DK173128B1 - Vaccine against the porcine reproductive and respiratory syndrome method for its preparation, as well as a virus and a DNA - Google Patents

Description

DK 173128 B1

The invention relates to a vaccine capable of preventing the porcine reproductive and respiratory syndrome, in particular an inactivated vaccine containing the virus causing this disease, in an inactivated form, a method of producing it, and a virus and a DNA sequence.

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The disease called the Porcine Reproductive and Respiratory Syndrome (PRRS) affects pregnant sows in which it can induce anorexia, miscarriage, stillbirths, mummified or desiccated fetuses, infirm pigs dying in a few hours or days, respiratory problems after birth, and breeding problems (Loula, T.: "Clinical Presentation of Mystery Pig 10 Disease in the breeding herd and suckling piglets," Proceedings of the Mystery Swine Disease Committee Meeting, 06 Oct1990, Denver, Colorado, Livestock Conservation Institute, Madison, WL, USA) . In some cases, the infected lakes have blue spots on their ears, which is why they are also called the "blue abortion" or "blue-ear swine disease" (Veterinary Record, b. 130, no. 3, 18 JAN 1992). The disease is also called "the mysterious swine disease" (Mystery Pig 15 Disease, MPD; Mystery Swine Disease, MSD), "the mysterious reproductive syndrome" (Mysterious Reproductive Syndrome, MRS), "Swine Infertility and Respiratory Syndrome," ) or "the Porcine Epidemic Abortion and Respiratory Syndrome" (Porcine Epidemic Abortion and Respiratory Syndrome. PEARS.

20 The first epizootic outbreaks of this disease occurred in the United States and Canada in 1987.1 Europe saw the first outbreak in Germany in 1990, from which it spread to the Netherlands and Belgium in late 1990 and early 1991. In Spain, the first cases were seen of the disease in mid-January 1991, when significant resplrative changes were observed in a pig population of 300 individuals imported from Germany (Plana et al., Med. Vet., b. 8, no. 11, 1991). Shortly thereafter, two breeding flocks were found about 500 m from the original problem stock, a disease that was characterized by an abnormally high number of abortions during the last phase of the gestation period and at a mortality rate of 70% among the small cubs. An analysis of the observed clinical symptoms, taking into account that these stocks (i) had undergone an intensive vaccination program against porcine parvovirus, Aujeszky's disease and swine flu, and (ii) laboratory studies had rejected the presence of other abortion diseases , led to suspicion that one was facing PRRS here. The agent that caused the disease has been isolated from samples from these farms, as described below.

A number of agents have been correlated with this course of infection. encephalomyocarditis 35 virus, swine flu, classical swine fever, African swine fever, mucosal disease, Aujeszky's disease, brucellosis, leptospirosis, Q fever, parvovlrosis and chlamydia disease, some even related it to mycotoxins (Loula, T: Disease in the breeding herd and suckling piglets, "Proceedings of the Mystery Swine Disease Com- 2 DK 173128 B1» mittee Meeting, supra; Mengeling, WL and Lager, KM: "Mystery Pig Disease; Evidence and considerations 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 problems," Proceedings of the Mystery Swine Disease Com-5 mittee 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), 2 3-33, 1991; Woolen, N. et al., J. Am. Vet. Assoc., 197, 600-601,1990).

10 WO-92/21375 in the name of Stitching Centraal Veterinary Institute (CDI) describes the isolation

of a virus called "Lelystad Agenset" (LA) or "Lelystad Virus" (LV), and described 3 as being the cause of the disease then called MSD. When inoculating Isolated LV

intranasally in pregnant sows, loss of appetite and even eating wakefulness are observed on days 4 to 10-12 after inoculation, reproductive disorders and blue on the ears of some of the 15 infected sows on days 9-10 after inoculation. However, it is also observed that the disease | is not reproduced in two of the eight experimentally infected sows (see Table 6 of the PCT application mentioned above), as in one of the sows the number of live-born piglets that survived the first week is high (six out of nine; so no. 1305), and another sow had two stillborn piglets, while the other nine survived the first week (sow # 1065). LV virus isolated by CDI belongs to 20 genus Arteriviridae and has a genome consisting of a polyadenylated RNA moiety with a length of 14.5 to 15.5 kb (determined in neutral agarose gel) which replicates by a set of subgenome RNAs at the 3 'end. The above PCT application specifies the LV genome nucleotide sequence, the eight possible open reading frames (ORFs), the amino acid sequence as derived from the identified ORFs, and the putative N-glycosylation sites.

25 Subsequently, other viruses causing PRRS have been isolated on German, French and Spanish farms. The virus that isolated in Tubingen, Germany (TV) (Virology, 193, 329-339, 1993) is 99.3% homologous with nucleotide level LV in ORF # 2-7 (there are 24 different base pairs (bp) out of a total of 3316 bp in this area). The derived TV amino acid sequence is 99.2% homologous to LV (there are only ten different amino acids in the derived amino acid sequence as encoded in ORF Nos. 2-5, as there is no difference in those from ORF Nos. 6 and 7 derived sequences).

On the other hand, the homology is less between LV and TV compared to the virus isolated in our laboratory (the Spanish strain, SV). So far, only the nucleotide and amino acid sequences corresponding to ORF # 3-7 have been compared and with the following results: - 35 1. There is 95.5% homologous! at the nucleotide level of SV versus LV or TV (out of a total of 2599 bp, 144 are different); and ® 2. There are 94.9% homologous! at the amino acid level of SV versus LV or TV (out of a total of 955 amino acids, 47 amino acids are observed which are different).

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These amino acid variations may be related to higher pathogenicity of some strains than others, among others, the vims isolated in our laboratory (SV) are more pathogenic than other known PRRS viruses. If Example 8 is considered below, it will be seen that the percentage of surviving piglets in a sow infected with the French strain is 75%, while the corresponding percentage-5 proportion of a sow infected with the Spanish strain is 9.5% (Table 12 and 14). The percentage is 61%. when the sows are infected with LV (Table 6, WO-92/21375), as 58 live piglets are born out of a total number of 95.

The disease is the cause of severe losses in the pig industry, since in acute outbreaks it can cause 10 mortality of 70% of the pigs in a litter. The problem could be solved by an appropriate vaccination program to prevent the disease from occurring. Thus, vaccines that are effective against PRRS are needed.

WO-93/06211 in the name of Collins, J.E. and Benfield, D.A. discloses a vaccine against MSD containing an infectious agent Isolated from the lungs of pigs. who are infected with MSD. However, the PCT application does not characterize and identify the infectious agent that has not been deposited with any recognized depository. Therefore, there are serious problems with the reproducibility of the information in this PCT application, as the product described cannot be verified and the results obtained cannot be compared with, for example, the present application results. Similarly, in this PCT application neither the antigen nor the adjuvant used in the formulation of the vaccine has been clearly defined, which, as will be seen below, plays an important role. Finally, there are also no examples showing the potency and efficacy of vaccines.

25 WO-93/07989 reviews e.g. the identification of the agent that causes PRRS, and hence of guided vaccines. The isolated vims have characteristics similar to LV. but unlike the virus that is Isolated in our laboratory, it is not able to grow to detectable ST cells (swine testis cell). Apparently the vaccine has had an effect, but the degree of protection has been sufficient in only about 52% of the vaccinated animals, which must be considered a relatively low level, especially when considering the high viral titers used in the vaccination dose. Furthermore, this PCT application does not mention anything about the organization of the viral genome or about the proteins it encodes, why one cannot or only with an undue investigative effort can compare this virus with the virus that we have isolated in our laboratory.

35 Consequently, vaccines against PRRS are still needed. This problem is solved according to the present invention by providing a vaccine which is capable of providing effective protection of sows against the infectious disease. The antigenic part of the vaccine consists of an inactive 4 Isolated Spanish Isolate. The invention also provides combinations of the isolated PRRS viral antigen (the Spanish strain) and various porcine pathogens for the purpose of providing bi- or multivalent vaccines.

5 The invention will be explained in more detail with reference to the sequence list and with the aid of the drawing, in which Figures 1-5 show the homology and the existing differences between LV and the virus isolated in our laboratory at the amino acid level derived from ORF no. 3 to 7 (SEQ ID NO: 1-5). The amino acids are expressed by a one-letter code. The upper of each line set consisting of two lines corresponds to the amino acid sequence of LV. while the bottom line corresponds to the virus isolated in our laboratory (the Spanish strain). The homologous amino acids are shown with two vertical dashes placed vertically over one another, while two dots placed vertically above one another Indicates that there has been a conservative substitution of some amino acids with others, with the conservative assuming that substituted by a functionally equivalent amino acid, Absence of such streaks and dots between two amino acids indicates that non-conservative substitutions have occurred.

In the following detailed description of the invention, the sections are numbered for clarity.

J 20 1. Samples 1.1 Selected DVRs for virus isolation

Mummified fetuses, stillborn weaners, and live but infirm weaners of the age of 1–10 days who are offspring of lakes with clinical problems due to PRRS are selected.

25 1.2 Preparation of Samples

Using necropsy, samples of lung, spleen, liver, kidney, brain and heart are taken from the piglets. In a special case, samples from the lungs are taken from a stillborn pig, born of a sow suspected of having a PRRS infection. With this lung is prepared a homogenize with DMEM (GIBCO; 30 g of lung in 90 ml of DMEM), supplemented with an antibiotic solution (PEG) consisting of 1000 IU / ml penicillin, 1 mg / ml streptomycin and 0.5 mg / ml gentamicin. The resulting suspension is left at 4 ° C for 1 hour, frozen and thawed twice, centrifuged, and the resulting supernatant is stored at -70 ° C for use in infecting alveolar macrophages from pig lungs. In another case, samples are prepared from "35 the lungs of a live-born pig, who died a few hours after birth.

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Furthermore, btod is taken from the animals by puncturing the vena cava, thereby obtaining (i) blood plasma stored at -70X and used for virus isolation, and (i ·) serum used for antibody titration.

5 2. The alveolar macrophages from graft lungs 2.1 Acquisition

Pigs that are 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 pigs used are between 7 and 8 weeks old. The animals are anesthetized with pheno-10 barbital, sodium before extraction of the lungs, which is done by first binding the trachea (trachea) under the epiglottis (larynx) and sectioning over the bandage. After extraction of the lung, wash externally with physiological saline and introduce an antibiotic solution into PBS and PEG at successful rinses. The cells resulting from these rinses are centrifuged for 10 min. at 300 g and resuspended in the DMEM medium (DMEM medium supplemented with non-essential amino acids, GIBCO, 1% 1 mM sodium pymvate and 1% 2mM glutamine), 10% fetal calf serum (FCS) and a PEG solution of antibiotics 1% island. The cells are counted in Newbauer chambers.

2.2 Sterility check

Appropriate testing procedures confirm that the alveolar macrophages from pig lungs are uncontaminated with bacteria, fungi and other viruses. Likewise, the good general condition of the cells is confirmed by optical microscopy.

The absence of mycoplasma contamination is confirmed using a cytochemical detection method with DAPI (4,6-diamidin-2-phenylindole), which selectively binds to DNA and forms DNA-DAPI complexes with high fluorescence and high specificity.

3. Isolation of viruses 3.1 Isolation of viruses and growth of viruses and alveolar macrophages from orifices A culture of alveolar macrophages from pig lungs previously produced in DMEM medium 30 and FCS at a concentration of 10% is infected with a homogenate of a sample suspected of containing the agent that causes PRRS. The test consists in a case of a lung isolate from a small pig, stillborn of a sow with clinical symptoms of PRRS, in other cases a lung isolate from a live pig, who died a few hours after birth, is the progeny of a sow with PRRS symptoms, as well as an isolate from blood plasma. The homogenate is kept in contact with the 35 macrophage culture for 1 hour at 37 ° C, then Inoculum is removed and fresh DMEM medium with 2% FCS and 1% o PEG solution is added, buffered with CO 2 and incubated at 37 ° C. C for several days during which, under the microscope, the cytopathic effect (CPE) induced by virus in the macrophages is observed: 3-4 days after the infection (dpi = days 6 DK 173128 B1 post infection), the CPE was 70-80% (appearance of giant cells and deformed cells). The cultures are frozen at -80 ° C.

At the same time, a culture of alveolar macrophages from pig lungs not infected with PRRS is being prepared for use as a negative control.

Subcultures are prepared with the isolated virus and it is observed that there is 100% CPE from the second dpi. Virus is frozen at -80 ° C for later identification and characterization.

; a 1 o; a Similarly, viruses are isolated from blood plasma and other specimens from stillborn piglets or 'live but weak pigs born of infected sows.

i] One of the isolated viruses, namely the virus bearing the designation PRRS-CY-218-JPD-P5-6-91, "15 is isolated from the lung of a stillborn pig. It is capable of experimentally reproducing disease, has the characteristics mentioned in Section 4 and has been deposited with the European Collection of Animal Cell Cultures (ECACC), Salisbury, United Kingdom, on June 29, 1993 under Accession number V93070108.

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- 20 3.2 Viral growth in other cell systems

The isolated virus (Spanish strain) infections are carried out in co-cultures of alveolar macrophages from pig lungs and ST cells (swine testis continuous cell line) ATCC CRL 1745 ST, as the first step towards adapting virus ST cells. After multiple serial passages (5-6) on ST cells co-cultures and ST macrophages and pure cultures, a virus infection titer of the order of 106 TCIDgo / ml is obtained in the case of co-infection. cultures of macrophages and ST cells, and of the order of 104 5 TCID ^ / ml for virus I pure ST cells. (TCID ^, tissue culture infectious dose means 50%, tissue culture infection dose 50%).

Furthermore, alveolar macrophages from pig lungs can also be immortalized (immortalized) = 30 by fusion with ST cells by hybridization.

Alternatively, the alveolar macrophages from pig lungs can be immortalized by fusion with the L-14 cell line (porcine peripheral blood B cells) ECACC No. 91012317 or with cell line No. Jag-1 (porcine trophoblast cell line) obtained from Dr. I'm Ramsonas ().

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The merger takes place as is customary in this area.

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Alternatively, viruses can be caused to grow on ST cells or any other porcine cell line that has introduced the genes encoding the membrane receptors for PRRS virus from alveolar macrophages from pig lungs.

The viruses produced with these cell systems can be used to formulate both live and inactivated vaccines.

4. Identification and characterization of viruses 41 Designation and deposition of viruses 10 The virus isolated from the lung of a stillborn pig and referred to as PRRS-CY-218-JPD-P5-6-91 is deposited with the European Collection of Animal Cell Cultures (ECACC), Salisbury,

United Kingdom, June 29, 1993 with the deposit number V93070108. In this specification, this virus is occasionally referred to as "the Spanish stem virus" (SV) 15 4.2 Characterization of the isolated virus

This virus (PRRS-CY-218-JPD-P5-6-91) has the following characteristics: a) Production of a moderate CPE in a continuous ST cell line (ATCC CRL 1748 ST) (fetal swine testis) with an average titer of 104'5TCID5O / ml and on alveolar macrophages from pig lungs with a mean titer of 105 5 TCID ^ / ml; B) by infection of co-cultures of alveolar macrophages from pig lungs and ST cells, an average titer of 106 · 3 TCID ^ / ml is obtained (which is a logarithmic unit larger (1 log10) than when a pure culture of alveolar macrophages from piglets are infected); c) cytoplasmic replication; d) production of cytoplasmic vacuoles; E) lipid envelope; f) a size of 40-50 nm; g) no hemadsorption or hemagglutination is observed with chicken, guinea pig, swine or human red blood cells of group O; h) loss of infectivity at acidic pH (pHs5); (I) induction of micro (interstitial pneumonia) and macroscopic lesions in 2-month-old pups; (j) adversely affecting the reproductive performance of pregnant sows with stillbirths, mummified fetuses, and living but weak pigs; k) cross-reaction with Lelystad reference serum (IPMA * Immuno Peroxidase Monolayer 35 Assay); l) cross-reaction with sera from animals with clinical infections (IPMA); m) serum from sows infected with this virus cross-reacts with LV; n) polyadenylated RNA genome approximately 15,000 bp in length (neutral agarose gel); O) replication by sub-genomic RNA sets at the 3 'end; p) nucleotide sequence with 8 ORFs; q) in a purified suspension which is electrophoresed in polyacrylamide gel and subsequent transfer by immunoblot is detected by means of a specific serum prepared in sows from our own laboratory and cross-reacting with the Lylysstad PPRS vims, 4 larger bands corresponding to proteins with an apparent molecular weight of 15000, 23000, 54000 and 66000 Dalton, and which are not detected in the negative control-1 control samples (uninfected macrophages); r) When ORF # 3-7 of this virus is compared to LVs and / or TVs, 10 95.5% homology at nucleotide level (114 different bp out of a total of 2599 bp) and 94.9% amino acid level homology are observed ( 51 different amino acids out of a total of 955); and s) vims belong to the genus Arterivims.

4.3 Applied Technique for Identifying Viruses p 15 4.3.1 Experimental Reproduction of the Disease in Drottioe Lakes I Sows bred as a cross breed breed of German landrace and large whites and tribes from farms with systematic serological control of Aujeszky's diseased vims, foot-and-mouth disease 1 vims, porcine parvovirus, classical swine fever virus, swine flu vims (types H1N1 and: H3N2) and transmissible gastroenteritis vims. In addition, the antibody titration assay is performed against the PRRS-causing virus. The animals are infected at a time between day 77 and day 90 of gestation (gestational period) with an isolate from alveolar macrophages from pig lungs, in one case intravenously (IV) and intranasally (IN), while in another case only intranasally (Example 2.1 ). Throughout the experiment, the animals' food intake, rectal temperature and clinical condition were monitored. In order to exclude the above-mentioned agents, blood samples are taken from 1 25 all the sows before the infection. They turn out to be seronegative against all agents. Similarly, after the experimental infection, all lakes are also found to be seronegative against 1 of all the viruses mentioned and seropositive to PRRS (measured against reference LV). The results obtained are shown in Table 1.

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Table 1

Surveys performed

Infectious HAI (1) HAI (2) NPLA (3) ELISA (4) SN (5) SN (6) IPMA (7) agent AS__Ur__Ml__Lu .__: __ j.u .__ Lu .__ i.u, MKS__Ml__Ml__Lu .__ Ml __.____ Lu. __I.u.

PP __: __ Ml__Ml ____ Ml__Ml ___ Ml__i.u, KSF__Ml__I.u. -__ Ml__Lu .___ Ml__i.u.

SI__Ml __-__ Ml__Lu ___ M ___ M ___ i.u.

TG I.u .__ Ml ___ Ml__I.u, I.u, ____-___ I.u.

PPRS i.u. i.u. i.u. i.u. I.u. I.u. + 5 Abbreviations used: AS Aujeszky's Sick MKS Foot And Mouth Disease PP Porein parvovirus KSF Classic Swine Flu 10 SI S Flu Influenza TC Transmittable Gastroenteritis PRRS The Porcine Reproduction and Respiratory Syndrome HAI Hasmagglutlnatlon peroxidase-linked assay perisidase-linked assay NPLA immunosorbent assay SN Serone neutralization assay IPMA Immuno peroxidase monolayer assay (t) Vannier et al. Rec Med Vet. 155 (2), 151-158 (1979) (2) Charley, B, Doctoral dissertation, Alfort (1976) 20 (3) Trepsta et al. Vet. Mcrob., 9, 113-120 (1984) (4) Elliot, M., J. Rech. Pore., 20,141-146 (5) Lucam, F., "Diagnostic sero-immunology of human and animal freezing," F. Bricout, L. Jouberf, J. M. Hureaux (1977) (6) Jiménez et al., J. Wot .. 60.131-139 (1986) 25 (7) Wensvoort et al. Vet QUARTERLY, b. 13. No. 3 (July 1991) negative + positive Iu not performed 30 4.3.2 Experimental reproduction of the disease in weaners

The purpose of this study is to investigate whether the virus that causes reproductive changes in sows is capable of inducing resplrative symptoms and macro- and microscopic lesions at lung level in 2-month-old piglets. A number of piglets are infected with this virus (the Spanish strain), intranasally, and are then killed at a specific time after the infection (Example 2.2).

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The most relevant results show that this virus at the macroscopic level gives rise to multiple (multiple) consolidation foci as well as microscopic interstitial pneumonia (Table 4). From a health point of view, no relevant clinical symptoms are observed.

5 4.3.3 Sensitivity to chloroform

This study is done to determine if the Isolated virus has a lipid capsule (envelope). The method is used according to Feldman, H. and Wang, S. 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 TCID ^ / ml, while the treatment with chloroform leads to a reduced titer of less than 101 · 3 TCID ^ / ml, on the basis of which it can be determined that the isolated virus has lipid capsule.

4.3.4 Sequencing of the viral genome This is done in five steps: 15 i) Purification of viruses

ii) Purification of the viral RNA

iii) Synthesis of cDNA

iv) Cloning and Characterization of the cDNA Clones * v) Sequencing and Comparison of the Sequences with LV's Sequences 1 20 j ad i) Purification of Virus I Viruses replicated on alveolar macrophages from pig lungs are "clarified" and up - concentrated by filtration on MILLIPORE® filters. Thereafter, virus is centrifuged in a 10-50% methyl trizamide gradient (SIGMA). After centrifugation, a band is obtained which is concentrated by centrifugation. With this purified virus, an electrophoresis is run in polyacrylamide gel, and an immunoblot is elicited with a specific serum, showing proteins with an apparent molecular weight of 15, 23, 54 and 66 kilo-Daltons (kDa).

ad iii Purification of viral RNA

A technique for selecting and purifying RNA is based on the fact that the RNA contains a poly (A) sequence at the 3-end. A commercial kit (Pharmacia) is used which only allows binding of the RNA-poly (A) chain to a cellulose oligo (dT) matrix and its subsequent elution.

35 ad iih Synthesis of cDNA

- A commercial kit (Boehringer Mannheim) is used, following the instructions provided. Briefly, the viral genomic RNA is incubated in the presence of an _L 'oligo (dT), dATP, dCTP, dGTP, dTTP and reverse transcriptase.

11 DK 173128 B1 by ivl Klonino and characterization of the cDNA clones cDNA is cloned into a vector derived from pUC18 and a series of clones are obtained Containing the complete nucleotide sequence corresponding to ORF # 3-7, cf. the sequence list. SEQ ID NO: 1-5.

5 ad v) Sequence series and sequence comparison with LV v.a. sequencing

The sequences SEQ ID NO. 1-5 corresponding to ORF # 3-7 of the virus isolated in our laboratory has been completely sequenced. The sequence list lists the resulting 10 cDNA sequences, as well as the amino acid sequences encoded by each ORF. The length of the total sequenced region is approximately 2599 nucleotides (nt). As can be seen, ORF 3 has a length of about 798 nt and encodes a protein of 266 amino acids.

ORF 4 has a length of approximately 552 nt and encodes a protein of 183 amino acids. The start of this ORF 4 is located in the ATG code. which is approximately 540 bp from the ATG start code of ORF 3. ORF 3 and ORF 4 share a sequence of about 246 nt.

ORF 5 has a length of about 606 nt and encodes a protein of 200 amino acids. In practice, the start code for this ORF 5 overlaps with the end code of the ORF 4 (they share the TG nudeotides, the 20 ATG code at the beginning of ORF 5, and the TGA end code in ORF 4). The ATG start code in ORF 5 is located about 1092 nt from the ATG start code in ORF 3.

ORF 6 has a length of about 522 nt and encodes a protein of 173 amino acids. This ATG start code for ORF 6 is located 8 nt upstream from the beginning of the TAG end code in ORF 5 (at about 1682 nt from the ATG start code in ORF 3).

ORF 7 has a length of about 387 nt and encodes a protein of 129 amino acids. The ATG start code for ORF 7 is located 5 nt upstream from the beginning of the TAA end code for ORF 6 (at about 2193 nt from the ATG start code of ORF 3).

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

v.b.1 Comparison with LV

When comparing the cDNA sequences of ORF # 3-7 of LV with the corresponding sequences of the virus isolated in our laboratory, we see the following: i) At nucleotide level, 114 nucleotides out of 2599 are possible differently, which corresponds ii) at the amino acid level, 47 amino acids out of 955 are possible differently, which corresponds to a homology of about 94.9%, iii) of the 47 different amino acids there are 35, must be considered as non-conservative substitutions, of which there are: 5 12 in the product of the ORF-3 gene (Fig. 1); 9 in the product of the ORF-4 gene (Fig. 2); 10 in the product of the ORF-5 gene (Fig. 3). although it should be noted that the product of the LV-ORF-5 gene contains an amino acid more than the product of ORF-5 from the Spanish virus, more specifically, amino acid # 35 (Asn) in LV's 10 ORF-5 product is not present in the product expressed by the Spanish virus; 4 in the product of the ORF-6 gene (Fig. 4); whereas the product of the ORF-7 gene contains no non-conservative substitution (Fig. 5).

(Iv) the partial homology for each of the products expressed by the different ORFs from the Spanish virus and LV is 93.6% for the ORF-3 and ORF-5 products, - 94.0% for the ORF The -4 product, 96.6% for the ORF-6 product and 99.2% for the ORF-7 product.

As mentioned above, the differences in amino acid sequence may be related to the higher pathogenicity of one strain relative to the other, as the virus isolated in our laboratory (the Spanish strain) is more pathogenic than other known PRRSs. viruses such as for example the French virus (Example 8) and LV (Table 6 of WO-92/21375).

The vaccine provides a vaccine capable of preventing the porcine reproductive and respiratory syndrome (PRRS). The vaccine has been shown to be effective in preventing reproductive disorders in lakes such as the birth of stillbirths, mummified or live but weak piglets, re-covering and similar problems as a result of the virus that causes PRRS. The vaccine induces cellular immunity in the vaccinated animals has also been confirmed.

The vaccine contains an appropriate amount of the viral PRRS antigen, from the Spanish strain and inactivated form, as well as an adjuvant and a preservative.

35 Studies conducted with these vaccines have confirmed the efficacy as shown in - Examples 7 and 8.

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Furthermore, the vaccine has been shown to be effective in avoiding re-coverage, which is otherwise required in infected sows.

In fact, sows vaccinated with the vaccines of the invention and infected with the PRRS-causing mucus were covered and became pregnant at the first ovulation after the previous birth and weaning of the piglets.

5.1 Ingredients 5.1, a The antigenic part 10 As an active component, the vaccine contains inactivated Spanish viral PRRS antigen at a concentration greater than or equal to 105 · 5 TCID ^ per vaccine dose. The inactivation may take place chemically, inter alia, by treatment with β-propiolactone or other conventional inactivating agents such as ethyleneimine or formaldehyde, or physically.

15 5.1 .b Adiuvanser

Although it is possible to use both aluminum hydroxide type, Quil A or mixtures thereof, and oil adjuvants in the formulation of the vaccine. It has been found (Example 4) that the best results are obtained with an oil adjuvant. In particular, an oil adjuvant consisting of a mixture of Marcol 52, Simulsol 5100 and Montanide 886 has produced very good results · 20 clays.

Marcol 52 is a low-density mineral oil sold by ESSO Espaftola, S.A.; Simulsol 5100 is a polyethoxyoleate ether sold by SEPiC and Montanide 888 is anhydrous mannitol ether octadecenoate of high purity which is sold by SEPIC.

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It has been found that the adjuvant plays a significant role in the effectiveness of the vaccine. Thus, a Comparative Example (Example 6) describes the vaccination of sows with two different vaccines, one sow was vaccinated using the oil adjuvant mentioned above (Ref. 1), and another sow was vaccinated using the adjuvant Munokynin® (Ref. 2). ). Although 30 both vaccinations led to seroconversion, it was found in an experimental infection that the sows that had been vaccinated with the vaccine Ref. 1 was protected against experimental infection with PRRS virus, while the other lakes that had been vaccinated with the vaccine Ref. 2, were not protected despite having antibodies against the current virus at the time of infection. This suggests or proves that an appropriate adjuvant may be of great importance in the modulation and stimulation of the immune response, in principle at the cellular immunity level. This has been further confirmed in comparative experiments (Example 7, Table 8) using the Spanish strain of PRRS virus as sows vaccinated and re-vaccinated with the vaccine Ref. 1 did not provide a serologic response at the time of infection, but was nonetheless protected.

However, it is also conceivable that the vaccine Ref. 2 (with Munokynin®) is capable of eliciting 5 cellular immunity. To this would be necessary to add substances that enhance the cellular response (CRP), ie. substances that strengthen the T helper cell subpopulations (Th-j and Th2), such as, for example, IL-1 (interleukin-1). IL-2, IL-4, IL-5, IL-6, IL-12, g-IFN (gamma-interferon), the tissue necrosis factor and similar substances. Obviously, it is also possible to add these PRC substances to vaccines with oil adjuvants, which would then control their cell-immune effect.

Other types of adjuvant may also be used which modulate and immunostimulate the cellular response, for example, MDP (muramyl dipeptide), ISCOM (Immuno Stimulating Complex) or liposomes.

jj 15 5.1 .c Preservatives m Any preservative that is usually used in the formulation of vaccines may be used. These include Thimerosal (the sodium salt of (2-carboxy-phenylthio) -ethyl mercury (ALDRICH).

20 5.2 Preparation of the Vaccine i

The vaccines of the invention can be prepared by mixing the antigenic portion containing inactivated viral antigen and another portion, the adjuvant, which may be oil-based, but not

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needs to be, depending on the adjuvant of choice. Optionally, CRP-25 substances may also be added to any of the (o parts or phases. In the case of an oil adjuvant, an emulsion, preferably a double w / o / w (water / oil / water), is obtained. emulsion (when the adjuvant is a mixture of Marcol 52, Simulsol 5100 and Montanide 888).

5.3 Control of the vaccine 30 In addition to the usual studies that the vaccine must be submitted before administration, ie. for (i) purity (bacteria, fungi, mycoplasmas and other viruses), (ii) identification, (iii) safety, (lv) strength and (v) physicochemical control, a number of field studies have been performed (Example 5 .b) in relation to the safety and efficacy of a total of 5 farms, where 508 sows were vaccinated and re-vaccinated with one of the vaccines of the invention, while the remaining 472 sows 35 were not vaccinated and included as control sows for the simultaneous assessment of the safety and efficacy of the vaccine; on one of the farms, after the vaccination process, the naturally occurring disease PRRS was detected in unvaccinated animals.

ICE

: 3f * j 15 DK 173128 B1 5.4 PosolQQi (dosage) and instructions regarding the vaccine's Intake

It has been found that a dose of 2 ml of oil vaccine with a concentration of inactivated viral antigen greater than or equal to 10 administered deeply intramuscularly, provides the protection of a very high percentage of vaccinated animals against PRRS.

5 The following vaccination program is recommended: • First vaccination: Vaccination of all breeding animals (sows and omer) with re-vaccination after 21 days. Then give a dose during each lactation (sows) and (for omer) every 6 months.

• Subsequent vaccinations: 10 - Animals to be used for breeding: First vaccination at 6 months of age, re-vaccination after 21 days.

- Sows: Vaccination is recommended during lactation, if possible 15 days before cover - Omer: Vaccination twice a year (every 6 months).

Alternatively, if the vaccine does not contain any CRP substances, these may be injected elsewhere than the inoculation but simultaneously.

Polyvafent Vaccines In a further aspect of the invention, bi- or multivalent combination vaccines of various porcine pathogens are provided which contain, in addition to the inactivated viral PRRS antigen (the Spanish strain), one or more of the pathogens listed below.

Thus, bi- or multivalent vaccines containing the inactivated viral PRRS antigen and one or more of the following pathogens may be prepared: Actinobacillus pleuropneumonlae, Haemophilus parasuls, Porcin parvovirus, Leptospira, Escherichia coli, Erysipelothrix rhuslopathchla, Paste , Porcine respiratory coronavirus, Rotavirus or the pathogens that cause Aujeszky's disease, swine flu or transmissible gastroenteritis.

30 EXAMPLES

Example 1 Virus Isolation 1.A Sample Preparation

From the lungs of a stillborn pig, offspring of a sow with classic symptoms of PRRS (sow 35 did not have antibodies to Aujeszky's disease, porcine parvovirus, foot-and-mouth disease, classical swine fever, swine flu (types H1N1 and H3N2) and transmissible gastroenteritis), a 10% suspension is prepared in the culture medium DMEM supplemented with an antibiotic solution (PEG) consisting of 1000 IU / ml penicillin, 1 mg / ml streptomycin and 0.5 mg / ml gentamicin in a lung ratio: DMEM solution of 1:10 (W / V). The resulting suspension is homogenized and left for 1 hour at room temperature (20-22 ° C). The homogenate is frozen and thawed twice, centrifuged and the supernatant stored at -70 ° C for use in infecting the alveolar macrophages from pig lungs.

5 Similarly, specimens from the lung are prepared from a live-born pig that died a few hours after birth. Furthermore, blood is either withdrawn which is mixed with anticoagulant for the isolation of blood plasma virus or which is not mixed with anticoagulant to obtain serum.

10 1 B Preparation of alveolar macrophages from ariseluna

Alveolar macrophages are obtained from the lungs of pigs that are seronegative to Aujeszky's disease, porcine parvovirus, foot-and-mouth disease, classical swollen fever, swine flu (types H1N1 and H3N2) and transmissible gastroenteritis. The pigs used are between 7 and 8 weeks old. Prior to the ^ 15 extraction of the lungs, the animals are anesthetized with phenobarbital sodium and sacrificed. Immediately after, after binding under the larynx, the lungs are extracted along with the trachea. The extra cured lung is washed externally with physiological saline solution and washed successively | with 50 ml of PBS supplemented with 2% PEG solution of antibiotics until a total of 500 ml of PBS is used. The cells resulting from these rinses are centrifuged for 10 min. at 300 g.

The centrifugation washing step is repeated two more times. The resulting cells are washed with PBS

.L

and PEG solution of antibiotics and resuspended in the DMEM medium (DMEM supplemented with non-essential amino acids (GIBCO), 1% 1 mM sodium pyruvate and 1% 2mM glutamine), 10% fetal calf (FCS) and a 1% o PEG solution of antibiotics. The cells are counted in Newbauer chambers and for this purpose, a 1/1 O dilution of the macrophage suspension is prepared by adding I 25 of 0.4 ml of DMEM medium and 0.5 ml of trypan blue solution to 0.1 ml of macrophage suspension. A cell number of 1 -1.2 x 10 9 is obtained.

^ 1 .C Virus Isolation

A 25 cm2 culture bottle containing a culture of the previously prepared alveolar macrophages from pig lungs (3 x 10 6 cells / ml) in DMEM medium and 10% FCS is infected with 1 ml of the homogenate of a sample derived from the lungs on a stillborn pig (Example 1 A). The homogenate is left in contact with the macrophage culture for 1 hour at 37 ° C, pH 7.0-7.4 buffered with CO 2, and incubated at 37 ° C for several days, observing the CPE induced by virus on the macrophages. 3-4 days dpi, a CPE of 70-80% is observed, which is why the 35 cultures were frozen at -80 ° C.

-ym

TEN

: ί = At the same time, a culture of alveolar macrophages is produced from uninfected pig lungs, which culture is used as negative control.

IBS

17 DK 173128 B1

The isolated virus is subcultured and it is observed that the CPE from the second dpi is 100%. Viruses are frozen at -80 ° C for later identification and characterization. After the fourth passage in macrophages, similar titrations are performed in 96 well / well microplates 5 and an average titer of 1056TCID5o / ml is obtained according to the method of Reed & Muench (Am. J. Hyg., 27, 493-497, 1938 (it must be 1983 !!).

A sample of the Isolated Virus (Spanish strain) designated PRRS-CY-218-JPD-P5-6-91, which is Isolated from a Pig's Lung, is able to experimentally reproduce the disease and is deposited with ECACC on June 29, 1993 with number V93070108.

Similarly, the vims are isolated from live and stillborn piglets, offspring of lakes that are experimentally infected.

Example 2

Identification and characterization of viruses

Example 2.1 Experimental reproduction of the disease in pregnant sows Twelve sows are used, cross-breeding of German landrace and large whites, derived from farms with systematic serological control of viruses from Aujeszky's disease, foot-and-mouth disease, porcine parvovl-20 intoxication, classical swine fever, swine flu (types H1N1 and H3N2) and transmissible gastroenteritis. Furthermore, the antibody evaluation test is performed against the virus that causes PRRS.

The lakes are transferred to the research center's security stables one week before the infection and placed in separate stables. At a time between days 77 and 90 of the gestation, so Nos. 53, 76, 8, 25 62, 91, 93 and 19 are infused intravenously (IV) and intranasally (IN) twice with 5 ml of the Spanish strain of PRRS virus , isolated on alveolar macrophages from pig lungs and bearing the designation PRRS-CY-218-JPD-P5-6-91. from the fourth pass on macrophages filtered through a 200 nm filter and with a titer of 105 · 6 TCID ^ / ml. The remaining 5 lakes (Nos. 14, 40, 13, 30 and 85) are inoculated between days 65 and 85 in the 5 ml virus station, only IN.

30

During the experiment, food intake, rectal temperature and clinical condition of the animals are monitored daily, as well as reproductive disorders or changes (premature or late gestation, live but weak piglets, stillborn piglets, mummified fetuses and healthy piglets).

In order to exclude the pathogens mentioned above, blood samples were taken from the sows before and after the infection, showing that the animals were seronegative before and after the infection, and seropositive to PRRS after the infection (see Table 1, section 4.3.1).

35 18 DK 173128 B1

The reproductive results are shown in Tables 2 and 3. As can be seen in Table 2, among the 93 piglets born there were 15 mummified, 35 stillborn, 22 who were born alive but died on the third day, and 21 who survived the first week.

5

Some lakes showed signs of lack of appetite for 2 * 4 days, on days 6 and 8 after infection, while other lakes showed signs of lack of appetite on day 2 after the infection. No cases of elevated temperature (hypertemia) were observed. 4 lakes (Nos. 8, 62, 92 and 93) departed prematurely (1-6 days premature), while 3 lakes departed 1-2 days late.

10

Among the live-born piglets, 1 or 2 pr. litter eye-edema. The weak piglets showed a lack of coordination, paresis (paralysis) of the hindquarters, teasing brushes and myoclonia (muscle twitching). By necroscopy of some of the stillborn and infirm piglets, the presence of a clear fluid in copious quantities in the thoracic cavity (thoracic 15 cavity) was observed. Healthy piglets born of Infected mothers and killed on 8-12. the day of their lives showed gray areas of consolidation. By microscopy, the most significant change was observed to be mild multifoci interstitial pneumonia with enlargement of the alveolar septi as a result of infiltration of or with mononuclear cells. These lesions were seen in all the animals examined in this experiment.

20

As can be seen in Table 3, out of 65 piglets, there were 36 stillborns, 26 alive but weak-borns who died on the 2nd day after birth, and 3 who survived the first week. One sow died 12 days prematurely (# 40), while the others departed 1 or 2 days prematurely. The clinical symptoms of weak piglets are similar to those observed with IN + IV. Interstitial pneumonia was also observed. The infected lakes showed no signs of appetite or hyperthermia.

The most relevant difference between the two infection systems is that in infectious IN + IV mummified piglets are observed and that 1-2 of the live-born piglets in each litter exhibit eye distress.

30

In summary, it is concluded that at about 80 days of gestation via both infection pathways (IN and IV), by infection with the virus isolated from animals that have been naturally infected (the Spanish strain) , reproduction of the disease PRRS is observed in pregnant sows and a high proportion of mummified fetuses, stillborn piglets and live but weak piglets are observed in a condition very similar to that observed in acute natural infectious outbreaks. It is advisable to infect artificial IN as this is the natural field infection.

(M

In this way, and therefore the most suitable way to assess the effectiveness of the vaccine.

li In T? i 19 DK 173128 B1 By this experiment it has also been possible to establish a model for experimental infection in pregnant sows which allows assessment of the effectiveness of the vaccine.

Table 2 5 ___ IN + IV___

So Infection Faring Number of Mummy- Weak, Dead- Apparent Interstitial Time (Gestational Pig in Fice-Dead Born Healthy Pigs Tiel No. (Gestational- all accounted for 48 hours dead live pneumonia days)) after 1 monl and 7th week day 53 77 115 9 3 2 0 - 4 4/4 76 77 116 11 2 4 1 - 4 fU

8 77 110 12 2 4 1 - 5 5/5 62 80 113 16 3 6 4 3 3/3 91 90 109 14 1 8 1 4 i.u.

93 88 110 17 4 2 11 - - i.u.

19 1 88 116 14 4__1__8 - 1 I.u.

93 15 17 35 5 I 21

Table 3 | _ | _ | _i__IN ____

So Infection Faring Number of Mummy- Weak, Dead- Apparent Interstitial Time (Gestational Pig in Fice-Dead Born Healthy Pigs Tiel No. (Gestational- all accounted for 48 hours dead live pneumonia days)) after 1 moni 2nd and 7th week day 14 65 111 15 - - 12 - 3 iu

40 82 102 12 - 12 i.u.

13 80 113 12 10 2 i.u.

30 80 113 15 12 3 i.u.

85 I 85 112 11__4__7 - - i.u.

65 - 26 1 36 1 - 3 10 i.u. In both tables, "not investigated" means 20 DK 173128 B1

Example 2.2 Experimental reproduction of the disease in petty theories

The purpose of this experiment is to investigate whether the isolated virus (Spanish strain) that causes reproductive disorders in lakes is capable of causing clinical symptoms as well as macroscopic and microscopic lesions in the lungs of 2-month-old 5 piglets. In 10 piglets, IN is infected with 5 ml virus, the Spanish strain, with a titer of 105 · 6 TCIDjo / ml, and 6 other piglets act as control animals (all derived from 2 litters). The animals are killed on day 3.7. 8, 9 and 11 µl (after the infection). Table 4 shows the results obtained.

Table 4

Pig No. Infected / Control dpi (days after Antibodies Interstitial Isolation of (I / K) infection) pneumonia virus 2 I 3-2 + lu.

12 I 3-2 + i.u.

11 K 3 - i.l. i.u.

1 I 7 1:80 4+ + 14 I 7 1:80 2+ + 13 K 7 - i.l.

10 I 8 1: 160 4+ + 15 I 8 1: 160 2+ + 1 8 K 8 - i.l.

17 K 8 i.l.

4 I 9 1 160 4+ + ,, 5 K 9 - i.l.

] 20 I 11 1: 160 4+ + i 18 I 11 1: 320 3+ + 9 I 11 1: 320 3+ 6 K 11 - i.l.

10 i.l. means "no lesions" i.u. means "unexpressed" 2+ means mild interstitial pneumonia Z 3+ means intermediate interstitial pneumonia 4+ means severe Interstitial pneumonia 15

During the 11 days of the experiment, neither clinical respiratory symptoms nor hyperthermia were observed, although the infected animals lost weight compared to the uninfected animals. By microscopy, the most relevant aspect is found to be the presence of multiple consolidation foci in the lungs, crowded or swollen lymph nodes in the mandibola (lower jaw), 20 and any intestinal hemorrhagia (bleeding). At the microscopic level, interstitial pneumonia is observed.

21 DK 173128 B1

Viruses are isolated from the solutions obtained by rinsing the infected animals' lungs in a fresh culture of macrophages, but no viruses are isolated from the control animals, where no seroconversion or macro- or microscopic lesions at lung level are isolated.

5 At cell counts on the rinse fluid from the infected animals' lungs, 30% of dead cells (macrophages) are found, which can be a crucial factor in secondary infections due to the destruction of an immunological key defense element (macrophages).

The absence of respiratory symptoms may be due to the fact that the experimental infections have been carried out in 10 stables, where they are disinfected continuously, and therefore the bacterial concentration is much lower than in flocks living under field conditions.

Example 2.3 Sensitivity to chloroform

The method according to Feldman, H. and Wang, S. (Section 4.3.3) is used and the result is that the isolated virus has a lipid capsule as a titer drop of 4 log10 occurs from the control cultures to the chloroform-treated.

Example 2.4 Sequencing of the Viral Aenome Purification of Vim 20 The virus replicated on alveolar macrophages from pig lungs is purified by filtration and centrifugation in a 10-50% metrizamide gradient (SIGMA), resulting in a band which is again centrifuged, cf. Section 4.3.4. An electrophoresis I polyacrylamide gel is run with the purified virus and an immunoblot is developed with a specific serum showing proteins with an apparent molecular weight of 15, 23, 54 and 66 kDa (kilo-Dalton).

25

ίΠ Oprensnlno of the viral RNA

The viral RNA is purified using a commercial kit (PHARMACIA), binding the poly (A) tail of RNA to a cellulose oligo (dT) matrix and subsequently eluting it.

30 ίίΩ Synthesis of cDNA

A commercial kit (BOEHRINGER MANNHEIM) is used, cf. section 4.3.4 iii. ivl Cloning qq characterization of the cDNA clones cDNA is cloned into a vector derived from pUC18, and a series of clones are obtained containing the complete nucleotide sequence corresponding to ORF # 3-7.

V) Sequencing and comparison of the sequences with LVs

The results of the sequencing of cDNA derived from the virus isolated in our laboratory (ORF # 3-7), as well as the comparison with the LV sequence, are discussed in section 4.3.4 v. amino acid level is about 94.9% homologous! and a total of 5 47 different amino acids, 35 of which represent non-conservative substitutions. These differences in amino acid levels may be responsible for the different pathogenicity of various isolated PRRS virus strains.

Example 3 Formulation of a vaccine

An anti-PRRS vaccine is prepared in the form of an emulsion, following the procedure described below.

A culture of alveolar macrophages from pig lungs is infected with MOI (multiplicity order infection) of 0.001 and incubated at 37 ° C for 24 hours, after which the culture medium is replaced with the infectious medium (DMEM supplemented with 2% FCS). The culture is incubated for 4 days at 37 ° C until 1 is observed 70-80% CPE. An IPMA test is then performed to confirm the identification. Viruses are collected by vacuum aspiration and frozen at -80 ° C.

The viral suspension intended to be included in the vaccine should have a minimum tear of 105 · 5 TCID 50 / ml (before Inactivation) and must not be contaminated with bacteria, fungi, 1 mycoplasmas or other viruses. If the titer is lower, it must be adjusted by concentrating the antigen.

To inactivate the viral suspension, 2% * β-propiolactone solution is added and stirred overnight at 4 ° C, keeping the pH of 7.4 by adding 0.5 N NaOH. After the inactivation period is completed, the viral suspension is stored at 37 ° C for 1 hour.

Next, the following are prepared: - (a) an antigenic phase of the inactivated viral antigen at a minimum concentration of - 105 · 5 TCID / dose and the preservative; and b) an oil phase consisting of Marcol 52, Simulsol 1500 and Montanid 888.

The constantly stirred aqueous phase is slowly added to the reaction vessel containing the oil phase, which is also stirred. After addition is complete, stirring is continued for 10 minutes.

Particularly preferred vaccines against PRRS (which are capable of preventing PRRS) comprise per dose of 2 ml: m »mi 23) 17% of an antigenic phase containing: i) the viral PRRS antigen in the DMEM culture medium, the Spanish strain, inactivated with β-propiolactone at a minimum concentration of ........................... ........................ 0.5 x 106 TCIDgo, and 5 ii) thimerosal .............. .................................................. .................... 0.01%; and b) 47% of an oil phase Containing: i) Marcol 52 ......... .................................................. ...................... 790.0 mg ii) Simulsol 5100 .................... .................................................. ....... 70.0 mg iii) Montanide 888 ................................... ......................................... 80.0 mg 10

The oil / water phase ratio is given as weight / volume ratio (W / V).

This vaccine is designated MSDRef. 1. The vaccine is subjected to appropriate control prior to use.

Similarly, another vaccine containing adjuvanted Munokynin® (aluminum hydroxide and Quil A, supplier; American Cyanamid) is prepared, maintaining the amount of inactivated virus. This vaccine is called MSD Ref. 2nd

Example 4

Evaluation of the adjuvant

A field study is carried out with a total of 128 sows, of which 49 are vaccinated with a dose of the vaccine designated MSDRef. 1, 50 lakes are vaccinated with a dose of the vaccine designated MSD Ref. 2 (Munokynin®) and the remaining 29 are not vaccinated and act as 25 control animals. After 22 days, the sows are re-vaccinated with a dose of the vaccine in question.

The following parameters are examined: 1. Serological response by IPMA determination at the following times T0: Vaccination and blood sampling 30 T22 'Re-vaccination and blood sampling

Tsi: Blood sampling 50 days after vaccination 2. General type reactions (appetite, hyperthermia, etc.)

The results obtained are shown in Tables 5 and 6, which show the proportion of sows with positive serological response (Table 5) and the arithmetic mean of the serological titres obtained (Table 6).

24 DK 173128 B1

Table 5

Proportion of animals with serological reaction + T0 T22 T51 MSP Ref. 1 __; ___ 59% ______ 100% _ MSP Ref, 2 ___: ___ 40% __ 87% _

Control 5

Table 6

Arithmetic mean of the serological titres MSP Ref. 1 __; __ 58 _200_ MSP Ref. 2 ____; __ 37___133_

Control - 10 No significant local or general reactions are observed.

The results show that a positive seroconversion is achieved with both vaccines, albeit somewhat Ί higher with MSD Ref. 1 (olle adjuvant). Upon re-vaccination, a higher serotonin version percentage is observed and the arithmetic mean of the titers obtained is higher than in animals vaccinated with MSD Ref. First

m

Example 5 Safety for lakes

Example 5A At the Laboratory Level 20 Example 5A.1 First aneurysm of lakes

18 first-born lakes (German landrace X large white) are selected from a pig farm, and are divided into two stables with 9 lakes per day. barn in such a way that the sows vaccinated with the same vaccine (MSD Ref. 1 or -2) in Example 3 above are placed in the same barn.

25 - Nine sows are vaccinated deep IM with a dose of 2 ml of the vaccine MSD Ref. 1 containing 105 · 5 TCIDg / dose of inactivated virus titer, and re-vaccinated with a second dose of the same titer zim 25 days later. The other nine lakes are vaccinated and re-vaccinated on the same days with the vaccine MSD Ref. 2 (dose of 2 ml, 105 · 5 TCID + / dose inactivate virus titre).

During the first 5 days after inoculation, the following is observed: 5 a) Local reaction

Macroscopic observation of the site of inoculation and palpation, comparing the degree of inflammation with objects of known size, b) General reaction

Macroscopic observation of the animals and assessment of their appetite. In negative cases, check the 10 rectal temperature every 12 hours until the hyperthermia or other negative symptoms have disappeared.

It turns out that there is a slight inflammatory reaction in some lakes, predominantly in the inoculation site, but it disappears in all cases within a few days; no purulent formation (pus-) is observed. Only one of the sows refused to eat all the feed at the first feeding after inoculation, but the food intake was normal at the subsequent feeding so that no rectal temperature was required. No significant differences are observed in response to the different vaccines being tested, and on this basis it can be concluded that both vaccines are safe.

20

Example 5A.2 Thirty-three lakes

Seven pregnant sows (German landrace X large white) from a pig farm are selected at random: 6 first-born sows of about 9 months and 1 multi-term sow of 3 years and 7 months.

25

Only the vaccine designated MSD Ref. 1 is used.

The sows are deeply vaccinated IM with a dose of 2 ml vaccine containing inactivated virus with a titer of 105 · 5 TCID® / dosIs and 15 days later re-vaccinated with a dose of the same 30 titer.

In the first 5 days after inoculation, the following observations are made: a) Local reaction

Macroscopic observation of the inoculation site and palpation, the degree of inflammation being noted and compared to objects of known size.

b) Loss of appetite

Macroscopic observation of the animals and examination of appetite c) Rectal temperature 26 DK 173128 B1 Measurement of rectal temperature 24 hours after vaccination and 24 hours after re-vaccination.

A slight local reaction is observed in two of the animals, but which is not serious due to small size and disappears within a few days. No cases of appetite and hyperthermia 5 were observed. On this basis, it can be established that the vaccine is safe.

Example 5.B Safety qq field efficiency

This study is done on 5 farms listed below. A variable number of sows from each farm are deeply vaccinated IM with a dose of 2 ml of the vaccine MSD Ref. 1 containing a titer of 10 inactivated virus of 105 · 5 TCID ^ / dose of re-vaccination 21 days later with a different dose of the same titer, while the other sows are not vaccinated and used as control animals:

Farm Number of vaccinated Number of control animals Total j RAMON DEL QUINTA 19 11 30 _ (Banyoles) _________ CAL SABATER 46 34 80 _ (Orriols) ________ E. CANELA 153 147 300 - (Preixana) _____ R. CUNILLERA 127 123 250 _ (L 'AIbi) ___________________ INVERSORS PICBER 163 157 320 (Bellpuig) ____ SUM 508 472 980

After observation of local and general reactions, the vaccine must be described as safe. Local reactions are observed in only 1% of animals. No changes were observed on the farms mentioned above with respect to the observed productive parameters when compared to their clinical history.

Regarding serologic response, some farms seroconversion to or in response to the vaccine, while the response to other farms is negative. However, this does not indicate a low degree of protection. since seronegative animals by experimental infection in the laboratory resist experimental infection (Examples 7 and 8).

In conjunction with the transfer of immunity from vaccinated mother sows to their offspring, there is a “25-fold decrease in antibody titers at 1 month of age.

TTI

ffi mim 27 DK 173128 B1 In vaccinated and re-vaccinated sows that are serologically positive, there is a large decrease in antibody titers 2 months after re-vaccination.

Example 6

Investigation of cell immunity

Five pregnant lakes (German landrace X large white) are used from a pig farm. The animals are transferred to the research center's security stables.

10 Randomly two sows who are vaccinated with the vaccine designated MSD Ref. 1. Another sow is vaccinated with the vaccine MSD Ref. 2. The two remaining lakes are not vaccinated.

The lakes are deeply vaccinated IM with a dose of 21 of the vaccine MSD Ref. 1 or -2 containing inactivated virus with a titer of 105 · 5 TCID +, and 20 days after, the sows are vaccinated with a different dose of the same titer.

Later, between days 77 and 90 of gestation, all sows IN are infected with 5 ml of virus PRRS-CY-218-JPD-P5-6-91 with a titer of 105 θ TCID ^ / ml. At the time of infection, it is confirmed that all of the vaccinated sows have antibodies to the PRRS-causing virus (positive serology). Table 7 shows the total reproductive results:

Table 7

Number Vaccine Number Number of healthy, Number of infirm, Number of pigs Number of sows and piglets live births live births still alive after stillborn piglets piglets the first week piglets 2 MSD 23 20 - 20 3 __Ref. 1_______ 1 MSD 12 - 6 6 __Ref. 2_________________ 2 - 24 7 - 17 25 It appears that the sows vaccinated with the vaccine MSD Ref. 1 (oil adjuvant) resists infection better than the sows vaccinated with the vaccine MSD Ref. 2 (aqueous adjuvant), which could mean that the adjuvant plays a significant role in establishing cellular immunity.

28 DK 173128 B1

Example 7

Effectiveness in Pregnant Viewers

Eleven breeding sows (German landrace X large white) are used from a pig farm. The animals are transferred to the research center's safe sheds.

5

Randomly, three lakes (So Nos. 57, 63 and 74) are selected, which are vaccinated with the vaccine MSD Ref. First

Three lakes (Nos. 15, 18 and 23) are vaccinated with the vaccine MSD Ref. 2, and the remaining 5 lakes (Nos. 14,40,13, 30 and 85) are not vaccinated.

10 The lakes are deeply vaccinated IM with a dose of 2 ml of the vaccines MSD Ref. 1 or -2 containing inactivate! virus with a titer of 10 ^ TCID ^ dose, and re-vaccinated with a second dose of the same titer 20 days later.

Local and general reactions are noted.

15

The serological response of the animals is examined by the IPMA test according to the following scheme: TQ: Blood sampling and vaccination

Tjo: Blood sampling and re-vaccination 1 T42: Blood sampling] 20 T7e: Blood sampling and experimental infection 'T8: Blood sampling after the experimental infection T25: Blood sampling after the experimental infection

Tgo: Blood sampling after the experimental infection * 25 The experimental infection is carried out in the research center's security stables. All the animals

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IN is infected with 5 ml of the virulent virus PPRS-CY-218-JPD-P5-6-91 with a titer of 10 ^ TCIDsc / ml. The serological response, as well as the number of live and stillborn piglets from each sow, are noted. Blood samples are taken from the pre and post colostrum piglets, and from the stillborn and the piglets killed at various times, samples of lung 30 and brain are taken for virus isolation and histological examination.

Table 8-10 below shows the results obtained: -ϊϋ 29 DK 173128 B1

Table 8

Serological results

No. To T20 T42 T78 T8 T25 T50 _15 _____: ___ 1/160 1/160 1/640 1/1280 i.u.

18 __, ___-___ 1/80. _ 1/80 1/320 1/640 1/320 23 __: __ 1/160 1/160 i.d .__ Ιό, __ Ld .__ Ld.

57 __-__ 1/160 1/160 1/320 ___ 1/320 1/1280 1/160 63 __-___ 1/80 1/80 1/160 1/640 1/1280 1/160 74 ____: __ 1/80 i.u. 1/160 1/640 1/2560 Lu._ c14 __- ___-_________ 1/1280 1/2560 i.u.

c40 __-__-__-__-__ i.u. 1/1280 i.u.

c13 __ ___-____-____-____ Lir ___ ___ 1/320 i.u.

c30 __: __- -___-___ i.u. 1/320 Lu .__ c85 i.u. 1/320 i.u.

I.u. means "not examined" 5 i.d. means "not pregnant" DK 173128 B1 0 g £

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Table 10

The serology of piglets A. Vaccinated sows

Animals Offspring No. Serology Serology Isolation of No. (number in pre-colo- post-colostrum virus birth order) strum 15® i.u. i.u. i.u.

_ ^ __ 3 days__10 days__ 18__1 ___: ___ Lu .___ Lu .___: _ __2 __- _____ i.u .__ Lu ___: _ __7 i.u. 1/640 1/160 __- __8__i.u. 1/2560 1/320 - __9 ___ i.u, 1/1260 1/640 - __10 i.u. 1 / 1280__1 / 320 __11__i.u .___ 1 / 1280-1 / 2560 1/640 -_ __12 iu__1 / 2560 ___ 1/640 _ "_ ____ 13 ___ Lu .__ £ 1 / 2560__1 / 160 - __14 Lu .__ 1/1280 1 / 160 ^ ___ 15 Lu. 1/2560 1/160 23 lay. 'LcL id Ld' ^^ __ 4 days__8 days__ 57 I 1 I - 1 / 320-1 / 640__1 / 320 __- __2___ 1 / 320-1 / 640 1 / 160-1 / 320 - __3 _____; ___ 1/640 ___ Lu __- __4 ___-__ 1 / 320-1 / 640 ___ Lu ___- 5 - 1/640 iu

____1 day__8 days__ 63 I 1 I i.u. £ 1/2560 __1 / 640 ______-_ __2 Ju ___ £ 1/2560 1/320 -_ __3 ___ | U__1 / 2560 1 / 320-1 / 640 __4__iu__1 / 2560 1 / 640-1 / 1280 __- __5__ 1 / 1280__ 1 / 320__ - __6__ 1 / 2560__1 / 640 ___- __7 ___ Lu__1 / 640-1 / 1280 1/320 ____- 8 iu 1/1280 Lu.

74a Lu - * 32 DK 173128 B1 B. Control lakes (not vaccinated)

Animal No. Offspring No. Serology Serology Isolation (birth-pre-colo- post-colo- of four-stream strum intoxication) __ = 4— _i___12t__38t__5 days 9 days__ C14 I 1 I i.u. ^ 1/2560> 1/2560 1/1280 1/640 __2 -> 1/2560> 1/2560 1/1280 1/1280 3> 1/2560> 1/2560 1/1280 1/1280 1/1280 c40 - iu

____9 days__ c13 I 1 I - ____ 1/160 __ + __2 __: __ 1/160 __- __3 __-___ 1/320 __- 4 - 1/320 c30 - I.u. i.u.

c85 i.u. i.u. i.u.

a: These lakes died due to a too high temperature in the environment. Caesarean sections corresponding to 1 112 gestation days 5 Lu: Not examined 1 ·: Negative J +: Positive 'I: Hours i.d. Not pregnant 10

The results obtained show that there is positive seroconversion against the virus that causes PRRS. In addition, there is a satisfactory response to experimental infection when compared to the control animals in which most fetuses died.

1 15 Thus, one must conclude that this vaccination is an effective precaution against PRRS.

Example 8

Efficacy of the vaccine against experimental infection when using another PRRS · causative agent (cross-protection) 20

The purpose of this experiment is to investigate the efficacy of the vaccine designated ~ MSD Ref. 1 in an experimental infection using 2 pathogenic strains of the PRRS-inducing virus.

! M

~ 25 The PRRS strains used are: = i) the Spanish strain, PRRS-CY-218-JPD-P5-6-91; and ΊΒΪ

H

33 DK 173128 B1 ii) the French tribe, SDRP II 8B, supplied by Dr. E. Albina at the Taboratoire Central de Recherches Avicole et Porcine, ”Ploufragan, France.

The vaccine used is MSD Ref. 1, the formulation of which is given in Example 5.

5

30 sows that are seronegative to the PRRS-inducing viruses are used and are at a stage in their cycle that does not include the period 10 days before to 10 days after cover, nor the period 10 days until 10 days after farrowing.

10 The animals are divided into four groups: A: 10 sows are vaccinated and re-vaccinated! M and infected IN with the Spanish strain PRRS-CY-218-JPD-P5-6-91.

B: 5 sows are not vaccinated and infected with the Spanish strain PRRS-CY-218-JPD-P5-6-91.

15 C: 10 sows are vaccinated and re-vaccinated iM and infected IN with the French strain SDRP II 8B.

D: 5 sows are not vaccinated and infected with the French strain SDRP II8B.

Twenty sows from groups A and C above are vaccinated with a dose of 2 ml of the vaccine designated MSD Ref. 1 deep IM and re-vaccinated 21 days later with the same dose of vaccine.

Thereafter, between days 70 and 80 at the gestation, all sows were experimentally infected IN with Group A and B: 5 ml of virus PRRS-CY-218-JPD-P5-6-91 with a titer of 105 · 8 TCID ^ ml; and Group C and D: 5 ml of virus SDRP II 8B with a titer of 105 · 8 TCID 2 / ml.

25

Parameters examined 1. Serological response by IPMA assay to the time points: T0: Blood sampling and vaccination Τ2Γ · Blood sampling and re-vaccination 30 T41: Blood sampling T ,: Blood sampling and infection Τμ7: Blood sampling 7 days

2. Measurement of rectal temperature, local reaction and general reaction for 4 days after vaccination and re-vaccination, or until (the elevated) temperature) or any clinical symptoms disappear.

3. Measurement of rectal temperature, observation of food intake and clinical symptoms for 6 days after the experimental infection.

34 EN 173128 B1 4. Detection of antibodies in serum and isolation of virus I serum and in whole blood monocytes.

5. Reproduction parameters at the time of farrowing, for example the number of live-born piglets, the number of stillborn or mummified piglets and the number of live but weak piglets that die within the first week.

6. Determination of serum concentration of virus present by titration on macrophages based on CPE.

7. Determination of viruses in the piglet's pleural fluid (pteura = mesothelioma) and lungs.

The results of the reproduction parameter study are shown in Tables 11-12 (infection with PRRS-CY-218-JPD-P5-6-91) and 13-14 (infection with SDRP in 8B).

Table 11

Vaccinated Lakes 15 Infection with PRRS-CY-218-JPD-P5-8-91

Sow No. Number of small- Of which live- Of which live, Of which live pigs in all healthy, equine bony stillbirths after the first '_______ (splay-legged) week I 37a 14_________________] 39__6___6 -: __: ______ 5_ II 86__12___8________2 1_______1____7 45________ _______ 2 _________ 8, ___ 19__10 ___ 9 ________ 1 __: _________-___________ 8__ _38__9__8 _____________ ^ ____- 1___8 41__7__5 __: ________ 2___5 71__y ______ 6 ______ 1 ____; _________ 4___6 36 10__8_____1__1_____2_____6____ 26b

Total 75 55 6 2 12 51 a: Another disease (not PRRS) b: Sick. died before infection% share of live piglets: 88% (51 survivors out of 75) 20

Here, a protection ratio of 68% is obtained when comparing the number of live-born pigs to the number of pigs surviving the first week. The fact that the experimental infection is far more powerful than the natural infection in the field, when considering the results above, gives even greater expectation of future protection possibilities.

59 τπ mem 35 DK 173128 B1

Table 12

Non-vaccinated lakes Infection with PRRS-CY-218-JPD-P5-6-91

Sow No. Anlal small- Of which live- Of which live, Of which live pigs in all healthy equine bony stillbirths after the first ____ (splay-legged) week 63__9 ____-______ - 1 _____ 8 __-_ 88__6 ___-__ 3 ___: ___ 3 __-_ -_______ 3 ____-__ 5 ______-_ 22__12____2________ - ______-______ 10__ ________1_ 28__7__3 __: __-__ 4__3 Total 42 5 6 1 30 4% share of live piglets: 9.5% (4 survivors out of 42)

The Spanish strain used in the infection is extremely pathogenic (90.5%) as only 4 pigs out of 42 survive the first week.

Table 13

Vaccinated lakes

Infection with SDRP II 8B

Sun No. Number of small- Of which Of which- Of which live. Of which, in live pigs, total healthy bony stillborns after the first ________________ r __ (splay-legged) __ week 51__13___10____2_____________1__10 27__14______12___1 -____ 1__11 16 __9 ___ 8 __: __: _ 1__7 17 __15 ____ 13 ____: ____ : __ 2___13 57__10__9 ___-__-__ 1___8 61__12__8__1 ____; __ 3 8 78__10 I 10 I - _I - I 10 52 ° _______ Total 105 I 83 | 4 | - | 11 | 86 0: Sick, dead from other cause% share of live piglets: 82% (86 survivors out of 105) 36 DK 173128 B1

About 82% protection is observed.

Table 14

Non-vaccinated lakes

5 Infection with SDRP II 8B

Sow No. of small- Of which Of which, Of which live, Of which live, Pigs alive, all healthy, equine bony stillbirths after the first (splay-legged) _week 62__12___7_______1_______4 -? ______ 81__15 ___ 13 ___............ ................ 2____________J3_______ 4__12 12L ___; ___________: ____________; __ 11 94__14___9____1 _______-______ 4___9 1__13__8__1__4 __: __ 8 Total 66 49 3 8 6 50% share of live piglets: 75.7% (50 survivors out of 66) m

Mortality of about 24.3% is observed. The pathogenicity of the French strain is 10 very weak (24.3%), compared with the corresponding results for the Spanish strain (mortality 90.5%). With regard to comparison of vaccinated and unvaccinated lakes in the case of the French tribe, the results are not very significant. At any rate, however, it can be noted that the pathogenicity of the vaccinated animals has been reduced to 17.8%.

|

"TH

37 GB 173128 B1 SEQUENCE LIST Number of sequences. 5

SEQ ID NO: 1 (i) Sequence Properties: (A) Length: 798 Base Pair 265 (266) Amino Acids (B) Type:

Nucleotide and related amino acid sequence (ii) Molecule type: cDNA from genome RNA and protein (vi) Natural origin: (A) Organism:

Arterivlrus sp; PRRS virus (PRRS = Porcine Reproductive and Respiratory Syndrome) (B) Tribe: "The Spanish Tribe" (C) 1 ndfvid / isolate: PRRS-CY-218-JPD-P5-6-91 corresponding to V93070108 filed with ECACC, European Collection of Animal Cell Cultures on 29 JUN 1993 (ix) Properties (A) Name / Code: ORF # 3 (ORF = Open Reading Frame) (D) Subcellular location of the gene product:

Membrane protein (x) Sequence description: SEQ ID N0: 1 38 DK 173128 B1 ATG GCT CAT CAG TGT GCA CGC TTC CAT TTT TTC CTC TGT AGC TTC ATC 48

With Ala His Gin Cys Ala Arg Phe His Phe Phe Leu Cye Ser Phe Ile 15 10 15 TGT TAC C TT GTf CAT AGT GCT TTG GCT TCG AAT TCC AAT TCT ACG CTA 96

Cys Tyr Leu Val His Ser Ala Leu Ala Ser Asn Ser Asn Ser Thr Leu 20 25 30 TGT TTT TGG TTT CCA TTG GCC CAC GGC AAC ACA TCA TTC GAG CTA ACC 144

Cys Phe Trp Phe Pro Leu Ala His Gly Asn Thr Ser Phe Glu Leu Thr 35 40 45 ATC AAC TAC ACC ATA TGT ATG CCC TGC TCT ACC AGT CAA GCG GCT CAC 192

Ile Asn Tyr Thr Ile Cys With Pro Cys Ser Thr Ser Gin Ala Ala Kis 50 55 60 CAA AGA CTC GAG CCC GGT CGT AAC ATG TGG TGC AGA ATA GGG CAC GAC 240

Gin Arg Leu Glu Pro Gly Arg Asn Met Trp Cys Arg Ile Gly His Asp 65 70 75 80 AGG TGT GAG G AA CGT GAC CAT GAT GAG TTG TCA ATG TCC ATT CCG TCT 288

Arg Cys Glu Glu Arg Asp His Asp Glu Leu Ser With Ser Ile Pro Ser Θ5 90 95 GGG TAC GAT AAC CTC AAA C TT GAG GGT TAT TAT GCT TGG C TG GCC TTT 336

Gly Tyr Asp Asn Leu Lys Leu Glu Gly Tyr Tyr Ala Trp Leu Ala Phe 100 105 110 TTG TCC TTT TCC TAC GCG GCC CAA TTC CAT CCG GAG TTG TTC GGA ATA '384

Leu Ser Phe Ser Tyr Ala Ala Gin Phe His Phe Glu Leu Phe Gly Ile 115 120 125 GGA AAC GTG TCG CGC GTC TTC GTG GAC AAG CAA CAC CAG TTC ATT TGC 432

Gly Asn Val Ser Arg Val Phe Val Asp List Gin Hi? Gin Phe Ile Cys 130 135 140 GCC GAG CAT GAT GGA CGA AAT TCA ACC ATA TCT ACC G AA TAT AAC ATC 480

Ala Glu His Asp Gly Arg Asn Ser Thr Ile Ser Thr Glu Tyr Asn Ile 145 150 155 160 j TCC GCA TTA TAT GCG TCG TAC TAC CAT CAC CAA ATA GAC GGG GGC AAC 528! Ser Ala Leu Tyr Ala Ser Tyr Tyr His His Gin Ile Asp Gly Gly Asn 165 170 175 TGG TTC CAT TTG GTG CTC AAC ATT TCA G AA TGG C TG CGG CCA TTC TTT 576

Trp Phe His Leu Glu Trp Leu Arg Pro Phe Phe Ser Ser Trp Leu Val 180 185 190 TCC TCC TGG CTG TGG TTT CTG AGG CGT TCG CCT GTA AGC CCT G TT TCT 624

Leu Asn Ile Ser Trp Phe Leu Arg Arg Ser Pro Val Ser Pro Val Ser 195 200 205 CGA CGC ATC TAT CAG ATA TTA AGA CCA ACA CGA CCG CGG CTG CCG GTT 672

Arg Arg Ile Tyr Gin Ile Leu Arg Pro Thr Arg Pro Arg Leu Pro Val 210 215 220 TCA TGG TCC TTC AGA ACA TCA ATT GTC TCC GAC CTC ACG GGG TCT CAA 720

Ser Trp Ser Phe Arg Thr Ser Ile Val Ser Asp Leu Thr Gly Ser Gin 225 230 235 240 CAG CGC AAG AGA ACA TTT CCT TCG GGA AGC CGT CTC AAT GTC GTG AAG 768

Gin Arg List Arg Thr Phe Pro Ser Gly Ser Arg Leu Asn Val Val List * 245 250 225 'Xi »CCG TCG GTA TTC CCC AGT ACA TTA CGA TAA 798

Pro Ser Val Phe Pro Ser Thr Leu Arg End 260 265 S | 39 DK 173128 B1

SEQ ID NO: 2 (i) Sequence Properties: (A) Length: 552 base pairs 183 amino acids (B) Type:

Nucleotide and related amino acid sequence (ii) Molecule type: cDNA from genome RNA and protein (vi) Natural origin: (A) Organism:

Arterivims sp .; PRRS virus (PRRS = the Porcine Reproductive and Respiratory Syndrome) (B) Tribe: "The Spanish Tribe * (C) Individual / isolate: PRRS-CY-218-JPD-P5-6-91 corresponding to V93070108 filed with ECACC , European Collection of Animal Cell Cultures on June 29, 1993 (ix) Features: (A) Name / Code: ORF # 4 (ORF = Open Reading Frame) (B) Location: about 540 base pairs from starting ATG- the code of ORF # 3 (SEQ ID NO: 1) shares a sequence of about 246 base pairs with SEQ ID NO: 1 (D) Subcellular location of the gene product:

Membrane Protein (x) Sequence Description: SEQ ID NO: 2 40 DK 173128 B1 ATG GCT GCG GCC ATT CTT TTC CTC CTG GCT GGT GCT CAA CAT TTC ATG 48

With Ala Ala Ala Ile Leu Phe Leu Leu Ala Gly Ala Gin His Phe Met 15 10 15 GTT TCT GAG GCG TTC GCC TGT AAG CCC TGT TTC TCG ACG CAT CTA TCA 96

Val Ser Glu Ala Phe Ala Cys List Pro Cys Phe Ser Thr His Leu Ser 20 25 30 GAT ATT AAG ACC AAC ACG ACC GCG GCT GCC GGT TTC ATG GTC CTT CAG 144

Asp Ile List Thr Asn Thr Thr Ala Ala Ala Gly Phe With Val Leu Gin 35 40 45 AAC ATC AAT TGT CTC CGA CCT CAC GGG GTC TCA ACA GCG CAA GAG AAC 192

Asn Ile Asn Cys Leu Arg Pro His Gly Val Ser Thr Ala Gin Glu Asn 50 55 60 ATT TCC TTC GGG AAG CCG TCT CAA TGT CGT G AA GCC GTC GGT ATT CCC 240

Ile Ser Phe Gly List Pro Ser Gin Cys Arg Glu Ala Val Gly Ile Pro 65 70 75 80 CAG TAC ATT ACG ATA ACG GCT AAT GTG ACC GAT GAA TCG TAT TTG TAC 288

Gin Tyr Ile Thr Ile Thr Ala Asn Val Thr Asp Glu Ser Tyr Leu Tyr 85 90 95 AAC GCG GAC TTG CTG ATG CTT TCT GCG TGC CTT TTC TAC GCT TCA GAA 336

Asn Ala Asp Leu Leu With Leu Ser Ala Cys Leu Phe Tyr Ala Ser Glu, 100 105 110 ^ ATG AGC GAA AAA GGC TTC AAA GTT ATC TTT GGG AAC GTC TCT GGC GTT 384

With Ser Glu List Gly Phe List Val Ile Phe Gly Asn Val Ser Gly Val 1 115 120 125 Ί GTT TCT GCT TGT GTC AAT TTT ACA GAT TAT GTG GCC CAT GTG ACC CAA 432! Val Ser Ala Cys Val Asn Phe Thr Asp Tyr Val Ala His Val Thr Gin 130 135 140 i CAT ACC CAG CAG CAT CAT CTG GTA ATT GAT CAC ATT CGG TTG CTG CAT 480

His Thr Gin Gin His His Leu Val Ile Asp His Ile Arg Leu Leu His 145 150 155 160 TTC TTG ACA CCA TCT ACA ATG AGG TGG GCT ACA ACC ATT GCT TGT TTG 528

Phe Leu Thr Pro Ser Thr With Arg Trp Ala Thr Thr Ile Ala Cys Leu 165 170 175 TTC GCC ATT CTC TTG GCG ATA TGA 552

Phe Ala Ile Leu Leu Ala Ile End 180 m 41 DK 173128 B1

Information on SEQ 10 no: 3 (i) Sequence properties: (A) Length: 603 (606) base pairs 200 amino acids (B) Type:

Nucleotide and associated amino acid sequence (ii) Molecule type: cDNA from genome RNA and protein (vi) Natural origin: (A) Organism:

Arterivirus sp .; PRRS virus (PRRS = Porcine Reproductive and Respiratory Syndrome) (B) Tribe: "The Spanish Tribe" (C) Individual / isolate: PRRS-CY-218-JPD-P5-6-91 corresponding to V93070108 deposited with ECACC , European Collection of Animal Cell Cultures on 29 JUN 1993 (ix) Properties: (A) Name / Code: ORF # 5 (ORF 1 Open Reading Frame) (B) Location: starting ATG code located around 1092 base pairs from the starting ATG code of ORF # 3 (SEQ ID NO: 1); the start ATG code partially overlaps the TGA end code in ORF # 4 = SEQ ID NO: 2 (TG common) (D) Subcellular location of the gene product:

Membrane protein (x) Sequence description: SEQ ID NO: 3 42 DK 173128 B1 ATG AGA TGT TCT CAC AAA TTG GGG CGT TTC TTG ACT CCT CAC TCT TGC 48

With Arg Cys Ser His List Leu Gly Arg Phe Leu Thr Pro His Ser Cys 15 10 15 TTC TGG TGG C TT TTT TTG CTG TGT ACC GGC TTG TCC TGG TCC TTT GTC 96

Phe Trp Trp Leu Phe Leu Leu Cys Thr Gly Leu Ser Trp Ser Phe Val 20 25 30 GCT GGC GGC AGC AGC TCG ACA TAC CAA TAC ATA TAT AAC TTA ACG ATA 144

Ala Gly Gly Ser Ser Ser Thr Tyr Gin Tyr Ile Tyr Asn Leu Thr Ile 35 40 45 TGC GAG CTG AAT GGG ACC GAC TGG TTG TCC AAC CAT TTT GAT TGG GCA 192

Cys Glu Leu Asn Gly Thr Asp Trp Leu Ser Asn His Phe Asp Trp Ala 50 55 60 GTC GAG ACC TTT GTG CTT TAC CCG GTT GCC ACT CAT ATC CTC TCA CTG 240

Val Glu Thr Phe Val Leu Tyr Pro Val Ala Thr His Ile Leu Ser Leu 65 70 75 80 GGT TTT CTC ACA ACA AGC CAT TTT TTT GAC GCG CTC GGT CTC GGC GCT 288

Gly Phe Leu Thr Thr Ser His Phe Phe Asp Ala Leu Gly Leu Gly Ala 85 90 95 I GTG TCC ACT ATA GGA TTT GTT GGC GGG CGG TAT GTA CTC AGC AGC GTG 336

Val Ser Thr Ile Gly Phe Val Gly Gly Arg Tyr Val Leu Ser Ser Val 100 105 110

Tj TAC GGC GCT TGT GCT TTC GCA GCG TTC GTA TGT TTT GTC ATC CGT GCT 384

Tyr Gly Ala Cys Ala Phe Ala Ala Phe Val Cys Phe Val Ile Arg Ala 115 120 125 GTT AAA AAT TGC ATG GCT TGC CGC TAT GCC CAC ACC CGG TTT ACC AAC 432

Fall List Asn Cys With Ala Cys Arg Tyr Ala His Thr Arg Phe Thr Asn 130 135 140 TTC ATT GTG GAC GAC CGG GGG AGA ATC CAT CGG TGG AAG TCT CCA ATA 480

Phe Ile Val Asp Asp Arg Gly Arg Ile His Arg Trp List Ser Pro Ile 145 150 155 160 GTG GTA GAG AAA TTG GGC AAA GCT G AA GTC GGT GGC GAC CTT GTC ACC 528

Val Val Glu Lys Leu Gly Lys Ala Glu Val Gly Gly Asp Leu Val Thr 165 170 175 T ATC AAA CAT GTC GTC CTC GAA GGG GTT AAA GCT CAA CCC TTG ACG AGG 576

Ile Lys His Val Val Leu Glu Gly Val Lys Ala Gin Pro Leu Thr Arg 180 185 190 1 in ACT TCG GCT GAG CAA TGG GAA GCC TAG 603

Thr Ser Ala Glu Gin Trp Glu Ala End 195 200 43 DK 173128 B1

SEQ ID NO: 4 (i) Sequence Properties: (A) Length: 522 base pairs 173 amino acids (B) Type:

Nucleotide and related amino acid sequence (ii) Molecule type: cDNA from genome RNA and protein (vi) Natural origin: (A) Organism:

Arterivirus sp .; PRRS virus (PRRS = Porcine Reproductive and Respiratory Syndrome) (B) Tribe: "The Spanish Tribe" (C) Individual / isolate: PRRS-CY-218-JPD-P5-6-91 corresponding to V93070108 deposited with ECACC , European Collection of Animal Cell Cultures on JUN 29, 1993 (ix) Features: (A) Name / Code: ORF # 6 (ORF = Open Reading Frame) (B) Location: about 1682 base pairs from starting ATG- the code in ORF # 3 (SEQ ID NO: 1) or 8 base pairs upstream from the beginning of the TAG end code in ORF # 5 = SEQ ID NO: 4 (D) Subcellular location of the gene product:

Membrane Protein (x) Sequence Description: SEQ ID NO: 4 44 DK 173128 B1 ATG GGA AGC CTA GAC GAT TTT TGC AAT GAT TCT ACC GCC GCA CAA AAG 48

With Gly Ser Leu Asp Asp Phe Cys Asn Asp Ser Thr Ala Ala Gin List 15 10 15 CTT GTG CTA GCC TTT AGC ATT ACA TAT ACA CCT ATA ATG ATA TAC GCC 96

Leu Val Leu Ala Phe Ser Ile Thr Tyr Thr Pro Ile With Ile Tyr Ala 20 25 30 CTT AAG GTG TCA CGC GGC CG A CTC CTG GGG CTG TTG CAC ATC CTA ATA 144

Leu Lys Val Ser Arg Gly Arg Leu Leu Gly Leu Leu His Ile Leu Ile 35 40 45 TTC CTG AAT TGT TCT TTC ACA TTC GGA TAC ATG ACA TAT GTG CGT TTT 192

Phe Leu Asn Cys Ser Phe Thr Phe Gly Tyr With Thr Tyr Val Arg phe 50 55 60 CAA TCC ACC AAC CGT GTC GCA CTT ACT CTG GGG GCT G TT GTC GCC CTT 240

Gin Ser Thr Asn Arg Val Ala Leu Thr Leu Gly Ala Val Val Ala Leu 65 70 75 80 CTG TGG GGT GTT TAC AGC TTC ACA GAG TCA TGG AAG TTT GTT ACT TCC 288

Leu Trp Gly Val Tyr Ser Phe Thr Glu Ser Trp Lys Phe Val Thr Ser 85 90 95 AGA TGC AGA TTG TGT TGC CTA GGC CGG CGA TAC ATT CTG GCC CCT GCC 336

Arg Cys Arg Leu Cys Cys Leu Gly Arg Arg Tyr Ile. Leu Ala Pro Ala 100 105 110 CAT CAC GTA G AA Eight GCT GCA GGT CTC CAT TCA ATC CCA GCG TCT GGT 384 2 His His Val Glu Ser Ala Ala Gly Leu His Ser Ile Pro Ala Ser Gly i 115 120 125 j AAC CGA GCA TAC GCT GTG AGA AAG CCC GGA CTA ACA TCA GTG AAC GGC 432 in Asn Arg Ala Tyr Ala Val Arg Light Pro Gly Leu Thr Ser Val Asn Give ,, 130 135 140 yim fli STT SP S? A CGG AGC CTC GTG CTG GGC GGC AAA CGA GCT 480 L u Val Pro Giy Leu Ser Leu Val Leu Gly Gly List Arg Ala 145 150 155 160 GTT AAA CGA GGA GTG GTT AAC CTC GTC AAG TAT GGC CGG TAA 522

Val Lys Arg Gly Val Val Asn Leu Val Lys Tyr Gly Arg End 165 170 m 45 DK 173128 B1

SEQ ID NO: 5 (i) Sequence Properties: (A) Length: 387 base pairs 128 (129) amino acids (B) Type:

Nucleotide and associated amino acid sequence (ii) Molecule type: cDNA from genome RNA and protein (vi) Natural origin: (A) Organism:

Arterivlrus sp; PRRS virus (PRRS = Porcine Reproductive and Respiratory Syndrome) (B) Tribe: "The Spanish Tribe" (C) Individual / isolate: PRRS-CY-218-JPD-P5-6-91 corresponding to V93070108 filed with ECACC , European Collection of Animal Cell Cultures on JUNE 29, 1993 (ix) Features: (A) Name / Code: ORF # 7 (ORF = Open Reading Frame) (B) Location: about 2193 base pairs from starting ATG- the code in ORF # 3 (SEQ ID NO: 1); The ATG start code is located about 5 base pairs from the start of the TAA end code in ORF # 6 = SEQ ID NO: 4; shares a sequence of about 246 base pairs with SEQ ID NO: 1 (D) Subcellular location of the gene product: nucleocapsid protein (x) Sequence description: SEQ ID NO: 5 46 DK 173128 B1 ATG GCC GGT AAA AAC CAG AGC CAG AAG AAA AAG AAA AGT GCA GCT CCG 48

With Ala Gly List Asn Gin Ser Gin List List List List Ser Ala Ala Pro 15 10 15 ATG GGG AAT GGC CAG CCA GTC AAT CAA CTG TG C CAG TTG C TG GGT GCA 96

With Gly Asn Gly Gin Pro Val Asn Gin Leu Cys Gin Leu Leu Gly Ala 20 25 30 ATG ATA AAG TCC CAG CGC CAG CAA CCT AGG GGA GGA CAG GCC AAA AAG 144

With Ile List Ser Gin Arg Gin Gin Fro Arg Gly Gly Gin Ala List List 35 40 45 AAA AAG CCT GAG AAG CCA CAT TTT CCC TTA GCT GCT G AA GAT GAC ATC 192

List List Pro Glu List Pro His Phe Pro Leu Ala Ala Glu Asp Asp Ile 50 55 60 CGG CAC CAC CTC ACC CAG ACC G AA CGT TCC CTC TGC TTG CAA TCG ATC 240

Arg His His Leu Thr Gin Thr Glu Arg Ser Leu Cys Leu Gin Ser Ile 65 70 75 80 CAG ACG GCT TTT AAT CAA GGC GCA GGA ACT GCG TCG CTT TCA TCC AGC 288

Gin Thr Ala Phe Asn Gin Gly Ala Gly Thr Ala Ser Leu Ser Ser Ser 85 90 95 GGG AAG GTC AGT TTT CAG GTT GAG TTC ATG CTG CCG GTT GCT CAT ACG 336

Gly List Val Ser Phe Gin Val Glu Phe With Leu Pro Val Ala His Thr 100 105 110 GTG CGC CTG ATT CGC GTG ACT TCT ACA TCC GCC AGT CAG GGT GCA AGC 384

Val Arg Leu Ile Arg Val Thr Ser Thr Ser Ala Ser Gin Gly Ala Ser 115 120 125 TAA 387

Than

Claims (30)

Vaccine against the porcine reproductive and respiratory syndrome (PRRS), characterized by comprising an appropriate amount of the viral PRRS antigen or virus, from the Spanish strain strain PRRS-CY-218-JPD-P5-6. 91, which is deposited with ECACC with the deposit number V93070108, in an inactivated form, as well as an appropriate adjuvant and, if desired, a preservative. Vaccine according to claim 1, characterized in that it contains a dose of inactivated virus 10 of at least 105 · 5 TCID / µg / dose. Vaccine according to claim 1, characterized in that viruses have been grown in a culture of alveolar macrophages from pig lungs. Vaccine according to claim 1, characterized in that viruses have been cultured in a co-culture of alveolar macrophages from murine and ST cells (ATCC CRL1746 ST). Vaccine according to claim 1, characterized in that viruses have been grown in a culture of ST cells (ATCC CRL 1746 ST). 20 Vaccine according to claim 1, characterized in that viruses have been grown in hybrid cells of alveolar macrophages from pig lungs which are fused to ST cells by hybridization. Vaccine according to claim 1, characterized in that viruses have been grown in hybrid cells of alveolar macrophages from pig lungs which are fused with the L-14 cell line (ECACC No. 91012317) or with the cell line Jag-1. Vaccine according to claim 1, characterized in. that virus has been cultured in ST cells or in another porcine cell line that has introduced the genes encoding membrane receptors for PRRS virus from the alveolar macrophages from pig lungs. Vaccine according to claim 1, characterized in that the adjuvant is an oil adjuvant. Vaccine according to claim 9, characterized in that the oil adjuvant consists of a mixture of Marcol 52, Simulsol 5100 and Montanide 888. DK 173128 B1 Vaccine according to claim 1, characterized in that it is an emulsion of (i) an aqueous antigenic phase containing the inactivated vim and (ii) an oil phase containing adjuvant. Vaccine according to claim H. characterized in that the emulsion consists of 53% by volume 5 of an aqueous phase containing the inactivated virus and 47% by weight of an oil phase containing the adjuvant. Vaccine according to claims 1-12, characterized in that it is capable of inducing cellular: immunity in vaccinated animals. 10 Vaccine according to claim 13, characterized in that it further contains substances for enhancing the cellular response (CRP, Cell Response Potentiation) which enhance the immune effect of the cell such as IL-1, IL-2, IL-4, IL -5, li-6, IL-12, g-IFN, the tissue necrosis factor and similar substances. 15 Vaccine according to claim 1, characterized in that the adjuvant is aqueous. Vaccine according to claim 15, characterized in that it further contains substances for! enhancement of the cellular response (CRP), which induces cellular immune effect of the cell, such as IL-1, IL-2, IL-4, IL-5, li-6, IL-12, g-IFN , the tissue necrosis factor and similar substances. Vaccine according to claim 1, characterized in that the adjuvant is capable of modulating and immunostimulating the cellular response, for example MDP, ISCOM or liposomes. 25 Vaccine against the porcine reproductive and respiratory syndrome (PRRS), characterized in that it is an emulsion comprising a) 53% of an aqueous phase containing the viral PRRS antigen of the Spanish strain designated PRRS-CY-218-JPD -P5-6-91, deposited with ECACC with the deposit number V93070108, inactivated form in the culture medium DMEM In a mlmum Concentratlon of 105 · 5 TCID 50 / dose, and b) 47% of an oil phase containing a mixture of Marcol52, Simulsol5100 and Monta-nide 888. Vaccine according to claim 18, characterized in that it can induce cell immunity in * vaccinated animals. 5 * 9 m DK 173128 B1 ECACC with the deposit number V93070108, inactivated form, as well as an appropriate adjuvant and, if desired, a preservative, characterized by comprising the following steps: 1. cultivation of the PRRS-inducing virus bearing the designation PRRS-CY-218-JPD P5-6-91, and deposited with ECACC with the deposit number V93070108, in an appropriate cell system, 2) collection of virus from the cell system when a minimum titre of 105 · 5 TCID 2 / ml is obtained, 3. inactivation of virus by physical or chemical methods; and 4. mixing the Inactivated virus with the adjuvant and preservative. Vaccine according to claim 19, characterized in that it further contains substances for enhancing the cellular response (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, the tissue necrosis factor and similar substances. 21. Bi- or multivalent vaccine against the porcine reproductive and respiratory syndrome and one or more other porcine infections, characterized in that it contains an appropriate amount of the viral PRRS antigen or virus, from the Spanish strain designated PRRS-CY -218-JPD-P5-6-91, deposited with ECACC with the deposit number V93070108, inactivated form plus one or more porcine pathogens. 10 Vaccine according to claim 21, characterized in that it contains at least one porcine pathogen selected from the group consisting of Actinobacillus pieuropneumoniae, Haemophilus parasuis, Porcin parvovirus, Leptospira, Escherichia coli, Erysipelothrix rhusiopathiae, Pasteurelia multo-cida , Rotavirus, or against the pathogens that cause Aujeszky's disease, swollen infections or transmissible gastroenteritis. A method of preparing a vaccine against the porcine reproductive and respiratory syndrome (PRRS), which contains an appropriate amount of PRRS virus, from the Spanish strain, designated PRRS-CY-218-JPD-P5-6-91 , and which is deposited with The method according to claim 23, characterized in that viruses have been cultured in: i) a culture of alveolar macrophages from pig lungs, or ii) a co-culture of alveolar macrophages from pig lungs and ST cells (ATCC CRL1746 ST) or (iii) a ST cell culture (ATCC CRL 1746 ST), or (iv) a culture of hybrid cells of alveolar macrophages from pig lungs fused with ST cells, or (v) a culture of hybrid cells of alveolar macrophages from pig lungs fused with cell line L-14 (ECACC No. 91012317) or with cell line Jag-1, or in DK 173128 B1 vi) ST cells or any other porcine cell line that has had the genes encoding PRRS the viral membrane receptors of the alveolar macrophages from pig lungs. 25. PRRS-inducing virus, the Spanish strain having characteristics substantially similar to those of the virus bearing the designation PRRS-CY-218-JPD-P5-6-91 and deposited with ECACC with the deposit number V93070108. The virus of claim 25, characterized by comprising a substantial portion of at least one of the 10 DNA sequences of FIG. 1-5. The virus of claim 25 or 26 in inactivated form and useful vaccine formulations against the porcine reproductive and respiratory syndrome. A virus according to claim 25 or 26 in inactivated form and useful in bi or mutant vaccine formulations against the porcine reproductive and respiratory syndrome and other porcine infections. 29. DNA sequence derived from PRRS quadruple virus, the Spanish strain designated 20 PRRS-CY-218-JPD-P5-6-91, deposited with ECACC with the deposit number: | "V93070108. The DNA sequence of claim 29, characterized by comprising a substantial portion of at least: one of the DNA sequences of FIG. 1-5. 25 Μ mm