CA2410694A1 - Porcine reproductive and respiratory syndrome virus (prrsv) dna vaccines - Google Patents

Abstract

DNA vaccines comprising expression vectors and nucleic acid sequences encoding ORF proteins from porcine reproductive and respiratory syndrome virus (PRRSV) are described. The invention also provides methods of using these vaccines to immunize swine against porcine reproductive and respiratory syndrome (PRRS) and to detect PRRSV infection. The present invention also relates to host cells transformed with these vectors. The present invention further relates to the use of these vaccines to immunize swine against PRRSV infection, as transfer vectors to construct other expression vectors, and for diagnostic purposes.

Description

PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME
VIRUS (PRRSV) DNA VACCINES
FIELD OF THE INVENTION
The present invention relates to swine reproductive and respiratory syndrome.
BACKGROUND OF THE INVENTION
PRRS
Porcine reproductive and respiratory syndrome (PRRS) is a viral disease characterized by inappetence and severe reproductive failure, including late term abortions, increased numbers of still-born, mummified, and weak-born piglets, and respiratory problems affecting pigs of all ages (Goyal (1993) J. Vet. Diagn. Invest. 5:656-664). It is an economically important viral disease that affects swine worldwide.
PRRSV
Porcine reproductive and respiratory syndrome virus (PRRSV) has been found to be the causative agent of PRRS. The PRRSV is a small enveloped positive-stranded RNA virus and is presently classified within the family Arteriviridae, along with lactate dehydrogenase elevating virus (LDV), equine arteritis virus (EAV), and simian haemorrhagic fever virus (Meulenberg et al., (1993) Virology 192:62-72). Together with members of the family Coronaviridae, these viruses have been recently grouped into the order Nidovirales (Cavanagh (1997) Arch. Virol.
142:629-633).
The genome of PRRSV is about 15 kb in length and contains eight open reading frames (ORFs).
ORF 1 a and ORFIb, situated at the 5' end of the genome, represent nearly 75%
of the viral genome and code for proteins with apparent replicase and polymerase activities (Meulenberg et al., (1993)

2 Virology 192:62-72; Conzelmann et al., (1993) Virology 193:329-339). Six putative structural proteins have been identified and assigned to distinct smaller ORFs, namely, ORFs 2 to 7, located at the 3' end of the genome (Mardassi et al., (1995) Arch. Yirol. 140:1405-1418; Mardassi et al., (1996) Virology221:98-112;Meulenbergetal., (1995) Virology206:155-163). The major structural proteins consist of a 25 kDa envelope glycoprotein (GPS), an 18-19 kDa unglycosylated membrane protein (M), and a 15 kDa nucleocapsid (N) protein, encoded by ORFs S, 6, and 7, respectively (Mardassi et al., (1995) Arch. Yirol. 140:1405-1418; Mardassi et al., (1996) Virology 221:98-112;
Meulenberg et al., (1995) Virology 206:155-163). In addition, the expression products of ORFs 2,

3, and 4, with respective apparent molecular masses of 30, 45, and 31 kDa, are also incorporated into virus particles as membrane-associated glycoproteins and are designated as GPz, GP3, and GP4, respectively (Meulenberg et al., (1996) Virology 225:44-51; Van Nieuwstadt et al., (1996) J.
Virology 70:4767-4772).
Strains of PRRSV
Although the clinical syndromes associated with PRRS V infection are similar in North America and Europe, strains from the two continents are distinct. North American and European serogroups have been established on the basis of reactivities both to polyclonal pig sera (Wensvoort et al., (1992) J. Yet. Diagn. Invest. 4:134-138) and to MAbs directed to both the N and M
proteins (Nelson et. al., (1993) J. Clin. Microbiol. 31:3184-3189; Drew et al., (1995) J. Gen. Virol.
76:1361-1369; Dea et al., (1996) J. Clin. Microbiol. 34:1488-1493).
DNA sequence analysis of both North American and European strains revealed high genomic variations (Mardassi et al., (1994) J. Gen. Virol. 75:681-685; Mardassi et al., (1994) .I. Clin.
Microbiol. 32:2197-2203; Meng et al., (1995) Arch. Yirol. 140:745-755;
Murtaugh et al., (1995) Arch. Yirol. 140:1451-1460).
The prototype European strain of PRRSV is the Lelystad virus (LV). This virus was described in EP 0 587 780 B1.

significant antigenic differences have also been reported among North American isolates (Nelson et. al., (1993)J. Clin. Microbiol. 31:3184-3189; Deaetal., (1996).1. Clin.
Microbiol. 34:1488-1493;
Yoon et al., (1995).1. Vet. Diagn. Invest. 7:386-387). Furthermore, genomic variations have also been reported among North American virulent and non-virulent isolates, in spite of limited antigenic variations (Kapur et al., (1996) .I. Gen. Virol. 77:1271-1276; Meng et al., (1995) J. Gen. Virol.
76:3181-3188). As yet, a correlation between genomic and antigenic variations among North American isolates remains to be demonstrated.
North American and European strains of PRRS V display a high degree of variability in their ORF
2, 3, 5, and 7 coding regions with less than 60% amino acid identities (Mardassi et al., (1995) Arch.
Virol. 140:1405-1418; Meng et al., (1995) J. Gen. Yirol. 76:3181-3188; Meng et al., (1995) Arch.
Yirol. 140:745-755; Murtaugh et al., ( 1995) Arch. Yirol. 140:1451-1460).
Among North American isolates, the ORFs 3, 4, and 5 show the highest degrees of diversity (Meng et al., (1995) .I. Gen.
Virol. 76:3181-3188; Kapur et al., (1996) J. Gen Yirol. 77:1271-1276).
Unique PRRSV strains have been isolated in Quebec (Dea et al., (1992) Can.
Vet. J. 33:801-808;
Mardassi et al., (1994) Can. J. Vet. Res. 58:55-64; Mardassi et al., (1994) J.
Gen. Yirol. 75:681-685). One such strain was adapted to grow in cell culture and is known as the Quebec reference cytopathic strain IAF-Klop (Mardassi et al., (1995) Arch. Virol. 140:1405-1418).
ORFS
ORES encodes a 25 kDa glycosylated envelope protein (GPs). GPs is present rather abundantly in the virion and is partially exposed in association with the lipidic envelope (Mardassi et al., (1996) Virology 221:98-112; Meulenberg et al., (1995) Virology 206:155-163). It has been demonstrated that GPS is the major viral envelope glycoprotein (Loemba et al., (1996) Arch.
Yirol. 141:751-761;
Nelson et al., ( 1993) J. Clin. Microbiol. 31:3184-3189; Meulenberg et al., (1995) Virology 206:155 163). A hypervariable region with antigenic potential has been identified within the N-terminal half of GPS of North American field isolates (Meng et al., (1995) Arch. Virol.
140:745-755).

4 'The amino acid sequence identity between the Quebec IAF-Klop strain and the reference US strain is 89% for the ORFS-encoded glycoprotein, whereas the predicted product of the Quebec and LV
strains displays only 52% amino acid identity (Mardassi et al., (1995) Arch.
Virol. 140:1405-1418;
Meng et al., (1995) Arch. Virol. 140:745-755).
Recently, it has been demonstrated that the Quebec IAF-Klop strain can be differentiated from the US modified live vaccine strain (MLV) derived from the ATCC VR-2332, in two ways: (1) serologically using monoclonal antibodies directed towards the N and M major structural proteins;
and (2) by restriction fragment length polymorphisms of the ORF 6 and 7 genes (Gagnon and Dea ( 1998) Can. J. Vet. Res. 62:110-116); Pirzadeh et al., ( 1998) Can. J. Yet.
Res. 62:in press; Dea et al., (1996).1. Clin. Microbiol. 34:1488-1493).
A hypervariable region with antigenic potential has been identified within the N-terminal half of the ORES gene productof North American field isolates (Pirzadeh et al., (1998) Can. J. Vet. Res. 62:in press).
Vaccines A number of commercial vaccines exist against PRRSV infection. Vaccines comprising inactivated or attenuated infectious agent ATCC VR-2332 are claimed in the international patent application WO 93/03760. One such vaccine, RespPRRSTM (Boehringer Ingelheim Inc., St-Joseph, MO), a modified live-attenuated virus vaccine derived from the US reference strain following successive passages in monkey kidney cells, is described in Murtaugh et al., (1995) Arch.
Yirol. 140:1451-1460. This vaccine, also known as IngelvacTMMLV, which is presently used widely in pig farms of the U.S. and Canada, protects pigs against respiratory signs following PRRSV
infection. Unfortunately, many problems have been reported following the use of this vaccine in pregnant sows, and it is not approved for use in adult breeding stock. Also, it is unknown whether the vaccine strain gains virulence following consecutive passages in pigs.
Vaccines similar to RespPRRSTM have been developed using different strains of PRRSV. U.S.

5 Patent No. 5587164 describes a vaccine formulated using a PRRSV isolate designated as ISU-P and having Accession number ATCC VR 2402. Another vaccine, PrimePacrM, (Schering-Plough Corp., New Jersey, USA) has been derived from a North American strain that does not possess the epitope of the N protein defined by the SDOW 17 anti-N Mab (Nelson et al., (1993) J.
Clin. Microbiol.
31:3184-3189). These vaccines present the same problems as the RespPRRSTM
vaccine.
The vaccine CyBIueTM (Cyanamid, Spain), is an inactivated vaccine from a Spanish PRRSV grown in primary pig alveolar macrophage culture. This vaccine uses macrophage, which can be a source of infection by an advantageous agent,. Also, one cannot discriminate between animals vaccinated with this vaccine and naturally-infected animals. Furthermore, this vaccine is only used against European strains: it does not protect against North American strains.
The use ofbaculovirus-expressed ORF7 and ORF3 proteins of a Spanish strain of PRRSV have been reported to induce protective immunity in pregnant sows (Plana-Duran et al., ( 1997) Virus Genes 14:19-29). Virus challenge studies have shown that pregnant sows were partially protected against reproductive failure without developing a noticeable neutralizing humoral response. Since, however, only the protein is given to the animal, the animal does not have a cellular response, merely a humoral response.
U.5 Patent No. 5,690,940 describes PRRSV vaccines involving PRRSV strains of low pathogenicity that do not cause clinical symptoms of PRRS. One such virus, designated MN-Hs, has ATCC
Accession No. VR2509.
U.S Patent No. 5,695,766 describes the "Iowa strain" of PRRSV and a vaccine comprising an inactivated or attenuated virus of this strain. The use of live attenuated viral vaccines is generally quite effective as the viruses mimic a natural infection. A serious disadvantage of such vaccines, however, is their pathogenicity in immunosuppressed recipients exposed to environmental stress, such as poor housing and over-crowding often prevalent in intensive animal raising operations. This can be of great concern in veterinary medicine where clinical outbreaks are sometimes reported

6 shortly after prophylactic immunization. These vaccines also require special handling to maintain viability and to avoid tissue culture contaminants.
Inactivated vaccines, however, require additional immunizations, disadvantageously contain adjuvants, are expensive to produce, and are labourious to administer.
Furthermore, some infectious virus particles may survive the inactivation process and may cause disease after administration to the animal.
Administration of live modif ed vaccines are also problematic because they may result in virus persistence, which in turn contributes to generation of mutants due to the selective immune pressure on the resident variants. Persistently-infected animals may eventually shed newly generated mutants, particularly in the case of unstable pathogens such as RNA viruses.
Such mutants may be responsible for new outbreaks.
Also, it is well known that such traditional attenuated live virus vaccines can revert to virulence resulting in disease outbreaks in inoculated animals and the possible spread of the pathogen to other animals.
Monoclonal antibodies to the ORES product of the Quebec IAF-Klop strain have been produced and shown to have virus-neutralizing activity in vitro (Pirzadeh and Dea (1997) J.
Gen. Virol. 78:1867-1873). Of the five monoclonal antibodies produced, only two reacted with the modified live attenuated vaccine strain ATCC VR-2332, while none reacted to the prototype European strain LV.
This suggests that ORES contains serotype-specific linear neutralizing epitopes. Immunizing pigs with recombinant ORFS protein failed to trigger the immune system to produce neutralizing antibodies (Loemba et al., ( 1996) Arch. Virol. 141:751-761; Pirzadeh and Dea (1998) J. Gen. Virol.
79:289-299).
Production and purification of large quantities of viral particles for use in whole viral inactivated vaccines or their immunogenic structural proteins is economically unfeasible for low yield viruses

7 such as PRRSV.
Improved vaccines might be constructed based on recombinant DNA technology.
These vaccines would contain only the necessary and relevant immunogenic material needed to elicit a protective immune response against the PRRSV pathogens, or the genetic information encoding such material, and would not display the above-mentioned disadvantages of the live or inactivated vaccines.
DNA Immunization The term DNA immunization refers to the induction of an immune response to a protein antigen expressed in vivo subsequent to the introduction of sequences encoding an antigenic polypeptide (Davis and Whalers Use plasmid DNA for direct gene transfer and immunization G. Dickson, ed.
(London: Chapman & Hall, 1995)368-387). Direct gene transfer by intramuscular and/or intradermic injection of DNA encoding an antigenic protein may be used for the purpose of immunization. The resulting in situ production of the protein can involve biosynthetic processing and post-translational modifications (glycosylation processing, adequate protein folding).
Consequently, both humoral and cell-mediated immune responses to viral surface antigen have been obtained after the expression of a transferred gene, and these are dose dependent (Tighe et al., ( 1998) Immunology Today 19: 89-97). Alternatively, DNA may be used to transfect cells in culture (ex vivo), which are then reintroduced into the body (indirect gene transfer).
Since genetic immunization does not require the isolation of proteins, it is especially valuable for proteins that may lose conformational epitopes when extracted and purified biochemically.
The use of DNA immunization against viral pathogens has obtained encouraging results in laboratory animals such as mice (Davis et al., (1994) Vaccine 12:1503-1509;
Martins et al., (1995) ,l. Virol. 69:2574-2582; Ulmer et al., (1993) Science 259:1745-1748; Yokoyama et al., (1995) J.
Virol. 69:2684-2688), guinea pigs (Bourne et al., (1996) J: Inf. Dis. 173: 800-807), and rabbits (Sundaram et al., (1996) NA. Res. 24:135-137). Recently, the use of DNA
immunization has been reported in pigs for protection against Aujeszky's disease (Gerdts et al., (I997) .l. Gen. Virol.
78:2147-2151 ).

8 Diagnostics The tests presently used for serological diagnosis of PR.RSV infection cannot discriminate between vaccinated and naturally-infected pigs. There is a need for serological and molecular procedures that would allow for distinction between a vaccine strain and emerging variants possibly responsible for new outbreaks or persistence of the disease in vaccinated herds.
Increasing commercial exchanges between Europe and North America also require that effective diagnostic tools be made available to prevent transmission of serotypes that do not exist in the importing countries.
Considering the increased incidence of PRRSV infection through the world, a need remains for an effective vaccine against PRRSV infection. The vaccine should be safe for use in swine, including pregnant sows and suckling, unweaning, and growing pigs. As well, a test is needed for the serological diagnosis of PRRSV infection that can differentiate between vaccination and naturally-occurring strains of PRRSV.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention. Publications referred to throughout the specification are hereby incorporated by reference in their entireties in this application.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide porcine reproductive and respiratory syndrome virus (PRRSV) DNA vaccines. In accordance with an aspect of the present invention there is provided a DNA vaccine comprising an expression vector and a nucleic acid sequence encoding one or more PRRSV ORFs or fragments thereof, wherein upon administration into a swine free from PRRSV infection, said swine is protected from infection by PRRSV.

9 In accordance with another aspect of the present invention there is provided a DNA vaccine selected from the group comprising: pRc/CMV2; pRc/CMV3; pRc/CMV4; pRc/CMVS; pRc/CMV6;
pRc/CMV7; pAdCMVS/ORF2; pAdCMVS/ORF3; pAdCMVS/ORF4; pAdCMVS/ORFS;
pAdCMVS/ORF6; pAdCMVS/ORF7; AdCMVS/ORF2; AdCMVS/ORF3; AdCMVS/ORF4;
AdCMVS/ORFS; AdCMVS/ORF6; and AdCMVS/ORF7.
In accordance with another aspect of the present invention there is provided a composition comprising (i) a DNA vaccine comprising an expression vector and a nucleic acid sequence encoding one or more PRRSV ORFs or fragments thereof, wherein upon administration into a swine free from PRRSV infection, said swine is protected from infection by PRRSV; and (ii) a pharmaceutically acceptable carrier, buffer, solvent, or diluent.
In accordance with another aspect of the present invention there is provided a kit for the administration of a DNA vaccine, wherein said DNA vaccine comprises an expression vector and a nucleic acid sequence encoding one or more PRRSV ORFs or fragments thereof, wherein upon administration into a swine free from PRRSV infection, said swine is protected from infection by PRRSV, comprising: (i) the DNA vaccine, either lyophilized or in solution;
(ii) contained in a container, such as a syringe, pipette, eye dropper, vial, nasal spray, or inhaler; and (iii) instructions for use.
In accordance with another aspect of the present invention there is provided a serum suitable for treatment of swine infected with PRRSV, comprising the semi-purified blood serum of a mammal inoculated with a DNA vaccine comprising an expression vector and a nucleic acid sequence encoding one ormore PRRSV ORFs or fragments thereof, wherein upon administration into a swine free from PRRSV infection, said swine is protected from infection by PRRSV.
In accordance with another aspect of the present invention there is provided a method of using a DNA vaccine to protect swine against PRRS, comprising administering to said swine an effective amount of a PRRS V DNA vaccine, wherein said DNA vaccine comprises an expression vector and a nucleic acid encoding one or more PRRSV ORFs or fragments thereof, wherein upon administration into a swine free from PRRS infection said swine is protected from infection.
In one embodiment, the present invention provides DNA vaccines comprising an expression vector and a nucleic acid sequence encoding one or more PRRS V ORFs or fragments thereof, wherein upon administration into a swine free from PRRSV infection, said swine is protected from infection by PRRSV.
In a specific embodiment of the present invention, the expression vector may be a plasmid. The expression vector may further comprise transcription regulatory elements operably linked to the nucleic acid sequence, including the CMV promoter or bovine growth hormone terminator. In a preferred embodiment, the plasmid is pRc/CMV or pAd/CMVS. In specific preferred embodiments, the PRRSV DNA vaccine is pRc/CMV2, pRc/CMV3, pRc/CMV4, pRc/CMVS, pRc/CMV6, pRc/CMV7, pAdCMVS/ORF2, pAdCMVS/ORF3, pAdCMVS/ORF4, pAdCMVS/ORFS, pAdCMVS/ORF6, or pAdCMVS/ORF7.
In another specific embodiment, the expression vector may be a replication-defective adenovirus.
Preferably, the adenovirus lacks at least a portion of the E1 region. The expression vector may further comprise adenovirus encapsidation and packaging signals, adenovirus tripartite leader sequences, and major late enhancer sequences. In a preferred embodiment, the adenovirus is Ad/CMVIacZ. In specific preferred embodiments, the PRRSV DNA vaccine is AdCMVS/ORF2, AdCMVS/ORF3, AdCMVS/ORF4, AdCMVS/ORFS, AdCMVS/ORF6, or AdCMVS/ORF7 The PRRSV ORFs or fragments thereof may be from any strain of PRRSV. In a preferred embodiment, the PRRSV strain is IAF-Klop.
The ORFs may be any ORF or combination of ORFs from a PRRSV virus, including ORFs 2, 3, 4, 5, 6, and 7, or any combination thereof.

The nucleic acid may be cDNA, genomic DNA, or a cDNA/genomic DNA hybrid. The nucleic acid may further comprise the first ATG colon of the ORF, a Kozak motif , and/or the C-terminal stop colon of the ORF. The nucleic acid is under the transcriptional control of a promoter functional in eukaryotic cells. In a preferred embodiment, the promoter is the CMV promoter.
S Another embodiment of the present invention provides for a composition comprising the PRRSV
DNA vaccines of the present invention and a pharmaceutically acceptable carrier, buffer, solvent, or diluent.
In a further embodiment, the present invention provides for host cells that has been transformed by PRRSV DNA vaccines.
In yet another embodiment, the present invention provides for kits for the administration of PRRS V
DNA vaccines and for use in the detection of PRRSV in a sample.
In yet a further embodiment, the present invention provides for serum suitable for treatment of swine infected with PRRSV, comprising the semi-purified blood serum of a mammal inoculated with a DNA vaccine comprising an expression vector and a nucleic acid sequence encoding one or more PRRSV ORFs or fragments thereof, wherein upon administration into a swine free from PRRSV
infection, said swine is protected from infection by PRRSV.
A further embodiment ofthe present invention provides a method of using a DNA
vaccine to protect swine against PRRS, comprising administering to said swine an effective amount of a PRRSV DNA
vaccine, wherein said DNA vaccine comprises an expression vector and a nucleic acid encoding one or more PRRSV ORFs or fragments thereof, wherein upon administration into a swine free from PRRS infection said swine is protected from infection. The DNA vaccine may be administered by intramuscular injection, subcutaneous injection, intradennal introduction, impression through the skin, inhalation, intraperitoneally, or intravenously. The method may comprise the additional step of administering single or multiple booster vaccinations to the swine.

A further embodiment of the present invention provides a method of using a serum suitable for treatment of swine infected with PRRSV to protect swine against PRRS, comprising administering to said swine and effective amount of said serum.
Various other objects and advantages of the present invention will become apparent from the detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1. Ultrathin section of a MARC-145 cell infected with the reference Quebec IAF-Klop strain of PRRS virus. Intracellular particles can be observed in the lumen of the cytoplasmic vesicles, but not in the nucleus (arrow). Virions consist of empty (electron translucent) or complete (electron dense center) enveloped particles, and possess a central isometric core approximately 25-30nm in diameter (arrows). Bar represents 100 nm.
Fig. 2. SDS-PAGE analysis of PRRSV-induced polypeptides. The figure represents radioimmunoprecipitation profiles of extracellular virions of the PRRSV strain IAF-Klop collected from supernatants of ['SS] methionine-labelled MARC-145 cells after maximum cytopathic effect was achieved. The homologous porcine hyperimmune serum was used for precipitation of viral structural proteins after treatment (Lane 3) or no treatment with glyco F
(Lane 2). Positions of molecular size markers (Lane 1 ) are shown on the left and the viral structural proteins are indicated by their molecular mass (in kilodaltons) on the right (GPS: 24.5 kDa; M: 19 kDa; and N: 14 to 15 kDa). Only the GPS protein was sensitive to glyco F treatment and thus represents a glycosylated protein. The arrow indicates the possible unglycosylated form of the 24.5 K
protein, regularly observed after glyco F treatment.
Fig. 3. Identification of proteins encoded by PRRSV ORFs 5, 6, and 7 in purified viral preparations.
(A) Reactivity of monospecifc antisera a5, a6, and a7 were first analyzed by western immunoblotting assay. Proteins from sucrose-gradient purified virus were separated in denaturing 12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Strips of nitrocellulose were probed with either a5, a6, or a7 monospecific antiserum or their corresponding preserum collected prior to immunization (pas, pa6, or pal, respectively). As controls, nitrocellulose strips were reacted with a hyperimmune porcine anti-PRRSV serum (aV) or its preserum (paV). (B) Gel analysis of PRRSV proteins immunoprecipitated from Triton X-100 lysates of [35S]methionine-labelled and sucrose gradient-purified virus. Viral proteins were solubilized with LB-1 lysis buffer containing 1% Triton X-100 followed by immunoprecipitation with aV, a5, a6, or a7 antiserum.
The M,s of ['4C]methylated size marker protein bands (lane M) are to the left of gel B, and positions of the three PRRSV major structural proteins N, M, and E, are indicated in the left and right margins of A and B, respectively.
Fig. 4. Immunofluorescent staining of COS7 cells at 24 h post-transfection with pRc/CMVS
plasmid. Expression of GPS of PRRSV (IAF-Klop strain) was confirmed by indirect immunofluorescence (IIF) following incubation in the presence of the rabbit anti-ORFS
monospecific serum. A similar fluorescent profile was obtained following incubation with the autologous anti-PRRSV porcine hyperimmune serum. Expressed GPS protein mostly accumulated in the perinuclear region.
Fig. 5. Reactivity by immunoblotting of the serum of DNA-immunized pigs and mice towards the GPS of PRRSV and the recombinant ORFS-pH protein expressed in E. coli. Lane l:
Immunoblot showing reactivity of a convalescent pig serum towards three major structural proteins (N, M, and GPS ) of PRRSV (IAF-Klop strain). Lanes 2 and 3: reactivity towards GPS of pig and mouse sera at day S 1 post-immunization with pRe/CMVS. Lanes 4 and S: reactivity of pRc/CMVS immunized mouse and pig sera with ORFS-pH recombinant protein expressed in E. coli. Lane 6: reactivity of porcine convalescent serum with ORFS-pH recombinant protein.
Fig. 6. Ex-vivo blastogenic response of porcine PBMCs following incubation in the presence of different concentrations of Concanavaline A or ORFS-pH recombinant protein antigen at variable times post-immunization. (a) PBMCs obtained from both GST-ORFS or pRc/CMVS
immunized pigs underwent specific blastogenesis in the presence of ORFS-pH, and stimulation indexes of 7 to 12 were calculated. (b) No significant difference was observed in non-specific mitogen-induced blastogenesis response of PBMCs obtained from vaccinated and unvaccinated pigs at variable times S post-immunization.
Fig. 7. Histological findings in lungs of control (a) and unvaccinated PRRSV-challenged (b,c,d) pigs. (a) Spongiform aspect of the lung of a normal pig showing clear airway passages (bronchiole and alveolar duct indicated by arrows) and well delineated interalveolar septae. (b) General aspect of interstitial pneumonitis with alveolar septae thickened by lymphomononuclear cells infiltration.
(c) Free mononuclear cells (arrows), necrotic cell debris, and proteinaceous exudate within the bronchioles lumen. (D) Presence of mononuclear cells lining the epithelium of a large bronchi. Note also the focal mild hyperplasia of the respiratory epithelium. HPS staining.
Fig. 8. Histopathological findings in lungs of GST-ORES (a, b) and pRc/CMVS
(c, d) immunized pigs 14 days after challenge with PRRSV (IAF-Klop strain). (a) Localized region of intensive 1 S interstitial pneumonitis, and accumulation of macrophages and necrotic cell debris within the lumen of a bronchiole with apparently no apparent damages to the epithelium. (b) Normal aspect of the epithelium of a large branchi with no accumulation of inflammatory exudate within the lumen. Note at the left, the presence of significant lesions of interstitial pneumonitis with alveolar septae thickened by lymphomononuclear cells infiltration. (c, d) Moderate interstitial pneumonitis (ip) with no apparent damages to the epithelium of the bronchioles and alveolar ducts (arrows). Absence of mononuclear cells and necrotic cell debris within the alveolar lumen. HPS
staining.
Fig. 9. Genomic map of the shuttle vector pAdCMV S/ORFS used for construction of recombinant adenoviruses carrying PRRSV structural protein genes. The pAdCMVS/ORFS
construct contains the following: an adenovirus encapsidation and packaging signal; an adenovirus tripartite leader 2S sequence and major late enhancers; the adenovirus major late promoter enhancer (enh MLP); an HCMV promoter (pro) and enhancer (enh); a gene that confers resistance to ampicillin (Amp); an E. coli replicon pML2; a polyadenylation site (pA); SS 1 splicing signal (ss);
inverted terminal repeats (ITR); tripartite leader (tpl); an origin of replication in E. coli cells (Ori); and human adenovirus type 5 portions involved in homologous recombination with genomic DNA of the wild type virus (Ad5).
DETAILED DESCRIPTION OF THE INVENTION
The following terms and abbreviations are used throughout the specification and in the claims:
"expression construct" means any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. Thus, in certain embodiments, expression includes both transcription of an ORF gene and translation of an ORF
mRNA into an ORF gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding an ORF;
"porcine reproductive and respiratory syndrome (PRRS)" refers to the disease syndromes Mystery Swine Disease, Mystery Pig Disease, Mystery Disease, Mystery Reproductive Syndrome, swine plague, New Pig Disease, Wabash syndrome, abortus blau, Blue Eared Pig Disease, Porcine Epidemic Abortion and Respiratory Syndrome (PEARS), swine infertility and respiratory syndrome (SIRS), Epidemic Late Abortion of Swine (ELAS), Hyperthermia, Anorexia and Abortion Syndrome of the Sow (HAASS), PNP, EMCV, Interstitial Pneumonia, Porcine Arterivirus, the disease caused by the Iowa strain of PRRS V, and closely-related variants of these diseases which have appeared and which will appear in the future;
"porcine reproductive and respiratory virus (PRRSV)" refers to the causatory agent of a porcine reproductive and respiratory syndrome, as described above;

"vaccine" means an agent that protects a swine against PRRS caused by a PRRSV.
Vaccine can additionally mean an agent whereby, after administration of the agent to an unaffected swine, lesions in the lung or symptoms of the disease do not appear or are not as severe as in infected, unprotected swine, and if, after administration of the agent to an affected swine, lesions in the lung or symptoms of the disease are eliminated or are not as severe as in infected, unprotected swine. An unaffected swine is a swine that has either not been exposed to a PRRSV, or that has been exposed to a PRRSV
but is not showing symptoms of the disease. An affected swine is a swine that is showing symptoms of the disease; and "nucleic acid sequence" as used herein refers to a polymeric form of nucleotides of any length, both to ribonucleic acid sequences and to deoxyribonucleic acid sequences. In principle, this term refers to the primary structure of the molecule; thus, this term includes double and single stranded DNA, as well as double and single stranded RNA, and modifications thereof.
It is readily apparent to those skilled in the art that variations or derivatives of the nucleotide sequence encoding a protein can be produced which alter the amino acid sequence of the encoded protein. The altered expressed protein may have an altered amino acid sequence, yet still elicits immune responses which react with a PRRSV, and are considered functional equivalents. In addition, fragments of the full length genes which encode portions of the full length protein may also be constructed. These fragments may encode a protein or peptide which elicits antibodies which react with a PRRSV, and are considered functional equivalents.
The present invention resides in the discovery that the expression of a PRRSV
ORF is sufficient to elicit neutralizing antibodies in swine.
The present invention describes the use of expression constructs encoding PRRSV ORF nucleic acid sequences to immunize swine against PRRS. The PRRSV ORF nucleic acid sequences can also be used to detect the presence of PRRSV infection in swine.

ORF Nucleic Acids Any PRRSV ORF nucleic acid sequence can be used in the present invention, including ORFs 2, 3, 4, 5, 6, and 7.
The nucleic acid according to the present invention may encode an entire ORF
gene, a functional ORF protein domain, or any ORF polypeptide, peptide, or fragment that is sufficient to effect an immune response against a PRRSV infection. The expression construct may contain more than one ORF nucleic acid sequence. The ORF may be derived from genomic DNA. In preferred embodiments, however, the nucleic acid encoding an ORF comprises complementary DNA (cDNA).
The term "cDNA" is intended to refer to DNA prepared using messenger RNA
(mRNA) as template.
The advantage of using a cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, non- or partially-processed RNA template, is that the cDNA does not contain any non-coding sequences but, rather, contains only the coding region of the corresponding protein. There may be times when the full or partial genomic sequence is preferred, such as where the non-coding regions are required for optimal expression or where non-coding regions such as introns are to be targeted.
It is also contemplated that a given ORF nucleic acid may be represented by natural variants that have slightly different primary sequences but, nonetheless, are biological functional equivalents of each other. As is well known in the art, the degeneracy of the genetic code permits substitution of bases in a codon resulting in another codon but still coding fox the same amino acid, e.g. the codon for the amino acid glutamic acid is both GAT and GAA. In order to function according to the present invention, all that is required is that the expressed ORF cause an immune response against PRRSV. Immune reactivity may include both production of antibodies by B-cells (humoral immunity) and activation of T-cells (cellular immunity). Humoral immunity may be demonstrated by the induction of antibody production by B-cells in vivo or in vitro. Cell-mediated immunity may be demonstrated by T-cell activation, for example by increased T-cell protein synthesis, or by the stimulation of B-cells by activated T-cells. Assays for both types of immunity are well known in the art.
Furthermore, fragments of the nucleic acid sequences encoding the PRRSV ORFs or functional variants thereof as mentioned above are included in the present invention. The term "fragment" as used herein means a DNA or amino acid sequence comprising a subsequence of the nucleic acid sequence or polypeptide of the invention. Said fragment is or encodes a polypeptide having one or more immunogenic determinants of the PRRSV ORF protein, i.e. has one or more epitopes of the ORF protein that are capable of eliciting an immune response in swine, as described above.
Fragments can inter alia be produced by enzymatic cleavage of precursor molecules, using restriction endonucleases for the DNA and proteases for the polypeptides. Other methods include chemical synthesis of the fragments or the expression of polypeptide fragments by DNA
fragments.
Nucleic acid sequences may be derived from any isolate of a PRRS strain. The ORF nucleic acid sequences for numerous PRRS strains have been submitted to GenBank, the NIH
genetic sequence database. These nucleotide sequences are available at Internet address http://www.ncbi.nhn.nih.gov/Entrez/nucleotide.html.
Other PR.RSV ORF nucleic acid sequences may be obtained using generally applied Southern blotting technique or colony hybridization (Experiments in Molecular Biology, Slater (ed.), (1986:
Clifton, U.S.A.); Singer-Sam et al., (1983) Proc. Natl. Acad. Sci. U. S. A.
80:802-806; Maniatis et al., "Molecular Cloning, A laboratory Manual" 2"d ed. (1989: Cold Spring Harbor Laboratory Press, USA)). For example, restriction enzyme digested DNA fragments derived from a specific PRRSV
strain is electrophoresed and transferred, or "blotted" thereafter onto a piece of nitrocellulose filter.
The nucleic sequences encoding the ORFs can be identified on the filter by hybridization to a defined labelled DNA or "probe" of a known ORF sequence, under specific conditions of salt concentration and temperature that allow hybridization of the probe to any homologous DNA
sequences present on the filter. After washing the filter, hybridized material may be detected by autoradiography. Once the relevant sequence is identified, DNA fragments can be recovered after agarose gel electrophoresis.

In another way, PRRSV cDNA may be cloned into a ~.gtl 1 phage as described by Huynh et al., In:
D. Glover (ed.) "DNA Cloning: A Practical Approach" (1985: IRL Press Oxford) 49-78, and expressed into a bacterial host. Recombinant phages can then be screened with polyclonal serum raised against a purified ORF protein, determining the presence of corresponding immunological regions of the variant polypeptide.
A nucleic acid sequence according to the invention may be isolated from a particular PRRSV strain and multiplied by recombinant DNA techniques including polymerase chain reaction (PCR) technology. Alternatively, the nucleic acid sequence may be chemically synthesized in vitro by techniques known in the art.
DNA sequences encoding the polypeptides which are functional equivalents of the said PRRSV
ORFs can readily be prepared using appropriate synthetic oligonucleotides in primer-directed site-specific mutagenesis, as described by Morinaga et al. (1984) Biotechnology 2:636.
In a preferred embodiment, the ORF nucleic acid sequence is derived from the Quebec reference cytopathic strain IAF-Klop (Mardassi et al., ( 1994) Can. J. Vet. Res. 58:55-64). The IAF-Klop ORF
(IAF-exp91) nucleotide sequences are published in (Mardassi et al., (1995) Arch. Virol. 140:1405-1418). The nucleotide sequences for ORFs 3-7 of the IAF-Klop strain of PRRSV
are available from GenBank at accession numbers U64928 (IAF-Klop ORF) and L40898 (IAF-exp91) .
Vectors:
Once identif ed and isolated, the ORF nucleic acid sequences of this invention can be inserted into an appropriate expression vector, which contains the elements necessary for transcription and translation of the inserted gene sequences, in such a way that the inserted nucleic acid sequences are expressed to elicit an immune response in the infected host. Useful cloning vehicles may consist of segments of chromosomal, nonchromosomal, and synthetic DNA sequences, such as various known bacterial plasmids, virus DNA (such as retroviruses and vaccina virus), phage DNA, combinations of plasmids and viral or phage DNA such as plasmids which have been modified to employ phage DNA or other expression control sequences, or yeast plasmids.
Plasmids Plasmid DNA offers many potential advantages. First, it is much quicker and easier to make and purify plasmid DNA than viral vectors. This fact, combined with easier quality control, facilitates technology transfer and reduces cost ofproduction. In addition, some viral vectors necessarily result in integration of the DNA into the genome ( e.g. retrovirus); this introduces important safety concerns. Plasmid DNA is designed to remain nonintegrated and to be expressed from an episomal location.
An equally important consideration is an immune response to the injected material itself. Since DNA
does not seem to induce an immune response, there is a lack of immunogenicity of the vector itself.
Vector immunogenicity could preclude the use of the same or similar vector for subsequent immunization (Wolff et al. (1993) Journal Cell Sci. 103:1249-1259). Following DNA
immunization, however, only the expressed protein triggers the immune system for the production of a specif c immune response. Also, adverse reactions can be prevented (antibody dependent 1 S enhancement, hypersensibility, autoimmune racoons) by introducing only genomic regions encoding major antigenic determinants. Furthermore, since DNA is stable, it can be lyophylized to be used in less developed areas of the world where refrigeration is scarce and expensive. This is in contrast to most vaccine preparations.
Plasmids that can be used in the present invention include, but are not limited to, those derived from Escherishia coli (eg., pBR322, pBR325, pUC 12, pUC 13, and the like) or those derived from Bacillus subtilis (eg., pUB 110, pTPS, pC194 and the like). Specific plasmids that can be used in the present invention include, but are not limited to, pRc/CMV (In Vitrogen), pCDNA-3 (InVitrogen), pAdCMVS, and pAdTRS (Massie et al., (1998) J. Virol. 72:2289-2296). In a preferred embodiment of the present invention, the eukaryotic expression vectors pRc/CMV
(Invitrogen) and pAdCMVS
(Pirzadeh and Dea (1998) Journal of General Virology 79: 989-999) are used.

Adenoviruses In certain embodiments of the invention, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells. The first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma and adenoviruses (Ridgway, "Mammalian Expression Vectors" in Rodriguez & Denhardt eds, Vectors: A Survey ofMolecular Cloning Vectors and Their Uses (Stoneham: Butterworth, 1988) 467-492; Baichwal and Sugden, "Vectors For Gene Transfer Derived From Animal DNA Viruses: Transient and Stable Expression of Transferred Genes" in R. Kucherlapati, ed., Gene Transfer (New York: Plenum Press, 1986) 117-148). They can accommodate up to 8 kilobases of foreign genetic material and can be readily introduced in a variety of cell lines and laboratory animals.
Knowledge of the genetic organization of adenovirus, a 36 kb, linear and double-stranded DNA
virus, allows substitution of a large piece of adenoviral DNA with foreign sequences up to 8 kb (Grunhaus and Horwitz (1992) Virology 3:237-252). The infection of adenoviral DNA into host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.
Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage.
Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range, and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted terminal repeats (ITR), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA
replication. The E1 region (E 1 A and E 1 B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA
replication, late gene expression, and host cell shut off (Prevec et al., (1989) Journal of General Virology 70 (Pt2):429-434; Ascadi et al., (1995) J. Mol. Med. 73:165-180; Graham and Prevec (1991), "Manipulation of AdenovirusVector" in E.J. Murray, ed., Methods in Molecular Biology: Gene Transfer and Expression Protocols (Clifton, NJ: Humana Press) 7:109-128). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5' tripartite leader (tpl) sequence which makes them preferred mRNAs for translation.
In the current system, recombinant adenovirus is generated by homologous recombination between a shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process; therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.
Use of the VAC system is an alternative approach for the production of recombinant adenovirus.
Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses E 1 proteins (Graham et al., ( 1977) J. Gen. Virol. 36:59-72). Since the E3 region is dispensable from the adenovirus genome, the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the E1, the E3 or both regions (Graham and Prevec (1991) Meth. Mol. Biol. 7:109-128).
Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g. , Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293 (Graham et al., (1977),1. Gen.

Virol. 36:59-72).
Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the method of the present invention.
As stated above, the typical vector according to the present invention is replication defective and will not have an adenovirus E1 region; thus, it will be most convenient to introduce the nucleic acid encoding an ORF at the position from which the E 1 coding sequences have been removed. The position of insertion of the ORF coding region within the adenovirus sequences, however, is not critical to the present invention. The nucleic acid encoding an ORF
transcription unit may also be inserted in lieu of the deleted E3 region in E3 replacement vectors or in the E4 region where a helper cell line or helper virus complements the E4 defect.
Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g.,109 -10" plaque-forming unit (PFII)/ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome (Graham and Prevec (1991) Meth. Mol. Biol. 7:109-128). The foreign genes delivered by adenovirus vectors are episomal, and therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Top et al., (1971) J. Infect.
Diseases 124:148-154; Prevec et al., (1989) J. Gen. Virol. 70:429-434), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
Adenovirus vectors have been used in eukaryotic gene expression and vaccine development (Grunhaus and Horwitz (1992) Virology 3:237-252; Graham and Prevec (1992) Biotechnology 20:363-390), particularly in farm animals (Tones et al., (1995) Virology 213:503-516; Ebata et al., (1992) Virus Res 24:21-33; Breker-Klassen et al., (1995) J. Virology 69:4308-4315).
The preferred adenovirus vectors for use in the present invention are pAdBMI, pAdBMS, pAdCMVS, and pAdTRS (Ascadi et al., (1994) Human Mol. Genet. 3:578-584; Jani et al., (1997) J. Virol. Meth. 64:111-124; Massie etal., (1998)J. Virol. 72:2289-2296; U.S.
Patent No. 5,518,913).
Vector Elements In preferred embodiments, the nucleic acid encoding an ORF is under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression ofthe gene.
The term promoter will be used to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polyrnerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 by of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA
box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 by upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements can be flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 by apart before activity begins to decline.
Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
The particular promoter that is employed to control the expression of a nucleic acid encoding an ORF is not believed to be important, so long as it is capable of expressing the nucleic acid in the targeted cell; thus, where a swine cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a swine cell. Generally speaking, such a promoter might include either a mammalian or viral promoter.
In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, or the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of an ORF. The use of other viral or mammalian cellular or bacterial phage 1 S promoters to achieve expression of an ORF is contemplated as well, provided that the levels of expression are suff cient for a given purpose.
Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions ofDNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.
The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
The following are examples of enhancers that could be used in combination with a nucleic acid encoding an ORF in an expression construct: BHK enh, CMV enh, and AdMLP enh.
Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the ORF transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed. The inventors have employed the SV40L polyadenylation signal in the pRc/CMV vector, and the rabbit globin poly A signal in the adenovirus shuttle vectors, because they are convenient and known to function well in the target cells employed. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
There are many embodiments of the instant invention which those skilled in the art can appreciate from the specification; thus, different transcriptional promoters, terminators, carrier vectors, or specific gene sequences may be used successfully.
Construction The insertion of the ORF nucleic acids into a cloning vector is easily accomplished when both the genes and the desired cloning vehicle have been cut with the same restriction enzyme or enzymes, since complementary DNA termini are thereby produced. If this cannot be accomplished, it may be necessary to modify the cut ends that are produced by digesting back single-stranded DNA to produce blunt ends, or by achieving the same result by filling in the single-stranded termini with an appropriate DNA polymerase. In this way, blunt-end ligation with an enzyme such as T4 DNA ligase may be carried out. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini. Such linkers may comprise specific oligonucleotide sequences that encode restriction site recognition sequences. The cleaved vector and the ORF
nucleic acid sequences may also be modified by homopolymeric tailing, as described by Morrow ( 1979) Methods in Enzymology 68:3.
Once the ORF nucleic acid is inserted into the expression vector, the expression construct may be replicated in and isolated from an appropriate host organism. The selection of an appropriate host organism is affected by a number of factors known in the art. These factors include, for example, compatibility with the chosen vector, toxicity of proteins encoded by the hybrid plasmid, ease of recovery of the desired protein, expression characteristics, biosafety, and costs. A balance of these factors must be considered, and it must be understood that not all hosts will be equally effective for expression of a particular recombinant DNA molecule.
Suitable host microorganisms that can be used in this invention include but are not limited to plant, mammalian, or yeast cells, or bacteria such as Escherichia coli, Bacillus subtilis, Bacillus stearothermophilus and Actinomyces. Transfer of the recombinant cloning vector into the host cell may be carried out in a variety of ways. Depending upon the particular vector/host cell system chosen, such transfer may be effected by transformation, transduction, transfection or electroporation. Once such a modified host cell is produced, the cell can be cultured and the expression construct may be isolated from the culture.
In one embodiment of the present invention, the LAF-Klop PRRSV ORFS is incorporated into the pAdCMVS expression vector. Briefly, viral genomic RNA was extracted from PRRSV
IAF-Klop infected MARC-145 cells by the guanidinium isothiocyanate-acid phenol method (Chomczynski &
Sacchi ( 1987) Analytical Biochem. 162:156-159). The ORES gene was then reverse-transcribed and amplified by polymerase chain reaction (PCR) using the following primers: ETS
5 (forward primer) 5'- AAGCTT GCC GCC GCC ATG TTG GGG AAA TGC TTG ACC- 3' , which comprises the first ATG codon of the ORES gene downstream of a Kozak motif for initiation of translation in vertebrates (Kozaic (1987) Molecular Biology 196:947-950), and ETRS (reverse primer) 5'-TCTAGAGGCAAAAGTCATCTAGGG-3', which comprises the C-terminal stop codon of the viral gene. The nucleotide sequence accession number (EMBL/GenBankIDDBJ libraries) of IAF-Klop strain is U64928.
For directional cloning, Hind III and Xba I restriction sites were added at the 5' ends of the sense and antisense oligonucleotide primers, respectively (Pirzadeh and Dea (1987) J. Gen. Virol.
79:989-999). The ORES encoding region was further cloned into the Hind III and Xba I cloning sites of the eukaryotic expression vector pRc/CMV (Invitrogen), down-stream of the human cytomegalovirus (CMV) promoter, producing the plasmid pRc/CMVS.
In another embodiment of the present invention, the PCR amplified ORFS gene was inserted into the unique BamHl site of the adenovirus transfer vector pAdCMVS to generate pAdCMVS/ORFS, I O which was used for eukaryotic transient expression assays, DNA
immunization experiments, and recombinant adenovirus construction. In this shuttle vector, the gene is driven by an optimized human cytomegalovirus (CMV) promoter. The expression cassette is flanked on one end by the encapsidation and packaging signals of the human adenovirus type 5 and on the other end by an adenovirus sequence allowing recombination and generation of replication-defective recombinant virus in which El gene is replaced by the expression cassette. In this cassette, which was derived from pAdBMS (IJS Patent No. 5, 518,913), expression of heterologous genes is optimized by the presence of the Adenovirus tripartite leader sequence and the Adenovirus major late enhancer flanked by splice donor and acceptor sites.
The generation of recombinant adenovirus was done as detailed in Jani et al.
(1997) J. virological Methods 64:1 I 1-124. Briefly, AdCMVIacZ DNA was rendered non-infectious by CIaI digestion and co-transfected in 293 cells with the same amount of pAdCMVS/ORFS (or pAdTRS/ORFS) DNA
that was linearized by digestion of the unique CIaI site. Transfected cells were cultivated and viral plaques were picked and expanded. Recombinant AdCMV/ORFS viruses were identified by PCR
and by indirect immunofluorescence in 293 cells using the rabbit a5 monospecific antiserum (Mardassi et al., (1996) virology 221:98-112). These expression constructs were subsequently subcloned twice to ensure their purity.

Ex-vivo expression of pRc/CMVS and pAdCMVS/ORFS constructs was tested in transient expression experiments in COS7 and 293 cells maintained as confluent monolayers. Cells were transfected with plasmid DNA by calcium phosphate coprecipitation (Graham &
van der Eb, (1973) Virology 52:456-467). For indirect immunofluorescence (IIF), cells were reacted with anti-s ORFS rabbit monospecific hyperimmune serum (Mardassi et al., (1996) Virology 221:98-112) and the immune reaction was revealed following incubation with fluorescein-conjugated goat anti-rabbit Ig (Boehringer Mannheim), as previously described (Loemba et al., (1996) Archives of Virology 141:751-761).
The pRc/CMVS vector (In Vitrogen) or pcDNA3 (In Vitrogen) eukaryotic vectors used for genetic immunization both offer the following features: promoter sequences from the immediate early gene of the human cytomegalovirus (CMV) for high-level transcription;
polyadenylation signal and transcription termination sequences from the bovine growth hormone (BHG) gene to enhance RNA
stability; SV40 origin for episomal replication and simple vector rescue in cell lines expressing S V40 large T antigen (e.g. COS1, COS7, NIH3T3 and human 293 cells); T7 and SP6 RNA
promoters flanking the multiple cloning site for in vitro transcription of sense and antisense RNA; the fl origin for rescue of the sense strand for mutagenesis and single-stranded sequencing;
and the ampicillin resistance gene and ColEl origin for selection and maintenance in E. coli.
The pAdCMVS/ORFS construct contains the following: an adenovirus encapsidation and packaging signal; an adenovirus tripartite leader sequence and major late enhancers; the adenovirus major late promoter enhancer (enh MLP); an HCMV promoter (pro) and enhancer (enh); a gene that confers resistance to ampicillin (Amp); an E. coli replicon pML2; a polyadenylation site (pA); SS 1 splicing signal (ss); inverted terminal repeats (ITR); tripartite leader (tpl); an origin of replication in E. coli cells (Ori); and human adenovirus type 5 portions involved in homologous recombination with genomic DNA of the wild type virus (Ads) (see Figure 9).
Gene Transfer In order to effect expression of ORF constructs, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, in vivo, or ex vivo (see below). As described above, the preferred mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.
Experiments in administering recombinant adenoviruses to different tissues include trachea instillation ( Rosenfeld et al., (1992) Cell 68:143-155), muscle injection (Ragot et al., (1993) Nature 361:647-650), peripheral intravenous injection (Herz and Gerard (1993) Proc. Natl. Acad.
Sci. USA 90:2812-2816), and stereotactic inoculation into the brain (Le Gal La Salle et al., (1993) Science 259:988-990).
In a preferred embodiment, adenovirus constructs would be delivered to swine via intradermal I O and/or intramuscular injection using the GenGun transfection system (Johnston and De-chu (1994) "Gene Gun Transfection of Animal Cells and Genetic Immunization" In Methods in Cell Biology, 43:353-366), or using a 26 gauge needle and tuberculin syringe. The intramuscular inj ection is given into the tibialis cranalis muscle, whereas the intradermal injection is given into the dorsal surface of the ear.
Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present invention. These include calcium phosphate co-precipitation technique, DEAF-dextran, electroporation, direct microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection, aII of which are known in the art. Some of these techniques may be successfully adapted for in vivo or ex vivo use.
Once the expression construct has been delivered into the cell, the nucleic acid encoding ORF may be positioned and expressed at different sites. The nucleic acid encoding ORF
may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
In one embodiment of the invention, the expression construct may consist of DNA plasmids.
Transfer of the construct may be performed by any of the methods mentioned above that physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro, but it may be applied to in vivo use as well. Dubensky et al. (1984) Proc.
Natl. Acad. Sci. USA
81:7529-7533 successfully injected polyomavirus DNA in the form of CaP04 precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection.
Benvenisty and Neshif (1986) Proc. Natl. Acad. Sci. USA 83:9551-9555 also demonstrated that direct intraperitoneal injection of CaP04 precipitatedplasmids results in expression ofthe transfected genes. It is envisioned that DNA encoding an ORF could also be transferred in vivo in a similar manner to express ORF.
Another embodiment of the invention for transferring DNA expression constructs into cells could involve particle bombardment (Johnston and De-chu (1994) Methods in Cell Biology 43:353-366).
This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them.
Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force.
The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads. Selected organs, including the liver, skin, and muscle tissue of rats and mice, have been bombarded in vivo. This rnay require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment. Again, DNA encoding an ORF may be delivered via this method within the scope of the present invention.
In a further embodiment of the invention, the expression construct may be entrapped in a liposome.
Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium.
They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Also contemplated are lipofectamine-DNA
complexes. Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful in cultured chick embryo, HeLa and hepatoma cells.
Nicolau et al., (1987) Methods in Enzymology 149:157-176 accomplished successful Iiposome-mediated gene transfer in rats after intravenous injection.
In certain embodiments, gene transfer may more easily be performed under ex vivo conditions. Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into the animal. This may involve the surgical removal of tissue or organs from an animal or the primary culture of cells and tissues.
U.5. Pat. No. 5,399,346 disclose ex vivo therapeutic methods.
Primary mammalian cell cultures may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the expression construct, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture techniques are well documented. During in vitro culture, the expression construct may deliver and express a nucleic acid encoding an ORF into the cells. The cells may then be reintroduced into the original animal, or administered into a different animal, in a pharmaceutically acceptable form by any of the means described below.
Administration The vaccines of the present invention can be administered in a conventional active immunization scheme: single or repeated administration in a manner compatible with the dosage formulation and in such amount as will be prophylactically and/or therapeutically effective and immunogenic, i.e.
the amount of expression construct capable of expressing an ORF that will induce immunity in an animal against challenge by a virulent PRRSV. Immunity is defined as the induction of a higher level of protection in a population of animals after vaccination compared to an unvaccinated group.

The amount of expression construct to be introduced into a vaccine recipient will have a very broad dosage range and will depend on the strength of the transcriptional and translational promoters used.
In addition, the magnitude of the immune response will depend on the level of protein expression and on the immunogenicity of the expressed ORF gene product. In general, an effective dose ranges of about SO to 500 pg, and preferably about 50 to 100 p.g of plasmid DNA is administered.
Intramuscular injection, subcutaneous injection, intradermal introduction, impression through the skin, and other modes of administration such as intraperitoneal, intravenous, or inhalation delivery are also suitable. The preferable modes of administration are intramuscular and intradermal.
It is also contemplated that booster vaccinations may be provided.
The therapeutic compositions of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to inj ection may also be prepared. These preparations (recombinant adenoviruses) may also be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain

10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per millilitre of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients including salts, preservatives, buffers, stabilizers (such as skimmed milk or casein hydrolysate), and the like.
Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents, and inert gases. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to well known parameters.
Kits All the essential materials and reagents required for vaccinating swine, transforming cells, or detecting PRRSV infection, may be assembled together in a kit. This generally will comprise selected expression constructs. Also included may be various media for replication of the expression constructs and host cells for such replication. Such kits will comprise distinct containers for each individual reagent.
When the components of the kit are provided in one or more liquid solutions, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred. For in vivo use, the expression construct may be formulated into a pharmaceutically acceptable syringeable composition. In this case, the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the animal, such as the lungs, injected into an animal, or even applied to and mixed with the other components of the kit.
The components of the kit may also be provided in dried or lyophilized forms.
When reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means.
Irrespective of the number or type of containers, the kits of the invention also may comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal. Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
Use The vaccines of the present invention can be used for the immunization of swine against porcine reproductive and respiratory syndrome (PRRS). The expressed recombinant proteins can also be used as antigens for the development of diagnostic procedures.
The plasmid expression constructs of the present invention can be used as transfer vectors to construct other expression vectors, such as adenovirus expression constructs described previously.
The plasmid expression constructs of the present invention can also be used to inoculate swine, following which PRRSV-neutralizing serum is obtained from these swine. This serum can then be used for the passive immunization of other swine. Indeed, vaccinated pregnant sows could secrete large amounts of specific anti-PRRSV antibodies via their colostrum and milk which would protect suckling piglets. Also, serum from vaccinated animals, or purif ed gammaglobulin preparations, can be injected parenterally (intramuscular, intraveinous or intraperitoneal injection) to naive or immunodeprived pigs in order to protect them temporarily from natural PRRSV
infection.
The expression constructs of the present invention can also be used for diagnostic purposes.
Recombinant adenoviruses or recombinant plasmid DNA vectors carrying the foreign PRRSV viral genes (e.g. PRRSV structural protein) may be used to infect or transfect cell cultures (e.g. COS7, 293, A549, MARC-145, or KB cells). The foreign PRRSV ORF proteins carrying major antigenic determinants are then effectively expressed or synthesized in the cell cultures. These cells cultures may be used for diagnostic purposes; for example, one can determine whether an animal contains specific antibodies in its serum that are directed to the expressed PRRSV
proteins using immunochemieal techniques such as indirect immunolluorescence, immunoperoxydase, immunogold silver staining, or enzyme linked immunosorbant assay (ELISA). The recombinant PRRSV proteins may also be recovered from the supernatant fluids of homogenates of the adenovirus-infected cell cultures and used as a source of antigens for ELISA, radioimmuno assay (RIA), and agglutination assays.
Advantages The present invention describes vaccines for nucleic acid immunization against PRRSV. There are several advantages to immunization with nucleic acids, rather than with viruses or proteins. The first is the relative sirnplieity with which native or nearly native antigen can be presented to the immune system. Mammalian proteins expressed recombinantly in bacteria, yeast, or even mammalian cells often require extensive treatment to ensure appropriate antigenicity. A second advantage of DNA

immunization is that it can evoke both humoral and cell-mediated immune responses; the immunogen can enter the MHC class I pathway and evoke a cytotoxic T cell response.
The use of live attenuated viral vaccines is generally quite effective as the viruses mimic a natural infection. A serious disadvantage of such vaccines, however, is their pathogenicity in immunosuppressed recipients exposed to environmental stress, such as poor housing and over-crowding often prevalent in intensive animal raising operations. This can be of great concern in veterinary medicine where clinical outbreaks are sometimes reported shortly after prophylactic immunization. These vaccines also require special handling to maintain viability and to avoid tissue culture contaminants.
Administration of live modified vaccines are also problematic because they may result in virus persistence, which in turn contributes to generation of mutants due to the selective immune pressure on the resident variants. Persistently-infected animals may eventually shed newly generated mutants, particularly in the case of unstable pathogens such as RNA viruses.
These mutants may be responsible for new outbreaks.
Production and purification of large quantities of viral particles for use in whole viral inactivated vaccines or their immunogenic structural proteins is economically unfeasible for low yield viruses such as PRRSV.
A further advantage of the vaccines of the present invention is that the animals immunized with these vaccines can be differentiated from naturally-infected swine. Animals infected naturally by a virus or other pathogen produce antibodies to the major antigenic determinants of the various proteins of these pathogens; for example, in the case of PRRSV, infected pigs usually produce antibodies directed to the three major structural proteins N, M, and GPS. In the case of animals vaccinated by genetic immunization or with recombinant virus vectors, the animals produce antibodies directed only to the proteins expressed in the cells by the recombinant DNA plasmids or recombinant viruses.
The use of diagnostic tests that permit detection of antibodies specific to the targeted proteins, together with tests that permit detection of antibodies to the various proteins of the pathogens (e.g.
Western imunoblotting, indirect immunofluorescence using cells infected with the recombinant virus, or ELISA using recombinant proteins), allows for the differentiation between vaccinated and naturally-infected animals.
The present invention is described in further detail in the following non-limiting examples. It is to be understood that the examples described below are not meant to limit the scope of the present invention. It is expected that numerous variants will be obvious to a person skilled in the art to which the present invention pertains, without any departure from the spirit of the present invention.
The appended claims, properly construed, form the only (imitation upon the scope of the present invention.
EXAMPLES
Materials and Methods:
Experimental animals:
Twelve crossbred Fl (Landrace X Yorkshire) SPF piglets weaned at 3 weeks of age were obtained I S from a breeding farm in the province of Quebec. The breeding stock and piglets were tested and proven to be seronegative for PRRSV, encephalomyocarditis virus (EMCV), porcine parvovirus (PPV), haemagglutinating encephalomyelitis virus (HEV), transmissible gastroenteritis virus (TGEV) and Mycoplasma hyopneumoniae. The piglets used in this study were from two different litters and randomly divided into 4 experimental groups. Each group of three piglets were allocated to separate isolation rooms in facilities equipped with microorganism free filtered in-flowing and out-flowing air system. They were fed commercial food and water ad libitum.
Six week-old female BALB/c and CD-1 mice were purchased from Charles River Laboratories and separated in groups of 5 mice per cage which were equipped with individual filtered air channels.

YIYIIS SLYaIn:
The Quebec cytopathic strain IAF-Klop (Mardassi et al., ( 1994) Can. J. Vet.
Res. 58:55-64) used in this study was initially isolated from an acute case of PRRS and propagated in MARC-145 cells, a clone of MA-104 cells highly permissive to PRRSV (Kim et al., (1993) Archives of Virology S 133:477-483), graciously provided to us by J. Kwang (US Meat Animal Research Centre, USDA, Agricultural Research Service, Clay Centre, Nebraska). Virus titration was done by end-point dilution using immunoperoxydase on monolayer assay (IPMA) (Wensvoort et al., ( 1991 ) Veterinary Quarterly 13:121-130) and virus titres were expressed in tissue culture infective dose 50 (TCIDso) per ml, as previously described (Dea et al., (1992) Canadian Veterinary Journal 33:801-808). In order to verify the virulence of the virus strain and eliminate MARC-145 cellular proteins that are copurified with the virus, IAF-Klop strain of PRRSV was subjected to one passage on primary culture of porcine alveolar macrophages (PAMs) followed by two successive in vivo passages in pigs. The animals used for providing PAMs and for virus adaptation were obtained from the same farm, treated and reared under the same conditions, as mentioned above.
Piglets used for in vivo passages received via intratracheal inoculation 10 ml of cell culture supernatant, adjusted to 106 TCIDso of virus/ml. On the seventh day post-inoculation, when respiratory distress became evident, the piglets were euthanised and their lungs were aseptically collected and homogenized in RPMI
medium supplemented with 200 U/ml of penicillin, 200 ~g/ml of streptomycin and 50 pg/ml gentamycin. Each challenged piglet received via intratracheal injection, 10 ml of clarified 5% lung homogenate in sterile RPMI corresponding to m infectious dose of 5 X 105 TCIDso of virus, as established by back titration. The inoculum was further tested for the presence of the above indicated porcine pathogens and no organism other than PRRSV could be isolated.
ORF Nucleic Acids:
Viral RNA was extracted from PRRSV-infected MARC-145 cells, as previously described (Mardassi et al., (1996) Virology 221:98-112). The ORF encoding regions were amplified by RT-PCRTM using the oligonucleotide primers listed in Table 1.

Table 1. Sequences of the Oligonucleotide Primers used for RT-PCR and Size of the Expected Amplified Products ORF genes Sense Sequence (5' to 3') Genome locationz Product (bases) Size (bp) ORF2-S + ATGAAATGGGGTCTATGC 28 - 45 783 768 ORF3-S + ATGGCTAATAGCCGTACA 651 - 668 765 762 ORF4-S + ATGGCTGCGTCCCTTCTT 1196 - 1213 537 534 ORFS-S + ATGTTGGGGAAATGCTTGACC 1743 - 1763 612 600 ORF6-S + ATGGTGTCGTCCCTAGATGAC 2337 - 2357 579 522 ORF7-S + CTAAATATGCCAAATAACAAC 2845 - 2865 396 369 1 Sizes of individualgenes refer to those deduced from the nucleotide viral sequences of the tissue culture-adapted Quebec IAF-Klop strain of PRRSV
(EMBL/GenBank accession numbers are given in the text).

z Nucleotide location on the PRRSV genome was given in reference to the sequences of the prototype North American strains ATCC
VR-2385 (EMBL/GenBank accession numbers U20788 and U03040) and ATCC VR-2332 (EMBL/GenBank accession number U00153).

Plasmid Constructs:
The amplified ORF encoding regions were cloned into pGEX-4T1 plasmid (Pharmacia).
Recombinant fusion proteins, consisting of glutathion sulfotransferase (GST) joined to the 1~1 terminal of the ORF7, ORF6, ORFS, ORF4, and ORF3 proteins (GST-ORF7, GST-ORF6, GST
ORFS, GST-ORF4, and GST-ORF3, respectively), were expressed inE. coli and purified by affinity chromatography on glutathion sepharose columns, as previously described (Mardassi et al., (1996) Virology 221:98-112; Pirzadeh & Dea (1997) Journal of General Virology 78:1867-1873).

Subsequent SDS-PAGE analysis of the purified proteins confirmed that no contaminants from bacterial proteins were present in the purified recombinant fusion proteins (data not shown).
Plasmidic DNAs were purified from bacterial lysates by anion exchange chromatography on hydroxyapatite columns {QTAGEN Inc, Chatsworth, CA) and then precipitated by isopropanol, effectively eliminating bacterial protein contaminants.
The amplified ORF encoding regions were also cloned into pET 21, a prokaryotic plasmid (Novagen), to produce recombinant proteins in E. coli consisting of the ORFS, ORF4, and ORF3 encoded proteins, each fused at their C-terminal to 6 histidine residues (ORFS-pH, ORF4-pH, and ORF3-pH, respectively).
The ORFS encoding region was further cloned into the Hind III and Xba I
cloning sites of the eukaryotic expression vector pRc/CMV (Invitrogen), down-stream of the human cytomegalovirus (HCMV) promoter to produce pRc/CMVS. The sequence of the oligonucleotide primers used for this amplification were as follows:
ETS 5 (forward primer) : 5'- AAGCTT GCC GCC GCC ATG TTG GGG AAA TGC TTG ACC-3' (SEQ ID
NO: ), which comprises the first ATG codon of the ORFS gene downstream of a Kozak motif for initiation of translation in vertebrates (Kozak (1987) Nucleic Acids Research 15:8125-8132), and ETRS (reverse primers): 5'- TCTAGAGGCAAAAGTCATCTAGGG-3' (SEQ ID NO: ), which comprises the C-terminal stop codon of the viral gene.
The nucleotide sequence accession number (EMBL/GenBank/DDBJ libraries) of IAF-Klop strain is U64928. For directional cloning, Hind III and Xba I restriction sites were added at the S' ends of the sense and antisense oligonucleotide primers, respectively. Both strands of pRe/CMVS were sequenced in an Automated Laser Fluorescent DNA sequencer (Pharmacia LKB) in order to confirm that no error has occurred as a result of PCR amplif canon.
The PCR amplified ORFS gene was also inserted into the unique BamHl site of the adenovirus transfer vector pAdCMVS (Massie et al., (1998) J. Virology 72:2289-2296) to generate pAdCMVS/ORFS, which was used for eukaryotic transient expression assays, DNA
immunization experiments, and recombinant adenovirus construction. In this shuttle vector, the gene is driven by an optimized human cytomegalovirus (CMV) promoter. The expression cassette is flanked on one end by the encapsidation and packaging signals of the human adenovirus type 5, and on the other end by an adenovirus sequence allowing recombination and generation of replication-defective recombinant virus in which the E 1 gene is replaced by the expression cassette. This cassette was derived from pAdBMS (Ascadi et al., (1994) Human Mol. Genet. 3:578-584).
Expression of heterologous genes is optimized in this cassette by the presence of the adenovirus tripartite leader sequence and the adenovirus major late enhancer Clanked by splice donor and acceptor sites (Jani et al., (1997) J. VirologicaT Methods 64:11 I-124).
Similar strategies were used for cloning PCR amplified ORFs 2, 3, 4, 6, and 7 into the pAdCMVS
transfer vector to create shuttle vectors for the construction of recombinant replication defective adenoviruses.
Adenovirus Constructs:
The generation ofrecombinant adenovirus was done, as described in Jani et al., ( 1997) J. Yirological Methods 64:I 11-I24. Briefly, Ad/CMVIacZ DNA was rendered non-infectious by Clal digestion and co-transfected in 293 cells with the same amount of pAdCMVS/ORF2, ORF3, ORF4, ORFS, ORF6, or ORF7 DNA that was linearized by digestion of the unique CIaI site.
Transfected cells were cultivated in 6 cm tissue culture plates in DMEM medium containing 1% Sea-Plaque agar (FMC Products). Viral plaques were picked 10 to 20 days later and expanded.
Recombinant AdCMVS/ORF2, AdCMVS/ORF3, AdCMVS/ORF4, AdCMVS/ORFS, AdCMVS/ORF6, and AdCMVS/ORF7 viruses were identified by PCR and by IIF in 293 cells using the rabbit S
monospecific antiserum for each ORF oncoprotein (Mardassi et al., (1996) Virology 221:98-112;
Mardassi et al., (1998) J. Virology (in press); Gonin et al., (1998) Archives of Virology (in press)) and were subsequently subcloned twice to ensure purity.
Transient Expression of ORES:
Ex vivo expression of pRc/CMVS and pAdCMVS/ORF2 to 7 constructs were tested in transient expression experiments in COS7 and 293 cells maintained as confluent monolayers. Cells in 6 cm tissue culture plates were transfected with 15 ug plasmid DNA by calcium phosphate coprecipitation (Graham and van der Eb ( 1973) Virology 52:456-467). For indirect immunofluorescence (IIF), cells were incubated at 37 ° C and fixed with 80% cold acetone for 20 minutes at 4 °C at variable times ( 18 to 72h) post-transfection. The monolayers were then reacted for 30 minutes with rabbit S monospecific hyperimmune sera (Mardassi et al., ( 1996) Virology 221:98-112;
Gonin et al., (1998) Archives of Virology (in press)). The immune reactions were revealed following incubation with fluorescein-conjugated goat anti-rabbit Ig (Boehringer Mannheim) as previously described (Loemba et al., (1996) Archives of Virology 141:751-761).
Immunization:
In vivo expression of pRc/CMVS was verified by immunizing groups of 5 CD-1 or BALB/c mice with SO~cg of pRc/CMVS diluted in 50 p1 of phosphate buffered saline (PBS), and injected in the tibialis cranialis muscle with a 27 gauge-needle. The mice were boosted twice with the same quantities of DNA at 2 week-intervals. Control mice received the same amounts of parental pRc/CMV vector via an identical route and frequency, or three doses intraperitoneally of SOpg of GST-ORFS in Freund's complete or incomplete adjuvant.
Groups of 3 piglets were injected three times at two week intervals with 100 pg of pRc/CMVS
diluted in 0.5 ml of PBS. Two thirds of the volume was injected using a 26-gauge needle in the tibialis cranialis muscle of the right leg and one third was administered intradermally into the dorsal surface of the ear. Control piglets received either 100 pg of the parental vector via an identical route and frequency, or 300gg of GST-ORFS.
Virus neutralization and serological tests:
Mice and pig sera were tested for the presence of specific anti-GPS antibodies by virus neutralization (VN), IIF, ELISA and Western immunoblotting (WB) tests. The VN test was performed in triplicates with 100 p1 of serial dilutions of heat-inactivated (56° C, 45 min) test sera, incubated for 60 min at 37 °C in the presence of I00 TCIDso of the virus in DMEM. The mixtures were then put in contact with confluent monolayers of MARC-145 cells seeded in 96 well-microtitration plates 48-72 h earlier. Cell monolayers were incubated at 37°C in a humidified atmosphere containing 5% CO2, and observed daily for up to 5 days for the appearance of cytopathic effects (CPE). The monolayers were then fixed with a solution of 80% methanol containing 0.05% HzOz , and tested for expression of the PRRSV nucleocapsid protein by IPMA (Wensvoort et al., (1991) Veterinary Quarterly 13:121-130), using N protein specific MAb IAF-ICS (Pirzadeh & Dea (1997) Journal of General Virology 78:1867-1873). The immune reaction was revealed following subsequent incubation with peroxydase-labelled goat anti-mouse IgG (Boehringer Mannheim). Neutralizing titres were expressed as the reciprocal of the highest dilution that completely inhibited the expression of viral N protein. The IIF was performed on PRRSV-infected and acetone-fixed MARC-145 cells, as previously described (Loemba et al., (1996) Archives of Virology 141:751-761).
Indirect ELISA
was essentially performed as previously described (Pirzadeh & Dea (1997) Journal of General IO Virology 78:1867-1873) with minor modifications. Gel-purified ORES-pH
protein (0.1 ug of protein/well) in O.OSM-sodium carbonate buffer, pH 9.6, was used to coat flat-bottomed microtitration plates; peroxydase labelled-goat anti-porcine IgG was used to detect the captured antibodies. The substrate solution consisted of 0.1% urea peroxide and 0.02%
3,3',5,5'-tetramethyl benzidine in 10 mM citrate buffer, pH 5.0, mixed in equal volumes. The absorbance values were IS determined at 450 nm. WB was also performed as previously described (Pirzadeh & Dea (1997) Journal of General Virology 78:1867-1873) using either ORFS-pH protein or sucrose gradient purified-PRRSV as antigen.
Blastogenic transformation test:
At regular post-immunization intervals, pigs were medicated with Xylazine (Bayers) at a dose of 20 lmg/Kg and blood samples were collected from the anterior vena cava in vacuum tubes containing 1/10 volume 150 mM sodium citrate in PBS, and then diluted 1:3 in sterile RPMI. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Paque (density 1.077;
Pharmacia) centrifugation at 1,200 g for 20 min. The mononuclear cells were collected from the buffy coat and pelleted. The residual red blood cells were lysed by incubating cells with 0.53% ammonium 25 chloride for 10 min at 37°C. After Z washes in RPMI, the leukocytes were adjusted to a suspension of 2 X 106 cells per ml in RPMI containing 20% homologous heat inactivated PRRSV negative porcine serum, 50 U/ml of penicillin, and 50 ~eg/ml of streptomycin. The antigen-specific proliferation was determined by incubating PBMC in microtitration plates (4 X
105 cells in 200p1/weil in triplicates) during 72 h in the presence of variable concentrations (0, 0.1, 10, and 30 25p.g/ml) of ORES-pH protein. Blastogenic capacity of the PBMC under test conditions was confirmed by including control triplicates containing 2. S, 5, or 10 pg/ml of Concanavaline A (ConA, Sigma Chemicals). After a 72 h stimulation period, the cells were labelled for 18 h with 0.1 ~eCi of [jH]thymidine (Amersham) per well, harvested with a semiautomatic cell harvester (Skatron Instruments). The incorporated radiolabelled nucleotide was measured by scintillation counting after addition of a fluorescent liquid scintillator (Cytoscint, ICN). The level ofproliferation was expressed as the mean of counts per minute (CPM) of the test wells minus the mean of the background CPM
in control wells. Control for background levels consisted of PBMC cultures in media alone.
Virus isolation:
After collection of blood samples, pigs were euthanised by rapid intravenous injection of sodium pentobarbital (MTC Pharmaceuticals). Specimens were aseptically collected from lungs, spleen, liver, kidneys, and mediastinal and mesenteric lymph nodes. Tissue homogenates were prepared in DMEM to final concentrations of 1:20 and 1:100. Following clarification by centrifugation at 10,000 g for 10 min, tissue homogenates were inoculated onto monolayers of MARC-14S cells in 24 well-culture plates or PAMs seeded in 96 well-microtitration plates. Cells were harvested by 2 1 S freeze-thaw cycles at 4-S days post-inoculation. Tissue culture supernatants were clarified and used for a second passage. Cultures were observed daily for CPE until day S post-inoculation, at which time infected monolayers were fixed with cold acetone for IIF.
RT PCR:.
Total RNA was extracted from tissues collected from challenged animals and from MARC-14S cells inoculated with tissue homogenates. RT-PCR was performed using oligonucleotide primers 1006PS+1007PR and 1008PS+1009PR to amplify ORF6 and ORF7 genomic regions of PRRSV
respectively, as previously described (Mardassi et al., (1995) Archives of Virology 140:1405-1418).
Histopathological examination:
Thin sections (S pm thick) of formaline fixed, paraffin embedded tissues from the lungs, spleen, liver, kidneys, and thoracic and mesenteric lymph nodes of all pigs were routinely processed for the hematoxylin-phloxin-safran (HPS) staining, as described previously (Dea et al., (1991) Journal of Veterinary Diagnostic Investigation 3:275-282).

Results Transient expression of cloned ORFS gene:
Expression of the ORFS product was demonstrated in both COS7 and 293 cells lines at 24 and 36 h post-transfection. The identification of GPS was confirmed by IIF using monospecific anti-ORFS
rabbit antiserum or the porcine anti-PRRSV serum. As shown in Fig. 4, an intense cytoplasmic fluorescence could be observed in approximately 10 to 15% of the cells, and the expressed GPS
tended to accumulate near the perinuclear region. Similar findings were observed for the expressed products of ORFs 3, 4, and 7 (Gonin et al., (1988) Archives of Virology (in press); Gagnon et al., ( 1997) 781" Annual Meeting of the CRWAD Chicago, Nov.B-12).
Antibody response of mice and pigs:
Sera collected at various times post-immunization (Table 2) were positive for the presence of anti-PRRSV antibodies by IIF. The protein specificity of mice and pigs sera to GPS
was established by immunoblotting with purified whole virus and E. coli- expressed recombinant ORFS-pH fusion protein (Fig. 5) and by ELISA (Table 2). BALB/c mice inoculated with the GST-ORFS or pRc/CMVS developed neutralizing antibodies which could be first detected two weeks after the second booster injection. The VN titres of BALB/c mice sera were estimated between 32 and 64 by the 8th week post-immunization and persisted through the end of the 12 week-observation period.
In contrast, the CD-1 mice did not develop neutralizing antibodies to PRRSV
despite a significant anti-ORFS specific antibody response detected by ELISA and IIF. Seroconversion was also demonstrated by IIF and ELISA in both groups of vaccinated pigs (Table 2) 15 days after first injection of either GST-ORFS or pRc/CMVS. Neutralizing antibodies were detected in sera of the DNA-immunized pigs only 2 to 3 weeks after the second booster injection (8 to 9 weeks after first inoculation of plasmidic DNA), and 2 weeks after PRRSV challenge, with estimated titres close to 128. None of the virus challenged animals in the unvaccinated or GST-ORES
immunized group developed detectable neutralizing antibodies (VN titres <8) to PRRSV 2 weeks after infection (Table 2). Control animals tested negative to PRRSV and ORFS-pH protein in IIF
and ELISA
throughout the observation period.
Specific blastogenic response to ORES pH:
PBMCs obtained from both groups of immunized pigs underwent specific blastogenic transformation ex-vivo in a dose dependent way in the presence of ORFS-pH
protein, whereas [3H]-thymidine incorporation of the PBMCs obtained from unvaccinated animals remained at basal level (Fig. 6A). Blastogenic transformation indexes of 7-12 and 10-12 were calculated 2 weeks after the second booster injection of GST-ORFS and pRc/CMVS, respectively.
Concentrations higher than 10 ~tg of the ORES-pH protein per ml of culture medium did not increase [3H]-thymidine incorporation levels of PBMCs from both groups of pigs. No significant variations were observed in blastogenic response to ConA of vaccinated pigs compared to unvaccinated controls (Fig. 6B).
Table 2.
Antibody Response of DNA and GST-ORFS
Immunized Mice and Pigs Animal Immunogen SerologicalImmunization a.nd Sample Collection Schedule (days) Group and Dose Tests 1 151 371 5123 652 G1:50~.g ELISA - 140149 5601196 640f196 6401196 pRc/CMVS IIF - 3516 S1t16 51116 51116 mice' G2:50~g ELISA - 320198 8960131351024013135>12800 _ _ _ -Gl:SOgg ELISA - 2601120 5601196 5601196 960196 pRc/CMVS IIF - 2218 45116 10231 102131 BALB/c VN - - <8 51137 58f31 mice" G2:50pg ELISA - 6401196 256017844800f202425601784 VN - - <g 45116 102131 G1:100pg ELISA - 133147 5331189 6671189 6671189 pRc/CMVS IIF - 64f0 10730 5331189 6671189 Pigs VN - - <8 <g 107f30 G2:300pg ELISA - 40010 42671508 >12800 >12800 VT( _ _ <g <g <g ~ Groups of 5 mice or 3 piglets were immunized by pRc/CMVS plasmid or GST-ORFS
expressed in E. Coli on the mentioned days. Blood samples were collected from the retro-orbital vein of mice or the anterior vena cava of pigs prior to each immunization.
Z Sample collection only.
3 Pigs were challenged with 5 x lOs TCID50 by infra-tracheal inoculation.
' Control animals consisted of 5 BALB/c mice, 5 CD-1 mice and 3 F1 piglets.
Each control animal was injected with corresponding quantities of parental pRc/CMV plasmid via identical route and frequency. Control animals remained seronegative throughout the observation period.
ELISA: Reciprocal of highest serum dilution reacting with the recombinant ORFS-pH expressed in E. Coli.
IIF: Reciprocal of highest serum dilution at which specific cytoplasmic fluorescence was observed in PRRSV-infected MARC- 145 cells.
VN: Reciprocal of highest serum dilution which inhibited 100% of CPE and expression of N viral protein in PRRSV
(IAF-Klop strain) infected MARC 145 cells stained by IPMA.
Antibody titres correspond to the average titres t standard deviation.
Clinical observations:
Unvaccinated pigs developed clinical signs of respiratory disease, beginning 2 to 3 days after virus challenge and persisting through the end of the 2 week observation period. The principal signs included a marked drop in feed consumption, hyperthermia (40.2 to 41.7°C) that persisted for 10 to 14 days, eyelids oedema, laboured breathing (abdominal respiration) in two pigs accompanied by rasping and crowing sounds heard during inspiration. Apart from a transitory mild fever {39.8-40.4 °C) that lasted not more than 2 to 3 days, all vaccinated pigs remained clinically healthy during the 2 week-observation period following virus challenge. The average feed consumption and growth rate of vaccinated pigs remained identical to those of unvaccinated unchallenged controls.
Yirus isolation:
As summarized in Table 3, after a single passage on MARC-145 cells, virus was recovered from tissue homogenates (dilutions 1/20 and 1/100) of several organs (lungs, spleen, kidneys, liver, lymph nodes) of unvaccinated animals two weeks a8er virus challenge. In contrast, apart from lungs and mediastinal lymph nodes, no virus was isolated from other organs of DNA immunized pigs after two successive passages. This is indicative of the generalized viremia of unvaccinated pigs compared to respiratory tract localization of virus in DNA immunized animals. PRRSV was also recovered from the spleen and kidneys of one of the 3 GST-ORFS immunized pigs. Presence of the viral genome in the lungs of all three groups of animals was demonstrated by RT-PCR;
however, RT-PCR revealed the presence of the viral genome in the spleens of only unvaccinated challenged pigs and those pigs that had been immunized with GST-ORFS, not in the spleens of DNA immunized animals. Furthermore, PRRSV burden was lower in lungs and mediastinal lymph nodes of DNA vaccinated pigs since it could not be recovered after two passages on MARC-145 cells from the 1/100 dilution of tissue homogenate. The presence of viral antigen could only be detected in the 1/20 dilution of lung and mediastinal lymph nodes homogenates, with delayed appearance of CPEs suggestive of low virus titres.
Necropsy findings:
Unvaccinated virus challenged pigs euthanised at day 14 post-inoculation had gross lesions that were confined to the respiratory tract and thoracic cavity. Portions of the lungs were tan and partly collapsed, with occasional anteroventral areas of congestion and consolidation. The mediastinal lymph nodes were enlarged and congested. Adherence of the pleura to the thoracic cage was observed in one pig, with slight accumulation of non-suppurative exudate within the thoracic cavity (hydrothorax) and pericardium (hydropericardium). No significant gross lesions were observed in the other organs. Pulmonary hepatisation and glandular aspect at lung section was remarkable in one of the GST-ORFS vaccinated animals. Apart from mild tumefaction of mediastinal lymph nodes in one of the DNA vaccinated pigs, no significant gross abnormalities were observed in this group of animals. Microscopic lesions observed in unvaccinated-virus challenged pigs were confined to the lungs and consisted of macrophage infiltration, pyknotic cell debris and protein rich exudate in the lumen of large bronchi and bronchioli, a peribronchiolar and perivascular lymphomononuclear cells infiltration, the presence of lymphomononuclear cells within the alveolar lumen with hyperplasia of type 2 pneumocytes, mononuclear cells invasion, and presence of pyknotic cells in alveolar septae (Fig. 7 b, c, and d). The GST-ORFS immunized pigs developed intense interstitial pneumonitis, characterized by hyperplasia of bronchiolar epithelium and pneumocytes type II of the alveolar endothelium, perivascular cuffing, lymphomononuclear cells infiltration, and thickening of alveolar septae (Fig. 8a and b). A remarkably milder interstitial pneumonitis was observed in the DNA vaccinated pigs. In those pigs, large airways (bronchi and bronchioli), as well as alveolar ducts, were normal in appearance with an absence of cells and cellular debris within the lumen (Fig. 8c and d).

Our results show that DNA immunization with a plasmid encoding the ORFS of PRRSV protected pigs from developing intensive PRRSV-induced lesions observed in unvaccinated virus challenged controls. Virus dissemination to organs other than the lungs and the accessory lymph nodes was not observed in DNA-vaccinated animals after a massive virus challenge, and these animals had remarkably lower virus burden in their respiratory system as compared to the GST-ORFS vaccinated animals or unvaccinated controls. These results show that an expression vector encoding PRRSV
ORFS is an effective DNA vaccine against PRRSV infection.
Results have also been obtained using other ORFs. Balb/c mice immunized genetically with pAdCMVS/ORF3 and pAdCMVS/ORF4 had specific humoral immune responses triggered as demonstrated by indirect immunofluorescence and Western immunoblotting (Gonin et al., (1997) 16'" Annual Meeting of the American Society for Virology (ASV), Montana State University, Bozeman, MT, July 19-23; Gagnon et al., (1997) 78'" Annual Meeting of the CRWAD, Chicago, IL, November 8-12). Accordingly, expression vectors encoding a PRRSV ORF provide DNA vaccines against PRRSV infection.
1 S The foregoing has outlined some of the more pertinent objects of the present invention. These objects should be construed as being merely illustrative of some of the more prominent features and applications of the invention. Many other beneficial results can be obtained by applying the disclosed invention in a different manner or modifying the invention within the scope of the invention. Accordingly other objects and a full understanding of the invention may be had by refernng to the summary of the invention, the detailed description describing the preferred embodiments in addition to the scope of the invention defined by the claims taken in conjunction with the accompanying drawings.

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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i)APPLICANT: Institut National de la Recherche Scientifique (ii) TITLE OF INVENTION: Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) DNA Vaccines (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: MBM & CO.
(B) STREET: P.O. BOX 809, STATION B
(C) CITY: OTTAWA
(D) PROVINCE: ONTARIO
(E) COUNTRY: CANADA
(F) POSTAL CODE: K1P 5P9 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: Windows (D) SOFTWARE: WordPerfect 9.0 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: n/a (B) FILING DATE: 06-16-1998 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SWAIN, Margaret (B) REGISTRATION NUMBER: 10926 (C) REFERENCE/DOCKET NUMBER: 255-118DIV
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 613/567-0762 (B) TELEFAX: 613/563-7671 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: ETS 5 forward primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
AAGCTTGCCG CCGCCATGTT GGGGAAATGC TTGACC
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: ETR 5 reverse primer (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:

Claims (34)

1. THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
   OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
   A DNA vaccine comprising an expression vector and a nucleic acid sequence encoding one or more PRRSV ORFs or fragments thereof, wherein upon administration into a swine free from PRRSV infection, said swine is protected from infection by PRRSV.
2. 2. The DNA vaccine of claim 1, wherein the expression vector is a plasmid.
3. 3. The DNA vaccine of claim 2, additionally comprising transcription regulatory elements operably linked to said nucleic acid sequence.
4. 4. The DNA vaccine of claim 3, wherein said regulatory elements comprise a promoter sequence and a terminator sequence.
5. 5. The DNA vaccine of claim 4, wherein said promoter sequence is CMV.
6. 6. The DNA vaccine of claims 4 or 5, wherein said terminator sequence is bovine growth hormone.
7. 7. The DNA vaccines of claims 2 to 6, wherein said plasmid comprises pRc/CMV.
8. 8. The DNA vaccine of claims 2 or 6, wherein said plasmid is pAd/CMV5.
9. 9. The DNA vaccine of claim l, wherein the expression vector is a replication defective adenovirus.
10. 10. The DNA vaccine of claim 9, additionally comprising one or more nucleic acid sequences selected from the group comprising encapsidation signals, packaging signals, tripartite leader sequences, and major late enhancer sequences.
11. 1 l . The DNA vaccine of claim 9, wherein the adenovirus is AdCMV5/ORF5.
    5?
12. 12. The DNA vaccines of any one of claims 1 to 11, wherein said PRRSV ORFs or fragments thereof are selected from the group comprising ORF 2, ORF 3, ORF 4, ORF 5, ORF
    
    and ORF 7.
13. 13. The DNA vaccine of claim 1, wherein said strain of PRRSV is IAF-Klop.
14. 14. The DNA vaccine of claim 1, wherein said nucleic acid sequence is selected from the group comprising cDNA, genomic DNA, or a cDNA/genomic DNA hybrid.
15. 15. The DNA vaccine of claim 1, wherein said nucleic acid sequence further comprises the first ATG codon of the ORF, a Kozak motif, and/or the C-terminal stop codon of the ORF.
16. 16. A host cell that has been transformed or transfected by the DNA vaccine of claim 1.
17. 17. A DNA vaccine selected from the group comprising:
    
    (i) ~pRc/CMV2;
    
    (ii) pRc/CMV3;
    
    (iii) pRc/CMV4;
    
    (iv) pRc/CMV5;
    
    (v) pRc/CMV6;
    
    (vi) pRc/CMV7;
    
    (vii) pAdCMV5/ORF2;
    
    (viii) pAdCMV5/ORF3;
    
    (ix) pAdCMV5/ORF4;
    
    (x) pAdCMV5/ORF5;
    
    (xi) pAdCMV5/ORF6;
    
    (xii) pAdCMV5/ORF7;
    
    (xiii) AdCMV5/ORF2;
    
    (xiv) AdCMV5/ORF3;
    
    (xv) AdCMV5/ORF4;
    
    (xvi) AdCMV5/ORF5;
    
    (xvii) AdCMV5/ORF6; and (xviii) AdCMV5/ORF7.
18. 18. A composition comprising (i) a DNA vaccine comprising an expression vector and a nucleic acid sequence encoding one or more PRRSV ORFs or fragments thereof, wherein upon administration into a swine free from PRRSV infection, said swine is protected from infection by PRRSV; and (ii) a pharmaceutically acceptable carrier, buffer, solvent, or diluent.
19. 19. A host cell that has been transformed or transfected by the DNA vaccine of claim17.
20. 20. A kit for the administration of a DNA vaccine, wherein said DNA vaccine comprises an expression vector and a nucleic acid sequence encoding one or more PRRSV ORFs or fragments thereof, wherein upon administration into a swine free from PRRSV
    infection, said swine is protected from infection by PRRSV, comprising:
    (i) the DNA vaccine, either lyophilized or in solution;
    (ii) contained in a container, such as a syringe, pipette, eye dropper, vial, nasal spray, or inhaler; and (iii) instructions for use.
21. 21. A kit for detecting an antibody in a sample, said antibody specifically recognizing PRRSV, comprising a cell line that has been transformed or transfected by a DNA
    vaccine of claims I or 17, which is capable of expressing one or more of the PRRSV
    ORFs or fragments thereof containing an antigenic part or component of a PRRSV.
22. 22. A kit for detecting an antibody in a sample, said antibody specifically recognizing PRRSV, comprising one or more PRRSV ORF proteins or fragments thereof, containing an antigenic part or component of a PRRSV, recovered from cell cultures transformed or transfected by a DNA vaccine of claims 1 or 17.
23. 23. The kit of claim 20 and 21, wherein the sample is a biological sample, such as blood or blood serum, sputum, saliva, or tissue, derived from a swine.
24. 24. A serum suitable for treatment of swine infected with PRRSV, comprising the semi-purified blood serum of a mammal inoculated with a DNA vaccine comprising an expression vector and a nucleic acid sequence encoding one or more PRRSV ORFs or fragments thereof, wherein upon administration into a swine free from PRRSV
    infection, said swine is protected from infection by PRRSV.
25. 25. A method of using a DNA vaccine to protect swine against PRRS, comprising administering to said swine an effective amount of a PRRSV DNA vaccine, wherein said DNA vaccine comprises an expression vector and a nucleic acid encoding one or more PRRSV ORFs or fragments thereof, wherein upon administration into a swine free from PRRS infection said swine is protected from infection.
26. 26. The method of claim 25, wherein the DNA vaccine is administered by intramuscular injection, subcutaneous injection, intravenous injection, intradermal introduction, impression through the skin, inhalation, intraperitoneally, or by scarification.
27. 27. The method of claim 25, comprising the additional step of administering single or multiple booster vaccinations to the swine.
28. 28. The method of claim 25, wherein the DNA vaccine is administered topically via application of a solution, comprising the DNA vaccine, to the mucous membranes of the conjunctiva, the nasopharynx or the oropharynx.
29. 29. A kit of either claim 20 or 21, comprising the DNA vaccine, either lyophilized or in solution, contained in either a vial, a nasal spray or an inhaler, and instructions for use.
30. 30. A method of using a DNA vaccine claimed in either one of claims 1 or 17 to protect swine against PRRS, comprising administering an effective amount of said vaccine to a swine in need of protection.
31. 31. A method of using a serum claimed in claim 24 to protect swine against PRRS, comprising administering to said swine and effective amount of said serum.
32. 32. A method of using a DNA vaccine claimed in either of claims 1 or 17 to detect antibody in a sample, said antibody specifically recognizing PRRSV, comprising:
    (i) transforming or transfecting a cell culture with said DNA vaccine;
    (ii) expressing one or more of the PRRSV ORFs, or fragments thereof, of said DNA
    vaccine; and (iii) using said expressed PRRSV ORFs or fragments thereof as a source of antigen to detect the said antibody, using immunochemical techniques.
33. 33. The method of claim 32, wherein the sample is a biological sample, such as blood or blood serum, sputum, saliva, or tissue, derived from a swine.
34. 34. The method of claim 32, wherein the immunochemical technique is selected from the group comprising indirect immunofluorescence (IIF), immunoperoxydase (POD), western immunoblotting (WB), radioimmunoprecipitation (RIPA), enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and agglutination assays.