ZA200307620B - Vaccine against the nile fever virus - Google Patents

Description

> “4 I ® 1 ©. .2003/ 7520

RECOMBINANT VACCINE AGAINST WEST NILE VIRUS

The present invention relates to in vivo and in vitro expression vectors comprising and expressing at least one polynucleotide of the West Nile fever virus, as well as immunogenic compositions and vaccines against

West Nile fever. It aiso relates to methods for immunizing and vaccinating against this virus.

The West Nile fever virus (WNV) was first identified in man in 1937 in Ouganda in the West Nile Province (Zeller H. G., Med. Trop., 1999, 59, 490-494).

Widespread in Africa, it is also encountered in India, Pakistan and the Mediterranean basin and was identified for the first time in the USA in 1999 in New York City (Anderson J. F. et al., Science, 1999, 286, 2331-2333).

The West Nile fever virus affects birds as well as mammals, together with man.

The fever is characterized in birds by an attack of the central nervous system and death. The lesions include encephalitis, hemorrhages in the myocardium and hemorrhages and necroses in the intestinal tract.

In chickens, experimental infections by subcutaneous inoculations of the West Nile fever virus isolated on crows led to necroses of the myocardium, nephrites 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 fever virus also affects horses, particularly in North Africa and Europe (Cantile C. et al,

Equine Vet. J., 2000, 32 (1), 31-35). These horses reveal signs of ataxia, weakness of the rear limbs, paresis evolving towards tetraplegia and death. Horses and camels are the main animals manifesting clinical signs in the form of encephalitis.

Anti-WNV antibodies were detected in certain rodents, in livestock, particularly bovines and ovines, as well as in domestic animals, particularly in the dog (Zeller H. G., Med. Trop., 1999, 59, 490-494; Lundstrom J. O.,

Journal of Vector Ecology, 1999, 24 (1), 1-39).

The West Nile fever virus also affects with a number of symptoms the human species (Sampson B. A,,

Human Pathology, 2000, 31 (5), 527-531; Marra C. M., Seminars in Neurology, 2000, 20 (3), 323-327).

The West Nile fever virus is transmitted to birds and mammals by the bites of certain mosquitoes (e.g.

Culex, Aedes, Anopheles) and ticks.

Wild and domestic birds are a reservoir for the West Nile virus and a propagation vector as a result of their 40 migrations.

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The virions of the West Nile fever virus are spherical particles with a diameter of 50 nm constituted by a lipoproteic envelope surrounding an icosahedric nucleocapsid containing a positive polarity, single-strand

RNA.

A single open reading frame (ORF) encodes all the viral proteins in the form of a polyprotein. The cleaving and maturation of this polyprotein leads to the production of about ten different viral proteins. The structural proteins are encoded by the 5' part of the genome and correspond to the nucleocapsid designated C (14 kDa), the envelope glycoprotein designated E (50 kDa), the pre-membrane protein designated prM (23 kDa), the membrane protein designated M (7 kDa). The 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), 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), on the basis of the vaccinia virus, constructed in vivo expression vectors containing various inserts corresponding to nucleotide sequences } coding for non-structural proteins of the Kunjin virus, optionally associated with structural proteins. These vectors were administered to the mouse to evaluate the immune cell response. The authors stress the importance of the cell response, which is essentially stimulated by non-structural proteins and especially

NS3, NS4A and NS4B. These articles reveal the difficulty in providing a good vaccination strategy against

West Nile fever.

Hitherto there is no vaccine preventing infection by the WN virus.

The present invention relates to a means for preventing and/or combating diseases caused by the WN virus.

Another objective of the invention is to propose such a means usable in different animal species sensitive to the disease caused by said virus and in particular equine and avian species.

Another objective of the invention is to propose immunization and vaccination methods for the target species.

Yet another objective of the invention is to propose means and methods making it possible to ensure a differential diagnosis.

Thus, the first object of the invention is in vitro and/or in vivo expression vectors comprising a polynucleotide encoding the envelope protein E of the WN virus. These vectors also comprise the elements necessary for the expression of the polynucieotide in the host cell.

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In addition to the polynucleotide encoding E, the expression vectors according to the invention can comprise one or more other polynucleotides encoding other proteins of the WN virus, preferably structural proteins of the WN virus and said sequences are preferably chosen from among those encoding the pre-membrane protein prM and the membrane protein M.

The vector preferably comprises a polynucleotide forming a single encoding frame corresponding e.g. to prM-E, M-E and more particularly prM-M-E. A vector comprising several separate polynucleotides encoding the different proteins (e.g. prM and/or M and E) also falls within the scope of the present invention. The vector, more particularly in vivo, can also comprise polynucleotides corresponding to more than one WN virus strain, particularly two or more polynucleotides encoding E or prM-M-E of different strains. As will be shown hereinafter, the vector, particularly in vivo, can comprise one or more nucleotide sequences encoding immunogens of other pathogenic agents and/or cytokins.

According to a preferred embodiment of the invention, the expression vector comprises a polynucleotide encoding prM-M-E and preferably in a single reading frame.

The term polynucleotide encoding a protein of the WN virus mainly means a DNA fragment encoding said protein, or the complementary strand of said DNA fragment. An RNA is not excluded.

In the sense of the invention, the term protein covers fragments, including peptides and polypeptides. By definition, the protein fragment is immunologically active in the sense that once administered to the host, it is able to evoke an immune response of the humoral and/or cellular type directed against the protein.

Preferably the protein fragment is such that it has substantially the same immunological activity as the total protein. Thus, a protein fragment according to the invention comprises at least one epitope or antigenic determinant. The term epitope relates to a protein site able to induce an immune reaction of the humoral type (B cells) and/or cellular type (T cells).

Thus, the minimum structure of the polynucleotide is that encoding an epitope or antigenic determinant of the protein in question. A polynucleotide encoding a fragment of the total protein more particularly comprises a minimum of 21 nucleotides, particularly at least 42 nucleotides and preferably at least 57, 87 or 150 consecutive nucleotides of the sequence in question. Epitope determination procedures are well known to the one skilled in the art and it is more particularly possible to use overlapping peptide libraries (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 de Chiron) and algorithms (De Groot A. et al., Nature Biotechnology, 1999, 17, 533-561). in particular the polynucleotides according to the invention comprise the nucleotide sequence encoding one 40 or two transmembrane domains and preferably two of them, located in the terminal part C of the protein E.

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For the WNV NY99 strain, these domains correspond to amino acid sequences 742 to 766 and 770 to 791 of GenBank AF196835.

Elements necessary for the expression of the polynucleotide or polynucleotides are present. In minimum manner, this consists of an initiation codon (ATG), a stop codon and a promoter, as well as a polyadenylation sequence for the plasmids and viral vectors other than poxviruses. When the polynucleotide encodes a polyprotein fragment, e.g. prM-E, M-E, prM-M-E, an ATG is placed at 5' of the reading frame and a stop codon is placed at 3'. As will be explained hereinafter, other elements making i possible to control the expression could be present, such as enhancer sequences, stabilizing sequences and signal sequences permitting the secretion of the protein.

The present invention also relates to preparations comprising such expression vectors. It more particularly relates to preparations comprising one or more in vivo expression vectors, comprising and expressing one or more of the above polynucleotides, including that encoding E, in a pharmaceutically acceptable excipient or vehicle. oo According to a first embodiment of the invention, the other vector or vectors in the preparation comprise and express one or more other proteins of the WN virus, e.g. prM, M, prM-M.

According to another embodiment, the other vector or vectors in the preparation comprise and express one or more proteins of one or more other WN virus strains. In particular, the preparation comprises at least two vectors expressing, particularly in vivo, polynucleotides of different WN strains encoding the same proteins and/or for different proteins, preferably for the same proteins. This is more particularly a matter of vectors expressing in vivo E or prM-M-E of two, three or more different WN strains. The invention is also directed at mixtures of vectors expressing prM, M, E, prM-M, prM-E or M-E of different strains.

According to yet another embodiment and as will be shown in greater detail hereinafter, the other vector or vectors in the preparation comprise and express one or more cytokins and/or one or more immunogens of one or more other pathogenic agents.

The invention also relates to various combinations of these different embodiments.

The preparations comprising an in vitro or in vivo expression vector comprising and expressing a polynucleotide encoding prM-M-E constitute a preferred embodiment of the invention.

According to a special embodiment of the invention, the in vivo or in vitro expression vectors comprise as the sole polynucleotide or polynucleotides of the WN virus, a polynucleotide encoding the protein E, optionally associated with prM and/or M, preferably encoding prM-M-E and optionally a signal sequence of the WN virus.

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According to a special embodiment, one or more of the non-structural proteins NS2A, NS2B and NS3 are expressed jointly with the structural proteins according to the invention, either via the same expression vector, or via their own expression vector. They are preferably expressed together on the basis of a single polynucleotide.

Thus, the invention also relates to an in vivo or in vitro expression vector comprising the polynucleotide encoding NS2A, NS2B, NS3, their combinations and preferably for NS2A-NS2B-NS3. Basically said vector can be one of the above-described vectors comprising a polynucleotide encoding one or more structural proteins, particularly E or prM-M-E. As an alternative, the invention relates to a preparation as described hereinbefore, also incorporating at least one of these vectors expressing a non-structural protein and optionally a pharmaceutically acceptable vehicle or excipient.

In order to implement the expression vectors according to the invention, the one skilled in the art has various strains of the WN virus and the description of the nucleotide sequence of their genome, cf. particularly

Savage H. M. et al. (Am. J. Trop. Med. Hyg. 1999, 61 (4), 600-611), table 2, which refers to 24 WN virus strains and gives access references to polynucleotide sequences in GenBank.

Reference can e.g. be made to strain NY99 (GenBank AF196835). In GenBank, for each protein the corresponding DNA sequence is given (nucleotides 466-741 for prM, 742-966 for M, 967-2469 for E, or 466- 2469 for prM-M-E, 3526-4218 for NS2A, 4219-4611 for NS2B and 4612-6468 for NS3, or 3526-6468 for

NS2A-NS2B-NS3). By comparison and alignment of the sequences, the determination of a polynucleotide encoding such a protein in another WNV strain is immediate.

It was indicated hereinbefore that polynucleotide was understood to mean the sequence encoding the protein or a fragment or an epitope specific to the WN virus. Moreover, by equivalence, the term polynucleotide also covers the corresponding nucleotide sequences of the different WN virus strains and nucleotide sequences differing by the degeneracy of the code.

Within the family of WN viruses, identity between amino acid sequences prM-M-E relative to that of NYQ9 is equal to or greater than 90%. Thus, the invention covers polynucleotides encoding an amino acid sequence, whose identity with the native amino acid sequence is equal to or greater than 90%, particularly 92%, preferably 95% and more specifically 98%. Fragments of these homologous polynucleotides specific with respect to WN viruses, are also considered equivalents.

Thus, on referring to a polynucleotide of the WN virus, this term covers equivalent sequences within the sense of the invention.

It has also been seen that the term protein covers immunologically active peptides and polypeptides. For the requirements of the invention, it covers: 40 a) corresponding proteins of the different WN virus strains,

® 6 b) proteins differing therefrom, but maintaining with a native WN protein an identity equal to or greater than 90%, particularly 92%, preferably 95% and more specifically 98%.

Thus, on referring to a protein of the WN virus, this term covers equivalent proteins within the sense of the invention.

Different WN virus strains are accessible in collections, particularly in the American Type Culture Collection (ATCC), e.g. under access numbers VR-82 or VR-1267. The Kunjin virus is in fact considered to be a WN virus.

According to the invention, preferably the polynucleotide also comprises a nucleotide sequence encoding a signal peptide, located upstream of the expressed protein in order to ensure the secretion thereof. It can consequently be an endogenic sequence, i.e. the natural signal sequence when it exists (coming from the same WN virus or another strain). For example, for the NY99 WN virus, the endogenic signal sequence of E corresponds to nucleotides 922 to 966 of the GenBank sequence and for prM it is a matter of nucleotides 421 to 465. It can also be a nucleotide sequence encoding a heterologous signal peptide, particularly that oo encading the signal peptide of the 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-M-E.

According to a first embodiment of the invention, the in vivo expression vectors are viral vectors.

These expression vectors are advantageously poxviruses, e.g. the vaccinia virus or attenuated mutants of the vaccinia virus, e.g. MVA (Ankara strain) (Stick! 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 being obtained after more than 570 passages of the Ankara vaccine strain on chicken embryo fibroblasts) or NYVAC (its construction being described in US-A-5 494 807, particularly in examples 1 to 6, said patent aiso describing the insertion of heterologous genes in sites of this recombinant and the use of matched promoters - reference also to be made to WO-A-96/40241), avipox (in particular canarypox, fowlpox, pigeonpox, quailpox), swinepox, raccoonpox and camelpox, adenoviruses, such as avian, canine, porcine, bovine, human adenoviruses and herpes viruses, such as equine herpes virus (EHV serotypes 1 and 4), canine herpes virus (CHV), feline herpes virus (FHV), bovine herpes viruses (BHV serotypes 1 and 4), porcine herpes virus (PRV), Marek’s disease virus (MDV serotypes 1 and 2), turkey herpes virus (HVT or

MDV serotype 3), and duck herpes virus. When a herpes virus is used, the vector HVT is preferred for the vaccination of the avian species and the vector EHV for the vaccination of horses.

According to one of the preferred embodiments of the invention, the poxvirus expression vector is a canarypox or a fowlpox, whereby such poxviruses can possibly be attenuated. Reference can be made to the canarypox commercially available from ATCC under access number VR-111. Attenuated canarypox 40 viruses were described in US-A-5,756,103 and WO-A-01/05934. Numerous fowipox virus vaccination

, . ® 7 strains are available, e.g. the DIFTOSEC CT® strain marketed by MERIAL and the NOBILIS® VARIOLE vaccine marketed by Intervet.

For poxviruses, the one skilled in the art can 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 vaccinia virus, the insertion sites for the polynucieotide or polynucleotides to be expressed are in particular the gene of thymidine kinase (TK), the gene of hemagglutinin (HA), the region of the inclusion body of the A type (ATI). In the case of canarypox, the insertion sites are more particularly located in or are constituted by ORFs, C3, C5 and C6. In the case of fowlpox, the insertion sites are more particularly located in or constituted by the ORFs F7 and F8.

The insertion of genes in the MVA virus has been 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., 1994, Vaccine, 12 (11), 1032-1040, to which the one skilled in the art can refer. The compiete MVA genome is described in Antoine G., Virology, 1998, 244, 365-396, which enables the one skilled in the art to use other insertion sites or other promoters.

Preferably, when the expression vector is a poxvirus, the polynucieotide to be expressed is inserted under the control of a specific poxvirus promoter, particularly the vaccine promoter 7.5 kDa (Cochran et al., J.

Virology, 1985, 54, 30-35), the vaccine promoter I3L (Riviere et al., J. Virology, 1992, 66, 3424-3434), the vaccine promoter HA (Shida, Virology, 1986, 150, 451-457), the cowpox promoter ATI (Funahashi et al., J.

Gen. Virol., 1988, 69, 35-47), or the vaccine promoter H6 (Taylor J. et al., Vaccine, 1988, 6, 504-508; Guo

P. etal. J. Virol., 1989, 63, 4189-4198; Perkus M. et al., J. Virol., 1989, 63, 3829-3836).

Preferably, for the vaccination of mammals the expression vector is a canarypox. Preferably, for the vaccination of avians, particularly chickens, ducks, turkeys and geese, the expression vector is a canarypox ora fowlipox.

When the expression vector is a herpes virus HVT, appropriate insertion sites are more particularly located in the BamHI | fragment or in the BamHI M fragment of HVT. The HVT BamHI | restriction fragment comprises several open reading frames (ORFs) and three intergene regions and comprises several preferred insertion zones, namely the three intergene regions 1, 2 and 3, which constitute preferred regions, and ORF UL55 (FR-A-2 728 795, US-A-5 980 906). The HVT BamHI M restriction fragment comprises ORF

UL43, which is also a preferred insertion site (FR-A-2 728 794, US-A-5 733 554).

When the expression vector is an EHV-1 or EHV-4 herpes virus, appropriate insertion sites are in particular 40 TK, UL43 and UL45 (EP-A-668355).

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Preferably, when the expression vector is a herpes virus, the polynucleotide to be expressed is inserted under the control of a strong eukaryote promoter, preferably the CMV-IE promoter. These strong promoters are described hereinafter in the part of the description relating to plasmids.

According to a second embodiment of the invention, the in vivo expression vectors are plasmidic vectors known as plasmids.

The term plasmid covers any DNA transcription unit in the form of a polynucleotide sequence comprising a polynucleotide according to the invention and the elements necessary for its in vivo expression. Preferably there is a supercoiled or non-supercoiled, circular plasmid. The linear form also falls within the scope of the invention.

Each plasmid comprises a promoter able to ensure, in the host cells, the expression of the polynucleotide inserted under its dependency. In general, it is a strong eukaryote promoter. The preferred strong eukaryote promoter is the early cytomegalovirus promoter (CMV-IE) of human or murine origin, or optionaity having another origin such as the rat or guinea pig. The CMV-IE promoter can comprise the actual promoter part, which may or may not be associated with the enhancer part. 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. Preference is given to human CMV-IE (Boshart M. et al., Cell., 1985, 41, 521-530) or murine CMV-IE.

In more general terms, the promoter has either a viral or a cellular origin. A strong viral promoter other than

CMV-IE is the early/late promoter of the SV40 virus or the LTR promoter of the Rous sarcoma virus. A strong cellular promoter is the promoter of a gene of the cytoskeleton, such as e.g. 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, maintaining an adequate promoting activity are included within the present invention, e.g. truncated CMV-IE promoters according to WO-A-98/00166. The notion of the promoter according to the invention consequently includes derivatives and sub-fragments maintaining an adequate promoting activity, preferably substantially similar to that of the actual promoter from which they are derived. For CMV-IE, this notion comprises the actual promoter part and/or the enhancer part, as well as derivatives and sub-fragments.

Preferably, the plasmids comprise other expression control elements. It is in particular advantageous to incorporate stabilizing sequences of the intron type, preferably intron II of the rabbit p-globin gene (van

Ooyen et al., Science, 1979, 206: 337-344).

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

The other expression control elements usable in plasmids can also be used in herpes virus expression vectors.

According to another embodiment of the invention, the expression vectors are expression vectors used for the in vitro expression of proteins in an appropriate cell system. The proteins can be harvested in the culture supernatant after or not after secretion (if there is no secretion a cell lysis is done), optionally concentrated by conventional concentration methods, particularly by ultrafiltration and/or purified by conventional purification means, particularly affinity, ion exchange or gel filtration-type chromatography methods.

Production takes place by the transfection of mammal celis by plasmids, by replication of viral vectors on mammal cells or avian cells, or by Baculovirus replication (US-A-4 745 051; Vialard J. et al., J. Virol., 1990 64 (1), 37-50; Verne A., Virology, 1988, 167, 56-71), e.g. Autographa californica Nuclear Polyhedrosis Virus

AcNPV, on insect cells (e.g. Sf Spodoptera frugiperda cells, ATCC CRL 1711). Mammal cells which can be used are in particular hamster cells (e.g. CHO or BHK-21) or monkey cells (e.g. COS or VERO). Thus, the invention also covers expression vectors incorporating a polynucleotide according to the invention, the thus produced WN proteins or fragments and the preparations containing the same.

Thus, the present invention also relates to WN protein-concentrated and/or purified preparations. When the polynucleotide encodes several proteins, they are cleaved, and the aforementioned preparations then contain cleaved proteins.

The present 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 excipient or vehicle and optionally an adjuvant.

The immunogenic composition notion covers any composition which, once administered to the target species, induces an immune response directed against the WN virus. The term vaccine is understood to mean a composition able to induce an effective protection. The target species are equines, canines, felines, bovines, porcines, birds, preferably the horse, dog, cat, pig and in the case of birds geese, turkeys, chickens and ducks and which by definition covers reproducing animals, egg-layers and meat animals.

The pharmaceutically acceptable vehicles or excipients are well known to the one skilled in the art. For example, it can be a 0.9% NaCl saline solution or a phosphate buffer. The pharmaceutically acceptable vehicles or excipients also cover any compound or combination of compounds facilitating the administration 40 of the vector, particularly the transfection, and/or improving preservation.

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The doses and dose volumes are defined hereinafter in the general description of immunization and vaccination methods.

The immunogenic compositions and vaccines according to the invention preferably comprise one or more adjuvants, particularly chosen from among conventional adjuvants. Particularly suitable within the scope of the present invention are (1) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, (2) immunostimulating sequences (ISS), particularly oligodeoxyribonucleotide sequences having one ore more non-methylated CpG units (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 p 147 of “Vaccine Design, The Subunit and Adjuvant Approach” published by M. Powell, M. Newman, Plenum Press 1995, and the emulsion MF59 described on p 183 of the same work, (4) cation lipids containing a quaternary ammonium salt, (5) cytokins or (6) their combinations or mixtures.

The oil in water emulsion (3), which is particularly appropriate for viral vectors, can in particular be based on: - light liquid paraffin oil (European pharmacopoeia type), - isoprenoid oil such as squalane, squalene, - oil resulting from the oligomerization of alkenes, particularly isobutene or decene, - esters of acids or alcohols having a straight-chain alkyl group, - more particularly vegetable oils, ethyl oleate, propylene giycol, di(caprylate/caprate), glycerol tri(caprylate/caprate) and propylene glycol dioleate, - esters of branched, fatty alcohols or acids, particularly isostearic acid esters.

The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, particularly: . esters of on the one hand sorbitan, mannide (e.g. anhydromannitol oleate), glycerol, polyglycerol or propylene glycol and on the other hand oleic, isostearic, ricinoleic or hydroxystearic acids, said esters being optionally ethoxylated, - polyoxypropylene-polyoxyethyiene copolymer blocks, particularly Pluronic®©, especially L121.

Among the type (1) adjuvant polymers, preference is given to polymers of crosslinked acrylic or methacrylic acid, particularly crosslinked by polyalkeny! ethers of sugars or polyaicohols. These compounds are known under the name carbomer (Pharmeuropa, vol. 8, no. 2, June 1996). The one skilied in the art can also refer to US-A-2 909 462, which describes such acrylic polymers crosslinked by a polyhydroxyl compound having at least three hydroxyl groups, preferably no more than eight such groups, the hydrogen atoms of at least three hydroxyl groups being replaced by unsaturated, aliphatic radicals having at least two carbon atoms.

The preferred radicals are those containing 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals can also contain other substituents, such as methyl. 40 Products sold under the name Carbopol©® (BF Goodrich, Ohio, USA) are particularly suitable. They are in

® 11 particular crosslinked by allyl saccharose or by allyl pentaerythritol. Among them particular reference can be made to Carbopol®© 974P, 934P and 971P.

Among the maleic anhydride-alkenyi derivative copolymers, preference is given to EMA® (Monsanto), which are straight-chain or crossiinked ethyiene-maleic anhydride copolymers and they are e.g. crosslinked by divinyl ether. Reference can be made to J. Fields et al., Nature 186: 778-780, June 4, 1960.

With regards to their structure, the acrylic or methacrylic acid polymers and EMA® are preferably formed by basic units having the following formula:

I I onc —fors)---

COOH COOH in which:

CT - Ry and R,, which can be the same or different, represent H or CH; - x=0or1, preferably x = 1 - y=1or2,withx+y=2.

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

These polymers are dissolved in water or physiological salt solution (20 g/l NaCl) and the pH is adjusted to 7.3 to 7.4 by soda, in order to give the adjuvant solution in which the expression vectors will be incorporated.

The polymer concentration in the final vaccine composition can range between 0.01 and 1.5% wiv, more particularly 0.05 to 1% w/v and preferably 0.1 to 0.4% w/v.

The cationic lipids (4) containing a quaternary ammonium salt and which are particularly but not exclusively suitable for plasmids, are preferably those complying with the following formula:

CH, +

RO— CH CHCHN=ReX

OR CH, 1 in which R, is a saturated or unsaturated straight-chain aliphatic radical having 12 to 18 carbon atoms, R, is another aliphatic radical containing 2 or 3 carbon atoms and X is an amine or hydroxyl group.

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Among these cationic lipids, preference is given to DMRIE (N-(2-hydroxyethyl)-N,N-dimethyi-2,3- bis(tetradecyloxy)-1-propane ammonium ; WO-A-96/34109), preferably associated with a neutral lipid, preferably DOPE (dioleoyl-phosphatidyl-ethano! amine; Behr J. P., 1994, Bioconjugate Chemistry, 5, 382- 389) in order to form DMRIE-DOPE.

Preferably, the plasmid mixture with said adjuvant is formed extemporaneously and preferably, prior to its administration, the mixture formed in this way is given time to complex, e.g. for between 10 and 60 minutes and in particular approximately 30 minutes.

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

The DMRIE or DMRIE-DOPE adjuvant:plasmid weight ratio is between 50:1 and 1:10, particularly 10:1 and 1:5 and preferably 1:1 and 1:2.

The cytokin or cytokins (5) can be supplied in protein form to the composition or vaccine, or can be co- expressed in the host with the immunogen or immunogens. Preference is given to the co-expression of the cytokin or cytokins, either by the same vector as that expressing the immunogen, or by its own vector.

The cytokins can in particular be chosen from among: interleukin 18 (IL-18), interleukin 12 (IL-12), interleukin 15 (IL-15), MIP-1a (macrophage inflammatory protein 1a; Marshall E. et al., Br. J. Cancer, 1997, 75 (12), 1715-1720), GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor). Particular reference is made to avian cytokins, particularly those of the chicken, such as clL-18 (Schneider K. et al., J. Interferon

Cytokine Res., 2000, 20 (10), 879-883), clL-15 (Xin K. -Q. et al., Vaccine, 1999, 17, 858-866), and equine cytokins, particularly equine GM-CSF (WO-A-00/77210). Preferably, use is made of cytokins of the species to be vaccinated.

WO-A-00/77210 describes the nucleotide sequence and the amino acid sequence corresponding to equine

GM-CSF, the in vitro GM-CSF production and the construction of vectors (plasmids and viral vectors) permitting the in vivo equine GM-CSF expression. These proteins, plasmids and viral vectors can be used in immunogenic compositions and equine vaccines according to the invention. For example, use can be made of the plasmid pJP097 described in example 3 of said earlier-dated application or use can be made of the teaching of the latter in order to produce other vectors or for the in vitro production of equine GM-CSF and the incorporation of said vectors or said equine GM-CSF in immunogenic compositions or equine vaccines according to the invention.

The present invention also relates to immunogenic compositions and so-called subunit vaccines, incorporating the protein E and optionally one or more other proteins of the WN virus, particularly prM or M and preferably produced by in vitro expression in the manner described hereinbefore, as well as a pharmaceutically acceptable vehicle or excipient.

C 13

The pharmaceutically acceptable vehicles or excipients are known to the one skilled in the art and can e.g. be 0.9% NaCl saline solution or phosphate buffer.

The immunogenic compositions and subunit vaccines according to the invention preferably comprise one or more adjuvants, particularly chosen from among conventional adjuvants. Particularly suitable within the scope of the present invention are (1) an acrylic or methacrylic acid polymer, a maleic anhydride and alkenyl derivative polymer, (2) an immunostimulating sequence (ISS), particularly an oligodeoxyribonucleotide sequence having one or more non-methylated CpG units (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 emulsion SPT described on p 147 of "Vaccine Design, The Subunit and Adjuvant Approach”, published by M. Powell, M.

Newmann, Plenum Press 1995, and the emulsion MF59 described on p 183 of the same work, (4) a water in oil emulsion (EP-A-639 071), (5) saponin, particularly Quil-A, or (6) alumina hydroxide or an equivalent. The different types of adjuvants defined under 1), 2) and 3) have been described in greater detail hereinbefore in connection with the expression vector-based vaccines.

The doses and dose volumes are defined hereinafter in connection with the general description of immunization and vaccination methods.

According to the invention, the vaccination against the WN virus can be combined with other vaccinations within the framework of vaccination programs, in the form of immunization or vaccination kits or in the form of immunogenic compositions and multivalent vaccines, i.e. comprising at least one vaccine component against the WN virus and at least one vaccine component against at least one other pathogenic agent. This also includes the expression by the same expression vector of genes of at least two pathogenic agents, including the WN virus.

The invention also relates to a multivalent immunogenic composition or a multivalent vaccine against the

WN virus and against 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.

The thus expressed "immunogen" is understood to mean a protein, glycoprotein, polypeptide, peptide, epitope or derivative, e.g. fusion protein, inducing an immune response, preferably of a protective nature.

As was stated hereinbefore, these multivalent compositions or vaccines also comprise 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 is inserted and at least a second expression vector in which a polynucleotide encoding an immunogen of another pathogenic agent is inserted. As stated before, those multivalent compositions or vaccines also comprise a pharmaceutically acceptable vehicle or excipient, and optionally an adjuvant.

For the immunogenic compositions and multivalent vaccines, the other equine pathogens are more particularly chosen from among the group including viruses of equine rhinopneumonia EHV-1 and/or EHV-4 (and preferably there is a combination of immunogens of EHV-1 and EHV-4), equine influenza virus EIV, eastern encephalitis virus EEV, western encephalitis virus WEV, Venezuelan encephalitis virus VEV (preference is given to a combination of the three EEV, WEV and VEV), Clostridium tetani (tetanus) and their mixtures. Preferably, for EHV a choice is made of the genes gB and/or gD; for EIV the genes HA, NP and/or N; for viruses of encephalitis C and/or E2; and for Clostridium tetani the gene encoding all or part of the subunit C of the tetanic toxin. This includes the use of polynucleotides encoding an immunologically active fragment or an epitope of said immunogen.

The other avian pathogens are more particularly chosen from among the group including viruses of the

Marek’s disease virus MDV (serotypes 1 and 2, preferably 1), Newcastle disease virus NDV, Gumboro disease virus IBDV, infectious bronchitis virus IBV, infectious anaemia virus CAV, infectious laryngotracheitis virus ILTV, encephalomyelitis virus AEV (or avian leukosis virus ALV), virus of hemorragic enteritis of turkeys (HEV), pneumovirosis virus (TRTV), fowl plague virus (avian influenza), chicken hydropericarditis virus, avian reoviruses, Escherichia coli, Mycoplasma gallinarum, Mycoplasma gallisepticum, Haemophilus avium, Pasteurella gallinarum, Pasteurella multocida gallicida, and mixtures thereof. Preferably, for MDV a choice is made of the genes gB and/or gD, for NDV the genes HN and/or F; for IBDV the gene VP2; for IBV the genes S (more 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 and for fowl plague the genes HA, N and/or NP. This includes the use of polynucleotides encoding an immunologically active fragment or an epitope of said immunogen.

By way of example, in a multivalent immunogenic composition or a multivalent vaccine according to the invention, to which an adjuvant has optionally been added in the manner described hereinbefore and which is intended for the equine species, it is possible to incorporate one or more of the plasmids described in WO-

A-98/03198 and particularly in examples 8 to 25 thereof, and those described in WO-A-00/77043 and which relate to the equine species, particularly those described in examples 6 and 7 thereof. For the avian species, it is e.g. possible to incorporate one or more of the plasmids described in WO-A1-98/03659, particularly in examples 7 to 27 thereof.

The immunogenic compositions or recombinant vaccines as described hereinbefore can also be combined with at least one conventional vaccine (inactivated, live attenuated, subunits) directed against at least one other pathogen.

C) 1s

In the same way, the immunogenic compositions and subunit vaccines according to the invention can form the object of combined vaccination. Thus, the invention also relates to multivalent immunogenic compositions and multivalent vaccines comprising one or more proteins according to the invention and one or more immunogens (the term immunogen having been defined hereinbefore) of at least one other pathogenic agent (particularly from among the above list) and/or another pathogenic agent in inactivated or attenuated form. In the manner described hereinbefore, these multivalent vaccines or compositions also incorporate a pharmaceutically acceptable vehicle or excipient and optionally an adjuvant.

The present invention also relates to methods for the immunization and vaccination of the target species referred to hereinbefore.

These methods comprise the administration of an effective quantity of an immunogenic composition or vaccine according to the invention. This administration can more particularly take place by the parenteral route, e.g. by subcutaneous, intradermic or intramuscular administration, or by oral and/or nasal routes.

One or more administrations can take place, particularly two administrations.

Co The different vaccines can be injected by a needleless, liquid jet injector. For plasmids it is also possible to use gold particles coated with plasmid and ejected in such a way as to penetrate the cells of the skin of the subject to be immunized (Tang et al., Nature 1992, 356, 152-154).

The immunogenic compositions and vaccines according to the invention comprise an effective expression vector or polypeptide quantity. in the case of immunogenic compositions or vaccines based on plasmid, a dose consists in general terms about in 10 ug to about 2000 pg, particularly about 50 ug to about 1000 pg. The dose volumes can be between 0.1 and 2 ml, preferably between 0.2 and 1 ml.

These doses and dose volumes are suitable for the vaccination of equines and mammals.

For the vaccination of the avian species, a dose is more particularly between about 10 ug and about 500 ng and preferably between about 50 ug and about 200 pg. The dose volumes can in particular be between 0.1 and 1 mi, preferably between 0.2 and 0.5 ml.

The one skilled in the art has the necessary skill to optimize the effective plasmid dose to be used for each immunization or vaccination protocol and for defining the optimum administration route.

In the case of immunogenic compositions or vaccines based on poxviruses, a dose is in general terms between about 10? pfu and about 10° pfu.

C 16

For the equine species and mammals, when the vector is the vaccinia virus, the dose is more particularly between about 10* pfu and about 10° pfu, preferably between about 10° pfu and about 10° pfu and when the vector is the canarypox virus, the dose is more particularly between about 10° pfu and about 10° pfu and preferably between about 10%° pfu or 10° pfu and about 10° pfu.

For the avian species, when the vector is the canarypox virus, the dose is more particularly between about 10° pfu and about 107 pfu, preferably between about 10* pfu and about 10° pfu and when the vector is the fowlpox virus, the dose is more particularly between about 10? pfu and about 10° pfu, preferably between about 10° pfu and about 10° pfu.

In the case of immunogenic compositions or vaccines based on the viral vector other than poxviruses, particularly herpes viruses, a dose is generally between about 10° pfu and about 10° pfu. In the case of immunogenic compositions or avian vaccines a dose is generally between about 10° pfu and about 10° pfu.

In the case of immunogenic compositions or equine vaccines a dose is generally between about 10° pfu and about 10° pfu.

The dose volumes of the immunogenic compositions and equine vaccines based on viral vectors are generally between 0.5 and 2.0 mi, preferably between 1.0 and 2.0 ml, preferably 1.0 ml. The dose volumes of immunogenic compositions and avian vaccines based on viral vectors are generally between 0.1 and 1.0 mi, preferably between 0.1 and 0.5 ml and more particularly between 0.2 and 0.3 ml. Also in connection with such a vaccine, the one skilled in the art has the necessary competence to optimise the number of administrations, the administration route and the doses to be used for each immunization protocol. In particular, there are two administrations in the horse, e.g. at 35 day intervals.

In the case of immunogenic compositions or subunit vaccines, a dose comprises in general terms about 10 ug to about 2000 ug, particularly about 50 ug to approximately 1000 pg. The dose volumes of the immunogenic compositions and equine vaccines based on viral vectors are generally between 1.0 and 2.0 ml, preferably between 0.5 and 2.0 ml and more particularly 1.0 mi. The dose volumes of the immunogenic compositions and avian vaccines based on viral vectors are generally between 0.1 and 1.0 ml, preferably between 0.1 and 0.5 ml, and more particularly between 0.2 and 0.3 mi. Also for such a vaccine, the one skilled in the art has the necessary skill to optimise the number of administrations, the administration route and the doses to be used for each immunization protocol.

The invention also relates to the use of an in vivo expression vector or a preparation of vectors or polypeptides according to the invention for the preparation of 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 different characteristics indicated in the description are applicable to this object of the invention.

A vaccine based on plasmid or a viral vaccine expressing one or more proteins of the WN virus or a WN 40 subunit vaccine according to the present invention will not induce in the vaccinated animal the production of

® 17 antibodies against other proteins of said virus, which are not represented in the immunogenic composition or vaccine. This feature can be used for the development of differential diagnostic methods making it possible to make a distinction between animals infected by the WN pathogenic virus and animals vaccinated with vaccines according to the invention. In the former, these proteins and/or antibodies directed against them are present and can be detected by an antigen-antibody reaction. This is not the case with the animals vaccinated according to the invention, which remain negative. In order to bring about this discrimination, use is made of a protein which is not represented in the vaccine (not present or not expressed), e.g. protein

C or protein NS1, NS2A, NS2B or NS3 when it is not represented in the vaccine.

Thus, the present invention relates to the use of vectors, preparations and polypeptides according to the invention for the preparation of immunogenic compositions and vaccines making it possible to discriminate between vaccinated animals and infected animals.

It also relates to an immunization and vaccination method associated with a diagnostic method permitting such a discrimination. } The protein selected for the diagnosis or one of its fragments or epitopes is used as the antigen in the diagnostic test and/or is used for producing polyclonal or monoclonal antibodies. The one skilled in the art has sufficient practical knowledge to produce these antibodies and to implement antigens and/or antibodies in conventional diagnostic methods, e.g. ELISA tests.

The invention will now be described in greater detail using embodiments considered as non-limitative examples.

Examples

All the constructions are implemented using standard molecular biology methods (cloning, digestion by restriction enzymes, synthesis of a complementary single-strand DNA, polymerase chain reaction, elongation of an oligonucleotide by DNA polymerase...) 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) are isolated and purified using the Geneclean® kit (B10101 Inc. La Jolla, CA).

Example 1: Culture of the West Nile fever virus

For their amplification, West Nile fever viruses NY99 (Lanciotti R. S. et al, Science, 1999, 286, 2333-7)) are cultured on VERO cells (monkey renal cells), obtainable from the American Type Culture Collection (ATCC) under no. CCL-81.

® 1

The VERO celis are cultured in 25 cm? Falcon with eagle-MEM medium supplemented by 1% yeast extracts and 10% calf serum containing approximately 100,000 cells/ml. The cells are cultured at +37°C under a 5%

CO, atmosphere.

After three days the cellular layer reaches to confluence. The culture medium is then replaced by the eagle-

MEM medium supplemented by 1% yeast extracts and 0.1% cattle serum albumin and the West Nile fever virus is added at a rate of 5 pfu/cell.

When the cytopathogenic effect (CPE) is complete (generally 48 to 72 hours after the start of culturing), the viral suspensions are harvested and then clarified by centrifugation and frozen at -70°C. In general, three to four successive passages are necessary for producing a viral batch, which is stored at -70°C.

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

The viral RNA contained in 100 mi of viral suspension of the West Nile fever virus strain NY99 is extracted after thawing with solutions of the High Pure Viral RNA Kit Cat # 1 858 882, Roche Molecular Biochemicals, whilst following the instructions of the supplier for the extraction stages. The RNA sediment obtained at the end of extraction is resuspended with 1 to 2 mi of RNase-free, sterile distilled water.

Example 3: Construction of plasmid pFC 101

The complementary DNA (ADNC) of the West Nile fever virus NY99 is synthesized with the Gene Amp RNA

PCR Kit (Cat # N 808 0017, Perkin-Elmer, Norwalk, CT 06859, USA) using the conditions supplied by the manufacture.

A reverse transcriptase polymerase chain reaction (RT-PCR reaction) is carried out with 50 ul of viral RNA suspension of the West Nile fever virus NY98 (example 2) and with the following oligonucleotides:

CF101 (30 mer) (SEQ ID NO:1) 5STTTTTTGAATTCGTTACCCTCTCTAACTTC 3' and FC102 (33 mer) (SEQ ID NO:2)

STTTTTTTCTAGATTACCTCCGACTGCGTCTTGA 3'

This pair of oligonucleotides allows the incorporation of an EcoRI restriction site, a Xbal restriction site and a stop codon at 3' of the insert.

The synthesis of the first DNAc strand takes place by elongation of oligonucleotide FC102, following the hybridization of the latter with the RNA matrix.

The synthesis conditions of the first DNAc strand are a temperature of 42°C for 15 min, then 99°C for § min 40 and finally 4°C for 5 min. The conditions of the PCR reaction in the presence of the pair of oligonucleotides

Claims (57)

¢ PCT/FR02/01200 CLAIMS

1. A vaccine to protect animals susceptible to West Nile Virus, against WN virus, comprising one or several recombinant avipox, NYVAC or MVA viruses expressing WN virus proteins prM, M and E, said recombinant viruses comprising one or several polynucleotides encoding one or several proteins prM, M and E, and a pharmaceutically acceptable vehicle or excipient.

2. The vaccine according to claim 1, wherein it comprises a recombinant avipox virus expressing the three proteins prM, M and E.

3. The vaccine according to claim 1, wherein it comprises a recombinant avipox virus which contains a polynucleotide forming one reading phase so 15 encoding prM-M-E.

4. The vaccine according to claim 1, 2 or 3, wherein the recombinant virus is a canarypox.

5. The vaccine according to claim 4, wherein the canarypox virus is ALVAC.

6. The vaccine according to any one of claims 1-3, wherein the recombinant virus is a fowlpox.

7. The vaccine according to any one of claims 1-6, wherein the polynucleotide also encodes a signal peptide.

8. The vaccine according to claim 7, wherein it is a WN virus peptide signal.

9. The vaccine according to claim 7, wherein it is an heterologous peptide signal.

10. The vaccine according to claim 9, wherein it is the peptide signal of the human tissue plasminogen activator. AMENDED SHEET

PCT/FR02/01200

11. The vaccine according to any one of claims 1-10, wherein it comprises another polynucleotide encoding a protein from another WN strain and/or from another pathogenic agent, and/or for a cytokine.

12. The vaccine according to any one of claims 1-11, wherein it comprises another expression vector containing another polynucleotide encoding a protein from another WN strain and/or from another pathogenic agent, and/or for a cytokine.

13. The vaccine according to any one of claims 1-12, wherein it comprises further an adjuvant.

14. The vaccine according to claim 13, wherein the adjuvant is chosen among . {1) polymers of acrylic or methacrylic acid, and maleic anhydride and . alkenyl derivative polymers, (2) immunostimulating sequences, (3) an oil in water emulsion, (4) cationic lipids containing a quaternary ammonium salt, (5) cytokins.

15. The vaccine according to claim 13, wherein the adjuvant is a carbomer.

16. The vaccine according to claims 11 or 14, wherein the cytokine is an equine cytokine or an avian cytokine.

17. The vaccine according to claim 16, wherein the cytokine is chosen from among interleukin 18, interleukin 12, interleukin 15, MIP-1a, GM-CSF.

18. The vaccine according to claim 16, wherein the cytokine is chosen from among the avian cytokins clL-18 or clL-15.

19. The vaccine according to claim 16, wherein the cytokine is equine GM-

CSF.

20. The vaccine according to any one of claims 1-19, forming a multivalent vaccine, wherein it comprises a vaccine component directed against another pathogenic agent. AMENDED SHEET

¢ PCT/FR02/01200

21. The vaccine according to claim 20, wherein the component directed against another pathogenic agent contains an antigen as a sub-unit, an inactivated agent or an attenuated agent.

22. The vaccine according to claims 20 or 21, wherein it comprises a component vaccine against equine rhinopneumonia, against equine influenza virus, against eastern encephalitis virus, against western encephalitis virus, against Venezuelan encephalitis, against Clostridium tetani and their mixtures.

23. The vaccine according to claims 20 or 21, wherein it comprises a component vaccine against Marek's disease virus, against Newcastle : disease virus, against Gumboro disease virus, infectious bronchitis virus, . against infectious anaemia virus, against infectious laryngotracheitis virus, against encephalomyelitis virus, against virus of hemorragic enteritis, against pneumovirosis virus, against fowl plague virus, against chicken hydropericarditis virus, against avian reoviruses, against Escherichia coli, against Mycoplasma gallinarum, against Mycoplasma gallisepticum, against Haemophilus avium, against Pasteurella gallinarum, against Pasteurella multocida gallicida, and their mixtures.

24. The vaccine according to any of the preceding claims wherein the animals susceptible to West Nile Virus are selected from equines, canines, felines, bovines, porcines, and birds.

25. An immunogenic composition to induce an immune response against West Nile Virus (WNV) in an animal susceptible to WNV comprising a vector comprising a recombinant avipox virus or DNA plasmid that contains and expresses in vivo a nucleic acid molecule encoding WNV protein prM-M-E.

26. The immunogenic composition of claim 25, wherein the vector is the recombinant avipox virus. AMENDED SHEET

PCT/FR02/01200

27. The immunogenic composition of claim 26 wherein the avipox virus is a canarypox virus.

28. The immunogenic composition of claim 27 wherein the canarypox virus is an ALVAC canarypox virus.

23. The immunogenic composition of claim 25 wherein the vector is the DNA plasmid.

30. A method for inducing an immune response against WNV comprising administering to the animal the immunogenic composition of any one of claims 25-29. ~

31. The method of claim 30 wherein the animal is a horse.. ] } ) 15 :

32. A method for protecting a cat susceptible to WNV comprising administering to said cat a vaccine according to claim 6.

33. A method for protecting a horse susceptible to WNV comprising administering to said horse a vaccine according to claim 15.

34. Use of a vaccine in the manufacture of a preparation for protecting animals susceptible to West Nile Virus, said vaccine comprising one or several recombinant avipox, NYVAC or MVA viruses expressing WN virus proteins prM, M and E, said recombinant viruses comprising one or several polynucleotides encoding one or several proteins prM, M and E.

35. Use according to claim 34, wherein said vaccine is as defined in any one of claims 2 to 23.

36. Use according to claim 34 or claim 35, wherein the animals susceptible to West Nile Virus are selected from equines, canines, felines, bovines, porcines, and birds.

37. Use according to claim 34, wherein said vaccine is as defined in claim 6. AMENDED SHEET

PCT/FR02/01200

38. Use according to claim 37, wherein said animal is a cat.

39. Use according to claim 34, wherein said vaccine is as defined in claim 15.

40. Use according to claim 39, wherein said animal is a horse.

41. Use of an immunogenic composition of any one of claims 25 to 29 in the manufacture of a preparation for inducing an immune response against WNYV in an animal.

42. Use according to claim 41, wherein the animal is a horse.

43. A substance or composition comprising a vaccine for use in a method of Co 15 protecting animals susceptible to West Nile Virus, said vaccine comprising one or several recombinant avipox, NYVAC or MVA viruses expressing WN virus proteins prM, M and E, said recombinant viruses comprising one or several polynucleotides encoding one or several proteins prM, M and E, and said method comprising administering said substance or composition.

44, A substance or composition for use in a method of protection against a virus according to claim 43, wherein said vaccine is as defined in any one of claims 2 to 23.

45, A substance or composition for use in a method of protection against a virus according to claim 43 or claim 44, wherein the animals susceptible to West Nile Virus are selected from equines, canines, felines, bovines, porcines, and birds.

46. A substance or composition for use in a method of protection against a . virus according to claim 43, wherein said vaccine is as defined in claim 6.

47. A substance or composition for use in a method of protection against a virus according to claim 46, wherein said animal is a cat. AMENDED SHEET

PCT/FR02/01200

48. A substance or composition for use in a method of protection against a virus according to claim 43, wherein said vaccine is as defined in claim

15.

49. A substance or composition for use in a method of protection against a virus according to claim 48, wherein said animal is a horse.

50. A substance or composition for use in a method of inducing an immune response against WNV in an animal, said substance or composition comprising an immunogenic composition of any one of claims 25 to 29, and said method comprising administering said substance or composition.

51. A substance or composition for use in a method of treatment or oo prevention according to claim 50, wherein the animal is a horse. : — oT 15

52. A vaccine according to claim 1, substantially as herein described and illustrated.

53. A substance or composition for use in a method of treatment or prevention according to claim 25, or claim 50, substantially as herein described and illustrated.

B4. A method according to any one of claims 30 to 33, substantially as herein described and illustrated.

55, Use according to any one of claims 34 to 42, substantially as herein described and illustrated.

56. A substance or composition for use in a method of protection against a virus according to claim 43, substantially as herein described and illustrated.

57. A new vaccine; a substance or composition for a new use in a method of treatment or prevention; a new non-therapeutic method of treatment; a new use of a vaccine comprising one or several recombinant avipox, AMENDED SHEET

: PCT/FR02/01200

NYVAC or MVA viruses expressing WN virus proteins prM, M and E, said recombinant viruses comprising one or several polynucleotides encoding one or several proteins prM, M and E; or a new use of a composition of any one of claims 25 to 29; substantially as herein described.

AMENDED SHEET