 and early 1991 . In Spain, the first cases of the disease were detected in mid-January 1991 when major respiratory changes were observed in a load of 300 piglets imported from Germany (Plana et al., Med.

25 Fat., Vol. 8, No. 11, 1991). Shortly thereafter, a disease was detected in two reproductive herds located at a distance of 500 m from the herd where the original problem had occurred, which disease was characterized by an abnormally high abortion rate during the final phase of pregnancy and 70% piglet mortality. After analysis of the observed clinical symptoms and in view of the fact that i) these herds were subjected to an intensive vaccination program against swine parvovirus, Aujeszky's disease and swine influenza and that ii) laboratory tests excluded the presence of other abortion-causing diseases rest on clinical presence of PRRS. In samples from these farms, the causative agent of the disease was isolated as described in further detail below.

35 A number of viruses are correlated with this infectious process including encephalomyocarditis virus, swine influenza, classical swine fever, African swine fever, mucosal disease, Aujeszky's disease, brucellosis, leptospirosis, Q fever, parvovirosis and chlamydial disease, although some authors have related this have brought with mycotoxins (Loula, T., "Clinical Presentation of Mystery Pig Disease in the breeding and suckling piglets", Proceedings of the Mystery Swine Disease Committee Meeting, supra, 40 Mixture, WL and Lager, KM, "Mystery Pig Disease: Evidence and considerations for its aetiology ", in Proceedings of the Mystery Swine Disease Committee Meeting, supra; Dea et al.," Vims isolation from farms in Quebec experiencing severe outbreaks of respiratory and reproductive problems, "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 45 Meeting, supra; Loula, T. Agri-Practice, 12 (1). 23-33, 1991; Woolen, N. et al., J. Am. Fat. Assoc. 197: 600-601, 1990).

Fat. Microbiol. (1992) 33: 203 describes the isolation in Spain of a strain that causes PRRS. The Spanish isolate shows immunological similarity with the Lelystad virus (LV). No further characterization of the virus is given.

50 Animal Pharm. (1991) 238: 20 also describes the isolation in Spain of a virus responsible for PRRS, without giving details of the isolation method or isolate.

The PCT patent application published under publication number WO92 / 21375 in the name of Stichting Centraal Diergeneeskundig Instituut (CDI) describes the isolation of a virus called "Lelystad Agent" (LA) or "Lelystad Virus" (LV) that they if the causative virus of the disease identified as 55 MSD was identified The isolated LV causes loss of appetite during intranasal inoculation in pregnant sows and even a refusal to ingest for 4 to 10-12 days after inoculation, reproductive defects and blue coloration of the ears of some infected sows 194940 2 9-10 days after inoculation, however, it has also been observed that 2 out of 8 experimentally infected sows do not reproduce the disease (see Table 6 of the PCT patent application) given that in one of the sows the number of piglets born alive and surviving the first week was large (6 out of 9, sow 1305) and another sow two he still gave birth to piglets while the other 5 9 survived the first week (sow 1065). The virus isolated by CDI belongs to the genus Arteriviridae with a genome of at least one polyadenylated RNA molecule with a length of 14.5 to 15.5 kb (as determined in a neutral agarose gel), which replicates itself by of a group of subgenomic RNAs at the 3 'end. The PCT patent application cited above shows the nucleotide sequence of the LV genome with the 8 possible open reading frames (opwn reading frames = ORPs), the amino acid sequence derived from the identified ORFs and the possible N-glycosylation sites.

Other PRRS-causing viruses were then isolated in German, French and Spanish farms. The virus (TV) [(Virology, 193, 329-339 (1993)] isolated in Tubingen, Germany shows 99.3% homology with ORFs at nucleotide level of LV (where there are 24 base pairs (bp) from a total of 3316 base pairs in that region.) The deduced TV amino acid sequence shows 99.2% homology to 15 LV (in which only 10 amino acids in the deduced amino acid sequences encoded by ORFs 2 to 5 differ, while there are no differences in the sequences derived from ORFs 6 and 7).

On the other hand, the homology found between LV and TV compared to the virus (Spanish strain or SV) isolated in our laboratory is smaller. Up to now, only the nucleotide and amino acid sequences corresponding to ORFs 3 to 7 have been compared, resulting in the following results: 1. There is 95.5% homology of SV compared to LV or TV at the nucleotide level (with 144 differences on a total of 2599bp); and 2. there is 94.9% homology of SV to LV or TV at the amino acid level (from a total of 955 amino acids a total of 47 amino acids are found that differ).

These amino acid variations can be linked to the higher pathogenicity of a strain compared to another strain, making the virus (Spanish strain) isolated in our laboratories more pathogenic than other known PRRS viruses. For example, of the virus isolated in France (French strain), as shown in Example 8 of the present description, the percentage of piglets born from a sow infected with the French strain is 75% while the percentage is 9 Is 5% when sows with the Spanish strain (Tables 12 and 14) are infected. With infection with LV, the percentage is 61% when the sows are infected with it (Table 6 of PCT patent application WO 92/21375) since 58 piglets from a total of 95 piglets were born alive.

The disease causes serious losses in the pig industry because, in the event of an acute outbreak, it can cause a piglet mortality of 70% in a litter. A means to solve the problem created by this would be to carry out a suitable vaccination program that would allow the prevention of the disease. This would require vaccines that can effectively prevent PRRS.

WO 93/14196 describes a process for the industrial production of a myxovirus type virus, called virus A, and of a gown virus, called virus B. Both viruses cause PRRS.

The patent application published under WO 93/06211 in the name of Collins, J.E., and Benfield, D.A., relates to an anti-MSD vaccine containing an infectious agent isolated from lungs of pigs infected with MSD. The said PCT patent application, however, does not describe a characterization or identification of the infectious agent, nor a deposit thereof at any authorized depository institute. As a result, the realization of the knowledge that can be derived from the PCT patent application is very difficult because the product described in the PCT patent application cannot be verified and the results obtained cannot be compared with the results of the applicants for the application. PCT patent application. Neither in the said patent application are the antigen nor the adjuvant used in the formulation of the vaccine clearly described, while this will play a very important role as will be explained below. Nor are examples described describing the potency and efficacy of the vaccines.

The PCT patent application published under WO 93/07898 relates inter alia to the identification of the causative virus of PRRS and to vaccines derived therefrom. The isolated virus has characteristics similar to LV but different from the virus isolated in our laboratory, which is unable to grow at detectable levels on ST (boar testis) cells. Apparently the 55 vaccine has been effective, but protection has only been achieved in about 52% of the vaccinated animals, which can be considered as a relatively low level of protection, particularly in view of the high virus titer used in the vaccine dose. In addition, the PCT-3 194940 patent application does not provide information about the organization of the virus genome or proteins being encoded, so that comparison between this virus and the virus obtained in our laboratories is not, or only with too much research, can be performed.

Therefore, there remains a need for vaccines that are able to prevent PRRS. In order to solve this problem, the present invention provides a vaccine that is capable of effectively protecting sows against the infectious disease. The antigenic phase of this vaccine includes an inactivated Spanish isolate. The invention also provides combinations of the isolated PRRS viral antigen (Spanish strain) together with different porcine pathogens for the purpose of providing bi- or multivalent vaccines.

10

BRIEF DESCRIPTION OF THE FIGURES

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

Figures 6 to 10 show the homology and the existing differences between LV and the virus isolated in our laboratories at the level of amino acid sequences derived from ORFs 3 to 7. The amino acids are expressed in accordance with the 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 (Spanish strain) isolated at our laboratories. The homologous amino acids are indicated by two vertical lines while substitution of amino acids by other amino acids is indicated by two vertical dots in conservative substitutions, that is, when substitution of an amino acid with another amino acid that is functionally equivalent occurs. The absence of vertical lines and dots between two amino acids indicates non-conservative substitutions.

Detailed description of the invention 1. Samples 1.1. Animals selected for the isolation of the virus

Mummified fetuses, stillborn piglets and live but weak piglets from 1 to 10 days of age, and offspring of field sows with PRRS clinical problems were selected.

1.2. Preparation of the samples

Lung, spleen, liver, kidney, brain and heart samples from the piglets were obtained by necropsy. In one particular case, samples were prepared from the lung of a stillborn piglet thrown by a sow suspected of being infected with PRRS. A Salisbury (United Kingdom) with access number V93070108 was deposited with that lung.

3.2. Viral growth in other cell systems

Infections with the isolated virus (Spanish strain) have been carried out in co-cultures of swine testis continuous cell line ATCC CRL 1745 ST alveolar macrofa gene from a pig lung and ST cell as a first step to adjust the virus for ST cells. After a number of passes in series (5-6) at the ST cell and macrophage co-cultures and at cultures of ST separately, the virus infection titers were in the order of 10 6 TCID 50 / ml when macrophage ST cell co-cultures were infected and of the order of 101 · 2 TCIDgo / ml for the virus obtained separately in ST cells [TCIDgo, tissue culture infection dose 50%].

In addition, alveolar macrophages from a porcine lung can also be immortalized by fusion thereof with ST cells by hybridization. Alternatively, pig lung alveolar macrophages 45 can be immortalized by fusion thereof with peripheral blood L-14 cell line B cells (ECACC No. 91012317), or with Jag-1 cell line (pig trophoblast cell line) provided by Dr. Jag Ramsoondar Ramsoondar et al., Biol. Reprod. 49; 681-694.1993.

The merger rules include usual techniques in this area. Alternatively, viruses can be grown on ST cells or on other porcine cell lines, incorporating the genes encoding membrane receptors of alveolar macrophages of the porcine lung for the PRRS virus.

The viruses produced with these cell systems can be used in the formulation of both live and inactivated vaccines.

Virus identification and characterization 55 4.1. Appointment and deposit of the virus 2

The virus isolated from the lung of a stillborn piglet designated as PRRS-CY-218-JPD-P5-6-91 was submitted to the European Collection of Animal Cell Cultures (ECACC), Salisbury (United Kingdom) on 194940 4 June 29 1993 deposited under access number V93070108. In the present description, the virus is occasionally described without Spanish designation as Spanish virus (SV) or 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 low CPE on a continuous ST cell line (ATCC CRL 1746 ST) (fetal boar test) with an average titre of 104.5 TCID 50 / ml and on alveolar macrophages of a pig lung with average titers of 105.5 TCID 50 / ml; b) in the event of infection of alveolar macrophages from a pig lung and STC in co-culture therewith, average titers of 106 · 3 TCIDgo / ml are obtained [which titers represent a logarithm unit (1 log10) 10 higher than when only alveolar macrophages of a pig lung were infected separately]; c) cytoplasm replication; d) production of cytoplasmic vaccination; e) lipid shell; f) size of 40-50 nm; G) no haem adsorption or haemagglutination was observed with chicken, marmot, pig or red blood cells of human group O; h) loss of infectivity at acidic pH (pH £ 5); i) production of microscopic (interstitial pneumonia) and macroscopic lesions in piglets with an age of 2 months; (J) production of negative reproduction effects in pregnant sows with stillbirth, mummified fetuses and living but weak piglets; k) cross-reaction with a Lelystad reference serum (IPMA = Immunoperoxidase monolayer test); l) cross-reaction with sera from animals with clinical field infections (IPMA), m) serum from sows infected with this virus shows cross-reaction with LV; N) polydenylated RNA genome about 15,000 bp in length (neutral agarose gel); o) replication through a subgenomic RNA group present at the 3 'end; p) nucleotide sequence with 8 ORFs; q) 4 large bands were detected in a purified suspension which is subjected to electrophoresis in polyacrylamide gel and after transfer by immunoblot by means of a specific serum prepared in sows of our own laboratory and cross-reacting with the Lelystad PRRS virus. correspond to protein with molecular weights of 15,000, 23,000, 54,000 and 66,000 Daltons that were not detected in negative controls (uninfected macrophages); r) when ORFs 3 to 7 of this virus are compared to those of LV and / or TV, 95.5% homology is observed at the nucleotide level (with 114 bp different from a total of 2599 bp) and 94.9% homology at the amino acid level (51 amino acids differ on a total of 955); and s) the virus belongs to the genus Arterivirus.

4.3. Techniques used for virus identification 4.3.1. Experimental reproduction of the disease in pregnant sows 40 German Landrace x Large White Cross sows were used that originate from farms with systematic serological control against Aujeszky's disease, foot-and-mouth disease, swine parvovirus, classical swine fever, swine influenza (types H1N1 and H3N2) and transmissible gastroenteritis. In addition, an antibody titration test was performed against the PRRS causative virus. Between days 77 and 90 of pregnancy, the animals with an isolate obtained on alveolar macrophages from a pig lung 45 were infected intravenously and intranasally (IN) in one case while in the other case only infection via the intranasal route took place (example 2.1) ). Food intake, rectal temperature and the clinical condition of the animals were observed throughout the experiment. For the purpose of excluding the above viruses, blood samples from all sows were taken before infection. These were found to be seronegative to all viruses. Also, after experimental infection 50, the sows were all still seronegative for the said viruses, as well as seropositive for PRRS (as verified with reference LV). The results obtained are shown in Table 1.

5 194940 TABLE 1 Tests performed

INFE. HAI HAI NPLA ELISA SN SN IPMA

agent (1) (2) (3) (4) (5) (6) (7) 5 -

AD ND ND ND - ND ND ND

FMD ND ND ND ND - ND ND

PP - ND ND ND ND ND ND

CSF ND ND - ND ND ND ND

SF ND - ND ND ND ND ND

TG ND ND ND ND ND - ND

PPRS ND ND ND ND ND ND +

Legend ^ AD = Aujeszky's disease FMD = foot-and-mouth disease PP = swine parvovirosis CSF = classical swine fever SF = swine infuenza (H1N1, H2N3) 23 TG = transmissible gastroenteritis PRRS = swine reproduction and response syndrome HAI = haemagglutination linked to peroxidase NPLA = neutralizing peroxidase-linked test ELISA = enzyme-linked immunosorption test SN = seroneutralization IPMA = immunoperoxidase monolayer test (1) = Vannier et al., Ree. Méd. Fat. 155 (2), 151-158 (1979) (2) = Charley B., Thesis Doctoral, Alfort (1976) (3) = Trepsta et al., Vet. Microb. 9, 113-120 (1984) 30 (4) = Elliot M., J. Rech. Porc., 20, 141-146.

(5) = Lucam F., "Diagnostic sero-immunologique des viroses humains animals", F. Bricout; L. Joubert; J.M. Huraux (1977) (6) = Jimenez et al., J. Virol., 60, 131-139 (1986) (7) = Wensvoort et al., Vet. QUARTERLY, Vol. 13, no. 3 (July 1991) 35 (-) = negative (+) - positive ND = not performed.

40 4.3.2. Experimental reproduction of the disease in piglets

The purpose of this experiment was to confirm whether the causative virus of reproductive changes in sows was capable of causing 2 month old piglets to cause respiratory symptoms and macroscopic and microscopic lesions at the lung level. To this end, a number of piglets were infected with the virus via the IN route (Spanish strain), which piglets were subsequently killed several days after infection (example 2.2.) 45.

The most relevant data that result from this show that this virus caused multiple consolidation foci at macroscopic level and interstitial pneumonia at the microscopic level (Table 4). With regard to health, no relevant clinical signs could be observed.

4.3.3. Chloroform sensitivity test 50 This test was performed to determine if the isolated virus had a lipid mantle. To this end, the method of Feldman, H. and Wang, S., was used as described in "A manual of basic virological techniques", Prentice-Hall Inc., New Jersey, 146-148 (1978). The results obtained show that the untreated virus has a titer of 105.6 TOD ^ ml while after treatment with chloroform the titer is lower than 101 · 3 TOD ^ ml on the basis of which it can be said that the isolated virus has a lipid-55 has cloak.

4.3.4. Sequencing of the viral genome Five phases were performed: 194940 6 i) purification of the virus

ii) purification of the viral RNA

iii) cDNA synthesis iv) cloning and characterization of the cDNA clones

V) sequencing and comparing the sequences with those of LV

i) purification of the virus

Virus replicated on alveolar macrophages from a pig lung was purified and concentrated by filtration using MILLIPORE filters. The virus was then subjected to centrifugation in 10 to 50% metrizamide gradient (SIGMA). After completion of centrifugation, a band 10 was obtained which was concentrated by centrifugation. With the purified virus, a polyacrylamide gel electrophoresis was performed and an immunoblot was developed with a specific serum that exhibited proteins whose apparent molecular weights were 15.23, 54 and 66 K Daltons.

ii) Purification of the viral RNA

A technique for selection and purification of the RNA was applied based on the fact that the RNA contains a poly (A) sequence at the 3 'end. To this end, a commercially available kit (Pharmacia) was used that allowed exclusive binding of the RNA poly (A) chain to a cellulose oligo (dT) matrix as well as subsequent elution.

iii) cDNA synthesis A commercially available kit was used (Boehringer Mannheim) following the manufacturer's instructions. Briefly, the viral genomic RNA was incubated in the presence of an oligo (dT), dATP, dCTP, dGTP, dTTP and reverse transcriptase.

iv) Cloning and characterization of the cDNA clones The cDNA was cloned into a pUC18-derived vector and a series of clones was obtained that contained the complete nucleotide sequence corresponding to ORFs 3 to 7.

v) Sequencing and comparing the sequences with those of LV 25 v.a.) Sequencing

The area corresponding to ORFs 3-7 of the virus isolated at our laboratories is fully sequenced. Figures 1 to 5 show the sequences of obtained cDNA as well as the sequences derived from amino acids encoded by each ORF. The total length of the region whose sequence has been determined is approximately 2599 nucleotides (nt). As can be seen, ORF 3 has a length of approximately 789 nt and encodes a protein of 266 amino acid groups. ORF 4 is approximately 552 nt in length and encodes a protein of 183 amino acid groups. The onset of this ORF 4 is in the ATG codon which is approximately 540 bp from the initial ORF-3 ATG codon. ORF 3 and ORF 4 share a sequence of approximately 246 nt. ORF 5 is approximately 606 nt in length and encodes a protein of 200 amino acid groups. The initial codon of this ORF 5 practically overlaps the end codon of ORF 4 (they share the TG nucleotides, the ATG codon at the start of ORF 5 and the ORF 4 tGA end codon). The initial ATG codon of ORF 5 is 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 groups. This initial ATG codon of ORF 6 is located 8 nt downstream of the start of the termination TAG codon of ORF 5 (at about 1682 nt of the initial ATG codon of ORF 3). ORF 7 40 has a length of approximately 387 nt and codes for a protein of 129 amino acid groups. This initial ATG codon of ORF 7 is located 5 nt downstream of the start of the termination TAA codon of ORF 6 (at about 2193 nt of the initial ATG codon of ORF 3). The proteins encoded by ORFs 3 to 6 are membrane proteins, while the protein encoded by ORFs 7 is a nucleocapsid protein.

45 ex.) Comparison with LV

When comparing the cDNA sequences of LV ORFs 3-7 with those of the virus isolated in our laboratories, it can be observed that: i) at nucleotide level 114 the 2599 nucleotides differ, which corresponds to about 95, 5% homology, 50 ii) at amino acid level 47 amino acids of the 955 amino acids that are compared differ, representing approximately 94.9% homology, iii) of the 47 amino acids that differ 35 are considered non-conservative substitutions of which 12 are in the product of the ORF 3 gene (Figure 6); 9 are in the product of the ORF 4 gene (Figure 7); 10 are in the product of the ORF 5 gene (Figure 8), although it should be noted that the product of the LV-ORF 5 gene contains 1 amino acid more than the product of the Spanish virus ORF 5, specifically missing amino acid 35 (Asn) of the LV-ORF 5 product in the product expressed by the Spanish virus; 4 are in the product of the ORF 6 gene (Figure 9); 7 194940 while the product of the ORF 7 gene contains no non-conservative substitution (Figure 10), iv) partial homology of each product 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, 96.6% for the product of ORF 6 and 99.2% for the product of ORF 7.

As indicated above, the changes in the amino acids may be related to the higher pathogenicity of one strain compared to the other, since the virus that is more pathogenic in our laboratories (Spanish strain) than other known PRRS viruses, such as the virus isolated in France (Example 8) and LV (Table 6 of PCT patent application WO 92/21375).

5. Vaccines

The invention provides a vaccine that can prevent pig reproduction and respiration syndrome (PRRS). The vaccine has been shown to be effective in preventing reproductive changes in sows such as stillborn piglets, mummified piglets or live but weak piglets, "return to service" (recurrence of oestrus) and similar problems caused by the PRRS causative virus. Similarly, it has been confirmed that the vaccine induces cellular immunity in vaccinated animals.

The vaccine contains a suitable amount of inactivated Spanish strain PRRS viral antigen, as well as an adjuvant and a preservative. Tests performed with these vaccines have the efficacy of the vaccine, as shown by Examples 7 and 8. In addition, it has been shown that the vaccine is effective in preventing "return to service" (recurrence of oestrus) occurring in infected sows. Indeed, the sows vaccinated with the vaccines resulting from the present invention and with the causative PRRS virus were infected pairs and became pregnant with the first postpartum ovulation and suckling the piglets.

5.1. Components 25 5.1.a) Antigenic phase

As an active ingredient, the vaccine contains inactivated Spanish strain PRRS viral antigen at a concentration higher or equal to 105 · 5 TCIDgo per vaccine dose. The inactivation can be by chemical means comprising treatment with β-propiolactone or other conventional inactivating agents such as ethylene imine or formaldehyde, or by physical means.

5.1 .b) Adjuvants

Although it is possible to use aluminum hydroxide-type adjuvants, Quil A or mixtures thereof as well as oily adjuvants for formulating the vaccine, confirmation has been possible that the best results are obtained when using an oily adjuvant (Example 4).

In particular, it has been confirmed that an oily adjuvant formed by a mixture of Marcol 52, Simulson 5100 and Montanide 888 provides very good results. Marcol 52 is a low density inorganic oil produced by ESSO ESPANOLA, S.A. is being produced; Simulsol 5100 is a polyethoxyoleate ether marketed by SEPIC and Montanide 888 is a high purity anhydrous mannitol ether octadecanoate that is marketed by SEPIC.

Confirmation that the adjuvant plays an essential role in the efficacy of the vaccine has been possible. Thus, a challenge test (Example 6) using sows vaccinated with two different vaccines, one sow with the oily adjuvant indicated above (ref. 1) and the other sow using the adjuvant Munokynin® (ref. 2) ) has been vaccinated. Although both vaccines produce seroconversion, an experimental infection test showed that the sows treated with vaccine ref. 1 were vaccinated protected against 45 experimental infection with PRRS virus while the other sows vaccinated with vaccine ref. 2 were not protected, despite the fact that they had antibodies against the virus at the time of the challenge. This indicates that a suitable adjuvant can play a very important role in connection with the modulation and stimulation of immune response especially at the level of cellular immunity. In addition, this aspect has been confirmed through the challenge test conducted using 50 the Spanish strain of the PRRS virus, since the sows treated with ref. 1 vaccine had been vaccinated and re-vaccinated showed no serological response at the time of infection but were, notwithstanding, protected (Example 7, Table 8).

However, it is also possible that ref. 2 vaccine (m-et Munokynin®) can cause cellular immunity. To this end, it will be necessary to add agents that enhance cell response (CRP), that is, to add 55 agents that enhance helper T cell subpopulations (Th1 and Th2) such as IL-1 (interieukin-1), IL -2, IL-4, IL-5, IL-12, g-IFN (gamma interferon), cellular necrosis factor and similar substances. It will be clear that it is possible to add these CRP substances to vaccines with oily adjuvant in which case their cell immune effect will be enhanced. Other types of adjuvants that modulate and immunostimulate cell responses, such as MDP (muramyl dipeptide), ISCOM (Immuno Stimulant Complex) or liposomes can also be used.

5.1. c) Preservatives Any of the preservatives customarily used in the formulation of vaccines can be used. One of these is thimerosal [sodium salt of (2-carboxyphenylthio) ethyl-mercury] (ALDRICH).

5.2. Process for the preparation of the vaccine

The vaccines resulting from this invention can be obtained by mixing an antigenic phase that inactivates the viral antigen and another phase, such as adjuvant which may or may not be oily, depending on the adjuvant chosen. CRP substances can optionally be added to one of the two phases. When the adjuvant is oily, an emulsion can be formed that in a particular and preferred embodiment (when the adjuvant is a mixture of Marcol 52, Simulsol 5100 and Montanide 888) is a double w / o / w (water / oil / water) emulsion .

5.3. Vaccine control tests 15 In addition to the usual tests that the vaccine must pass before its administration in terms of (i) purity (with a view to bacteria, fungi and foreign viruses), (ii) identification, (iii) safety, (iv) strength and (v) physico-chemical controls, a number of field tests on a total of five farms (example 5b) regarding the safety and efficacy of the vaccine were performed. Hereby, 508 sows were vaccinated and re-vaccinated with one of the vaccines of the present invention, while the remaining 472 sows were not vaccinated and kept as control sows, the vaccine being observed to be safe and at the same time effective since on some of the farms the natural PRRS disease in unvaccinated animals was observed after vaccination.

5.4. Posology and instructions for administration of the vaccine It has been confirmed that one dose of 2 ml oily vaccine with a concentration of inactivated viral antigen equal to or higher than 10s, TCIDgo, administered by deep intramuscular route high percentage of vaccinated animals against PRRS. The following vaccination program is recommended: * 1st vaccination: 30 Vaccinate all breeding animals (sows and bears) and vaccinate again after 21 days. Then administer 1 dose during each lactation (sows), and every 6 months (bears).

* Post-vaccinations: - For animals intended for reproduction, the first vaccination should take place at the age of 6 months with re-vaccination after 21 days.

35 - It is advisable to vaccinate during the lactation period, if possible 15 days before coverage.

- Bears: vaccinate twice a year (every 6 months).

On the other hand, if no CRP substance is included in the vaccine, these substances can be injected simultaneously at a different location at the inoculation sites.

6. Multipurpose vaccines In order to achieve an additional object with the present invention, combinations of different porcine pathogens are provided which, in addition to the inactivated viral PRRS antigen (Spanish strain), contain one or more of the pathogens listed below in order to produce bi * or multivalent vaccines possible. In this way, bi- or multivalent vaccines can be prepared that inactivate viral PRRS antigen and one or more of the following pathogens: Actinobacillus pleuropneumoniae, 45 Haemophi lus parasuis, Swine parvovirus, Leptospira, Escherichia coli, Erysipelothrix rhusiopathiaida, Pasteuretellaellais, Pasticaellaiseptisellachis Pig Respiratory Coronavirus, Rotavirus or anything against the pathogens that cause Aujeszky's disease, swine influenza or transmissible gastroenteritis.

50 EXAMPLES

EXAMPLE 1

Isolation of the virus 55 1 .A. Preparation of the samples

From the lung of a stillborn pig, offspring of a sow with the classic PRRS symptoms [in which the sow was free from antibodies against Aujeszky's disease, swine parvovirosis, mouth and mouth disease, classical swine fever, swine influenza (types H1N1 and H3N2) and transmissible gastroenteritis] a 10% suspension was made in DMEM culture medium supplemented with an antibiotic solution (PEG) consisting of 1000 lU / ml penicillin, 1 mg / ml streptomycin and 0.5 mg / ml gentamicin, in a lung: DMEM solution of 1:10 (w / v). The produced suspension was homogenized and left at room temperature (20-22 ° C) for 1 hour. The homogenate was frozen and thawed 2 times, centrifuged and the resulting supernatant was stored at -70 ° C for use in the infection of alveolar macrophages from a pig lung. Similarly, samples were prepared from the lung of a pig that was born alive but died within a few hours. In addition, blood was removed and mixed with and without anticoagulant and used to isolate the virus (from blood plasma) and to obtain serum.

1 .B. Obtaining alveolar macrophages from a pig lung

Alveolar macrophages were obtained from the lungs of pigs that were seronegative to Aujeszki's disease, swine parvovirosis, foot-and-mouth disease, classical swine fever, swine influenza (types H1N1 and H3N2) and transmissible gastroenteritis. The age of the pigs that were used was between 7 and 8 weeks. For lung extraction, the animals were subjected to anesthesia with sodium phenol barbital and then killed. Immediately the lungs were removed along with the trachea after ligation under the epiglottis. The removed lung was washed externally with physiological saline and 50 ml PBS supplemented with 2% PEG solution of antibiotics was taken up in successive rinsing steps until a total of 500 ml PBS was taken up. The cells obtained from these washing steps were centrifuged at 300 g for 10 minutes. This washing step by centrifugation was repeated twice. The resulting cells were washed with PBS and a PEG solution of antibiotics and in DMEM medium [DMEM supplemented with non-essential amino acids (GIBCO), 1% sodium pyruvate 1 mM and 1% glutamine 2 mM], 10% fetal calf serum (FCS) and PEG solution of antibiotics resuspended at 1% o. Cell counting was performed in Newbauer chambers and for this purpose a 1/10 dilution of the macrophage suspension was prepared by adding 0.4 ml DMEM and 0.5 ml trypan blue solution to 0.1 ml macrophage suspension. A cell number between 1 and 1, 2 x 10® was obtained.

1.C. Isolation of the virus

A culture bottle with an area of 25 cm 2 containing a culture of alveolar macrophages from a pig lung previously prepared (3 x 10® cells / ml) in DMEM medium and 10% FCS was added with 1 ml of the homogenate of a sample from the lung of a stillborn pig infected (example 1.A.). The homogenate remained in contact with the macrophage culture for 1 hour at 37 ° C, was buffered with CO 2 at pH 7.0-7.4 and incubated for several days at 37 ° C and during this period it was virus infected on the macrophages produced CPE. After 3-4 dpi, a CPE of 70-80% was observed, after which the cultures were frozen at -80 ° C. At the same time, a culture of alveolar macrophages from uninfected porcine lung was prepared and used as a negative control. Subcultures with the isolated virus were performed where it was observed that the CPE was from the second DP1100%. The virus was frozen at -80 ° C for subsequent identification and characterization. After the fourth pass in macrophages, the corresponding titrations in 96-well microtiter plates were performed with an average titer of 105 · 640 TCID50 / ml in accordance with the Reed & Muench method [Am. J. Hyg., 27: 493-497 (1938)]. A sample of isolated virus (Spanish strain) designated PRRS-CY-218-JPD-P5-6-91 that was isolated from the lung of a stillborn piglet is capable of experimental reproduction of the disease and was deposited with the ECACC on June 29, 1993 under access number V93070108. Similarly, the virus was isolated from live and stillborn piglets, the offspring of sows 45 that were experimentally infected.

EXAMPLE 2

Virus identification and characterization 50 Example 2.1. Experimental reproduction of the disease in pregnant sows

Twelve sows, German Landrace x Large White Cross descended from farms with systematic serological control against the viruses of Aujeszki's disease, foot-and-mouth disease, swine parvovirosis, classical swine fever, swine influenza (types H1N1 and H3N2) and transmissible gastroenteritis applied. In addition, the antibody evaluation test against the PRRS 55 causative virus was performed.

The sows were moved to the safe stables of the research center 1 week before infection and placed in separate stables. Between days 77 and 90 of pregnancy, sows with 194940 became 10 numbers 53, 76, 8, 62, 91, 93 and 19, with 5 ml via the intravenous route (IV) and with 5 ml via the intra-nasal route (IN ) infected with PRRS virus, Spanish strain, that from the alveolar microfagen of the pig lung, that as PRRS-CY-218 JPD-P5-6-91 from a fourth pass of macrophages, filtered through a 200 nm filter and with a titer of 105.6 TCID 50 / ml was isolated. The 5 remaining sows (numbers 5, 14, 40, 13, 30 and 85) were infected with 5 ml of the virus via days IN between days 65 and 85 of pregnancy. During the experiment, daily food intake, rectal temperature and the clinical condition of the animals were observed as well as the reproduction changes (premature births, delayed births, live but weak piglets, stillborn piglets, mummified and healthy piglets).

In order to be able to further exclude the above-mentioned pathogens, blood samples were taken from the sows before and after infection with the result that the animals were seronegative to the pathogens before and after infection and were seropositive to PRRS after infection (see Table 1, 4.3.1. ).

The reproduction results appear in Tables 2 and 3. As shown in Table 2, 15 of the 93 piglets born were mummified, 35 were stillborn, 22 were born alive but died on the third day and 21 piglets survived 7 days of life.

Some sows showed a lack of appetite for 2 to 4 days, on days 6 and 8 after infection, while other sows showed a lack of appetite on the 2nd day after infection. Hyperthermia was not observed in any case. With 4 sows the delivery took place 1 to 6 days early (no. 8, 62, 92 and 93), while with the other 3 sows delivery 1 to 2 days was delayed. Of the piglets born alive, 1 or 2 showed edema of the eyes per litter. The weak piglets showed inco-ordination, paresis of the hind legs, upright hair and myoclonia. Necropsy performed on some of the stillborn and weak piglets revealed an abundance of clear fluid in the chest cavity. Healthy piglets born from infected mothers and killed after 8 to 12 days showed gray consolidation foci in the lungs. On microscopic observation, the most significant change was a low multifoci interstitial pneumonia with enlargement of alveolar septi due to the infiltration of mononuclear cells. These lesions appeared with all animals analyzed in the experiment.

As can be seen in Table 3, of the 65 piglets who gave birth, 36 were stillborn, 26 were born alive but weak, with death occurring on the second day of life and 3 surviving the first 30 weeks of life. One sow gave birth 12 days early (No. 4) while the other gave birth 1 or 2 days early. The clinical signs of the piglets born weak are similar to those obtained via IN IV-IV. Interstitial pneumonia was also observed. The infected sows showed no lack of appetite or hyperthermia.

The most relevant difference between the two infection systems is that piglets mummified through IN + IV were observed and that one or two of the piglets born alive in each nest showed edema around the eyes. In view of the results obtained, it can be concluded that the model for experimental infection in sows at approximately 80 days of pregnancy via both infection routes (IN and IV) with the virus isolated from animals naturally infected (Spanish strain) reproduces PRRS disease in pregnant sows and causes a high content of mummified 40 fetuses, stillborn piglets and live but weak piglets in a ratio similar to that occurring in acute natural outbreaks of infection. It is advisable to artificially infect via the IN route because this is the natural infection route in the field and is therefore the most appropriate way to evaluate the efficacy of the vaccine. Through this experiment it has been possible to set up a model for experimental infection in pregnant sows to test the efficacy of the vaccine.

TABLE 2

IN + IV

50 H

A B C D E F G hi h2 I

53 77 115 9 3 2 0 - 4 4/4 8 77 110 12 2 4 1 - 5 5/5 55 62 80 113 16 - 3 6 4 3 3/3

91 0 109 14 - 1 8 1 4 NT

11, 194940! TABLE 2 (continued)

IN + IV

H

5 A BCD E F Gh1h2l

93 88 110 17 4 2 11 - NT

19 88 116 14 4 1 8 - 1 NT

93 15 17 35 5 21 10 -

TABLE 3 IN

H!

i5 A B C D E F G hi h2 I

14 65 111 15 - 12-3

40 82 102 12 - - 12 - - NT

13 80 113 12 - 10 2 - - NT

20 30 80 113 15 - 12 3 - - NT; 85 85 112 11 - 4 7 - - NT! 65 - 26 36 - 3 25 A: Sow B: Time of infection (days of pregnancy) C: Delivery (days of pregnancy) D: Total piglets E: Mummified piglets 30 F: Weak piglets, dead after 48 hours G: Stillborn piglets H: Piglets with apparent good health h1: dead between 2 and 7 days h2: alive after 1 week 33 I: Interstitial pneumonia NT: Not tested.

Example 2.2. Experimental reproduction of the disease in piglets

This experiment was developed to confirm that the isolated virus (Spanish strain) that causes 40 reproductive changes in sows is able to produce clinical signs as well as macroscopic and microscopic lesions in the lungs of 2 month old piglets. For this purpose 10 piglets were infected via IN route with 5 ml Spanish strain virus with a titer of 105 · 6 TCID 1, ml and 6 other piglets were used as controls in which all piglets descended from two litters. The animals were killed on days 3, 7, 8, 9 and 11 after infection. The results obtained are shown in Table 4.

45 TABLE 4

Nr. I / C DPI Antibodies I.P. V.l.

50 2 I 3 - 2+ NT

12 I 3 2+ NT

11 C 3 - NL NT

11 7 1:80 4+ + 14 I 7 1:80 2+ +

55 13 C 7 - NL

10 I 8 1: 160 4+ + 194940 12 TABLE 4 (continued)

Nr. I / C DPI Antibodies I.P. V.l.

5 15 I 8 1: 160 2+ + 8 C 8 NL - 17 C 8 NL - 4 I 9 1: 160 4+ +

5 C 9 NL

10 20 I 11 1: 160 4+ + 18 I 11 1: 320 3+ + 9 I 11 1: 320 3+

6 C 11 - NL

No .: pig number l / C: infected (I) or control (C) DPI: days after infection IP: interstitial pneumonia VI: virus isolation 20 2+: only interstitial pneumonia 3+: intermediate interstitial pneumonia 4+: severe interstitial pneumonia NT: not tested NL: no lesions.

25 _

No clinical respiration signs or hyperthermia were observed during the 11 days of the experiment although weight loss occurred in the infected animals compared to the non-infected animals. Among microscopic observations, the most relevant aspect was the presence of multiple consolidation foci in the lungs, congested lymph nodes in the jaw and some intestinal bleeding. Interstitial pneumonia was observed at the microscopic level.

Virus was isolated from the solutions obtained from washing the lungs of infected animals on a fresh macrophage culture, but no virus was isolated from the control animals in which no seroconversion was observed, nor macroscopic or microscopic lesions at lung levels were observed. When cell counts were performed on the wax product from the lungs of infected animals, 30% dead cells (macrophages) were observed, which can be a factor of importance in secondary infections due to destruction of a key immune defense element (macrophages). The absence of respiration signs may be due to the fact that the experimental infections were carried out in stalls with continuous disinfection treatment, whereby the bacterial concentration was much smaller compared to that which exists in a herd under field conditions.

Example 2.3. Chloroform sensitivity test

The method of Feldman, H. and Wang, S. (4.3.3.) Was used, which provided the knowledge that the isolated virus has a lipid mantle, since a titre drop of 4 log10 at the control cultures compared to the cultures that treated with chloroform.

45 Example 2.4. Sequencing of the viral genome i) Purification of the virus

The virus that replicated on alveolar macrophages from a pig lung was purified by filtration and centrifugation in 10 to 50% metrizamide gradient (SIGMA) resulting in a band that was again centrifuged as mentioned in 4.3.4. An electrophoresis in 50 polyacrylamide gel was performed on the purified virus and an immunoblot was developed with a specific serum that showed proteins with an apparent molecular weight of 15, 23, 54 and 66 K Daltons.

ii) Purification of the viral RNA

The viral RNA was purified using a commercially available kit (PHARMACIA) that allows the binding of the RNA poly (A) tail to a cellulose oligo (dT) matrix and subsequent elution 55.

iii) cDNA synthesis

A commercially available kit was used (Boehringer Mannheim) (see 4.3.4. Iii).

13 194940 iv) Cloning and characterization of the cDNA clones

The cDNA was cloned into a vector derived from pUC18 and a series of clones was obtained containing the complete nucleotide sequence corresponding to ORFs 3-7

v) Sequencing and comparing the sequences with those of LV

The results of the cDNA sequencing of the virus isolated in our laboratories (ORFs 3-7), as well as the comparison between that sequence and the LV sequence are mentioned in 4.3.4.V where it can be seen that on amino acid level, approximately 94.9% homology exists with a total of 47 differences in amino acids, 35 of which correspond to non-conservative substitutions. These differences at the amino acid level may be responsible for the differences in pathogenicity that exist between the various isolated PRRS virus strains.

EXAMPLE 3 Formulation of a vaccine

A vaccine that can provide protection against PRRS is prepared in emulsion form according to the method set forth below. A culture of alveolar macrophages from a pig lung is infected with MOI (multiplicity order infection) of 0.001 and is incubated for 24 hours at 37 ° C with the culture medium replaced at the end with infection medium (DMEM supplemented with 2% FCS). The culture is incubated for 4 days at 37 ° C until 70-80% CPE is observed. Once this period is complete, an IPMA test is performed to confirm identification. The virus is collected by vacuum aspiration and frozen at -80 ° C. The viral suspension intended for vaccine must have 10ss TCIDgo / ml minimum titer prior to inactivation and must not be contaminated with bacteria, fungi, mycoplasmas or other viruses. If the titer is lower, it must be adjusted by concentrating the antigen. For the inactivation of the viral suspension, 2% o β-propiolactone solution is added and stirred overnight at 4 ° C, the pH being kept at 7.4 by the addition of 0.5 N NaOH. Once the inactivation period is complete, the viral suspension is maintained at 37 ° C for 1 hour. The following is then prepared: a) an antigenic phase of the viral antigen that is inactivated with a minimum concentration of 10 5 · 5 TCID 50 / dose and the preservative; and b) an oily phase consisting of Marcol 52, Simulsol 1500 and Montanide 888.

The aqueous phase, which is continuously stirred, is slowly added to the reactor which contains the oily phase which is also stirred. When the aqueous phase is completely added, stirring is continued for 10 minutes. In a particularly preferred embodiment, vaccines are prepared which are capable of preventing PRRS that per dose of 2 ml: a) 53% of an antigenic phase, which i) Spanish strain of PRRS viral antigen in DMEM culture medium, which is containing β-propiolactone inactivated with a minimum concentration of 105.5 TCID3, and ii) thimerosal contains 0.01%; and b) 47% of an oily phase comprising i) Marcol 52 ....... 790.0 mg 40 ii) Simulsol 5100 ... 70.0 mg iii) Montanide 888 .. 80.0 mg .

The oily phase / aqueous phase ratio is a weight / volume (w / v) ratio. This vaccine is referred to as MSD ref. 1. The vaccine obtained shall be subjected to the corresponding control tests prior to use. Another vaccine was prepared in a similar manner using Munokynin® (aluminum hydroxide and Quii A, as supplied by American Cyanamid) as an adjuvant using the same amount of inactivated virus. This vaccine is MSD ref. 2.

EXAMPLE 4 Evaluation of the adjuvant 50 A field test was performed on a total of 128 sows, 49 of which were vaccinated with 1 dose of the vaccine designated MSD Ref. 1 and 50 sows were treated with 1 dose of the vaccine designated MSD ref. 2 (Munokynin®) were vaccinated, while the remaining 29 were not vaccinated and kept as controls. After 22 days, the sows were again vaccinated with 1 dose of the corresponding vaccine. The following parameters were investigated.

55 1. Serological response by IPMA determination at the following times: T0: vaccination and blood collection T ^: vaccination again and blood collection 194940 14 TS1: blood collection 50 days after vaccination 2. General type of reactions (appetite for food, hyperthermia, etc.) The results obtained are shown in Tables 5 and 6, which indicate the percentages of sows with positive serological response (Table 5) and the arithmetic mean of the serological titers achieved (Table 6).

5 TABLE 5% animals with serological response (+) T0 ^ 22 T51 10 - MSD Ref. 1 - 59% 100% MSD Ref. 2 - 40% 87%

Check - - - 15 TABLE 6

Arithmetic mean of serological titers T0 T *> 2 T51 20 MSD ref. 1 - 58 200 MSD ref. 2 - 37 133 control - - - 25 No significant local or general type of reactions were observed. It can be confirmed on the basis of the results obtained that positive seroconversion can be achieved with both vaccines, although these are somewhat higher with the MSD ref vaccine. 1 (oily adjuvant). A higher seroconversion rate is observed during revaccination and the arithmetic mean of the titers obtained in animals treated with MSD ref. 1 have been vaccinated higher.

EXAMPLE 5

Sow safety Example 5.A. At laboratory level 35 Example 5.A.1. - Sows giving birth for the first time

Eighteen sowing (German Landrace x Large White Cross) sowing for the first time from a pig production farm were selected and distributed over two stables with 9 sows per stable, so that sows vaccinated with the same vaccine (MSD ref. 1 or MSD ref 2) obtained in Example 3 above were housed in the same stable. Nine sows were vaccinated via deep IM route with 40 1 dose of MSD ref vaccine. 1 of 2 ml containing 105.5 TCID 50 / dose of inactivated virus titer and were re-vaccinated 20 days later with another dose of the same titer. The other nine sows were vaccinated with the MSD ref 2 vaccine (2 ml dose 105.5 TCIDgo / dose inactivated virus titer) and re-vaccinated on the same days. The following observations were made during the first 5 days after inoculation: 45 a) Local reaction

This consists of macroscopic observation of the inoculation site and its exposure, whereby the degree of inflammation in comparison with objects of known size is observed, b) General reaction

This consists of macroscopic observation of the animals and verification of their appetite. In the negative case, rectal temperature is checked every 12 hours until hyperthermia or other adverse signs have disappeared. The results obtained indicate that a somewhat inflammatory reaction occurs in some sows, which is evident at the inoculation site and disappears in any case within a few days; no festering formations were observed. Only one of the sows refused to include all the food in the first feed after inoculation, but the food intake was normal with the next feed 55 so that rectal temperature measurement was unnecessary. There were no substantial differences in response to the different vaccines tested on the basis of which it can be confirmed that both vaccines are safe. Example 5.A.2. - Pregnant sows 15 194940

Seven pregnant sows (German Landrace x Large White Cross) from a pig production farm were selected at random: 6 first-born sows of about 9 months and 1 sow who had given birth more often, at the age of 3 years and 7 months.

The vaccine, referred to as MSD ref. 1 was the only one used. The sows were vaccinated via the 5 deep IM route with a 2 ml vaccine dose containing inactivated virus titer of 105-5 TCID / ml and after 15 days they were again vaccinated with 1 dose of the same titer. During the first 5 days after inoculation the following observations were made: a) local reactions consisting of making macroscopic observations of the size of inoculation and touching it, whereby the degree of inflammation is compared with objects with a well-known size noted.

b) Lack of appetite which consists of macroscopic observation of the animals and checking for loss of appetite.

c) Rectal temperature 15 Measuring the rectal temperature 24 hours after vaccination and 24 hours after re-vaccination. The results obtained show that there was a somewhat local reaction in two of the animals that was not serious due to the small size and disappearance after a few days. Lack of appetite or hyperthermia was not observed in any case. Based on this, it can be confirmed that the vaccine is safe.

Example 5.b. Safety and efficacy in a field trial

This experiment was performed on the 5 farms indicated below. A variable number of sows from each farm was vaccinated via deep IM with 1 dose of 2 ml of the MSD ref vaccine. 1 containing a titer of 105.5 TCID / dose of inactivated virus and was vaccinated again 21 days later with another dose of the same titer with the other sows not being vaccinated and used as controls:

Farm Vaccinated Control Total 30 I 19 11 30 II 46 34 80 III 153 147 300 IV 127 123 250 V 163 157 320 35 TL 508 472 980 TL = total I Farm '' Ramon del Quinta ”(Banyoles) II: Farm" Cal Sabater ”(Orriols) 40 III: Farm“ E. Canela ”(Preixana) IV Farm” R. Cunillera ”(L 'Albi) Q: Farm“ Inversors Picber ”(Bellpuig) 45 After observation of general and local reactions, it was confirmed that the vaccine is safe Local reactions were only observed in 1% of the animals No variations were observed in relation to their clinical history in relation to the production parameters observed in the farms mentioned above. seroconversion to the vaccine, while in others the response was negative, but this does not indicate a low level of protection, since in the laboratory experimental infection test seronegative animals exp prevent erotic infection (examples 7 and 8). In connection with the transfer of maternal immunity from vaccinated animals to their offspring, a large fall in antibody titers at 1 month of age is observed. In vaccinated and re-vaccinated sows that are serologically positive, a large decrease in antibody titers occurs 2 months after re-vaccination.

194940 16 EXAMPLE 6 Verification of cell immunity

Five pregnant sows (German Landrace x Large White Cross) from a pig production farm were used. The animals were moved to the safe stables of the research center. Two sows were chosen at random and were treated with the vaccine that was MFD ref. 1 is indicated vaccinated. Another sow was vaccinated with the vaccine designated MSD ref. 2. The two remaining sows were not vaccinated. The sows were vaccinated via the deep IM route with 1 dose of 2 ml MSD ref. 1 or vaccine MSD ref. 2 containing inactivated virus titer of 105.5 TCIDgo and 10 days later the sows were vaccinated with another dose of the same titer. Then all sows between days 77 and 90 of pregnancy were infected via the IN route with 5 ml of the PRRS-CY-218-JPD-P5-6-91 virus with a titer of 105-8 TCID ^ / ml. At the time of the infection, it was verified that all vaccinated sows showed antibody to the PRRS causative virus (positive serology). Table 7 shows the reproduction results obtained as a whole: TABLE 7 (A) (B) (C) (D) (E) (F) (G) 2 MSD ref. 1 23 20 - 20 3 1 MSD ref. 2 12 - 6 - 6 2 - 24 - 7 - 17 (A) number of sows 25 (B) vaccine used (C): total number of piglets (D): number of piglets born alive and in good health (E) number of piglets born live but weak (F) number of piglets that lived after the first week 30 (G): number of stillborn piglets

The results obtained demonstrate that the sows treated with the MSD ref. 1 (oily adjuvant) are vaccinated more resistant to infection than the sows treated with MSD ref. 2 (aqueous adjuvant) have been vaccinated, which may mean that the adjuvant plays an important role in establishing cell immunity.

EXAMPLE 7 40 Efficacy for pregnant sows

Eleven breeding sows were used (German Landrace x Large White Cross) from a pig production farm. The animals were moved to the safe stables of the research center.

Three sows were chosen at will (sows nos. 57, 63 and 74) and were treated with the vaccine that was designated as MSD ref. 1 is indicated vaccinated. Three sows (nos. 15,18 and 23) were treated with the vaccine as 45 MSD ref. 2 is vaccinated and the remaining five sows (Nos. 14, 40, 13, 30 and 85) were not vaccinated.

The sows were vaccinated via the deep IM route with 1 dose of 2 ml of the vaccine designated as MSD ref. 1 or the MSD vaccine ref. 2, it is indicated that inactivated virus with a titer of 10 s, TCID 50 / dose and were vaccinated again 20 days later with another dose with the same titer. Local and 50 general responses were observed. Serological response in the animals was verified by the IPMA test in accordance with the following program: T0 blood collection and vaccination T20: blood collection and revacdination 55 T42: blood collection T78: blood collection and experimental infection 5 17 194940 T8 blood collection after experimental infection T25: blood test after experimental infection

Tgo: blood collection after experimental infection

Experimental infection was carried out in the safe stables of the research center. All animals were infected with 5 ml of PPRS-CY-218-JPD-P5-6-91 virulent virus with a titer of 105-5 TCIDgo / ml by the IN route. Serological response was noted, as well as the number of piglets born alive and dead to each sow. Pre- and post-colostrum blood collection was performed on the piglets and lung and brain samples were taken from stillbirths and killed piglets on different days to isolate the virus and for histological preparations. The results obtained are shown in Tables 8-10 below.

TABLE 8 15 Serological results

No. T0 T20 T42 T78 Te "^ 25 Tgo

1/160 1/160 1/640 1/1280 NT

20 18 - - 1/80 1/80 1/320 1/640 1/320

23 - 1/160 1/160 NP NP NP NP

57 - 1/160 1/160 1/320 1/320 1/1280 1/60 63 - 1/80 1/80 1/160 1/640 1/1280 1/160

74 - 1/80 NT 1/160 1/640 1/2560 NT

C14 - - - - 1/1280 1/2560 NT

C40 - - - - NT 1/1280 NT

C13 - - - - NT 1/320 NT

C30 - - - NT 1/320 NT

C85 - - - - NT 1/320 NT

30 -: - NT: not tested NP: not pregnant TABLE 9 2 ^ Results of delivery

No. (A) (B) (C) (D) (E) (F) (G) (Η) (I) (J) 15 * 83 112 13 - - - 4Q 18 78 111 11 8 - 3 - - 2 23 ® 57 63 108 - 13 7 - 6 63 79 114 - 13 8 1 2 2 2 74 * 83 112 6 - - 45 c14 65 111 15 - - 3 12 - c40 82 102 - 12 - - 12 c13 80 113 12 10 2 - c30 80 113 - 15 - 12 - 3 11 c85 85 113 - 11 4 7 4 50 a not pregnant * these sows died due to exceptionally high ambient temperatures. Caesarean section was performed after 112 days of pregnancy.

(A) date of infection (days of pregnancy) 55 (B) giving birth (days of pregnancy) (C): total number of piglets (caesarean section) 194940 18 (D) total number of piglets (born) (E) piglets that are in normal health born (F) piglets born weak ^ (G): piglets born alive with legs turned outward (H): stillborn piglets (dead) (I) stillborn piglets (mummified) (J) piglets born within the first week were dead.

IQ TABLE 10 - Serology of the piglets A. Vaccinated sows 1 2 3 4 5 15 15a) NT NT NT - 3 days 10 days

18 1 - NT NT

2 - NT NT

7 NT 1/640 1/160 20 8 NT 1/2560 1/320 9 NT 1/1280 1/640 10 NT 1/1280 1/320 11 NT 1 / 1280-1 / 2560 1/640 12 NT 1/2560 1/640 25 13 NT £ 1/2560 1/160 14 NT 1/1280 1/160 15 NT 1/2560 1/160

23 NP NP NP

4 days 8 days NP

30 57 1 - 1 / 320-1 / 640 1/320 2 - 1 / 320-1 / 640 1 / 160-1 / 320

3 - 1/640 NT

4 - 1 / 320-1 / 640 NT

5 - 1/640 NT

35 1 day 8 days 63 1 NT £ 1/2560 1/640 2 NT £ 1/2560 1/320 3 NT 1/2560 1 / 320-1 / 640 4 NT 1/2560 1 / 640-1 / 1280 40 5 - 1/1280 1/320 6 - 1/2560 1/640 7 NT 1 / 640-1 / 1280 1/320

8 NT 1/1280 NT

74a) NT

45 - (TABLE 10) B. Control of piglets (not vaccinated) 1 2 3 4 5 50 ---------------- 12 H. 38 H. 5 days 9 days C14 1 NT £ 1/2560> 1/2560 1/1280 1/640 2 - £ 1/2560 £ 1/2560 1/1280 1/1280 3 £ / 2560 £ 1/2560 1/1280 1/1280 1/1280

55 C40 - NT

9 days 19 194940 (TABLE 10) (continued) B. Control piglets (not vaccinated) 1 2 3 4 5 5 ------——----- C13 1 - 1/160 + 2 - 1/160 3 - 1/320 4 - 1/320

10 C30 - NT NT

C85 NT NT NT

Legend: a> These sows died due to exceptionally high ambient temperatures. A caesarean section was performed after 112 days of pregnancy.

1 Number assigned to each animal 2 Piglets (the number assigned to each pig corresponds to the birth order) 3 Pre-colostrum serology 4 Post-colostrum serology 20 5 Isolation of the virus NT Not tested

Negative + Positive H Hours NP: Not pregnant.

It is clear from the results obtained that positive seroconversion occurs against the causative virus of PRRS. In addition, satisfactory behavior against experimental infection occurs in comparison with the control animals, with mortality being produced in the majority of fetuses. As a result, it can be confirmed that this vaccination is an effective measure to prevent PRRS.

EXAMPLE 8 35 Efficacy of the vaccine against experimental infections with another virus that causes PRRS (cross protection)

This experiment was developed with a view to verifying the efficacy of the vaccine designated as MSD Ref. 1 in an experimental infection test using 2 pathogenic strains of the PRRS causative virus. The pathogenic PRRS strains used were: i) Spanish strain, PRRS-CY-218-JPD-P5-6-91; and ii) French strain, SDRP II 8B, which Dr. E. Albina from "Laboratoire Central de Recherches Avicole et Porcine", Ploefragan, France was provided.

The vaccine used was the vaccine designated as MSD ref. 1 whose formula is given in Example 5. Thirty sows that were sero-negative for the PRRS causative viruses were used in a reproduction cycle that was not between 10 days before or 10 days after coverage, neither 10 days before nor 10 days after giving birth. Four groups of animals were formed: A: 10 sows vaccinated and re-vaccinated 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 that were not subjected to any vaccination and were infected with Spanish strain 50 PRRS-CY-JPD-P5-6-91 via the IN route C: 10 sows vaccinated and re-vaccinated via the IM route and were infected via the IM route with French strain SDRP II 8b D: 5 sows that had not been subjected to any vaccination and were infected via the IN route with French strain SDRP II 8b.

55 Experiment performed

Twenty sows of group A and C mentioned above were vaccinated with 1 dose of 2 ml of the vaccine designated as MSD ref 1 via the deep IM route and vaccinated again with 194940 the same dose of vaccine 21 days later. Thereafter, all sows were experimentally infected between days 17 and 80 of pregnancy with:

Groups A and B: 5 ml virus PRRS-CY-218-JPD-P5-6-91 with a titer of 105 · 8 TOID ^ ml via the IN route; and 5 Groups C and D: 5 ml of virus SDRP II 8B with a titer of 105 · 8 TCID50 / ml via the IN route.

The reproduction parameter results are shown in Tables 11-12 (challenge with PRRS-CY-218-JPD-P5-6-91) and 13-14 (challenge with SDRP II 8b).

TABLE 11 (vaccinated sows) 10 Challenge with PRRS-CY-218-JPD-P5-6-91 (1) (2) (3) (4) (5) (6) (7) 37a) 14 15 39 6 6 - - - 5 68 12 8 2 1 1 7 45 10 7 1 - 2 6 19 10 9 1 - 8 38 9 8 1 8 20 41 7 5 - - 2 5 71 11 6 1 - 4 6 36 10 6 1 1 2 6 26 «25 TL 75 55 6 2 12 51 TL = total% living piglets: 68% (75 born / 51 surviving) 30 68% protection is observed when comparing the piglets born alive with the piglets having 7 days survived. The fact that experimental infection is much more powerful than natural infection in the field, in addition to the above results, suggests that the prospects for protection are much better.

35 TABLE 12 (unvaccinated sows)

Challenge with PRRS-CY-218-JPD-P5-6-91 (1) (2) (3) (4) (5) (6) (7) 40 63 9 - - 1 8 88 6 - 3 - 3 - 79 8 - 3 - 5 - 22 12 2 - - 10 1 28 7 3 4 3 45 ----- TL 42 5 6 1 30 4 TL = total 50% living piglets: 9, 5% (born 42/4 born survive)

The Spanish strain used for infection has an extremely high pathogenic power (90.5%) since only 4 piglets out of 42 born were surviving the first week.

21 194940 TABLE 13 (vaccinated sows)

Challenge with SDRP II 8B

(1) (2) (3) (4) (5) (6) (7) 5 -------- 51 13 10 2 - 1 10 27 14 12 1 - 1 11 16 9 8 - 1 7 17 15 13 - 2 12 10 1 7 - - - 7 8 15 13 - 2 13 57 10 9 - - 1 8 61 12 8 1 - 3 8 78 10 10 - - - 10 15 52c) TL 105 83 4 - 11 86 TL = total 20% live piglets: 82% (105 born / 86 surviving) approximately 82% protection is observed.

i TABLE 14 (unvaccinated sows)

25 Challenge with SDRP II 8B

(1) (2) (3) (4) (5) (6) (7) 62 12 7 1 4 - 9 81 15 13 - - 2 13 30 4 12 12 - - - 11 94 14 9 1 - 4 9 1 13 8 1 4 - 8 TL 66 49 3 8 6 50 35 ----——— -: -!

tl = total I

live piglets: 75.7% (66 born / 50 surviving)

40 Approximately 24.3% mortality is observed. The pathogenicity of the French strain is very weak J

(24.3%) compared to the results obtained with the challenge with Spanish strain (mortality 90.5%). The results used for the evaluation of efficacy in comparing vaccinated and unvaccinated sows are not very significant in the case of the French strain. However, the pathogenicity in the vaccinated animals is reduced to 17.8%.

45

Legend for Tables 11-14 a> Another disease (not PRRS) (the piglets are not used) b> Sick, died before infection c) Died due to another cause 50 (1) Sow reference (2) Total number of piglets (3) Number of healthy piglets born (4) Number of weak piglets born (5) Number of live piglets with legs turned out 55 (6) Number of stillborn piglets (7) Number of piglets that lived after the first week

Claims (10)

194940 22 I. Virus that causes pig reproduction and respiratory syndrome (PRRSS) Spanish strain designated as PRRS-CY-218-JPD-P5-6-91 that has been deposited at ECACC with accession number V93070108. Virus according to claim 1, characterized in that it comprises a DNA sequence according to one of the sequences of figures I-5, which DNA sequence codes for a product with the activity of one of the open reading frames of figures 1-5 . 3. Vaccine for protection against pig reproduction and respiratory syndrome (PRRS), characterized in that it comprises a suitable amount of PRRS viral antigen or virus of the Spanish strain according to claim 1, which is inactivated as well as a suitable adjuvant and optionally a preservative . Vaccine according to claim 3, characterized in that it contains an amount of inactivated virus of at least 105.5 TCID 50 / dose. Vaccine according to claim 3 or 4, characterized in that an oily adjuvant is formed by a mixture of Marcol 52, Simulsol 5100 and Montanide 888. A vaccine according to claims 3-5, characterized in that it is an emulsion of (i) 53% by volume of an aqueous antigenic phase containing the inactivated virus and (ii) 47% by weight of an oily phase containing the adjuvant. A vaccine according to claims 3-6, characterized in that it also contains one or more cell response enhancing substances (CRP) that enhance the cell immune effect and are selected from: IL-1, IL-2, IL-4, IL- 5 IL-6, IL-12, g-IFN, cell necrosis factor. A vaccine according to any one of claims 3-7, characterized in that the adjuvant is an adjuvant that can modulate cell response and immuno-stimulate is selected from MDP, ISCOM or liposomes. 9. A bi or multivalent vaccine that is capable of preventing pig reproduction and respiratory syndrome and one or more other pig infections, that an appropriate amount of PRRS viral antigen or virus from the Spanish strain according to claim 1 or 2 which is inactivated, optionally including components according to any one of claims 3-8, characterized in that it also comprises one or more pig pathogens selected from: Actinobacillus pleuropneumoniae, Haemophilus parasuis, pig parvovims, Leptospira, Escherichia coli, Erysipelothrix rhusiopathiae, Pasteurella multocida, Bordetella bronchiseptica, porcine respiratory coronavirus, Rotavirus, or causative agents of Aujeszky swine influenza or transmissible gastroenteritis. A method for the preparation of a vaccine according to any one of claims 3-9, characterized in that it comprises the following steps: 1. culturing the PRRS causative virus, Spanish strain according to claim 1 or 2 in a suitable cell system, 2) collecting the virus from the cell system after a minimum titer of 10 5 TCIDgo / ml, 3. inactivation of the virus by physical or chemical methods and 4. mixing the inactivated virus with the adjuvant and the preservative. II. Method according to claim 10, characterized in that the virus is grown at 40 i. culture of alveolar macrophages from a pig lung or on ii. a co-culture of alveolar macrophages from a pig lung and ST cells (ATCCCRL 1746 ST) or on iii. ST cell culture (ATCCCRL 1746 ST), or on iv. hybrid cells of alveolar macrophages from a pig lung fused to ST cells (ATCC CRL 45 1746 ST) or to v. hybrid cells of alveolar macrophages from a pig lung associated with L-14 cell line (ECACC No. 910123 17) or with cell line Jag-1 have been merged or on vi. any other porcine cell line with membrane receptors against the PRRS virus of alveolar pig lung macrophages. Hereby 11 sheets of drawing