WO1999051622A1

WO1999051622A1 - Avian il-15 nucleotides and polypeptides, and methods of immunizing poultry using avian il-15

Info

Publication number: WO1999051622A1
Application number: PCT/US1999/007485
Authority: WO
Inventors: Kang Choi; Yoshinari Tsusaki; Koichi Kamogawa; Hyun S. Lillehoj
Original assignee: Nippon Zeon Co., Ltd.; The United States Of America As Represented By The Secretary Of Agriculture
Priority date: 1998-04-06
Filing date: 1999-04-06
Publication date: 1999-10-14
Also published as: JPH11346786A; AU3472099A

Abstract

The present invention relates to an isolated avian IL-15 polypeptide comprising: a) the amino acid sequence of SEQ ID NO:1; b) fragments of the amino acid sequence of SEQ ID NO:1, wherein said fragments stimulate growth of avian T lymphocytes expressing ηδTCR; or c) the amino acid sequence of SEQ ID NO:1 having one or more amino acid substitutions, mutations, deletions and insertions and to polynucleotides encoding the amino acid sequences. The present invention further encompasses methods of recombinantly producing said amino acid and polynucleotide sequences and methods of using the amino acid and polynucleotide sequences, particularly for avian vaccines. The sequence of chicken IL-15, SEQ ID Nos:1 and 2 are described.

Description

AVIAN IL-15 NUCLEOTIDES AND POLYPEPTIDES, AND METHODS OF IMMUNIZING POULTRY USING AVIAN IL-15

FIELD OF THE INVENTION

The present invention relates to a chicken-derived interleukin 15 polypeptide, a gene coding therefor, a recombinant bearing the gene and a composition for the prophylaxis or treatment of poultry diseases comprising the recombinant or a polypeptide or an adjuvant thereof as an effective ingredient

BACKGROUND OF THE INVENTION

Proliferation and differentiation of T lymphocytes is regulated by cytokines acting in concert with signals induced by engagement of the T cell receptor (TCR) for antigen. While the principal cytokine used during the immune response is IL-2 (S. L. Swain et al . Curr. Opin. Immunol., 3:304 (1991)), a number of other molecules also stimulate T cells. One of these is IL-15, a glycosylated monomeric cytokine identified as a new member of the four helical-bundle cytokine family (K. H. Grabstein et al. Science, 264:965 (1994); J. D. Burton et al. Proc. Natl. Acad. Sci . USA, 91:4935 (1996)). Recent evidence suggests that the IL-2 receptor (IL-2R) shares polypeptide subunits with other cytokine receptors, including IL-4R (M. Kondo et al . Science, 262:1874 (1993)) and IL-15R (Y. Tagaya et al. Immunity, 4:329 (1996); J. G. Giri et al . E BO J., 13:2822 (1994); W. E. Carson et al. J. Exp . Med., 180:1395 (1994)). In addition, IL-15, while lacking amino acid sequence homology to IL-2, can activate cells via the ΣL- 2R (K. H. Grabstein et al . Science, 264:965 (1994); Y. Tagaya et al. Immunity, 4:329 (1996); J. G. Giri et al. EMBO J., 13:2822 (1994); W. E. Carson et al. J. Exp . Med., 180:1395 (1994)).

Several mammalian IL-15 genes have been cloned and characterized (K. H. Grabstein et al. Science, 264:965 (1994); E. Mrozek et al . Blood, 87:2632 (1996); D. M. Anderson et al. Genomics, 25:701 (1995)). In the case of monkey IL-15, mRNA analysis revealed constitutive gene expression in a variety of tissues including placenta, skeletal muscle, kidney, lung, liver, and pancreas (K. H. Grabstein et al. Science, 264:965 (1994)). The diverse expression of IL-15, compared with the more restricted expression of IL-2 and IL-2R, suggests a heterogeneous repertoire of IL-15 activities (R. N. Bamford et al. J. Eeukoc. Biol . , 59:476 (1996)). However, limited information is available on the function of IL-15. Murine IL- 15 has been shown to be a growth factor for T cells expressing the γ,δτCR (H. Nishimura et al. J. Immunol., 156:663 (1996)). γ,δτCR+ cells mediate local defense against bacteria and viral infections, display spontaneous cytotoxicity, and are activated by thymidine kinase independent mechanism (W. Haas et al. Annu. Rev. Immunol., 11:637 (1993); H. Spits et al . J. Immunol., 144:4156 (1990)). There is no report of an avian gene encoding a polypeptide having the same function as IL-15. SUMMITRY OF THE INVENTION

The present invention is drawn to a novel avian cytokine, as well as recombinant variants thereof and uses thereof.

The present invention relates to a prophylactic composition or vaccine using cytokines of the present invention.

The present invention further relates to a method of immunizing an animal with a recombinant vaccine comprising a cytokine and a component of a pathogen of interest against which vaccination is desired.

The present invention relates more specifically to IL-15 having the amino acid sequence of SEQ ID NO:2 and a polynucleotide encoding the amino acid sequence of SEQ ID

NO:l, particularly the polynucleotide sequence of SEQ ID NO:l.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from the detailed description given hereinbelow, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein;

FIG. 1 shows the alignment between chicken IL-15 and mammalian IL-15 proteins;

FIG. 2 shows tissue expression of chicken IL-15;

FIG. 3 shows analysis of spleen lymphocytes for chicken IL-15 by RT-PCR (FIG. 3A) or Northern hybridization (FIG. 3B) ;

FIG. 4 shows Western blot analysis of transformed E . col i FIG. 5 shows the biological activity of the CM (FIG. 5A) or recombinant chicken IL-15 from E. coli (FIG. 5B) by growth stimulation of spleen lymphoblasts;

FIG. 6 shows proliferation of ConA stimulated spleen lymphoblasts in the presence of chicken IL-15;

FIG. 7 shows CD expression of chicken IL-15 exposed lymphocytes;

FIG. 8 shows flow cytometry analysis of CD expression of chicken IL-15 exposed lymphocytes; FIG. 9 shows chicken IL-15 induction of cytotoxicity; and

FIG. 10 shows Western blot analysis of fNZ29R/IL-15.

DETAILED DESCRIPTION OF THE INVENTION

IL-15 Polypeptide The present invention is drawn to a novel avian cytokine, as well as recombinant variants thereof and uses thereof. Interleukin 15 (IL-15) of the present invention is derived from chickens. More specifically, the present invention is drawn to IL-15 encoded by the amino acid sequence of SEQ ID NO: 1. The IL-15 of the present invention however, is not limited only to the sequence as designated, but may be those in which the amino acids are naturally or artificially modified by substitution, deletion, addition or insertion, so long as the biological activity of IL-15 is maintained.

Since alteration around the four conserved cysteine residues might reduce the biological activity of IL-15 polypeptide, alterations in sequence in that region should be made with care. Murine IL-15 has been shown to be a growth f'actor for T lymphocytes expressing the γ,δ-subunits of chicken T cell receptors (γδTCR) (Nishimura et al . , J. Immunol., 156, 663, 1996) . It has also been shown in monkey that mammalian IL-15 is expressed not only in the spleen but also in a variety of other tissues (Grabstein et al., Science, 264, 965, 1994). The present avian IL-15 is expressed in the skeletal muscle, caecal tonsil (appendix) , small intestine, heart, liver, oviduct and spleen of chicken, as will be later described in the working examples. With regard to activity the present IL- 15 has also been shown to be a growth factor of T lymphocytes expressing the γ,δ-subunits of chicken T cell receptors (γδτCR+ T cells) . The polypeptide obtained in the examples has thus been identified as IL-15. In one embodiment, the IL-15 polypeptide and gene coding therefor are not those of Sundick and Gill-Dixon (R.S. Sundick and C. Gill-Dixon, J. Immunol., 159:720 (1997)) which is incorporated herein in its entirety by reference. In another embodiment, the IL-15 does not have a threonine residue at position 15 of SEQ ID NO:2 and preferably has a methionine residue at this position.

Variants of the specifically exemplified polypeptides are also encompassed by the present invention. Possible variants include allelic variants and corresponding polypeptides from other organisms, particularly other organisms of the same species, genus or family. The present invention is particularly drawn to other avian variants. The variants may have substantially the same characteristics as the IL-15 polypeptides of SEQ ID N0:2. The biological activity of IL-15 referred to in the present invention is used to mean a growth WO 99/51622 _ g _ PCT/US99/07485

promoting activity for lymphocytes expressing γδTCR. The term "substantially the same biological activity" is used to mean that when assessed by the testing method in the example later described, the activity, at least 70% of the γδTCR activity for chicken T cell receptors is retained 29 days after stimulation with ConA, preferably at least 80%, more preferably at least 90% and the activity is 10 times or more than the activity of chicken T cell receptors for α, β-subunits ( βTCR₎ .

As possible variants of the above specifically exemplified polypeptide, polypeptides of the present invention may have additional individual amino acids or amino acid sequences inserted into the polypeptide in the middle thereof and/or at the N-terminal and/or C-terminal ends thereof so long as the polypeptide possesses the desired physical and/or biological characteristics. Likewise, some of the amino acids or amino acid sequences may be deleted from the polypeptide so long as the polypeptide possesses the desired physical characteristics. Amino acid substitutions may also be made in the sequences so long as the polypeptide possesses the desired physical and biochemical characteristics.

The variants of polypeptides contemplated herein should possess more than 75% sequence identity (sometimes referred to as homology) preferably more than 85% identity, most preferably more than 95% identity, even more preferably more than 98% identity to the naturally occurring and/or specifically exemplified polypeptides or fragments thereof described herein. To determine this homology, two polypeptides are aligned so as to obtain a maximum match using gaps and inserts. Two sequences are said to be "identical" if the sequence of residues is the same when aligned for maximum correspondence as described below.

Optimal alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman, Add. Appl . Ma th . , 2_:482 (1981), by the homology alignment method of Needleman and Wunsch, J. Mol . Biol . , _48_:443 (1970), by the search for similarity method of Pearson and Lippman, Proc .

Na tl . Acad. Sci . USA, J35_:2444 (1988), or the like. Computer implementations of the above algorithms are known as part of the Genetics Computer Group (GCG) Wisconsin Genetics Software Package (GAP, BESTFIT, BLASTA, FASTA and TFASTA) , 575 Science Drive, Madison, WI .

"Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e. "gaps") as compared to the reference sequence for optimal alignment of the two sequences being compared. The percentage identity is calculated by determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window and multiplying the result by 100 to yield the percentage of sequence identity. Total identity is then determined as the average identity over all of the windows that cover the complete query sequence .

The polypeptide of the present invention may be expressed as a fusion polypeptide or chimeric polypeptide with a second polypeptide. The second polypeptide will usually impart an additional property or characteristic to the fusion polypeptide which is not possessed by the polypeptide of the present invention.

Fragments of the full length polypeptides such as proteolytic cleavage fragments which contain at least one, and preferably all, of the above-listed physical and/or biological properties are also encompassed by the present invention.

The polypeptide of the present invention may be the full 143 amino acids of SEQ ID NO: 2. Alternatively, it may be a fragment of 123 amino acids wherein the signal peptide has been deleted. Other fragments of the polypeptide of the present invention are also encompassed so long as the four conserved cysteine residues of the protein are not deleted. The amino acid residues immediately around the conserved cysteines should also be maintained or only changed with very conservative substitutes.

The present invention is also directed to a new polypeptide and a method for producing the polypeptide. Production of recombinant polypeptide is discussed in detail below. However generally, the recombinant polypeptide should possess one or more of the above-described biological and/or physical properties. Recombinant polypeptide can be produced by a process which comprises culturing a transformed cell or microorganism described herein under conditions which allow expression of the polypeptide, optionally recovering the thus expressed polypeptide and optionally purifying the recovered polypeptide. In processes for the synthesis of the polypeptide, DNA which encodes the polypeptide is ligated into a replicable (reproducible) vector, the vector is used to transform host cells, and the polypeptide is recovered from the culture. Suitable replicable vectors will be selected depending upon the particular host cell chosen. Suitable processes are known in the art and are described, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. c. 1989 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Chapters 16, 17 and 18. The polypeptide produced in this manner may be different from natural polypeptide in that it may be free of other polypeptides or materials which occur in natural polypeptide. The polypeptide produced by recombinant techniques may also contain some small amounts of contaminating materials from the microorganism, cells and/or fermentation system in which it was produced.

Thus, the present invention is also directed to these new or isolated polypeptides which are produced by recombinant DNA techniques .

DNA

The DNA of the present invention encodes the aforesaid IL-15 polypeptide or a polypeptide having substantially the same activity as that of IL-15. A specific example of the polypeptide encoded by the present DNA includes a polypeptide having SEQ ID NO: 1. A nucleic acid sequence "encodes" or -■ "codes for" a polypeptide if it directs the expression of the polypeptide referred to. The nucleic acid can be DNA or RNA. Unless otherwise specified, a nucleic acid sequence that encodes a polypeptide includes both the transcribed strand and the mRNA or the DNA representative of the mRNA. An "antisense" nucleic acid is one that is complementary to a strand representative of mRNA, including untranslated portions thereof. The term "complementary" applies to nucleic acid sequences and is used herein to mean that the sequence is complementary to all or a portion of a reference polynucleotide sequence.

A specific example of a polynucleotide of the present invention is that of SEQ ID NO: 2. However, the DNA of the present invention is not limited only to that sequence but also includes DNA encoding fusion polypeptides, variants and fragments thereof. The present invention includes cDNA as • well as genomic DNA containing or comprising the requisite nucleotide sequences as well as corresponding RNA and antisense sequences .

Cloned DNA within the scope of the invention also includes allelic variants of the specific sequences presented in the attached Sequence Listing. An "allelic variant" is a sequence that is a variant from that of the exemplified nucleotide sequence, but represents the same chromosomal locus in the organism. In addition to those which occur by normal genetic variation in a population and perhaps fixed in the population by standard breeding methods, allelic variants can be produced by genetic engineering methods. A preferred allelic variant is one that is found in a naturally occurring organism, including a laboratory strain. Allelic variants are either silent or expressed. A silent allele is one that does not affect the phenotype of the organism. An expressed allele results in a detectable change in the phenotype of the trait represented by the locus.

In accordance with degeneracy of genetic code, it is possible to substitute at least one base of the base sequence of a gene by another kind of base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. Hence, the DNA of the present invention may also have any base sequence that has been changed by substitution in accordance with degeneracy of genetic code.

The DNA is readily modified by substitution, deletion or insertion of nucleotides, thereby resulting in novel DNA sequences encoding the polypeptide or its derivatives. These modified sequences are used to produce mutant polypeptide and to directly express the polypeptide. Methods for saturating a particular DNA sequence with random mutations and also for making specific site directed mutations are known in the art; see e . g. Sambrook et al supra , Chapter 15.

The DNA molecule can comprise a nucleotide sequence of SEQ ID NO: 1, or can comprise a nucleotide sequence selected from the group consisting of a nucleotide sequence that hybridizes to a DNA molecule encoding the amino acid sequence of SEQ ID NO-*2 under salt and temperature conditions equivalent to 5x SSC and 42°C, preferably 0.2 x SSC and 68 °C and that codes on expression for a polypeptide that has one or more or all of the above-described physical and/or biological properties.

The present invention also includes polypeptides coded for by these hybridizable variants. See Chapters 11 and 12 of Sambrook et al, supra .

The present invention is also drawn to fragments of the polynucleotides described above, particularly fragments of the DNA of SEQ ID NO: 2 or of DNA encoding the amino acid sequence of SEQ ID NO: 2. Such fragments may be used as probes to identify allelic and recombinant variants of the present IL-15 gene or related members of the same gene family.

The polynucleotides of the present invention (chicken-derived IL-15 genes) can be modified, if necessary and desired, into recombinant DNA molecules by adding a suitable linker thereto, to construct recombinant vectors, transformants or recombinant viruses, as will be later described.

These recombinant DNA constricts have sequences which do not occur in nature or exist in a form that does not occur in nature or exist in association with other materials that do not occur in nature. The DNA and/or RNA sequences described hereinabove are "operably linked" with other DNA and/or RNA sequences. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a WO 99/51622 _ _{± 3} _ PCT/US99/07485 presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous (or in close proximity to) and, in the case of secretory leaders, contiguous and in reading phase.

The linker may particularly be at least one or two nucleotides that are not connected to naturally occurring chicken-derived IL-15 genes. The linker may be appropriately designed depending on the site of a vector or virus to be inserted. The linker can be connected to the DNA of the present invention by conventional genetic manipulation. Ligation of the added DNA may be effected by any conventional method so long as the expression of each gene is not inhibited. The DNA is digested with an appropriate restriction enzyme (s) followed by connection directly or through a linker sequence.

Recombinant vectors

Recombinant vectors of the present invention are recombinant vectors bearing at least the DNA of the present invention defined above, and may be a vector in which a promoter (later described) or a gene marker used as a marker for lacZ gene, etc. is inserted together. The vector may be an integrating or non-integrating vector. A vector in which the polynucleotides of the present invention are integrated is optionally chosen from plasmid, cosmid, phage or the like that is generally employed as a vector. Typically, vectors such as plasmids like pBR322, pBR325, pUC7, pUC8, pUC18, pUC19, pBluescript or pGEM, phage such as M13 phage or cosmid such as pHC79 are digested with an appropriate restriction enzyme ⁽s⁾ and the DNA of the present invention or other necessary DNA is inserted therein by the standard procedure. Where the recombinant vector is used to construct the recombinant virus, a non-essential region later described is incorporated into the recombinant vector followed by insertion of the DNA therein.

Recombinant virus A recombinant virus of the present invention contains an avian IL-15 gene of the present invention, and is constructed by inserting the DNA into a region that is not essentially required for growth of the parent virus by standard procedures. If necessary and desired, promoters and marker genes may also be inserted into the non-essential region, in addition to the DNAs .

(1) Parent virus

The parent virus which can be used in the present invention is a virus for insertion of the DNA of the present invention.

The kind of the virus is not particularly limited as long as it is usable as a virus for the recombinant techniques. Specific examples of viruses include a pox virus such as fowlpox virus, quailpox virus, vaccinia virus, etc.; a WO 99/51622 _ ! 5 _ PCT/US99/07485 baculovirus such as Autographa calif ornica , Trichopl usia ni, Bonvix mori, etc . ; herpes virus such as turkey herpes virus, infectious laryngo-tracheitis virus, Marek's disease virus, herpes simplex virus, etc. Inter alia, pox viruses that infect fowl are preferred, more preferably, those belonging to fowl pox viridae (FPV) . Examples of particularly preferred, and FPV include ATCC VR-251, ATCC VR-229, ATCC VR-249, ATCC VR-288, Nishigahara strain, Shisui strain, CEVA strain, narrowly defined FPV such as CEVA vaccine strain-derived virus capable of forming a large plaque when infected with CEF (chicken embryonic fibroblast) , viruses such as NP strain (Nakano pigeonpox strain) which are akin to the narrowly defined FPV and used as a fowlpox live vaccine strain, and the like. These viruses are commercially available and thus readily available.

(2) Non-essential region

The non-essential region used in the present invention is the DNA region non-essential for amplification of the parent virus described above. Non-essential regions which can be used in the present invention include the polyhedrin gene region of baculovirus, the TK gene region of poxvirus and the non- essential region of poxvirus described in Japanese Patent Application Laid-Open No. 1-168279. Specific examples of the non-essential region of poxvirus include EcoRI fragment (7.3 kbp) , EcoRI-Hindlll fragment (ca. 5.0 kbp) , BamHI fragment (ca. 4.0 kbp) and Hindlll fragment (ca. 5.2 kbp) of APV NP strain DNA described in the Japanese Patent Application supra, the TK gene region of quailpox virus, the TK gene region of turkey pox virus, etc. and regions that cause homology recombination with these regions.

(3) Vector containing the non-essential region for amplification of virus

The vector containing the non-essential region of virus which can be used in the present invention to construct a vector for virus recombination can be the same as the recombinant vector described hereinbefore. After these vectors are digested with an appropriate restriction enzyme (s), the non-essential region for the virus may be incorporated by the standard procedure.

(4) Foreign gene

The foreign gene which is incorporated into the virus according to the present invention is a gene coding for the polypeptide of the present invention or a fragment thereof, or a DNA molecule in which a part of the amino acid sequence for the polypeptide encoded by the DNA molecule of the present invention is modified to such an extent that the γδτCR+ T cell growth promoting activity-inducing capability is not damaged, or a fragment thereof.

The foreign gene can be incorporated into one region of the viral genome. Alternatively, a plurality of foreign genes may be incorporated into a plurality of regions of the viral genome. Furthermore, lacZ gene, genes coding for surface antigenic protein of the genus Eimeria such as Eimeria acervulina , Eimeria tenella or Eimeria maxima , or fragments thereof; genes coding for HN, F, etc. of Newcastle disease virus (NDV) , or fragments thereof; genes coding for ILTV gB or gC, or fragments thereof; genes coding for MDV gB, gC, gD, gH, gl, gE, etc. or fragments thereof, can also be incorporated in combination. In addition to these native genes, genes having homology with these genes can also be employed. The homology used herein refers to the homology assessed by Fasta program of the Genetics Computer Group Sequence Analysis Software package .

(5) Vector for recombination

The vector for virus recombination which can be used in the present invention comprises at least a polynucleotide of the present invention, inserted with a promoter that can control the DNA molecule into the non-essential region of the virus. Such a vector for recombination can be obtained by inserting the chicken IL-15 gene and a promoter controlling the chicken IL-15 into the non-essential region for virus by the standard procedure. Alternatively, the vector for recombination may be obtained by incorporating such a vector-derived fragment containing the virus non-essential region inserted with the chicken IL-15 gene and the promoter into another vector. Furthermore, marker genes such as lacZ gene of E. coli and a promoter controlling the gene may also be inserted for purification of the recombinant virus.

(6) Promoter

The promoter used in the present invention is not particularly limited as far as it functions as a promoter in a host infected with the recombinant virus. Specific examples of the WO 99/51622 _ _{1 Q} _ PCT/US99/07485 promoter include a promoter for vaccinia virus gene encoding the 7.5 K polypeptide, a promoter for vaccinia virus gene encoding the 11 K polypeptide, a promoter for vaccinia virus encoding thymidine kinase, a polyhedrin promoter for baculovirus, an IE promoter for cytomegalovirus, etc. These promoters may be modified by partial deletion, etc., so long as they can function as promoters. Synthetic promoters may also be used for the present invention, with reference to Moss et al., J. Mol. Biol . , 210:749-776, 771-784 (1989).

(7) Construction of recombinant virus

Any conventional method can be used for construction of the recombinant virus and there is no particular limitation thereto. For example, the vector for recombination is introduced into cells previously infected with virus by means of, e.g., electroporation, thereby to cause homologous recombination between the vector and the viral genomic DNA of the infected cells. Thus, the recombinant virus is constructed. The resulting recombinant virus is infected with host cells incubated in an appropriate medium. The plaques formed are picked as candidates for the desired recombinant virus. The candidate strains are purified by hybridization using the incorporated avian IL-15 gene as a probe or by means of selecting positive plaques expressing the marker gene inserted in combination with the IL-15 gene. By using an immunoassay with an antibody against an antigen encoded by the avian IL-15 gene incorporated, it is confirmed that the desired recombinant virus is obtained. In the case of recombinant virus where, e.g., lacZ gene is incorporated as a marker gene, β-galactosidase is expressed to successively " form blue plaques in the presence of Bluo-Gal (GIBCO-BRL Inc.₎ which is one of the substrates.

Host cells are not particularly limited so long as they can be infected with the virus used and can be reproduce the virus. Where FPV is employed, CEF and e bryonated egg chorioallantoic membrane cells can be used as host cells; Spodoptera frugiperda , etc. in the case of using baculovirus; and CEF, duck embryonic fibroblasts, etc. in the case of using turkey herpes virus.

Trans forma nt

The transformant of the present invention is a cell or microorganism transformed at least by the polynucleotide of the present invention or by an expression vector containing at least the polynucleotide of the present invention. Vectors used to construct the expression recombinant vector containing at least the polynucleotide of the present invention are not particularly limited but those similar to the aforesaid vectors may be employed.

The expression recombinant vectors can be constructed by standard procedures to insert the polynucleotide of the present invention, etc. into these vectors. For example, the vectors are digested with a restriction enzyme (s) followed by directly binding the genes described above under the control of the promoter functioning in host cells. Specific examples of the promoter which can be used in the present invention are lac promoter/operator, trp promoter, tac promoter, lpp promoter, PL promoter, amyE promoter, Gal7 promoter, PGK promoter, ADH promoter, etc.

For constructing the recombinant vector to express the avian- derived IL-15 polypeptide, the DNA is once incorporated into an appropriate vector and the thus constructed recombinant vector is then subcloned, a method which is well known to one skilled in the art. The subcloned genes are excised by an appropriate restriction enzyme (s) and bound to the promoters described above to construct the expression vector capable of producing a desired protein.

Vectors which can be used for the subcloning are not particularly limited. Specific examples of those vectors include plasmids such as pUC8, pUC9, pUCIO, pUCll, pUC18, pUC19, pBR322, pBR325, pBR327, pDR540, pDR720, pUBHO, pIJ702, pBluescript, pGEM, YEpl3, YEp24, YCpl9, YCp50, pAC373, pACTMl, etc. Using the resulting expression vectors, a variety of hosts can be appropriately transformed to obtain a microorganism capable of producing a fused protein comprising the avian-derived IL-15 polypeptide having γδτCR+ cell growth promoting activity or an amino acid sequence thereof.

Hosts used herein can be chosen in terms of compatibility of expression vectors, stability of the products, etc. and may be either procaryotic or eucaryotic cells. Specific examples of the host include cells from the genus Escherichia (e.g., E. col i ) , the genus Salmonella (e.g., Salmonella typhimurium) , actinomycetes, yeast, insect cells, chicken cells, human cells, mouse cells, rat cells, Chinese hamster ovary cells (CHO cells) and the like. Expression in a eucaryotic expression is preferable where a glycosylated form of IL-15 is desired. The host transformed by transfection of an appropriate expression vector can be cultured and proliferated under incubation conditions well known to one skilled in the art .

In producing proteins, conditions for inducing the promoter function can be chosen. Taking lac promoter/operator as a specific example, it is achieved by adding an appropriate amount of isσpropyl-1-thio-^β-D-galactopyranoside to a medium.

Using the thus transformed host, a composition for the prophylaxis or treatment of poultry diseases can be prepared by the standard procedure. For example, the host can be incubated under conditions generally adopted for incubation of microorganisms. Where E. coli is employed, incubation is carried out in LB medium at 37° C under aerobic conditions.

Chicken IL-15 polypeptide

The chicken IL-15 polypeptide of the present invention is encoded by the DNA molecule (chicken IL-15 gene) of the present invention described above. More specifically, the polypeptide of the present invention has an amino acid sequence, designated SEQ ID NO. 1.

The chicken IL-15 polypeptide can be produced by culturing the transformant of the present invention as described above. Alternatively, the polypeptide can also be produced by culturing the recombinant virus of the present invention described above in an appropriate host cell. The produced chicken IL-15 polypeptide can be isolated and purified in accordance with a modification of the methods described in Methods in Enzymology, vol. 182 (Guide to Protein Purification, edited by Murry P. Deutscher, published by Academic Press, Inc.⁾. The thus produced chicken IL-15 polypeptide can be diluted by the standard procedure or mixed with an appropriate salt solution, etc. The resulting dilution or mixture can be provided as the composition for the prophylaxis or treatment of poultry diseases. The dilution or mixture can also be used as an adjuvant for the antigenic protein of the polypeptide and a fowl pathogen. Specific examples of antigens to be mixed with include HN or F of HDV, gB, gC or UL32 of ILTV, gB, gC, gH, gL, gl or gE of MDV, surface antigens possessed by protozoa of the genus Eimeria, VP2 of infectious Fabricius bursitis disease virus ₍IBDV₎ and the 40 kDa polypeptide of Mycoplasma gallicepticum . A mixing ratio with the antigenic protein is not particularly limited. The composition can be administered subcutaneously, intravenously, intramuscularly, intraperitoneally, etc. It is also possible to administer by spraying into the air passage for immunization, or through drinking water.

Composi tion for the prophylaxis or trea tment _{of poul try} diseases

The present invention is also generally drawn to a method of vaccinating an animal, such as a bird, and particularly a chicken, comprising concomitant administration of a component of a pathogen of interest against which protection is desired and a cytokine which stimulates the immune^' system. Such a vaccine may be in a recombinant form wherein the pathogen component and cytokine are inserted into either separate or the same plasmid vector using the techniques discussed above and administered to the animal to be vaccinated. Vaccination with viral vectors containing both a component of the pathogen of interest and a cytokine results in an improved vaccination by stimulating the immune response to the pathogen component.

More specifically, the composition for the prophylaxis or treatment of poultry diseases in accordance with the present invention comprises the recombinant microorganism or recombinant virus of the present invention. That is, the composition comprises the recombinant microorganism or recombinant virus of the present invention in which the present chicken IL-15 gene has been inserted. The present composition may be used in combination with recombinant microorganisms or recombinant viruses which contain other antigen genes (e.g., surface antigen genes possessed by protozoa belonging to the genus Eimeria, gB, gC or UL32 gene derived from ILTV, gB, gC, gH, gL, gl or gE gene of MDV, HN or F gene of NDV, VP2 gene of IBDV, the 40 kDa polypeptide gene of Mycoplasma gallicepticum, etc.). The prophylactic compositions of the present invention also include recombinant microorganisms or recombinant viruses inserted with the chicken IL-15 gene and other antigenic genes together. In addition to the recombinant microorganisms and recombinant viruses, the composition may further contain pharmacologically acceptable carriers such as physiological saline, etc.

The composition for the prophylaxis or treatment of poultry diseases in accordance with the present invention can be prepared without any particular limitation. For example, incubation is conducted and continued under such conditions that the cell or microorganism used in the present invention can grow, until the recombinant microorganism of the present invention proliferates. In the case of using the recombinant virus of the present invention, the recombinant virus infects to the cell where the virus can grow and the infected cell is incubated until the recombinant virus amplifies. Subsequently, in the case of using the recombinant microorganism, the culture broth is centrifuged to obtain the recombinant microorganism capable of expressing chicken IL-15.

These recombinant microorganisms can be used as the composition for the prophylaxis or treatment of poultry diseases by itself or suspended in pharmacologically acceptable saline. In the case of using the recombinant virus, cells in which the recombinant virus amplifies are recovered, disrupted and then centrifuged. The supernatant containing high titer cell-independent recombinant virus is separated from the precipitates . The supernatant substantially free of the host cells and containing the cell culture medium and the recombinant virus can be used as the composition for the prophylaxis or treatment of poultry diseases according to the present invention. The composition may also be reconstructed with pharmacologically acceptable saline, etc. and the resulting system may be provided for - practical use. Alternatively, the supernatant may be lyophilized or freeze dried and provided for use as a lyophilized preparation. The composition for the prophylaxis or treatment of poultry diseases in accordance with the present invention can be administered by any route, so long as the recombinant of the present invention such as the recombinant microorganism or recombinant virus is expressed in fowl so that the chicken IL- 15 polypeptide is biosynthesized. For example, the recombinant of the present invention can be inoculated by scratches formed on the fowl skin or inoculated by subcutaneous injection through a syringe or other appropriate equipments. It is also possible to suspend the recombinant of the present invention in drinking water for fowl or mixing the recombinant with solid feed and orally administer the suspension or the feed. The composition may also be administered by inhalation of the recombinant through an aerosol or spray preparation, intravenous inoculation, intramuscular inoculation, intraperitoneal inoculation, etc.

In the case of inoculating, the dose is generally in the range of 10³ to 10^δ pfu (plaque forming unit) per chick. When injected, the dose is adjusted to approximately 0.1 ml by dilution with a pharmacologically acceptable solution such as saline.

The composition for the prophylaxis or treatment of poultry diseases comprising as an effective ingredient the recombinant microorganism or recombinant virus of the present invention can be stored and provided for use under ordinary conditions. Thus, storage in liquid nitrogen and laborious handling and inoculation which are required for the existing prophylactic such as cell-dependent vaccine preparations, can be eliminated or minimized. For example, the recombinant virus of the present invention may be freeze dried so that the recombinant virus can be stored at room temperature (approximately 20-22 °C) over a long period of time, and handled or transported at room temperature. Since the recombinant microorganism or recombinant virus of the present invention in the composition for the prophylaxis or treatment can be stored also in its freeze dried form, a suspension of e.g., the recombinant virus, can be frozen and stored at -20 °C to -70 °C.

The present invention is further drawn to vaccines and pharmaceutical compositions of the present invention assembled as a kit. A kit of the present invention contains the avian IL-15 of the present invention in either a lyophilized form or as a pharmaceutically acceptable suspension. The present IL- 15 preparation of the kit is packaged in a vial for administration and packaged with instructions for use. Alternatively, the kits of the present invention contain prepackaged preparations of vectors containing the IL-15 DNA of the present invention. Such, kits may also contain vectors containing DNA encoding a component of a pathogen of interest against which vaccination is desired. The DNA encoding the IL-15 gene, or DNA encoding another cytokine of interest, and the pathogen-derived gene may be contained which the same vector. Alternatively, the DNA encoding the IL-15 gene or another cytokine of interest, and the pathogen-derived gene - may be contained in separate vectors. The individual vectors may be mixed in a single vial for administration or the kit may contain separate vials, each respectively containing the IL-15 gene or the pathogen-derived gene. By putting the vector containing the IL-15 gene or cytokine gene in a separate vial, the IL-15 or cytokine containing vector may be used at the user's discretion with a recombinant pathogen vaccine of choice.

EXEMPLIFIED EMBODIE ENTS OF THE INVENTION

Chickens

SC chickens, White Leghorn FI crosses from Hyline International Production Center (Dallas Center, IA) , were obtained as fertile eggs, hatched at the Immunology and Disease Resistance Laboratory, and kept in wire cages. Chickens were provided feed and water ad libi tum and used at 8-10 weeks of age.

cDNA Cloning and Sequence Analysis of IL-15

A cDNA library was prepared in the ZAP XR expression vector (Stratagene, La Jolla, CA) using mRNA from a CD4⁺ chicken T cell hybridoma, designated P34, as described (K. D. Song et al. Vet . Immunol . Immunopa thol . , 58:321 (1997)). The library was screened using a rabbit antibody against a protein fraction of P34 conditioned medium (CM) showing T cell growth promoting activity (T. J. Myers et al. Vet . Immunol . Immunopa thol . , 34:97 (1992)). After tertiary screening, positive plaques were picked and excised as plasmid ⁽pbluescript-insert⁾ by coinfection with ExAssist helper phage

⁽Stratagene⁾ as described by the manufacturer's instructions. One recombinant plasmid (pUC-chIL-15) was selected for further characterization. pUC-chIL-15 cDNA was prepared and sequenced using the Dye Terminator Cycle sequencing kit

⁽Perkin Elmer Cetus, Brachburg, NJ) and a model 373 DNA Sequencer ⁽Perkin Elmer Cetus) . Computer analysis of nucleotide and protein sequences was carried out using the Fasta program of the Genetics Computer Group (GCG) Sequence Analysis Software package, Version 8. In addition, GCG programs Pileup and Bestfit were used to perform multiple alignments, pairwise comparisons, and presentation of sequence data.

Using a rabbit antibody against a protein fraction of P34 cell CM previously shown to contain T cell growth promoting activity ⁽T. J. Myers et al . Vet . Immunol . Immunopa thol . 34:97 ⁽1992^{) )}, 58 out of 600,000 plaques screened in the P34 cDNA library were immunoreactive . Of these, only one clone ₍no. 54⁾ was shown to encode chicken IL-15. By restriction endonuclease mapping, a 0.8 kb cDNA insert was identified in this clone. Nucleotide sequence analysis revealed a 800 bp insert with a poly(A) tail (SEQ ID NO:l). An open reading frame of 429 bp was identified capable of encoding a 143 amino acid polypeptide (SEQ ID NO:2) . The predicted amino acid sequence was compared with other protein sequences using the Swissprot database and maximum homology with mammalian IL-15s was observed ⁽Fig. 1) . Based upon the sequence alignment of chicken IL-15 with mammalian IL-15s, a predicted signal sequence of 20 amino acids was identified. Cleavage of the" signal sequence at the position indicated (arrow, Fig. 1) would generate a mature protein 123 amino acids in length. The highest sequence homology was with bovine IL-15 (34% identity, 59% similarity) . Compared to other mammalian IL-15 sequences, despite similarity in m.w. (see below), the overall level of homology was low, particularly when compared to inter-mammalian IL-15 alignments (e.g. human and mouse IL-15 share 96% amino acid identity) . However, all 4 cysteine residues required for biological activity of mammalian IL-15s (K. H. Grabstein et al . Science 264:965 (1994); D. M. Anderson et al. Genomics 25:701 (1995)) are conserved in the chicken sequence (asterisks, Fig. 1) .

Characteriza tion of IL-15 Gene Transcripts

Spleen lymphocytes, prepared as described (B. Kaspers et al . Vet . Immunol . Immunopa thol . , 44:71 (1994)), were resuspended in RPMI-1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 1 mM sodium pyruvate, 5 X 10^"5 M 2-ME, 0.1 mM nonessential amino acids, 100 units/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES buffer, and 2 mM glutamine (all from Sigma, St. Louis, MO). A single cell suspension of 1 X 10⁷ cells /ml was activated with 10 μg/ml Con A (Pharmacia, Piscataway, NJ) in 162 cm² flasks (Costar, Cambridge, MA) at 41 in 5% C0₂- Total RNA was prepared, 5 μg resolved by 1% formaldehyde denaturing agarose gel electrophoresis, transferred to nylon membrane (Hybond-N, Amersham, Arlington Heights, IL) , and probed with ³²P labeled chIL-15 cDNA prepared using a random primer labeling kit (Boehringer Mannheim, WO 99/51622 ~ ³ ° " PCT/US99/07485

Indianapolis, IN) using routine techniques^' (Molecular Cloning. A Laboratory Manual. 2^nd Ed., J. Sambrook et al . eds . , Cold Spring Harbor Press) . Blots were prehybridized for 2 hr, hybridized for 16-18 hr, washed twice with 2X SSC (0.3 M NaCl, 0.06 M sodium citrate, pH 7.4), 0.2% SDS at room temperature, followed by 0.2X SSC, 0.2% SDS at 65°C, and exposed to X-ray film (Eastman Kodak, Rochester, NY) .

To perform RT-PCR, total RNA was added to reverse transcriptase buffer, dNTPs, MgCl₂, AMV reverse transcriptase (Promega, Madison, WI), and oligonucleotide primers (sense primer, 5' -GGAATTCACTGCCATGATGTGCAAAGTA-3' (SEQ ID NO:4), antisense primer, 5' -TTTCTAGATTATTTTTGCAGATATCTCAC-3' (SEQ ID NO:5), Bioserve Biotech, Laurel, MD) . PCR was conducted in a thermal cycler (Perkin Elmer Cetus) using the following program: 5 min at 95°C (first cycle) , and 30 cycles at 55°C for 1 min, 72°C for 2 min, 94°C for 1 min, and 72°C for 10 min (final cycle) . Alpha enolase served as a control using oligonucleotide primers (Amitof Inc., Boston, MA) designed on the basis of published sequence (M. Tanaka et al. J. Biochem . (Tokyo) , 117:554 (1995)). PCR products were electrophoresed in 2% Nusieve agarose gel (FMC BioProducts, Rockland, ME) and visualized by ethidium bromide staining.

The mammalian IL-15 gene is expressed in a variety of tissues including placenta, skeletal muscle, kidney, lung, liver, and pancreas (K. H. Grabstein et al . Science 264:965 (1994)). To assess the tissue expression of chicken IL-15, RT-PCR using IL-15 specific primers was performed on total RNA isolated from caecal tonsil, heart, intestine, liver, skeletal muscle, oviduct, and spleen. A 429 base pair IL-15 transcript was detected in all tissues examined (Fig. 2) . Spleen lymphocytes were further analyzed for chicken IL-15 mRNA by RT-PCR (Fig. 3A) and Northern hybridization (Fig. 3B) at various times following 10 μg/ml Con A stimulation. In both cases, an IL-15 transcript was detected at all time points examined (2-16 hr₎ with maximum levels of expression at 4 hr. Interestingly, chicken IL-15 mRNA was detected in unstimulated spleen lymphocytes (0 hr) by RT-PCR but not by Northern hybridization .

Expression of IL-15 cDNA in E. coli and CHO-K1 Cells

The IL-15 gene was amplified from 1-5 μg of cDNA by PCR using the conditions described above. EcoRI and Xbal restriction sites at the 5' end of the sense primer and 3' end of the antisense primer respectively allowed directional cloning of the PCR product by digestion with JϊcoRI and Xbal (Life Technologies), fractionation in 1% GTG agarose (FMC BioProducts), ligation to EcoRI-Xbal digested pUC18, and transformation of DH5-alpha by the recombinant plasmid pUC- chIL-15 by electroporation with a Gene Pulser II (BioRad, Hercules, CA) according to standard procedures (Molecular Cloning. A Laboratory Manual. 2^nd Ed., J. Sambrook et al . eds . , Cold Spring Harbor Press) .

To express chicken IL-15 in E. coli, the 0.43 kilobase (kb) £coRI-XbaI fragment from pUC-chIL-15 was subcloned into the pMAL-c2 plasmid (New England Biolabs, Beverly, MA) , to produce a maltose binding protein (MBP) -chIL-15 fusion protein, according to the manufacturer's instructions. Transformed E. coli harboring the pMAL-chIL-15 plasmid was grown in 10 mg/ml tryptone, 5 mg/ml yeast extract, 5 mg/ml NaCl, 2 mg/ml glucose, and 100 μg/ml ampicillin at 37 °C to ODeoo = 0.4, and induced with 0.3 mM isopropyl-beta-thiogalactopyranoside (New England Biolabs) for 3hr. The MBP-chIL-15 fusion protein was purified by amylose gel affinity chromatography (New England Biolabs) . Briefly, pMAL-chIL-15 transformed E. coli were lysed by freeze-thawing and sonication, cellular debris removed by centrifugation at 5,000 X g for 30 min, and the supernatant diluted to 2.5 mg/ml with 0.02 M Tris-HCl, pH 7.4, 0.2 M NaCl, 1 mM EDTA (column buffer). Two hundred milligrams of protein was loaded onto the column (2.5 X 10 cm), washed with 12 column volumes of column buffer, and bound proteins eluted with column buffer containing 10 mM maltose (Sigma) . Eluted protein fractions were monitored at OD₂₈o and pooled. Recombinant chicken IL-15 was separated from the MBP protein by cleavage of 100μg of MBP-chIL-15 fusion protein (1 mg/ml) with 2μg of Factor Xa (1 mg/ml, New England Biolabs) in column buffer plus 10 mM maltose for 6 hr at room temperature.

To express chicken IL-15 in eukaryotic cells, the 0.43 kb £coRI-XbaI fragment from pUC-chIL-15 was subcloned into the pcDNA3 vector (In Vitrogen, San Diego, CA) according to the manufacturer's instructions. The resulting plasmid pcDNA3- chIL-15 was transfected into CHO-K1 cells (ATCC, Rockville, MD) using the Lipofectamine reagent (Life Technologies) . Briefly, 2 X 10 cells were cultured in 35 mm diameter tissue culture plates (Costar) and incubated for 24 hr. Two micrograms of plasmid cDNA in 100 ml of Ham's F-12 medium (Life Technologies) was mixed with 8 ml of Lipofectamine reagent, diluted into 100 ml of F-12, and incubated at room temperature for 30 min. The volume was then brought to 1.0 ml with F-12, layered on top of CHO-K1 cells, incubated at 37°C for 5 hr, and the medium replaced with F-12 containing 10% (v/v) FCS. After 24 hr incubation, the medium was replaced again, and 72 hr after transfection the cells were diluted 1:10 (v/v) into medium containing 500μg/ml geneticin (Life Technologies) . Several stable CHO-K1 cell lines secreting biologically active chicken IL-15 were selected.

Antibodies against a synthetic peptide corresponding to the predicted amino acid sequence of chicken IL-15 encompassing residues 77-89 (TLKKETEDDTEIK) (SEQ ID NO: 6) was generated by biweekly immunization of rabbits with lOOμg of peptide-KLH conjugate in Freund' s adjuvant (Sigma). Conjugation of peptide to KLH was carried out using the Inject Immunogen EDC conjugation kit (Pierce, Rockford, IL) . Extracts from pMBP- chIL-15 transfected E. coli were subjected to electrophoresis on 15% SDS-PAGE gels (BioRad) under reducing conditions (U. K. Laemmli Na ture, 227:680 (1970)), the resolved proteins e^'lectroblotted to nitrocellulose (H. Towbin et al . Proc . Na tl . Acad. Sci . USA, 79:4350 (1979)), and analyzed by sequential incubations with rabbit anti-peptide antiserum, biotin-labeled goat anti-rabbit antiserum (Sigma) , and avidin-labeled peroxidase (Sigma) . Immunoreactive bands were visualized with DAB substrate (SIGMA FAST™, Sigma) . Rabbit antiserum against a synthetic peptide encompassing residues 77-89 of chicken IL-15 was used in Western blotting to characterize the expression of amylose column purified MBP- chIL-15 fusion protein from transformed E. coli before and after cleavage with Factor Xa (Fig. 4). Prior to Factor Xa treatment, a 59 kDa immunoreactive band was observed (lane 7) .

After Factor Xa digestion of the MBP-chIL-15 fusion protein, several new bands appeared, the most prominent being 16 kDa, the expected size of recombinant chicken IL-15 (lane 6) . Minor immunoreactive bands of smaller m.w. presumably represent over-digestion of IL-15 by Factor Xa . As a negative controls, untransfected E. coli extract (lane 1), and an irrelevant protein, chicken interferon (IFN)-γ, expressed as MBP fusion protein, purified on an amylose column and analyzed before (lane 4) or after (lane 3) Factor Xa treatment, did not produce a detectable immunoreactive band of the same M.W.

Expression of biologically active recombinant chicken IL-15 was demonstrated by the ability of CM from transfected cells to stimulate the growth of 3 day Con A spleen lymphoblasts in a dose dependent manner (Fig. 5A) . The optimum concentration of CM from transfected CHO-K1 cells was 25% (v/v) . Recombinant chicken IL-15 expressed in E. coli also showed growth promoting activity (Fig. 5B) . MBP-chIL-15 fusion protein purified by amylose affinity chromatography stimulated Con A spleen lymphoblasts to an extent equal to CM from CHO-K1 cells transfected with IL-15 cDNA or Con A. Furthermore, purified MBP-chIL-15 fusion protein retained its stimulatory activity following Factor Xa digestion. As negative controls, CM from Con A stimulated spleen cells, or CM from CHO-Kl cells expressing an irrelevant recombinant cytokine, chicken IFN-γ, (Song et al. Vet . Immunol . Immunopa thol . Inpress (1997)), showed minimal stimulatory activities.

To determine the stability of chicken 11-15, CM from CHO-Kl cells stably transfected with the chicken IL-15 cDNA were stored at 4°C or 41°C for various lengths of time and assayed for bioactivity. At 41°C the observed half-life was 60 hr and at 4°C the half-life was 360 hr (data not shown).

Development and Characteriza tion of IL-15 Dependen t Y, &TCR⁺ Cells To prepare IL-15 assay cells, spleen cell suspensions were prepared from SC chickens. The lymphocytes purified by centrifugation through Histopaque-1077 (Sigma), resuspended at 5 X 10⁶ cells/ml in Iscove's modified Dulbecco' s medium with 10% FCS (IMDM-10), 10 μg/ml Con A, and the cells incubated at 41 in 5% C0₂ for 48 hr (T. J. Myers et al . Vet . Immunol .

Immunopa thol . , 34:97 (1992)). Prior to IL-15 assay, dead cells were removed by centrifugation through Histopaque-1077, and the viable cells treated with 0.05 M alpha-methyl- mannoside (Sigma) . IL-15 activity was measured by a quantitative colorimetric assay as modified (T. Mosmann J. Immunol . Methods . , 65:55 (1983)). Samples to be tested for IL-15 activity were diluted with IMDM and dispensed in triplicate into 96-well round-bottomed tissue culture plates (Costar) at 100 μl per well. An equal volume of assay cells WO 99/51622 _ 3 g _ PCT US99/07485 resuspended in IMDM (5 X 10^δ cells/ml) was added, the cells" incubated at 41°C for 70 hr, 20 ml of 3- (4 , 5-dimethylthiazol- 2-yl) -2,5-diphenyltetrazolium bromide (20 μl MTT, 10 mg/ml, Sigma) added, and the cells incubated for 3 hr. The cells were centrifuged at 1,000 X g for 10 min at room temperature, the supernatant carefully removed, and 150 μl of a 10% saponin solution (Fisher Scientific, Fair Lawn, NJ) added to lyse the cells. The plates were shaken for 20 min (Bellco Biotechnology, Vineland, NJ) , the cells thoroughly resuspended by multiple pipetting, centrifuged at 1,000 X g for 10 min, and the supernatant removed. One hundred seventy five microliters of 0.04 N HCl in isopropanol was added to dissolve the formazan crystals, the plates shaken, the cells resuspended, and centrifuged as above. One hundred microliters of supernatant was transferred to new 96-well flat-bottomed plates and ODsso measured using an ELISA microtiter plate reader (model 3550, BioRad) . As a positive control for IL-15 activity, CM prepared from Con A stimulated spleen cells as described (T. J. Myers et al . Vet . Immunol . Immunopa thol . , 34:97 (1992)) was used. For negative control, IMDM medium alone was used. The protein concentration of CM and material from other purification steps was determined using a modified Lowry total protein determination kit (Sigma) . A minimum of 6 test wells containing 100 μl IMDM (negative control) was used for each IL-15 assay, and the mean and standard deviation for these wells were determined. The mean plus two times the standard deviation for negative wells was used as a positive/negative cut-off for evaluating mean sample data (T. J. Myers et al . Vet . Immunol . Immunopa thol . , 34 : 97 ( 1992 !

ConA stimulated T lymphoblasts were maintained in IMDM-10 medium supplemented with 1 μg/ml transferrin, 5 ng/ml insulin, 25 ng/ml sodium selenite, 100 U/ml penicillin, 100 μg/ml streptomycin (all from Sigma), containing 25-30% (v/v) chicken IL-15 derived from stably transfected CHO-Kl cells. Cells were cultured at 4 X 10⁵ cells/ml until reaching a saturation density of approximately 5 X 10⁶ cells/ml after 4-5 days. Lymphoblasts were centrifuged through Histopaque-1077 every 3- 5 days and plated in fresh IMDM-10 medium supplemented with 25-30% chicken IL-15. Table 1 lists the MAbs used.

TABLE 1

Monoclonal Antibody Antigen Specificity Reference

1-9B CD3 22

CTLA 4 CD4 23

CTLA 8 CD8 23

TCR1 γδTCR 24

TCR2 αβTCR 24

Bu-1 B lymphocytes 25

EP72 CD8α 26

EP42 CD8β 26

OMM-cjlβ CD25 (chT6, IL-2RCC) 27

HNK-1 CD57 (NK cells) 28

P2M11 MHC class II 29

Cells were incubated with mAbs in Hanks' balanced salt solution (Life Technologies) without phenol red supplemented with 3% FCS and 0.1% NaN₃ (FCA buffer) on ice for 30 min, washed 3 times, and resuspended in 50 ml FCA buffer (H. S. Lillehoj et al . Eur. J. Immunol . , 18:2059 (1988)). Fifty microliters of FITC labeled rabbit antibody to mouse IgG (Sigma) was added, and the cells incubated for 30 min on ice. For two-color immunofluorescence, the cells were first incubated with mAb followed by phycoerythrin (PE) labeled rabbit anti-mouse antibody (Sigma) . After washing, a second incubation with FITC conjugated TCR1 mAb (Southern Biotechnology Associates, Birmingham, AL) was performed. Cell fluorescence was assessed on an EPICS V Dual bench flow cytometer (Coulter, Hialeah, FL) with 10,000 viable cells.

Isotype matched negative control MAbs were used in all assays.

Spontaneous cytotoxicity mediated by γ,δτCR⁺ cells was carried out by an in vitro 4 hr NK cell assay using a chicken lymphoblastoid tumor cell line, LSCC-RP9, as described (J. Y. Chai and H. S. Lillehoj Immunology, 63:111 (1988)). LSCC-RP9 target cells were washed, adjusted to 2 X 10⁵ cells/ml, 100 μl placed in 96-well microtiter plates, 100 μl of effector cells added at various concentrations to give effector-to-target ratios of 16:1, 8:1, 4:1 or 2:1, and the cells incubated at 41°C for 4hr. Percent cytotoxicity was calculated as described (J. Y. Chai and H. S. Lillehoj Immunology, 63:111 (1988) ) . As shown in Fig. 6, Con A stimulated spleen lymphoblasts demonstrated high proliferative activity at 5, 12, and 29 days of continuous culture in the presence of 25-30% chicken IL-15. Con A alone also stimulated cell proliferation, although at a level consistently lower than chicken IL-15. Three day Con A stimulated cells, prior to exposure to chicken IL-5, constituted a heterogeneous population of lymphocytes with many cells expressing CD3, CD4, CD8, γ,δTCR, and alpha, beta- TCR antigens (Fig. 7, upper panel ) . After continuous culture of lymphoblasts for 29 days in chicken IL-15, the predominant cells expressed the CD3, CD8, and γ,δTCR antigens (Fig. 7, lower panel ) . Cells expressing the CD4 and α,βτCR antigens represented a very small percentage of the total population. By two-color immunofluorescence, most of the γ,δτCR⁺ cells present in 29 day chicken IL-15 culture co-expressed the CD3, CDδalpha, and CD57 antigens, but were negative for CD8beta, IL-2Ralpha (chT6) , and MHC class II (la) antigens (Fig. 8). Since mammalian γ,δτCR⁺ T cells have been shown to mediate NK cell activity (12), chicken IL-15 induced γ,δτCR⁺ cells were tested for their ability to spontaneously lyse tumor cells.

Chicken IL-15 induced γ,δτCR⁺ cells showed a very high level of spontaneous cytotoxicity against LSCC-RP9 cells at effector- to-target cell ratios of 16:1, 8:1, 4:1 and 2:1 (Fig. 9).

Cons truction of plasmid pNZ29R/IL -15 for recombinan t fowlpox virus

By coinfection of ZAP XR expression vector (Stratagene) capable of expressing chicken IL-15 into E. coli with ExAssist helper phage (Stratagene) , the inserted plasmid"^" ⁽pBluescript/IL-15) was excised as described by the manufacturer's instructions. Plasmid pBluescript/IL-15 was digested with restriction enzymes BamHI and Kpnl to obtain a DNA fragment containing the chicken IL-15 gene. The DNA fragment was inserted into plasmid pNZ1829R at the BamHI- Kpnl site to construct pNZ29R/IL-15.

Construction and purifica tion of recombinan t fow_lpox _viru_s Fowlpox virus CEVA strain was infected to monolayered CEF cells with m.o.i. of 0.1. Four hours after the infection, these cells were scraped by trypsin digestion and used as a cell suspension. The suspension was centrifuged to obtain cell pellets. The cell pellets were resuspended in Saline G (0.14 M NaCl, 0.5 mM KC1, 1.1 mM Na₂HP0₄, 0.5 mM

MgCl₂.6H₂0, 0.011% glucose) in 2 x 10⁷ cells/ml. To the fowlpox virus-infected cell suspension, was added 10 μg of plasmid pNZ29R/IL-15. The mixture was allowed to stand at room temperature and subjected to electroporation under conditions of 3.0 KV/cm, 0.4 msec and 25 μF, using Gene pulser ⁽BioRad⁾ . Subsequently, the plasmid transfected cells were incubated at 37°C for 72 hours. The cells were thawed by freezing and thawing twice. The released recombinant fowlpox virus was selected as described below.

The solution containing the recombinant virus released from the thawed cells was diluted into 1 : 10³, 1 : 10⁴ and 1 : 10^, respectively. Each dilution was transfected into CEF cells and growth medium-containing agar medium of 10 ml was layered on top of CEF cells. After the agar was solidified at room temperature, incubation was continued at 37^CC until typical plaques appeared. When the plaques were grown in about a week, another agar medium containing 70 μg/ml of Bluo-Gal ⁽GIBCO⁾ was layered on each culture plate followed by incubation at 37°C for further 24 hours. Blue plaques were withdrawn from the plate and the virus contained was recovered. The recombinant virus was subjected to further purification by the same procedures until all plaques formed were stained blue with Bluo-Gal. In general, the procedures are completed in 4 to 6 cycles . The thus purified recombinant virus was designated fNZ2.9R/IL-15.

Verifi ca tion for expression of recombinan t fowlpox virus fNZ29R/IL-15

The recombinant fowlpox virus fNZ29R/IL-15 was transfected into CEF cells. After incubation at 37°C for about a week, the culture supernatant was recovered and the transfected cells were washed twice with PBS. The cells were then scraped with a cell scraper and recovered. The culture supernatant and the transfected cells were suspended in the same volume of SDS sample buffer (Laemmli, Nature, 227, 680, 1970). The resulting suspension was boiled. After centrifugation, the supernatant was electrophoresed on SDS- PAGE (Laemmli, Nature, 227, 680, 1970) . Then the resolved polypeptide was electroblotted to a nitrocellulose membrane. The nitrocellulose membrane was then treated with anti-MBP-IL-15 rabbit antiserum, biotin-labeled goat anti-rabbit antiserum and avidin-labeled alkaline phosphatase. Immunoreactive bands were visualized with ^"NBT (nitroblue tetrazolium chloride) and BCIP (5-bromo-4- chloro-3-indolyl phosphate) (GIBCO) .

The results shown in Fig. 10 reveal that a specific band of about 16 kDa was detected in the recombinant fowlpox virus fNZ29R/IL-15 transfected cells and its culture supernatant.

The band was not detected from the intact fowlpox virus with no genetic recombination or from the culture supernatant. It is thus likely that the band would be the chicken IL-15 polypeptide in which the recombinant fowlpox virus fNZ29R/IL-15 was expressed. Lane 1, molecular weight standards; lane 2, proteins from CEFs infected by fNZ29R/IL-15 clone 1; lane 3, proteins from CEFs infected by fNZ29R/IL-15 clone 2; lane 4, proteins from CEF' s infected by intact fowlpox virus; lane 5, culture supernatant from CEFs infected by fNZ29R/IL-15 clone 1; lane 6, culture supernatant from CEFs infected by fNZ29R/IL-15 clone 2; lane 7, culture supernatant from CEFs infected by intact fowlpox virus.

-43 -

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: LILLEHOJ, HYUN S

(ii) TITLE OF INVENTION: NOVEL CYTOKINES AND RECOMBINANTS THEREOF AND USES THEREOF

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(A) NAME: MURPHY JR, GERALD M

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(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 45..473

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GGCACGAGTT TGAATACCAG CATACAGATA ACTGGGACAC TGCC ATG ATG TGC AAA 56

Met Met Cys Lys 1

GTA CTG ATC TTT GGC TGT ATT TCG GTA GCA ATG CTA ATG ACT ACA GCT 104 Val Leu He Phe Gly Cys He Ser Val Ala Met Leu Met Thr Thr Ala 5 10 15 20 -44 -

TAT GGA GCA TCT CTA TCA TCA GCA AAA AGG AAA CCT CTT CAA ACA TTA 152 Tyr Gly Ala Ser Leu Ser Ser Ala Lys Arg Lys Pro Leu Gin Thr Leu 25 30 35

ATA AAG GAT TTA GAA ATA TTG GAA AAT ATC AAG AAC AAG ATT CAT CTC 200 He Lys Asp Leu Glu He Leu Glu Asn He Lys Asn Lys He His Leu 40 45 50

GAG CTC TAC ACA CCA ACT GAG ACC CAG GAG TGC ACC CAG CAA ACT CTG 248 Glu Leu Tyr Thr Pro Thr Glu Thr Gin Glu Cys Thr Gin Gin Thr Leu 55 60 65

CAG TGT TAC CTG GGA GAA GTG GTT ACT CTG AAG AAA GAA ACT GAA GAT 296 Gin Cys Tyr Leu Gly Glu Val Val Thr Leu Lys Lys Glu Thr Glu Asp 70 75 80

GAC ACT GAA ATT AAA GAA GAA TTT GTA ACT GCT ATT CAA AAT ATC GAA 344 Asp Thr Glu He Lys Glu Glu Phe Val Thr Ala He Gin Asn He Glu 85 90 95 100

AAG AAC CTC AAG AGT CTT ACG GGT CTA AAT CAC ACC GGA AGT GAA TGC 392 Lys Asn Leu Lys Ser Leu Thr Gly Leu Asn His Thr Gly Ser Glu Cys 105 110 115

AAG ATC TGT GAA GCT AAC AAC AAG AAA AAA TTT CCT GAT TTT CTC CAT 440 Lys He Cys Glu Ala Asn Asn Lys Lys Lys Phe Pro Asp Phe Leu His 120 125 130

GAA CTG ACC AAC TTT GTG AGA TAT CTG CAA AAA TAAGCAACTA ATCATTTTTA 493 Glu Leu Thr Asn Phe Val Arg Tyr Leu Gin Lys 135 140

TTTTACTGCT ATGTTATTTA TTTAATTATT TAATTACAGA TAATTTATAT ATTTTATCCC 553

GTGGCTAACT AATCTGCTGT CCATTCTGGG ACCACTGTAT GCTCTTAGTC TGGGTGATAT 613

GACGTCTGTT CTAAGATCAT ATTTGATCCT TTCTGTAAGC CCTACGGGCT CAAAATGTAC 673

GTTGGAAAAC TGATTGATTC TCACTTTGTC GGTAAAGTGA TATGTGTTTA CTGAAAGAAT 733

TTTTAAAAGT CACTTCTAGA TGACATTTAA TAAATTTCAG TAATATATGA AAAAAAAAAA 793

AAAAAAA 800

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 143 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Met Cys Lys Val Leu He Phe Gly Cys He Ser Val Ala Met Leu 1 5 10 15

Met Thr Thr Ala Tyr Gly Ala Ser Leu Ser Ser Ala Lys Arg Lys Pro 20 25 30

Leu Gin Thr Leu He Lys Asp Leu Glu He Leu Glu Asn He Lys Asn 35 40 45

Lys He His Leu Glu Leu Tyr Thr Pro Thr Glu Thr Gin Glu Cys Thr 50 55 60

Gin Gin Thr Leu Gin Cys Tyr Leu Gly Glu Val Val Thr Leu Lys Lys -45 -

65 70 75 80

Glu Thr Glu Asp Asp Thr Glu He Lys Glu Glu Phe Val Thr Ala He 85 90 95

Gin Asn He Glu Lys Asn Leu Lys Ser Leu Thr Gly Leu Asn His Thr 100 105 110

Gly Ser Glu Cys Lys He Cys Glu Ala Asn Asn Lys Lys Lys Phe Pro 115 120 125

Asp Phe Leu His Glu Leu Thr Asn Phe Val Arg Tyr Leu Gin Lys 130 135 140

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 101 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "SYNTHETIC DNA"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AAGCTTTTTT XTT TTTTTT TTTTGGCATA TAAATAATAA ATACAATAAT TAATTACGCG 60 TAAAAATTGA AAAACTATTC TAATTTATTG CACTCGGATC C 101

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GGAATTCACT GCCATGATGT GCAAAGTA 28

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer" -46- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TTTCTAGATT ATTTTTGCAG ATATCTCAC 29

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii ) MOLECULE TYPE : peptide

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 6 :

Thr Leu Lys Lys Glu Thr Glu Asp Asp Thr Glu He Lys 1 5 in

Claims

-47-

WHAT IS CLAIMED IS: 1. An isolated avian IL-15 polypeptide comprising a) the amino acid sequence of SEQ ID N0:1; b) fragements of the amino acid sequence of SEQ ID N0:1, wherein said fragments stimulate growth of avian T lymphocytes expressing ╬│╬┤TCR; or c) the amino acid sequence of SEQ ID N0:1 having one or more amino acid substitutions, mutations, deletions and insertions wherein said polypepide has at least 70% of the biological activity of the polypeptide of SEQ ID NO:l for stimulating growth of avain T lymphocytes expressing ╬│╬┤TCR and wherein said polypeptide shares at least 85% sequence identity with SEQ ID NO:l.

2. The avian IL-15 polypeptide of claim 1, comprising SEQ ID NO:l.

3. The avian IL-15 polypeptide of claim 1, wherein said polypeptide is expressed in chickens.

4. The avian IL-15 polypeptide of claim 1, wherein said -48 - polypeptide is expressed in a tissue selected from the group consisting of the skeletal muscle, caecal tonsil (appendix), small intestine, heart, liver, oviduct and spleen of chicken.

5. An isolated polynucleotide encoding an avian IL-15 gene comprising a) a DNA molecule encoding the polypeptide of claim 1; or b) a DNA molecule which hybridizes to the DNA of a) under stringent conditions.

6. An isolated polynucleotide encoding an avian IL-15 gene comprising a) a DNA molecule encoded by SEQ ID NO: 2, or a fragment thereof; b) a DNA molecule which encodes the polypeptide of SEQ ID N0:1, or a fragment thereof; or c) a DNA molecule which hybridizes to a) or b) under stringent conditions.

7. The polynucleotide of claim 5 or 6, wherein said polynucleotide is from a chicken.

8. An isolated polynucleotide comprising a DNA molecule encoded by SEQ ID NO: 2.

9. A recombinant vector comprising the polynucleotide of claim 5 or 6.

10. A transformant comprising the polynucleotide of claim 5 or 6 .

11. A recombinant virus comprising the polynucleotide of claim 5 or 6.

12. A composition for the prophylaxis of poultry diseases comprising a transformant of claim 9 as an effective ingredient.

13. A composition for the prophylaxis of poultry diseases comprising a recombinant virus according to claim 10 as an effective ingredient.

14. A composition for the prophylaxis of poultry diseases comprising as an effective ingredient a polypeptide of claim 1 or a pharmacologically acceptable salt thereof.

15. The composition of claim 11 further comprising pathogen derived antigen.

16. The composition of claim 14 wherein said pathogen derived antigen is expressed in a recombinant virus vector.

17. The composition of claim 14 wherein said pathogen derived antigen is expressed on a separate vector as said IL- 15.

18. The composition of claim 14 wherein said pathogen derived antigen is expressed on the same vector as said IL-15.

19. The composition of claim 14 wherein said pathogen derived antigen is derived from a pathogen causing an avian disease.

20. The composition of claim 14 wherein said pathogen derived antigen is selected from the group consisting of HN or F of HDV; gB, gC or UL32 of ILTV; gB, gC, gH, gL, gl or gE of MDV; surface antigens of protozoa or Eimeria , VP2 of IBDV and the 40kD polypeptide of Mycoplama gallicepticum .

21. An adjuvant comprising a transformant of claim 9 as an effective ingredient.

22. An adjuvant comprising a recombinant virus of claim 10 as an effective ingredient.

23. An adjuvant comprising a polypeptide of claim 1 as an effective ingredient.

24. A method of avian immunization comprising administering to said avian an effective amount of a vaccine comprising an effective amount of a cytokine which stimulate the immune system and an avian pathogen derived antigen.

25. The method of claim 24 wherein said cytokine and pathogen derived antigen are in viral vectors, viruses, or transformants.

26. The method of claim 23 wherein said cytokine is that of SEQ ID N0:1.

27. The method of claim 24, wherein said avian is a chicken.