HK1219412B - Vaccine against west nile fever - Google Patents

Description

The present invention relates to in vivo and in vitro expression vectors comprising a polynucleotide in which the polynucleotide codes for the prM, M, and E proteins of the West Nile virus (WN), and the elements necessary for the expression of the polynucleotide in the host cell, wherein the vector is a viral vector, said viral vector being canarypox or fowlpox, as well as immunogenic compositions and vaccines against West Nile fever comprising said expression vector. It also relates to a method for preparing said composition, as well as said expression vector for use in methods of immunization and vaccination against this virus.

The West Nile virus (WNV) was first identified in humans in 1937 in Uganda, in the West Nile province (Zeller H. G., Med. Trop., 1999, 59, 490-494).

Widely spread in Africa, and also found in India, Pakistan, and the Mediterranean basin, it was first identified in the United States in New York City in 1999 (Anderson J. F. et al., Science, 1999, 286, 2331-2333).

The West Nile virus affects both birds, mammals, and humans.

The disease is characterized in birds by an attack on the central nervous system and death. Lesions include encephalitis, hemorrhages in the myocardium, and hemorrhages and necroses in the intestinal tract.

In chickens, experimental infections through subcutaneous inoculation of West Nile virus isolated from crows caused myocardial necrosis, nephritis, and pneumonia 5 to 10 days after inoculation, and moderate to severe encephalitis 21 days after inoculation (Senne D. A. et al., Avian Disease, 2000, 44, 642-649).

The West Nile virus also affects horses, particularly in North Africa and Europe (Cantile C. et al., Equine Vet. J., 2000, 32(1), 31-35). These horses show signs of ataxia, weakness in the hind limbs, paralysis progressing to tetraplegia and death. Horses and camelids are the main animals that exhibit clinical signs in the form of encephalitis.

Antibodies against WNV have been detected in certain rodents, livestock, particularly cattle and sheep, and in domestic animals, notably dogs (Zeller H. G., Med. Trop., 1999, 59, 490-494 ; Lundstrom J. O., Journal of Vector Ecology, 1999, 24(1), 1-39).

The human species is not spared by the West Nile virus, with many symptoms (Sampson B. A., Human Pathology, 2000, 31(5), 527-531; Marra C. M., Seminars in Neurology, 2000, 20(3), 323-327).

The West Nile virus is transmitted to birds and mammals through the bites of certain mosquitoes (such as Culex, Aedes, Anopheles) and ticks.

Wild and domestic birds are a reservoir for the West Nile virus and a vector for its spread through their migrations.

The virions of the West Nile virus are spherical particles with a diameter of 50 nm, composed of a lipoprotein envelope surrounding an icosahedral nucleocapsid containing a single-stranded positive-sense RNA.

An unique open reading frame (ORF) codes for all viral proteins in the form of a polyprotein. The cleavage and maturation of this polyprotein leads to the production of about ten different viral proteins. Structural proteins are encoded by the 5' part of the genome and correspond to the nucleocapsid named C (14 kDa), the envelope glycoprotein named E (50 kDa), the precursor membrane protein named prM (23 kDa), and the membrane protein named M (7 kDa). Non-structural proteins are encoded by the 3' part of the genome and correspond to the proteins NS1 (40 kDa), NS2A (19 kDa), NS2B (14 kDa), NS3 (74 kDa), NS4A (15 kDa), NS4B (29 kDa), and NS5 (97 kDa).

Parrish C. R. et al. (J. Gen. Virol., 1991, 72, 1645-1653), Kulkarni A. B. et al. (J. Virol., 1992, 66(6), 3583-3592) and Hill A. B. et al. (J. Gen. Virol., 1992, 73, 1115-1123) constructed in vivo expression vectors derived from the vaccinia virus containing various inserts corresponding to nucleotide sequences encoding non-structural proteins of the Kunjin virus possibly associated with structural proteins. These vectors were administered to mice to assess the cellular immune response. The authors emphasize the importance of the cellular response, which is primarily stimulated by non-structural proteins, particularly NS3, NS4A and NS4B. These articles highlight the difficulty in identifying a good vaccination strategy against West Nile fever. The document WO9837911 describes a live, infectious and attenuated chimeric yellow fever virus whose genome includes a nucleotide sequence encoding prM-E of a second flavivirus, which second flavivirus can be the West Nile virus.

To date, there is no vaccine available to prevent infection by the WN virus.

The present invention aims to provide a means for the prevention and/or control of diseases caused by the WN virus.

Another objective of the invention is to provide such a means that can be used in various animal species susceptible to the disease caused by this virus, and in particular, equine and avian species.

Another objective of the invention is to provide such a means for its use in immunization and vaccination methods for target species against the WN virus.

Another objective of disclosure is to provide such means and methods to ensure a differential diagnosis.

Summary of the Invention

The invention is as defined in the claims.

Thus, the invention has as its first object an in vitro and/or in vivo expression vector comprising a polynucleotide in which the polynucleotide codes for the prM, M, and E proteins of the West Nile virus (WN), and the necessary elements for expression of the polynucleotide in the host cell, the vector being a viral vector, said viral vector being a canarypox or fowlpox.

The vector preferably comprises a single coding phase polynucleotide corresponding to prM-M-E. A vector as defined in the claims and comprising multiple separated polynucleotides encoding different proteins (e.g., prM and/or M and E) also falls within the scope of the present invention. The vector as defined in the claims, particularly in vivo, may also comprise polynucleotides corresponding to more than one strain of WN virus, particularly two or more polynucleotides encoding E or prM-M-E from different strains. As will be seen later, the vector, particularly in vivo, may also include one or more nucleotide sequences encoding immunogens of other pathogens and/or cytokines.

By "polynucleotide encoding a West Nile virus protein," we mainly refer to a DNA fragment that codes for this protein, or the complementary strand of this DNA fragment. An RNA is not excluded.

In terms of disclosure, the term "protein" encompasses fragments, including peptides and polypeptides. By definition, a protein fragment is immunologically active in the sense that, when administered to the host, it is capable of triggering an antibody-mediated and/or cellular immune response directed against the protein. Preferably, the protein fragment has substantially the same immunological activity as the full-length protein. Therefore, a protein fragment according to the disclosure includes at least one epitope or antigenic determinant. The term "epitope" refers to a site on the protein capable of inducing an antibody-mediated (B-cells) and/or cellular (T-cells) immune reaction.

The minimal structure of a polynucleotide is therefore the one that codes for an epitope or antigenic determinant of the protein in question. A polynucleotide coding for a fragment of the entire protein includes, in particular, at least 21 nucleotides, preferably at least 42 nucleotides, and more preferably at least 57, 87, or 150 consecutive nucleotides from the sequence it originates from. Techniques for determining epitopes are well known to those skilled in the art; for example, overlapping peptide banks (Hemmer B. et al., Immunology Today, 1998, 19(4), 163-168), Pepscan (Geysen H. M. et al., Proc. Nat. Acad. Sci. USA, 1984, 81(13), 3998-4002; Geysen H. M. et al., Proc. Nat. Acad. Sci. USA, 1985, 82(1), 178-182; Van der Zee R. et al., Eur. J. Immunol., 1989, 19(1), 43-47; Geysen H. M., Southeast Asian J. Trop. Med. Public Health, 1990, 21(4), 523-533; Multipin® Peptide Synthesis Kits from Chiron), and algorithms (De Groot A. et al., Nature Biotechnology, 1999, 17, 533-561) can be used.

In particular, the polynucleotides include the nucleotide sequence encoding one or both of the transmembrane domains, preferably both, located in the C-terminal part of the E protein. For the WNV NY99 strain, these domains correspond to the amino acid sequences 742 to 766 and 770 to 791 of GenBank AF196835.

Essential elements for the expression of the polynucleotide(s) are present. These include at a minimum an initiation codon (ATG), a stop codon, and a promoter, as well as a polyadenylation sequence for plasmids and viral vectors other than poxviruses. When the polynucleotide codes for a fragment of the polyprotein, such as prM-E, M-E, or prM-M-E, an ATG is placed 5' to the reading frame and a stop codon is placed 3'. As will be explained later, other elements allowing control of expression may also be present, such as enhancer sequences, stabilizing sequences, and signal sequences enabling secretion of the protein.

The present invention also relates to preparations comprising an expression vector according to the invention and a pharmaceutically acceptable vehicle or excipient.

According to a mode of the invention, the preparation comprises the said expression vector according to the invention and other vectors comprising and expressing one or more proteins from one or more other strains of West Nile virus (WN). In particular, the preparation comprises at least two vectors expressing, preferably in vivo, polynucleotides from different WN strains, encoding the same proteins and/or different proteins, preferably the same proteins, one of said at least two vectors being the said expression vector according to the invention. It is preferably vectors expressing in vivo E or prM-M-E of two, three or more different WN strains, one of said vectors being the said expression vector according to the invention. The invention also relates to mixtures of vectors expressing prM, M, E, prM-M, prM-E or M-E from different strains, one of said vectors being the said expression vector according to the invention.

In another mode, as will be seen in more detail later, the other vector(s) in the preparation include and express one or more cytokines and/or one or more immunogens from one or more other pathogens.

Disclosure also refers to the various combinations of these different methods.

According to a particular method of realizing the invention, the polynucleotide of the expression vector according to the invention further includes a nucleotide sequence encoding a signal peptide located upstream of the expressed protein, preferably a signal sequence from the WN virus.

According to a particular method of production, one or more of the non-structural proteins NS2A, NS2B, and NS3 are co-expressed with structural proteins, either via the same expression vector or via a separate expression vector. They are preferably expressed together from a single polynucleotide.

Therefore, the disclosure also relates to an expression vector, either in vivo or in vitro, comprising the polynucleotide encoding NS2A, NS2B, NS3, their combinations, and preferably NS2A-NS2B-NS3. At its basis, this vector can be one of the vectors described above, including a polynucleotide encoding one or more structural proteins, notably E or prM-M-E. As an alternative, the invention relates to a preparation as described above, further comprising the expression vector as defined in the claims, at least one of these vectors expressing a non-structural protein and optionally a pharmaceutically acceptable vehicle or excipient.

To produce the expression vectors according to the invention, a person skilled in the art has access to various strains of the WN virus and the description of the nucleotide sequence of their genome. See, in particular, Savage H. M. et al. (Am. J. Trop. Med. Hyg. 1999, 61(4), 600-611), Table 2, which lists 24 WN virus strains and provides references for accessing polynucleotide sequences in GenBank.

For example, one can refer to the NY99 strain (GenBank AF196835). In GenBank, the corresponding DNA sequence is specified for each protein (nucleotides 466-741 for prM, 742-966 for M, 967-2469 for E, i.e., 466-2469 for prM-M-E, 3526-4218 for NS2A, 4219-4611 for NS2B, and 4612-6468 for NS3, i.e., 3526-6468 for NS2A-NS2B-NS3). By comparing and aligning sequences, it is straightforward to determine a polynucleotide encoding such a protein in another WNV strain.

It was mentioned above that by "polynucleotide," one refers to the sequence encoding the protein or a fragment or an epitope specific to the WN virus. Furthermore, by equivalence, the term "polynucleotide" also encompasses the corresponding nucleotide sequences of the different WN virus strains and the nucleotide sequences that differ due to the degeneracy of the code.

Within the West Nile virus (WNV) family, the identity of amino acid sequences prM-M-E compared to that of NY99 is equal to or higher than 90%. Therefore, the disclosure includes polynucleotides encoding an amino acid sequence having an identity with the native amino acid sequence of equal to or greater than 90%, preferably 92%, more preferably 95%, and even more particularly 98%. Fragments of these homologous polynucleotides that are specific to WNV viruses will also be considered as equivalents.

Thus, if one refers to the WN virus polynucleotide, this term encompasses the equivalent sequences in terms of disclosure.

It has also been noted that the term "protein" encompasses immunologically active polypeptides and peptides. For the purposes of disclosure, this includes: a) the corresponding proteins from different strains of the WN virus; b) proteins that differ but maintain an identity of at least 90%, preferably 92%, more preferably 95%, and particularly 98%, with a native WN protein.

Thus, if one refers to a West Nile virus protein, this term encompasses the equivalent proteins in terms of disclosure.

Different strains of the West Nile virus are available from culture collections, such as the American Type Culture Collection (ATCC), for example under the access numbers VR-82 or VR-1267. The virus named Kunjin is considered to actually be a West Nile virus.

According to the invention, it is preferred that the polynucleotide further comprises a nucleotide sequence encoding a signal peptide, located upstream of the expressed protein in order to ensure its secretion. It may thus be an endogenous sequence, i.e., the natural signal sequence when it exists (originating from the same WN virus or from another strain). For example, for the WN NY99 virus, the endogenous E signal sequence corresponds to nucleotides 922 to 966 of the GenBank sequence; for prM, it corresponds to nucleotides 421 to 465. It may also be a nucleotide sequence encoding a heterologous signal peptide, notably the sequence encoding the signal peptide of human tissue plasminogen activator (tPA) (Hartikka J. et al., Human Gene Therapy, 1996, 7, 1205-1217). The nucleotide sequence encoding the signal peptide is inserted in frame and upstream of the sequence encoding E or its combinations, e.g., prM-ME.

According to a first method of disclosure, in vivo expression vectors are viral vectors.

These expression vectors are advantageously poxviruses, for example the vaccinia virus or attenuated mutants of the vaccinia virus, e.g., MVA (Ankara strain) (Stickl H. and Hochstein-Mintzel V., Munch. Med. Wschr., 1971, 113, 1149-1153; Sutter G. et al., Proc. Natl. Acad. Sci. U.S.A., 1992, 89, 10847-10851; commercial strain ATCC VR-1508; MVA is obtained after more than 570 passages of the Ankara vaccinia strain on chicken embryo fibroblasts) or NYVAC (its construction is described in US-A-5 494 807, particularly in examples 1 to 6; this patent also describes the insertion of heterologous genes into specific sites of this recombinant and the use of appropriate promoters; see also WO-A-96/40241),Avipoxviruses (particularly canarypox, fowlpox, pigeonpox, quailpox), swinepox, raccoonpox, and camelpox, adenoviruses such as avian, canine, porcine, bovine, and human adenoviruses, and herpesviruses such as equine herpesviruses (EHV serotypes 1 and 4), canine herpesvirus (CHV), feline herpesvirus (FHV), bovine herpesviruses (BHV serotypes 1 and 4), porcine herpesvirus (PRV), Marek's disease viruses (MDV serotypes 1 and 2), turkey herpesvirus (HVT or MDV serotype 3), and duck herpesvirus. When a herpesvirus is used for vaccination of avian species, the HVT vector is preferred,And for horse vaccination, the EHV vector is preferred.

According to the invention, the expression vector is a canarypox or fowlpox, these poxviruses possibly being attenuated. A commercially available canarypox is mentioned, available from ATCC under access number VR-111. Attenuated canarypox strains are described in US-A-5,756,103 and in WO-A-01/05934. Many fowlpox virus vaccine strains are available, for example the DIFTOSEC CT® vaccine marketed by MERIAL and the NOBILIS® VARIOLA vaccine marketed by Intervet.

For the poxviruses, the specialist may refer to WO-A-90/12882, and more particularly for the vaccinia virus to US-A-4,769,330; US-A-4,722,848; US-A-4,603,112; US-A-5,110,587; US-A-5,494,807; US-A-5,762,938; for fowlpox to US-A-5,174,993; US-A-5,505,941; US-5,766,599; for canarypox to US-A-5,756,103; for swinepox to US-A-5,382,425; and for raccoonpox to WO-A-00/03030.

When the expression vector is a canarypox virus, the insertion sites are particularly located in, or composed of, the COLs C3, C5 and C6. When it is a fowlpox virus, the insertion sites are particularly located in, or composed of, the COLs F7 and F8.

The insertion of genes into the MVA virus is described in various publications including Carroll M. W. et al., Vaccine, 1997, 15(4), 387-394; Stittelaar K. J. et al., J. Virol., 2000, 74(9), 4236-4243; Sutter G. et al., Vaccine, 1994, 12(11), 1032-1040, which a skilled person can refer to. The complete genome of MVA is described in Antoine G., Virology, 1998, 244, 365-396, allowing the skilled person to use other insertion sites or other promoters.

According to the disclosure, when the expression vector is a poxvirus, the polynucleotide to be expressed is inserted under the control of a specific poxvirus promoter, notably the vaccine 7.5 kDa promoter (Cochran et al., J. Virology, 1985, 54, 30-35), the vaccine I3L promoter (Riviere et al., J. Virology, 1992, 66, 3424-3434), the vaccine HA promoter (Shida, Virology, 1986, 150, 451-457), the cowpox ATI promoter (Funahashi et al., J. Gen. Virol., 1988, 69, 35-47), or the vaccine H6 promoter (Taylor J. et al. Vaccine, 1988, 6, 504-508; Guo P. et al. J. Virol., 1989, 63, 4189-4198; Perkus M. et al. J. Virol., 1989, 63, 3829-3836).

Preferably for mammalian vaccination, the expression vector is canarypox. Preferably for avian vaccination, particularly in chickens, ducks, turkeys, and geese, the expression vector is canarypox or fowlpox.

When the expression vector is a herpesvirus HVT, appropriate insertion sites are particularly located in the BamHI I fragment or in the BamHI M fragment of HVT. The BamHI I restriction fragment of HVT includes several open reading frames (ORFs) and three intergenic regions, and contains several preferred insertion sites, namely the three intergenic regions 1, 2, and 3, which are the preferred regions, and the UL55 ORF (FR-A-2 728 795, US-A-5 980 906). The BamHI M restriction fragment of HVT includes the UL43 ORF, which is also a preferred insertion site (FR-A-2 728 794, US-A-5 733 554).

When the expression vector is an equine herpesvirus EHV-1 or EHV-4, appropriate insertion sites are particularly TK, UL43 and UL45 (EP-A-0668355).

Preferably, when the expression vector is a herpesvirus, the polynucleotide to be expressed is inserted under the control of a strong eukaryotic promoter, preferably the CMV-IE promoter. These strong promoters are described below in the section of the description relating to plasmids.

According to a second mode of disclosure, in vivo expression vectors are plasmid vectors called plasmids.

The term "plasmid" is intended to encompass any DNA transcription unit in the form of a polynucleotide sequence comprising a polynucleotide according to the disclosure and the necessary elements for its in vivo expression. The circular, supercoiled or non-supercoiled plasmid form is preferred. The linear form also falls within the scope of this disclosure.

Each plasmid comprises a promoter capable of ensuring, in host cells, the expression of the inserted polynucleotide under its control. This is generally a strong eukaryotic promoter. The preferred strong eukaryotic promoter is the early promoter of the cytomegalovirus (CMV-IE), which may be of human or murine origin, or possibly of another origin such as rat or guinea pig. The CMV-IE promoter may include the actual promoter region, associated or not with an enhancer region. Reference can be made to EP-A-260 148, EP-A-323 597, US-A-5 168 062, US-A-5 385 839, US-A-4 968 615, WO-A-87/03905. The human CMV-IE (Boshart M. et al., Cell, 1985, 41, 521-530) or murine promoter is preferably used.

More generally, the promoter is either of viral origin or of cellular origin. As a strong viral promoter other than CMV-IE, one can mention the early/late promoter of the SV40 virus or the LTR promoter of the Rous sarcoma virus. As a strong cellular promoter, one can cite the promoter of a cytoskeleton gene, such as the desmin promoter (Kwissa M. et al., Vaccine, 2000, 18(22), 2337-2344), or the actin promoter (Miyazaki J. et al., Gene, 1989, 79(2), 269-277).

By equivalence, the sub-fragments of these promoters, retaining an adequate promoter activity, are included in the present disclosure: e.g., the truncated CMV-IE promoters according to WO-A-98/00166. The concept of a promoter according to the disclosure therefore includes derivatives and sub-fragments retaining an adequate promoter activity, preferably substantially similar to that of the original promoter from which they originate. For the CMV-IE, this concept includes the actual promoter region and/or the enhancer region and their derivatives and sub-fragments.

Preferably, the plasmids include other elements controlling expression. In particular, it is advantageous to incorporate stabilizing sequences of the intron type, preferably intron II of the rabbit β-globin gene (van Ooyen et al., Science, 1979, 206: 337-344).

As a polyadenylation (polyA) signal for plasmids and viral vectors other than poxviruses, one can notably use that of the bovine growth hormone gene (bGH) (US-A-5 122 458), that of the rabbit β-globin gene, or that of the SV40 virus.

Other control elements used in plasmids can also be used in herpesvirus expression vectors.

According to another mode of disclosure, expression vectors are vectors used to express proteins in an appropriate cellular system in vitro. The proteins can then be collected from the culture supernatant, either after secretion or not (if there is no secretion, cell lysis is performed), and possibly concentrated using classical concentration techniques, such as ultrafiltration, and/or purified using conventional purification methods, such as affinity chromatography, ion exchange, or gel filtration.

The production is carried out by transfecting mammalian cells with plasmids, by replicating viral vectors in mammalian or avian cells, or by replicating Baculovirus (US-A-4 745 051; Vialard J. et al., J. Virol., 1990, 64(1), 37-50; Verne A., Virology, 1988, 167, 56-71), for example, Autographa californica Nuclear Polyhedrosis Virus AcNPV, in insect cells (for example Sf9 Spodoptera frugiperda cells, ATCC CRL 1711 deposit). Mammalian cells that can be used include, for example, hamster cells (such as CHO or BHK-21), and monkey cells (such as COS or VERO). Therefore, the disclosure also covers these expression vectors incorporating a polynucleotide according to the disclosure, the WN proteins or fragments thus produced, and the preparations containing them.

Therefore, this disclosure also relates to purified and/or concentrated preparations of WN proteins. When the polynucleotide encodes for multiple proteins, these proteins are cleaved, and the above preparations then contain the cleaved proteins.

The invention also relates to immunogenic compositions and vaccines against the WN virus, comprising at least one in vivo expression vector according to the invention, and a pharmaceutically acceptable vehicle or excipient, and optionally an adjuvant.

The concept of immunogenic composition refers to any composition capable, once administered to the target species, of inducing an immune response directed against the WN virus. By vaccine is meant a composition capable of inducing effective protection. The target species are equines, canines, felines, bovines, swine, and birds, preferably horses, dogs, cats, pigs, and for birds, geese, turkeys, chickens, and ducks, which by definition includes breeding animals, laying hens, and meat animals.

Pharmaceutically acceptable vehicles or excipients are well known to those skilled in the art. For example, they may be 0.9% sodium chloride saline solution or a phosphate buffer. Pharmaceutically acceptable vehicles or excipients also include any compound or combination of compounds that facilitate the administration of the vector, particularly transfection, and/or improve preservation.

The doses and volume of doses are defined later within the framework of the general description of immunization and vaccination methods.

Immunogenic compositions and vaccines according to the invention preferably include one or more adjuvants, selected particularly from conventional adjuvants. Particularly suitable within the scope of the present invention are: (1) acrylic or methacrylic acid polymers, maleic anhydride and alkenyl derivative polymers, (2) immunostimulatory sequences (ISS), notably oligodeoxyribonucleotide sequences containing one or more unmethylated CpG motifs (Klinman D. M. et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 2879-2883; WO-A1-98/16247), (3) oil-in-water emulsions, in particular the SPT emulsion described on page 147 of "Vaccine Design, The Subunit and Adjuvant Approach" edited by M. Powell, M. Newman, Plenum Press 1995, and the MF59 emulsion described on page 183 of the same publication, (4) cationic lipids containing a quaternary ammonium salt, (5) cytokines, or (6) combinations or mixtures thereof.

Water-in-oil emulsion (3), which is particularly suitable for viral vectors, can notably be based on: light liquid paraffin oil (European Pharmacopoeia type); isoprenoid oils such as squalane and squalene; oil resulting from the oligomerization of alpha-olefins, in particular isobutene or decene; esters of linear alkyl group acids or alcohols; more particularly vegetable oils, ethyl oleate, propylene glycol dicaprylate/caprate, glycerol tricaprylate/caprate, propylene glycol dioleate; esters of branched-chain fatty acids or alcohols, particularly esters of isostearic acid.

Oil is used in conjunction with emulsifiers to form an emulsion. Emulsifiers are preferably nonionic surfactants, particularly: on one hand, esters of sorbitan, mannitol (e.g., anhydromannitol oleate), glycerol, polyglycerol, or propylene glycol, and on the other hand, oleic, isostearic, ricinoleic, or hydroxystearic acids, these esters being optionally ethoxylated; block copolymers of polyoxypropylene-polyoxyethylene, in particular Pluronic® such as L121.

Among the adjuvant polymers of type (1), crosslinked acrylic or methacrylic acid polymers are preferred, particularly those crosslinked by polyalkylenic ethers of sugars or polyalcohols. These compounds are known under the term carbomers (Pharmeuropa, vol. 8, no. 2, June 1996). A person skilled in the art can also refer to US-A-2 909 462, which describes such crosslinked acrylic polymers using a polyhydroxy compound having at least 3 hydroxyl groups, preferably not more than 8, with the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals containing at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g., vinyl, allyl and other ethylenically unsaturated groups. The unsaturated radicals themselves may contain other substituents, such as methyl. Products sold under the name Carbopol® (BF Goodrich, Ohio, USA) are particularly suitable. They are notably crosslinked by allyl sucrose or allylpentaerythritol. Among them, Carbopol® 974P, 934P and 971P are particularly mentioned.

Among maleic anhydride and alkene derivatives copolymers, EMA® (Monsanto) are preferred; they are maleic anhydride and ethylene copolymers, either linear or crosslinked, for example, crosslinked with divinyl ether. Reference can be made to J. Fields et al., Nature, 186: 778-780, June 4, 1960.

In terms of their structure, acrylic or methacrylic acid polymers and EMA® are preferably formed from basic units of the following formula: wherein: R1 and R2, which may be the same or different, represent H or CH3; x = 0 or 1, preferably x = 1; y = 1 or 2, with x + y = 2.

For EMA®, x = 0 and y = 2. For carbomers, x = y = 1.

These polymers are dissolved in water or in physiological saline (NaCl at 20 g/l), and the pH is adjusted to 7.3–7.4 with sodium hydroxide, to obtain the adjuvant solution in which the expression vectors will be incorporated.

The polymer concentration in the final vaccine formulation can range, for example, from 0.01% to 1.5% w/v, more particularly from 0.05% to 1% w/v, preferably from 0.1% to 0.4% w/v.

Cationic lipids (4) containing a quaternary ammonium salt, which are particularly but not exclusively suitable for plasmids, are preferably those that meet the following formula: in which R1 is a linear aliphatic radical, saturated or unsaturated, having 12 to 18 carbon atoms, R2 is another aliphatic radical containing 2 or 3 carbon atoms, and X is a hydroxyl or amine group.

Among these cationic lipids, DMRIE (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanammonium; WO-A-96/34109) is preferred, preferably associated with a neutral lipid, preferably DOPE (dioleoyl-phosphatidylethanolamine; Behr J. P., 1994, Bioconjugate Chemistry, 5, 382-389), to form DMRIE-DOPE.

Preferably, the mixture of the plasmid with this adjuvant is prepared immediately before use, and it is preferred to allow the resulting mixture to complex, for example, for a period ranging from 10 to 60 minutes, preferably about 30 minutes, before administration.

When DOPE is present, the molar ratio of DMRIE:DOPE preferably ranges from 95:5 to 5:95, particularly 1:1.

The plasmid: adjuvant ratio of DMRIE or DMRIE-DOPE can range, for example, from 50:1 to 1:10, particularly from 10:1 to 1:5, and preferably from 1:1 to 1:2.

The cytokine(s) (5) can be provided in the form of a protein in the composition or vaccine, or can be co-expressed in the host with the antigen(s). Preference is given to the co-expression of the cytokine(s), either by the same vector that expresses the antigen, or by a separate vector.

Cytokines can notably be selected from: interleukin 18 (IL-18), interleukin 12 (IL-12), interleukin 15 (IL-15), MIP-1α (macrophage inflammatory protein 1α; Marshall E. et al., Br. J. Cancer, 1997, 75(12), 1715-1720), GM-CSF (granulocyte-macrophage colony-stimulating factor). In particular, avian cytokines can be mentioned, such as those from chickens, like chIL-18 (Schneider K. et al., J. Interferon Cytokine Res., 2000, 20(10), 879-883), chIL-15 (Xin K.-Q. et al., Vaccine, 1999, 17, 858-866), and equine cytokines, such as equine GM-CSF (WO-A-00/77210). Preferably, cytokines from the species to be vaccinated are used.

WO-A-00/77210 describes the nucleotide sequence and the corresponding amino acid sequence of equine GM-CSF, the production of GM-CSF in vitro, and the construction of vectors (plasmids and viral vectors) allowing the expression of equine GM-CSF in vivo. These proteins, plasmids, and viral vectors can be used in immunogenic compositions and equine vaccines according to the invention. For example, the plasmid pJP097 described in Example 3 of this earlier application may be used, or the teachings of this earlier application may be employed to produce other vectors or to produce equine GM-CSF in vitro, and to incorporate these vectors or this equine GM-CSF into immunogenic compositions or equine vaccines according to the invention.

The present disclosure further relates to immunogenic compositions and so-called subunit vaccines, comprising the E protein, and optionally one or more other proteins of the WN virus, notably prM or M, preferably produced by in vitro expression as described above, and on the other hand, a pharmaceutically acceptable vehicle or excipient.

Pharmaceutically acceptable vehicles or excipients are well known to the skilled person. As an example, they may be a 0.9% sodium chloride saline solution or a phosphate buffer.

Immunogenic compositions and subunit vaccines according to the disclosure preferably include one or more adjuvants, selected particularly from conventional adjuvants. Particularly suitable within the scope of the present disclosure are: (1) a polyacrylic or polymethacrylic acid polymer, a maleic anhydride and alkenyl derivative polymer, (2) an immunostimulatory sequence (ISS), notably an oligodeoxyribonucleotide sequence having one or more unmethylated CpG motifs (Klinman D. M. et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 2879-2883; WO-A1-98/16247), (3) an oil-in-water emulsion, particularly the SPT emulsion described on page 147 of "Vaccine Design, The Subunit and Adjuvant Approach" edited by M. Powell, M. Newman, Plenum Press 1995, and the MF59 emulsion described on page 183 of the same publication, (4) a water-in-oil emulsion (EP-A-639 071), (5) saponin, particularly Quil-A, or (6) aluminum hydroxide or equivalent. The various types of adjuvants defined under (1), (2) and (3) have been previously described in more detail in relation to vector-based vaccines.

The doses and volume of doses are defined later within the framework of the general description of immunization and vaccination methods.

According to the invention, vaccination against the WN virus can be combined with other vaccinations within vaccination programs, in the form of immunization kits or vaccination products, or as immunogenic compositions and multivalent vaccines, meaning they include at least one component of a vaccine against the WN virus and at least one component of a vaccine against at least one other pathogenic agent. This also includes the expression by the same expression vector of genes from at least two pathogens, including the WN virus.

Thus, the invention has as its object a multivalent immunogenic composition or a multivalent vaccine against the WN virus and at least one other pathogen of the target species, using the same in vivo expression vector containing and expressing at least one polynucleotide of the WN virus according to the invention and at least one polynucleotide expressing an immunogen of another pathogen.

By "immunogen" as thus expressed, one particularly refers to a protein, glycoprotein, polypeptide or peptide, epitope or derivative, e.g., a fusion protein, which induces an immune response, preferably a protective one.

As described previously, these compositions or multivalent vaccines also include a pharmaceutically acceptable vehicle or excipient, and optionally an adjuvant.

The invention also relates to a multivalent immunogenic composition or a multivalent vaccine comprising at least one in vivo expression vector in which at least one polynucleotide of the WN virus according to the invention is inserted, and at least one second expression vector in which a polynucleotide encoding an immunogen of another pathogenic agent is inserted. As described previously, these multivalent compositions or vaccines also include a pharmaceutically acceptable vehicle or excipient, and optionally an adjuvant.

For immunogenic compositions and multivalent vaccines, said other equine pathogens are preferably selected from the group comprising equine herpesvirus EHV-1 and/or EHV-4 (preferably, immunogens of both EHV-1 and EHV-4 are combined), equine influenza virus EIV, Eastern equine encephalitis virus EEV, Western equine encephalitis virus WEV, Venezuelan equine encephalitis virus VEV (preferably a combination of EEV, WEV, and VEV), Clostridium tetani (tetanus), and their mixtures. Preferably, for EHV, the genes gB and/or gD are selected; for EIV, the genes HA, NP and/or N; for the encephalitis viruses, the genes C and/or E2; for Clostridium tetani, the gene encoding all or part of the C subunit of the tetanus toxin. This includes the use of polynucleotides encoding an immunologically active fragment or an epitope of said immunogen.

These other avian pathogens are particularly selected from the group including the Marek's disease virus MDV (serotypes 1 and 2, preferably serotype 1), Newcastle disease virus NDV, Gumboro disease virus IBDV, infectious bronchitis virus IBV, infectious anemia virus CAV, infectious laryngotracheitis virus ILTV, avian encephalomyelitis virus AEV (also known as avian leukosis virus ALV), turkey hemorrhagic enteritis virus HEV, pneumovirus (TRTV), avian pest (avian influenza), infectious coryza virus, avian reoviruses, Escherichia coli, Mycoplasma gallinarum, Mycoplasma gallisepticum,Haemophilus avium, Pasteurella gallinarum, Pasteurella multocida gallicida, and their mixtures. Preferably, for MDV, the genes gB and/or gD are selected; for NDV, the genes HN and/or F; for IBDV, the gene VP2; for IBV, the genes S (and particularly S1), M and/or N; for CAV, the genes VP1 and/or VP2; for ILTV, the genes gB and/or gD; for AEV, the genes env and/or gag/pro; for HEV, the genes 100K and hexon; for TRTV, the genes F and/or G; for avian influenza, the genes HA, N and/or NP. This includes the use of polynucleotides encoding an immunologically active fragment or an epitope of this immunogen.

For example, in a multivalent immunogenic composition or a multivalent vaccine according to the invention, optionally adjuvanted as described above, intended for the equine species, one can incorporate one or more of the plasmids described in WO-A-98/03198, and in particular in examples 8 to 25 of this earlier application, and those described in WO-A-00/77043 relating to the equine species, particularly those described in examples 6 and 7 of this earlier application. For the avian species, one can, for example, incorporate one or more of the plasmids described in WO-A1-98/03659, and in particular in examples 7 to 27 of this earlier application.

Immunogenic compositions or recombinant vaccines as described above can also be combined with at least one conventional vaccine (inactivated, attenuated live, subunit) directed against at least one other pathogen.

Similarly, the immunogenic compositions and subunit vaccines according to the disclosure may be used in combination with other vaccinations. Therefore, the disclosure also relates to multivalent immunogenic compositions and multivalent vaccines, comprising one or more proteins according to the disclosure, and one or more immunogens (the term immunogen has been defined above) of at least one other pathogenic agent (notably from the list mentioned above), and/or another pathogenic agent in an inactivated or attenuated form. As described previously, these multivalent compositions or vaccines also include a pharmaceutically acceptable vehicle or excipient, and optionally an adjuvant.

The present invention also relates to methods for immunizing and vaccinating the target species mentioned above.

These methods include the administration of an effective amount of an immunogenic composition or a vaccine according to the invention. This administration can be performed, for example, by the parenteral route, such as subcutaneous, intradermal, or intramuscular administration, or by the oral and/or nasal route. One or more administrations can be carried out, notably two.

Different vaccines can be administered using a needle-free liquid jet injector. For plasmids, gold particles coated with plasmid DNA can also be used and projected in a way that they penetrate the skin cells of the subject to be immunized (Tang et al., Nature 1992, 356, 152-154).

The immunogenic compositions and vaccines according to the invention include an effective amount of the expression vector according to the invention.

In the case of immunogenic compositions or plasmid-based vaccines, a dose generally ranges from about 10 µg to about 2000 µg, preferably from about 50 µg to about 1000 µg. The dose volume can range from 0.1 to 2 ml, preferably from 0.2 to 1 ml.

These doses and volume of dose are well suited for the vaccination of horses and mammals.

For the vaccination of birds, a dose is particularly between approximately 10 µg and approximately 500 µg, and preferably between approximately 50 µg and approximately 200 µg. The dose volumes can be particularly between 0.1 and 1 ml, preferably between 0.2 and 0.5 ml.

The expert in the field has the necessary skills to optimize the effective dose of plasmid to be used for each immunization or vaccination protocol and to determine the best route of administration.

In the case of immunogenic formulations or poxvirus-based vaccines, a dose is generally between approximately 10² pfu and approximately 10⁹ pfu.

For the equine species and other mammals, when the vector is the vaccinia virus, the dose is particularly between approximately 10⁴ pfu and approximately 10⁹ pfu, preferably between approximately 10⁶ and approximately 10⁸ pfu; when the vector is the canarypox virus, the dose is particularly between approximately 10⁵ pfu and approximately 10⁹ pfu, preferably between approximately 10⁵·⁵ or 10⁶ and approximately 10⁸ pfu.

For the avian species, when the vector is the canarypox virus, the dose is particularly between approximately 10³ pfu and approximately 10⁷ pfu, preferably between approximately 10⁴ and approximately 10⁶ pfu; when the vector is the fowlpox virus, the dose is particularly between approximately 10² pfu and approximately 10⁵ pfu, preferably between approximately 10³ and approximately 10⁵ pfu.

In the case of immunogenic compositions or viral vector-based vaccines other than poxviruses, such as herpesviruses, a dose is generally between approximately 10³ PFU and approximately 10⁸ PFU. In the case of immunogenic compositions or avian vaccines, a dose is generally between approximately 10³ PFU and approximately 10⁶ PFU. In the case of immunogenic compositions or equine vaccines, a dose is generally between approximately 10⁶ PFU and approximately 10⁸ PFU.

The dose volumes of the immunogenic compositions and viral vector-based equine vaccines are generally between 0.5 and 2.0 ml, preferably between 1.0 and 2.0 ml, more preferably 1.0 ml. The dose volumes of the immunogenic compositions and viral vector-based avian vaccines are generally between 0.1 and 1.0 ml, preferably between 0.1 and 0.5 ml, more preferably between 0.2 and 0.3 ml. Those skilled in the art also have the necessary expertise to optimize the number of administrations, the route of administration, and the doses to be used for each immunization protocol. In particular, two administrations are planned for horses, for example at an interval of 35 days.

In the case of immunogenic compositions or subunit vaccines, a dose generally comprises about 10 µg to about 2000 µg, preferably about 50 µg to about 1000 µg. The dose volumes for immunogenic compositions and viral vector-based equine vaccines are generally between 1.0 and 2.0 ml, preferably between 0.5 and 2.0 ml, more preferably 1.0 ml. The dose volumes for immunogenic compositions and viral vector-based avian vaccines are generally between 0.1 and 1.0 ml, preferably between 0.1 and 0.5 ml, more preferably between 0.2 and 0.3 ml. Those skilled in the art also have the necessary expertise to optimize the number of administrations, the route of administration, and the doses to be used for each immunization protocol.

The invention also relates to the use of an in vivo expression vector according to the invention for preparing an immunogenic composition or a vaccine intended to protect target species against the WN virus and possibly against at least one other pathogenic agent. The various features mentioned in the rest of the description apply to this other object of the invention.

A viral vaccine according to the present invention will not induce in the vaccinated animal the production of antibodies against other proteins of this virus that are not represented in the immunogenic or vaccine composition. This characteristic can be used for the development of differential diagnostic methods allowing the distinction between animals infected with the pathogenic WN virus and animals vaccinated using vaccines according to the invention. In the latter, these proteins and/or antibodies directed against them are present and can be detected by an antigen-antibody reaction. This is not the case for animals vaccinated according to the invention, which remain negative. To achieve this discrimination, a protein not represented in the vaccine (not present or not expressed) is used, for example, the C protein or the NS1, NS2A, NS2B, or NS3 proteins when they are not represented in the vaccine.

Therefore, the present invention relates to the use of a vector according to the invention for preparing immunogenic compositions and vaccines that allow distinguishing between vaccinated animals and infected animals.

It also relates to an immunization and vaccination method associated with a diagnostic method that enables this differentiation.

The selected protein for diagnosis or one of its fragments or epitopes is used as an antigen in the diagnostic test, and/or is used to produce antibodies, polyclonal or monoclonal. The skilled person has the knowledge and experience to produce these antibodies and to implement antigens and/or antibodies in classical diagnostic techniques, e.g., ELISA tests.

The invention will now be described in more detail using embodiments as non-limiting examples.

Examples:

All constructions were carried out using standard molecular biology techniques (cloning, restriction enzyme digestion, synthesis of single-stranded complementary DNA, polymerase chain reaction amplification, extension of an oligonucleotide by a DNA polymerase, etc.) described by Sambrook J. et al. (Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory. Cold Spring Harbor. New York. 1989). All the restriction fragments used for the present invention, as well as the various polymerase chain reaction (PCR) amplification fragments, were isolated and purified using the "Geneclean®" kit (BIO101 Inc., La Jolla, CA).

Example 1: Cultivation of the West Nile virus

For their amplification, New York virus of the West Nile (NY99) (Lanciotti R. S. et al., Science, 1999, 286, 2333-7) is cultivated on VERO cells (monkey kidney cells, available from the American Type Culture Collection (ATCC) under number CCL-81).

VERO cells are cultured in Falcon 25 cm² flasks using Eagle's MEM medium supplemented with 1% yeast extract and 10% calf serum, containing approximately 100,000 cells per ml. The cells are cultured at +37°C under an atmosphere of 5% CO₂.

After 3 days, the cell layer reaches confluence. The culture medium is then replaced with Eagle-MEM medium supplemented with 1% yeast extract and 0.1% bovine serum albumin, and the West Nile virus is added at a dose of 5 pfu per cell.

When the cytopathic effect (CPE) is complete (usually 48–72 hours after the start of cultivation), viral suspensions are collected, then clarified by centrifugation and frozen at -70°C. Three to four successive passages are generally required for the production of a viral batch. The viral batch is stored at -70°C.

Example 2: Extraction of viral RNA from the West Nile virus

The viral RNA contained in 100 ml of the NY99 West Nile virus strain viral suspension is extracted after thawing using the "High Pure™ Viral RNA Kit" (Cat # 1 858 882, Roche Molecular Biochemicals), following the manufacturer's instructions for the extraction steps. The resulting RNA pellet is resuspended with 1 to 2 ml of sterile RNase-free distilled water.

Example 3: Construction of the plasmid pFC101

The complementary DNA (cDNA) of the NY99 West Nile virus was synthesized using the "Gene Amp RNA PCR Kit" (Cat # N 808 0017, Perkin-Elmer, Norwalk, CT 06859, USA) under the conditions provided by the manufacturer.

An reverse transcription reaction followed by a chain amplification reaction (Reverse Transcription-Polymerase Chain Reaction, or RT-PCR) is carried out using 50 µl of the viral RNA suspension from the New York 1999 West Nile virus (Example 2), and with the following oligonucleotides: FC101 (30-mer) (SEQ ID NO: 1) 5' TTTTTTGAATTCGTTACCCTCTCTAACTTC 3' and FC102 (33-mer) (SEQ ID NO: 2) 5' TTTTTTTCTAGATTACCTCCGACTGCGTCTTGA 3'.

This pair of oligonucleotides allows the incorporation of an EcoRI restriction site, an XbaI restriction site, and a stop codon downstream of the insert.

The synthesis of the first DNA strand is carried out by elongating the oligonucleotide FC102 after hybridization of the latter to the RNA template.

The synthesis conditions for the first strand of cDNA are a temperature of 42°C for 15 minutes, followed by 99°C for 5 minutes, and finally 4°C for 5 minutes. The conditions for the PCR reaction in the presence of the oligonucleotide pair FC101 and FC102 are a temperature of 95°C for 2 minutes, then 35 cycles (95°C for 1 minute, 62°C for 1 minute, and 72°C for 2 minutes), and finally 72°C for 7 minutes to produce a 302 bp fragment.

This fragment is digested by EcoRI and then by XbaI to isolate, after agarose gel electrophoresis, the EcoRI-XbaI fragment of approximately 290 bp. This fragment is called fragment A.

The eukaryotic expression plasmid pVR1020 (C. J. Luke et al., J. of Infectious Diseases, 1997, 175, 95-97), derived from the plasmid pVR1012 (Figure 1 and Example 7 of WO-A-98/03199; Hartikka J. et al., 1997, Human Gene Therapy, 7, 1205-1217), contains the coding sequence of the signal sequence of human tissue plasminogen activator (tPA).

A pVR1020 plasmid is modified by digestion with BamHI and BgIII and insertion of a sequence containing multiple cloning sites (BamHI, NotI, EcoRI, XbaI, PmlI, PstI, BglII), which results from the annealing of the following oligonucleotides: PB326 (40-mer) (SEQ ID NO: 3) 5' GATCTGCAGCACGTGTCTAGAGGATATCGAATTCGCGGCC 3' and PB329 (40-mer) (SEQ ID NO: 4) 5' GATCCGCGGCCGCGAATTCGATATCCTCTAGACACGTGCT 3'.

The resulting vector, which is approximately 5105 base pairs (or bp) in size, is named pAB110.

Fragment A is ligated with the previously digested expression plasmid pAB110 by XbaI and EcoRI, to give the plasmid pFC101 (5376 bp). This plasmid contains, under the control of the early promoter of human cytomegalovirus or hCMV-IE (human Cytomegalovirus Immediate Early), an insert encoding the signal sequence of the tPA activator followed by the sequence encoding the prM protein.

Example 4: Construction of the pFC102 plasmid

The complementary DNA (cDNA) of the NY99 West Nile virus was synthesized using the "Gene Amp RNA PCR Kit" (Cat # N 808 0017, Perkin-Elmer, Norwalk, CT 06859, USA) under the conditions provided by the manufacturer.

An reverse transcription reaction, followed by a polymerase chain reaction (RT-PCR) is performed using 50 µl of the viral RNA suspension from the New York 1999 West Nile virus (Example 2), and with the following oligonucleotides: FC103 (30-mer) (SEQ ID NO: 5) 5' TTTTTTGAATTCTCACTGACAGTGCAGACA 3' and FC104 (33-mer) (SEQ ID NO: 6) 5' TTTTTTTCTAGATTAGCTGTAAGCTGGGGCCAC 3'.

This pair of oligonucleotides allows the incorporation of an EcoRI restriction site, an XbaI restriction site, and a stop codon downstream of the insert.

The synthesis of the first DNA strand is carried out by elongation of the FC104 oligonucleotide after hybridization of the latter to the RNA matrix.

The synthesis conditions for the first DNA strand are a temperature of 42°C for 15 minutes, followed by 99°C for 5 minutes, and finally 4°C for 5 minutes. The PCR reaction conditions in the presence of the oligonucleotide pair FC103 and FC104 are a temperature of 95°C for 2 minutes, followed by 35 cycles (95°C for 1 minute, then 62°C for 1 minute, and 72°C for 2 minutes), and finally 72°C for 7 minutes to produce a 252 bp fragment.

This fragment is digested by EcoRI and then by XbaI to isolate, after agarose gel electrophoresis, the EcoRI-XbaI fragment of approximately 240 bp. This fragment is then ligated with the expression plasmid pAB110 (Example 3), which has been previously digested by XbaI and EcoRI, to yield the plasmid pFC102 (5326 bp). This plasmid contains, under the control of the early promoter of the human cytomegalovirus or hCMV-IE (human Cytomegalovirus Immediate Early), an insert encoding the signal sequence of the tPA activator followed by the sequence encoding the M protein.

Example 5: Construction of the pFC103 plasmid

The complementary DNA (cDNA) of the NY99 West Nile virus was synthesized using the "Gene Amp RNA PCR Kit" (Cat # N 808 0017, Perkin-Elmer, Norwalk, CT 06859, USA) under the conditions provided by the manufacturer.

An reverse transcription reaction followed by a chain amplification reaction (Reverse Transcriptase-Polymerase Chain Reaction, or RT-PCR) is carried out using 50 µl of the viral RNA suspension from the New York 1999 West Nile virus (Example 2), and with the following oligonucleotides: FC105 (30-mer) (SEQ ID NO: 7) 5' TTTTTTGAATTCTTCAACTGCCTTGGAATG 3' and FC106 (33-mer) (SEQ ID NO: 8) 5' TTTTTTTCTAGATTAAGCGTGCACGTTCACGGA 3'.

This pair of oligonucleotides allows the incorporation of an EcoRI restriction site, a XbaI restriction site, and a stop codon downstream of the insert.

The synthesis of the first DNA strand is carried out by elongation of the FC106 oligonucleotide after hybridization of the latter to the RNA template.

The synthesis conditions for the first DNA strand are a temperature of 42°C for 15 minutes, followed by 99°C for 5 minutes, and finally 4°C for 5 minutes. The conditions for the PCR reaction in the presence of the oligonucleotide pair FC105 and FC106 are a temperature of 95°C for 2 minutes, followed by 35 cycles (95°C for 1 minute, then 62°C for 1 minute, and 72°C for 2 minutes), and finally 72°C for 7 minutes to produce a 1530 bp fragment.

This fragment is digested by EcoRI and then by XbaI to isolate, after agarose gel electrophoresis, the EcoRI-XbaI fragment of approximately 1518 bp. This fragment is then ligated with the expression plasmid pAB110 (Example 3), previously digested by XbaI and EcoRI, to yield the plasmid pFC103 (6604 bp). This plasmid contains, under the control of the human cytomegalovirus early promoter or hCMV-IE (human Cytomegalovirus Immediate Early), an insert encoding the signal sequence of the tPA activator followed by the sequence encoding the E protein.

Example 6: Construction of the pFC104 plasmid

The complementary DNA (cDNA) of the NY99 West Nile virus was synthesized using the "Gene Amp RNA PCR Kit" (Cat # N 808 0017, Perkin-Elmer, Norwalk, CT 06859, USA) under the conditions provided by the manufacturer.

An reverse transcription reaction followed by a polymerase chain reaction (RT-PCR) is carried out using 50 µl of the viral RNA suspension of the New York 99 strain of West Nile virus (Example 2), and with the following oligonucleotides: FC101 (30-mer) (SEQ ID NO: 1) and FC106 (33-mer) (SEQ ID NO: 8).

This pair of oligonucleotides allows the incorporation of an EcoRI restriction site, an XbaI restriction site, and a stop codon downstream of the insert.

The synthesis of the first DNA strand is carried out by elongation of the FC106 oligonucleotide after hybridization of the latter to the RNA template.

The synthesis conditions for the first DNA strand are a temperature of 42°C for 15 minutes, followed by 99°C for 5 minutes, and finally 4°C for 5 minutes. The conditions for the PCR reaction in the presence of the oligonucleotide pair FC101 and FC106 are a temperature of 95°C for 2 minutes, followed by 35 cycles (95°C for 1 minute, then 62°C for 1 minute, and 72°C for 2 minutes), and finally 72°C for 7 minutes to produce a 2031 bp fragment.

This fragment is digested by EcoRI and then by XbaI to isolate, after agarose gel electrophoresis, the EcoRI-XbaI fragment of approximately 2019 bp. This fragment is then ligated with the expression plasmid pAB110 (Example 3) previously digested with XbaI and EcoRI, resulting in the plasmid pFC104 (7105 bp). This plasmid contains, under the control of the early promoter of human cytomegalovirus or hCMV-IE (Human Cytomegalovirus Immediate Early), an insert encoding the signal sequence of the tPA activator followed by the sequence encoding the prM-M-E protein.

Example 7: Construction of the pFC105 plasmid

The complementary DNA (cDNA) of the NY99 West Nile virus was synthesized using the "Gene Amp RNA PCR Kit" (Cat # N 808 0017, Perkin-Elmer, Norwalk, CT 06859, USA) under the conditions provided by the manufacturer.

An reverse transcription reaction followed by a polymerase chain reaction (RT-PCR) is carried out using 50 µl of the viral RNA suspension of the New York 99 West Nile virus (Example 2), along with the following oligonucleotides: FC107 (36-mer) (SEQ ID NO: 9) 5' TTTTTTGATATCACCGGAATTGCAGTCATGATTGGC 3' and FC106 (33-mer) (SEQ ID NO: 8).

This pair of oligonucleotides allows the incorporation of an EcoRV restriction site, an XbaI restriction site, and a stop codon downstream of the insert.

The synthesis of the first DNA strand is carried out by elongating the oligonucleotide FC106 after it has hybridized to the RNA template.

The synthesis conditions for the first strand of cDNA are a temperature of 42°C for 15 minutes, followed by 99°C for 5 minutes, and finally 4°C for 5 minutes. The PCR reaction conditions in the presence of the oligonucleotide pair FC106 and FC107 are a temperature of 95°C for 2 minutes, followed by 35 cycles (95°C for 1 minute, then 62°C for 1 minute, and 72°C for 2 minutes), and finally 72°C for 7 minutes to produce a 2076 bp fragment.

This fragment is digested by EcoRV and then by XbaI to isolate, after agarose gel electrophoresis, the EcoRV-XbaI fragment of approximately 2058 bp.

This fragment is ligated with the expression plasmid pVR1012, previously digested with XbaI and EcoRV, to generate the plasmid pFC105 (6953 bp). This plasmid contains, under the control of the human cytomegalovirus early promoter or hCMV-IE (Human Cytomegalovirus Immediate Early), an insert encoding the prM-M-E polyprotein.

Example 8: Construction of the pFC106 plasmid

The complementary DNA (cDNA) of the NY99 West Nile virus was synthesized using the "Gene Amp RNA PCR Kit" (Cat # N 808 0017, Perkin-Elmer, Norwalk, CT 06859, USA) under the conditions provided by the manufacturer.

An reverse transcription reaction followed by a chain amplification reaction (Reverse Transcription-Polymerase Chain Reaction, or RT-PCR) is carried out using 50 µl of the viral RNA suspension from the New York 1999 West Nile virus (Example 2), along with the following oligonucleotides: FC108 (36-mer) (SEQ ID NO: 10) 5' TTTTTTGATATCATGTATAATGCTGATATGATTGAC 3' and FC109 (36-mer) (SEQ ID NO: 11) 5' TTTTTTTCTAGATTAACGTTTTCCCGAGGCGAAGTC 3'.

This pair of oligonucleotides allows the incorporation of an EcoRV restriction site and an Xbal restriction site, an ATG initiation codon in the 5' end, and a stop codon in the 3' end of the insert.

The synthesis of the first DNA strand is carried out by elongating the oligonucleotide FC109 after it has hybridized to the RNA template.

The synthesis conditions for the first strand of cDNA are a temperature of 42°C for 15 minutes, followed by 99°C for 5 minutes, and finally 4°C for 5 minutes. The PCR reaction conditions in the presence of the oligonucleotide pair FC108 and FC109 are a temperature of 95°C for 2 minutes, then 35 cycles (95°C for 1 minute, 62°C for 1 minute, and 72°C for 2 minutes), and finally 72°C for 7 minutes to produce a 2973 bp fragment.

This fragment is digested by EcoRV and then by XbaI to isolate, after agarose gel electrophoresis, the EcoRV-XbaI fragment of approximately 2955 bp.

This fragment is ligated with the expression plasmid pVR1012, previously digested with XbaI and EcoRV, to give the plasmid pFC106 (7850 bp). This plasmid contains, under the control of the human cytomegalovirus early promoter or hCMV-IE (Human Cytomegalovirus Immediate Early), an insert encoding the NS2A-NS2B-NS3 polyprotein.

Example 9: Construction of the donor plasmid for insertion into the C5 site of the canarypox virus ALVAC

Figure 16 of US Patent A-5,756,103 shows the sequence of a 3199 bp fragment of the canarypox virus genomic DNA. Analysis of this sequence revealed an open reading frame (ORF) named C5L, which starts at position 1538 and ends at position 1859. The construction of an insertion plasmid leading to the deletion of the C5L ORF and its replacement with a multiple cloning site flanked by transcription and translation termination signals was carried out as described below.

An ACP reaction was carried out using the matrix composed of the genomic DNA of the canarypox virus and the following oligonucleotides: C5A1 (42-mer) (SEQ ID NO: 12): 5' ATCATCGAGCTCCAGCTGTAATTCATGGTCGAAAAGAAGTGC 3' and C5B1 (73-mer) (SEQ ID NO: 13): to isolate a 223 bp ACP fragment (fragment B).

An ACP reaction was carried out using the matrix composed of the genomic DNA of the canarypox virus and the following oligonucleotides: C5C1 (72-mer) (SEQ ID NO: 14): and C5D1 (45-mer) (SEQ ID NO: 15): 5' GATGATGGTACCGTAAACAAATATAATGAAAAGTATTCTAAACTA 3' to isolate a 482 bp ACP fragment (fragment C).

The B and C fragments were hybridized together to serve as a template for an ACP reaction using the oligonucleotides C5A1 (SEQ ID NO: 12) and C5D1 (SEQ ID NO: 15), generating an ACP fragment of 681 bp. This fragment was digested with the restriction enzymes SacI and KpnI, and after agarose gel electrophoresis, a SacI-KpnI fragment of 664 bp was isolated. This fragment was then ligated with the pBlueScript® II SK+ vector (Stratagene, La Jolla, CA, USA, Cat # 212205), which had been previously digested with the restriction enzymes SacI and KpnI, resulting in the plasmid pC5L. The sequence of this plasmid was verified by sequencing. This plasmid contains 166 bp of sequences upstream of the COL C5L ("left flanking arm C5"), an early transcription termination signal, stop codons in all six reading frames, a multiple cloning site containing restriction sites for SmaI, PstI, XhoI, and EcoRI, and finally 425 bp of sequences downstream of the COL C5L ("right flanking arm C5").

The plasmid pMP528HRH (Perkus M. et al., J. Virol. 1989. 63. 3829-3836) was used as a template to amplify the complete sequence of the H6 vaccine promoter (GenBank Accession No. M28351) using the following oligonucleotides: JCA291 (34-mer) (SEQ ID NO: 16) 5' AAACCCGGGTTCTTTÀTTCTATACTTAAAAAGTG 3' and JCA292 (43-mer) (SEQ ID NO: 17) 5' AAAAGAATTCGTCGACTACGATACAAACTTAACGGATATCGCG 3' to amplify an ACP fragment of 149 bp. This fragment was digested with the restriction enzymes SmaI and EcoRI to isolate, after agarose gel electrophoresis, a SmaI-EcoRI restriction fragment of 138 bp. This fragment was then ligated with the plasmid pC5L, previously digested with SmaI and EcoRI, to give the plasmid pFC107.

Example 10: Construction of the recombinant virus vCP1712

An ACP reaction was carried out using the plasmid pFC105 (Example 7) as a template and the following oligonucleotides: FC110 (33-mer) (SEQ ID NO: 18): 5' TTTTCGCGAACCGGAATTGCAGTCATGATTGGC 3' and FC111 (39-mer) (SEQ ID NO: 19): 5' TTTTGTCGACGCGGCCGCTTAAGCGTGCACGTTCACGGA 3' to amplify an ACP fragment of approximately 2079 bp. This fragment was digested with the restriction enzymes NruI and SalI to isolate, after agarose gel electrophoresis, a NruI-SalI restriction fragment of approximately 2068 bp. This fragment was then ligated with the plasmid pFC107 (Example 9) previously digested with the restriction enzymes NruI and SalI to yield the plasmid pFC108.

The pFC108 plasmid was linearized with NotI, then transfected into primary chicken embryo cells infected with canarypox virus (ALVAC strain) using the previously described calcium phosphate precipitation technique (Panicali and Paoletti, Proc. Nat. Acad. Sci. 1982. 79. 4927-4931; Piccini et al. In Methods in Enzymology. 1987. 153. 545-563. Eds. Wu R. and Grossman L. Academic Press). Positive plaques were selected based on hybridization with a radiolabeled probe specific for the nucleotide sequence of the envelope glycoprotein E. These plaques underwent four successive rounds of plaque selection/purification until a pure population was isolated. A representative plaque resulting from in vitro recombination between the donor plasmid pFC108 and the ALVAC canarypox virus genome was then amplified, and the resulting recombinant virus stock was designated vCP1712.

Example 11: Construction of the recombinant virus vCP1713

The pFC104 plasmid (example 6) is digested with the restriction enzymes SalI and PmlI to isolate, after agarose gel electrophoresis, a Prrill-Sall restriction fragment of approximately 2213 bp. This fragment is then ligated with the pFC107 plasmid (example 9), which has been previously digested with the restriction enzymes NruI and SalI, to yield the pFC109 plasmid.

The pFC109 plasmid was linearized with NotI and then transfected into primary chicken embryo cells infected with canarypox virus (ALVAC strain), according to the technique described in Example 10. A representative range of in vitro recombination between the donor plasmid pFC109 and the ALVAC canarypox virus genome was selected based on hybridization with a radiolabeled probe specific for the nucleotide sequence of the envelope glycoprotein E, and was subsequently amplified. The resulting recombinant virus stock was designated vCP1713.

Example 12: Construction of the recombinant virus vCP1714

The plasmid pFC103 (example 5) is digested with the restriction enzymes SalI and PmlI to isolate, after agarose gel electrophoresis, a PmlI-SalI restriction fragment of approximately 1712 bp. This fragment is then ligated with the plasmid pFC107 (example 9), which has been previously digested with the restriction enzymes NruI and SalI, to generate the plasmid pFC110.

The pFC110 plasmid was linearized with NotI and then transfected into primary chicken embryo cells infected with canarypox virus (ALVAC strain) according to the technique described in Example 10. A representative range of in vitro recombination between the donor plasmid pFC110 and the ALVAC canarypox virus genome was selected based on hybridization with a radiolabeled probe specific for the nucleotide sequence of the envelope glycoprotein E, and was subsequently amplified. The resulting recombinant virus stock was designated vCP1714.

Example 13: Construction of the recombinant virus vCP1715

The plasmid pFC102 (example 4) is digested with the restriction enzymes SalI and PmlI to isolate, after agarose gel electrophoresis, a Pmll-Sall restriction fragment of approximately 434 bp. This fragment is then ligated with the plasmid pFC107 (example 9), which has been previously digested with the restriction enzymes NruI and SalI, to yield the plasmid pFC111.

The pFC111 plasmid was linearized with NotI and then transfected into primary chicken embryo cells infected with canarypox virus (ALVAC strain) according to the technique described in Example 10. A representative range of in vitro recombination between the pFC111 donor plasmid and the ALVAC canarypox virus genome was selected based on hybridization with a radiolabeled probe specific for the nucleotide sequence of the M membrane glycoprotein and was subsequently amplified. The resulting recombinant virus stock was designated vCP1715.

Example 14: Construction of the recombinant virus vCP1716

The pFC101 plasmid (example 3) is digested with the restriction enzymes SalI and PmlI to isolate, after agarose gel electrophoresis, a PmlI-SalI restriction fragment of approximately 484 bp. This fragment is then ligated with the pFC107 plasmid (example 9), which has been previously digested with the restriction enzymes NruI and SalI, to yield the pFC112 plasmid.

The pFC112 plasmid was linearized with NotI and then transfected into primary chicken embryo cells infected with canarypox virus (ALVAC strain) according to the technique of Example 10. A representative range of in vitro recombination between the donor plasmid pFC112 and the ALVAC canarypox virus genome was selected based on hybridization with a radiolabeled probe specific for the nucleotide sequence of the prM membrane glycoprotein and was subsequently amplified. The resulting recombinant virus stock was designated vCP1716.

Example 15: Construction of the donor plasmid for insertion into the C6 site of the canarypox virus ALVAC

Figure 4 of patent WO-A-01/05934 shows the sequence of a 3700 bp fragment of the canarypox virus genomic DNA. Analysis of this sequence revealed an open reading frame (ORF) named C6L, which starts at position 377 and ends at position 2254. The construction of an insertion plasmid leading to the deletion of the C6L ORF and its replacement with a multiple cloning site flanked by transcription and translation termination signals was carried out as described below.

An ACP reaction was carried out using the matrix composed of the genomic DNA of the canarypox virus and the following oligonucleotides: C6A1 (42-mer) (SEQ ID NO: 20): 5' ATCATCGAGCTCGCGGCCGCCTATCAAAAGTCTTAATGAGTT 3' and C6B1 (73-mer) (SEQ ID NO: 21): to isolate a 432 bp ACP fragment (fragment D).

An ACP reaction was carried out using the matrix composed of the genomic DNA of the canarypox virus and the following oligonucleotides: C6C1 (72-mer) (SEQ ID NO: 22): and C6D1 (45-mer) (SEQ ID NO: 23): 5' GATGATGGTACCTTCATAAATACAAGTTTGATTAAACTTAAGTTG 3' to isolate an ACP fragment of 1210 bp (fragment E).

Fragments D and E were hybridized together to serve as a template for an ACP reaction using the oligonucleotides C6A1 (SEQ ID NO: 20) and C6D1 (SEQ ID NO: 23), generating an ACP fragment of 1630 bp. This fragment was digested with the restriction enzymes SalI and KpnI, and after agarose gel electrophoresis, a SalI-KpnI fragment of 1613 bp was isolated. This fragment was then ligated with the vector pBlueScript® II SK+ (Stratagene, La Jolla, CA, USA, Cat # 212205), which had been previously digested with the restriction enzymes SalI and KpnI, resulting in the plasmid pC6L. The sequence of this plasmid was verified by sequencing. This plasmid contains 370 bp of sequences upstream of the COL C6L ("left flanking arm C6"), an early transcription termination signal, stop codons in all six reading frames, a multiple cloning site containing restriction sites for SmaI, PstI, XhoI, and EcoRI, and finally 1156 bp of sequences downstream of the COL C6L ("right flanking arm C6").

The plasmid pMPIVC (Schmitt J. F. C. et al., J. Virol., 1988, 62, 1889-1897; Saiki R. K. et al., Science, 1988, 239, 487-491) was used as a template to amplify the complete sequence of the vaccine I3L promoter with the following oligonucleotides: FC112 (33-mer) (SEQ ID NO: 24): 5' AAACCCGGGCGGTGGTTTGCGATTCCGAAATCT 3' and FC113 (43-mer) (SEQ ID NO: 25): 5' AAAAGAATTCGGATCCGATTAAACCTAAATAATTGTACTTTGT 3' to amplify a 151 bp ACP fragment. This fragment was digested with the restriction enzymes SmaI and EcoRI to isolate, after agarose gel electrophoresis, a SmaI-EcoRI restriction fragment of approximately 136 bp. This fragment was then ligated with the plasmid pC6L, previously digested with SmaI and EcoRI, to give the plasmid pFC113.

Example 16: Construction of the recombinant viruses vCP1717 and vCP1718

An ACP reaction was carried out using the plasmid pFC106 (Example 8) as a template and the following oligonucleotides: FC114 (33-mer) (SEQ ID NO: 26): 5' TTTCACGTGATGTATAATGCTGATATGATTGAC 3' and FC115 (42-mer) (SEQ ID NO: 27): 5' TTTTGGATCCGCGGCCGCTTAACGTTTTCCCGAGGCGAAGTC 3' to amplify an ACP fragment of approximately 2973 bp. This fragment was digested with the restriction enzymes PmlI and BamHI to isolate, after agarose gel electrophoresis, the PmlI-BamHI restriction fragment of approximately 2958 bp (fragment F). The plasmid pFC113 (Example 15) was digested with the restriction enzymes PmlI and BamHI to isolate, after agarose gel electrophoresis, the PmlI-BamHI restriction fragment of approximately 4500 bp (fragment G). The fragments F and G were then ligated together to give the plasmid pFC114.

The pFC114 plasmid was linearized with NotI, then transfected into primary chicken embryo cells infected with canarypox virus vCP1713 (Example 11) using the previously described calcium phosphate precipitation technique (Panicali and Paoletti, Proc. Nat. Acad. Sci. 1982, 79, 4927-4931; Piccini et al., In Methods in Enzymology, 1987, 153, 545-563. Eds. Wu R. and Grossman L., Academic Press). Positive plaques were selected based on hybridization with a radiolabeled probe specific for the nucleotide sequence of the envelope glycoprotein E. These plaques underwent four successive rounds of plaque selection/purification until a pure population was isolated. A representative plaque of the in vitro recombination between the donor plasmid pFC114 and the canarypox virus ALVAC genome was then amplified, and the resulting recombinant virus stock was designated vCP1717.

The linearized pFC114 plasmid was also used to transfect primary chicken embryo cells infected with canarypox virus vCP1712 (Example 10), according to the technique described above. The resulting recombinant virus stock was designated vCP1718.

Example 17: Construction of the pFC115 plasmid.

The complementary DNA (cDNA) of the NY99 West Nile virus was synthesized using the "Gene Amp RNA PCR Kit" (Cat # N 808 0017, Perkin-Elmer, Norwalk, CT 06859, USA) under the conditions provided by the manufacturer.

An reverse transcription reaction followed by a polymerase chain reaction (RT-PCR) is carried out using 50 µl of the viral RNA suspension of the New York 1999 West Nile virus (Example 2), and with the following oligonucleotides: FC116 (39-mer) (SEQ ID NO: 28) 5' TTTTTTGATATCATGACCGGAATTGCAGTCATGATTGGC 3' and FC106 (33-mer) (SEQ ID NO: 8).

This pair of oligonucleotides allows the incorporation of an EcoRV restriction site and a Xbal restriction site, an initiator codon in the 5' direction, and a stop codon in the 3' direction of the insert.

The synthesis of the first DNA strand is carried out by elongation of the FC106 oligonucleotide after hybridization of the latter to the RNA template.

The synthesis conditions for the first DNA strand are a temperature of 42°C for 15 minutes, followed by 99°C for 5 minutes, and finally 4°C for 5 minutes. The conditions for the PCR reaction in the presence of the oligonucleotide pair FC106 and FC116 are a temperature of 95°C for 2 minutes, followed by 35 cycles (95°C for 1 minute, then 62°C for 1 minute, and 72°C for 2 minutes), and finally 72°C for 7 minutes to produce a 2079 bp fragment.

This fragment is digested by EcoRV and then by XbaI to isolate, after agarose gel electrophoresis, the EcoRV-XbaI fragment of approximately 2061 bp.

This fragment is ligated with the expression plasmid pVR1012, previously digested with XbaI and EcoRV, to give the plasmid pFC115 (6956 bp). This plasmid contains, under the control of the human cytomegalovirus early promoter or hCMV-IE (human Cytomegalovirus Immediate Early), an insert encoding the prM-M-E polyprotein.

Example 18: Construction of recombinant viruses vCP2017

An ACP reaction was carried out using the plasmid pFC115 (Example 17) as a template and the following oligonucleotides: FC117 (36-mer) (SEQ ID NO: 29): 5' TTTTCGCGAATGACCGGAATTGCAGTCATGATTGGC 3' and FC111 (39-mer) (SEQ ID NO: 19) to amplify an ACP fragment of approximately 2082 bp. This fragment was digested with the restriction enzymes NruI and SalI to isolate, after agarose gel electrophoresis, a NruI-SalI restriction fragment of approximately 2071 bp. This fragment was then ligated with the plasmid pFC107 (Example 9) previously digested with the restriction enzymes NruI and SalI to yield the plasmid pFC116.

The pFC116 plasmid was linearized with NotI and then transfected into primary chicken embryo cells infected with canarypox virus (ALVAC strain) according to the technique of Example 10. A representative range of in vitro recombination between the pFC116 donor plasmid and the ALVAC canarypox virus genome was selected based on hybridization with a radiolabeled probe specific for the nucleotide sequence of the envelope glycoprotein E and was subsequently amplified. The resulting recombinant virus stock was designated vCP2017.

Example 19: Production of recombinant vaccines

For the preparation of equine vaccines, the recombinant canarypox virus vCP1712 (Example 10) is adjuvanted with carbomer solutions, namely Carbopol™ 974P manufactured by BF Goodrich, Ohio, USA (molecular weight of approximately 3,000,000).

A 1.5% stock solution of Carbopol™ 974P is initially prepared in distilled water containing 1 g/L of sodium chloride. This stock solution is then used to prepare a 4 mg/mL solution of Carbopol™ 974P in physiological water. The stock solution is mixed with the appropriate amount of physiological water, either in a single step or in several successive steps, and the pH is adjusted at each step using a 1N (or even more concentrated) sodium hydroxide solution to obtain a final pH value of 7.3–7.4.

The ready-to-use Carbopol™ 974P solution obtained is used to resuspend lyophilized recombinant viruses or to dilute concentrated stock solutions of recombinant viruses. For example, to obtain a viral suspension containing 10⁸ pfu per 1 ml dose, a stock viral solution is diluted to achieve a titer of 10⁸.³ pfu/ml, and then diluted in equal parts with the aforementioned ready-to-use Carbopol™ 974P solution at 4 mg/ml.

Recombinant vaccines can also be produced using recombinant canarypox viruses vCP1713 (example 11), vCP1717 (example 16), vCP1718 (example 16), vCP2017 (example 18), or a mixture of the three canarypox viruses vCP1714 (example 12), vCP1715 (example 13), and vCP1716 (example 14), according to the technique described above.

Example 20: Production of DNA vaccines for horses

A DNA solution containing the plasmid pFC104 (Example 6) is concentrated by ethanol precipitation as described in Sambrook et al. (1989). The DNA pellet is resuspended in a 0.9% NaCl solution to obtain a concentration of 1 mg/ml. A DMRIE-DOPE solution at 0.75 mM is prepared by resuspending a lyophilized DMRIE-DOPE sample with an appropriate volume of sterile H2O.

The formation of lipid-plasmid DNA complexes is carried out by equally diluting the 0.75 mM DMRIE-DOPE (1:1) solution with the 1 mg/ml DNA solution in 0.9% NaCl. The DNA solution is gradually introduced using a 26G needle along the wall of the flask containing the cationic lipid solution to avoid foam formation. Gentle mixing is performed as soon as the two solutions are combined. Finally, a composition containing 0.375 mM DMRIE-DOPE and 500 µg/ml plasmid is obtained.

It is advisable that all the solutions used are at room temperature for all the operations described above. The DNA/DMRIE-DOPE complexation is allowed to form at room temperature for 30 minutes before proceeding with animal immunization.

DNA vaccines can also be produced using DNA solutions containing the plasmids pFC104 (example 6) and pFC106 (example 8), or containing the plasmids pFC105 (example 7) and pFC106, the plasmids pFC115 (example 17) and pFC106, or containing the plasmids pFC101, pFC102 and pFC103 (examples 3 to 5), or containing the plasmid pFC105 or pFC115 according to the technique described in this example.

Example 21: In Vitro Expression Tests

The expression of WN proteins is tested for each construct using classical methods of indirect immunofluorescence and Western blot.

These tests are performed on 96-well plates containing CHO cells grown in monolayers and transfected with plasmids, or containing CEF cells grown in monolayers and infected with recombinant viruses.

WN proteins are detected using sera from infected horses or chickens and labeled antisera.

The size of the fragments obtained after migration on an agarose gel is compared to the expected sizes.

Example 22: Animal effectiveness

Recombinant vaccines and plasmid vaccines are administered twice, approximately two weeks apart, to EOPS chickens, about 7 days old, which are unvaccinated, by the intramuscular route with a volume of approximately 0.1 ml. A non-vaccinated control group is included in the study.

Chickens are tested by subcutaneous administration in the neck with 10³-4 TCID₅₀ of pathogenic West Nile virus.

On the one hand, viremia, the antibody response, and mortality are observed. Autopsies are performed to examine the lesions.

Example 23: Neutralizing antibody titration against WNV.

Serial dilutions are performed for each serum at a ratio of 3 in DMEM medium supplemented with 10% fetal bovine serum, in 96-well cell culture plates. To 0.05 ml of the diluted serum, 0.05 ml of culture medium containing approximately 100 TCID50/ml of WNV is added. This mixture is then incubated for 2 hours at 37°C in a CO2 incubator with an atmosphere containing 5% CO2.

0.15 ml of a Vero cell suspension containing approximately 100,000 cells per ml is then added to each mixture. The cytopathic effect (CPE) is observed by phase contrast microscopy after 4-5 days of cultivation at 37°C in an atmosphere containing 5% CO2. The neutralizing titers of each serum are calculated according to the Kärber method. The titers are given as the highest dilution that inhibits the cytopathic effect in 50% of the wells. The titers are expressed in log10 VN50. Each serum is titrated at least twice, preferably four times.

Example 24: Trial on vCP2017 horses.

Recombinant vaccines containing vCP2017 (Example 18), prepared extemporaneously with 1 ml of carbopol® 974P adjuvant (4 mg/ml), were administered twice, with an interval of 35 days, to horses older than 3 months that had not been vaccinated before, by the intramuscular route, with a volume of approximately 1 ml. Three groups of animals were vaccinated, with doses of 105.8 DICC50 (equivalent to 105.64 pfu) for Group 1, 106.8 DICC50 (equivalent to 106.64 pfu) for Group 2, and 107.8 DICC50 (equivalent to 107.64 pfu) for Group 3. A non-vaccinated control group was included in the study.

Serology was observed. Neutralizing antibody titers were determined and expressed in log10 VN50 as shown in Example 23.

1	< 0,6	< 0,78	2,66
2	< 0,6	1,14	2,58
3	< 0,6	1,16	2,26
témoin	< 0,6	< 0,6	< 0,6

SEQUENCE LISTING

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Claims (9)

1. An expression vector comprising:

   - a polynucleotide wherein the polynucleotide encodes the proteins E, prM and M of the West Nile Virus (WNV), and

   - the elements necessary for expression of the polynucleotide in a host cell,

   wherein the expression vector is a viral vector, further wherein the viral vector is a canarypox or fowlpox.
2. The expression vector according to claim 1, wherein the expression vector comprises a polynucleotide encoding prM-M-E from WNV in a single reading frame.
3. The expression vector according to any of claims 1-2 wherein the polynucleotide comprises in addition a nucleotide sequence encoding a signal peptide located upstream of the expressed protein.
4. An immunogenic composition comprising at least one expression vector according to any of claims 1-3 and a pharmaceutically acceptable carrier or excipient.
5. The immunogenic composition according to claim 4, wherein said composition further comprises an adjuvant.
6. The immunogenic composition according to claim 5, wherein the adjuvant is a carbomer.
7. A method of preparing an immunogenic composition according to any one of claims 4 to 6 comprising admixing at least one expression vector according to any of claims 1-3 with a pharmaceutically acceptable carrier or excipient.
8. The expression vector according to any of claims 1-3 or an immunogenic composition according to any of claims 4-6 for use in a method of inducing a protective immune response against WNV.
9. The use of an expression vector according to any of claims 1-3 or an immunogenic composition according to any of claims 4-6 for the manufacture of a medicament for inducing a protective immune response against WNV.