# Patent

## Title
Avian cytokines, such as IL-12, comprising a p40 and/or p35 subunit and vaccines

## Abstract

Embodiments of the present invention generally relate to novel avian cytokines, to DNA sequences encoding these novel cytokines and to their use in adjuvants for vaccine purposes.

## Background
RELATED APPLICATION
This application claims priority to provisional application Ser. No. 60/391,662 filed Jun. 26, 2002.


REFERENCE TO SEQUENCE LISTING
The material saved as “text document” under the file name “Substitute Sequence Listing” created on Apr. 4, 2007 is hereby incorporated by reference.
FIELD OF THE INVENTION
The present invention relates to novel avian cytokines, to DNA sequences encoding these novel cytokines and to their use in adjuvants for vaccine purposes.
BACKGROUND OF THE INVENTION
The group of soluble secreted molecules, collectively termed cytokines, represents critical communication signals among the cells of the immune system and between immune and non-immune system cells. Some of these cytokines have to form homodimers, such as interferon-gamma (IFN-γ), or homotrimers, such as tumor necrosis factor (TNF)-family molecules, in order to exert biological activity. Monomeric forms show minimal or no bioactivity.
Cytokines are at present used in drugs in cancer therapy and in the combat against chronic microbial infections. Cytokines are also evaluated for their use as immune stimulators in adjuvants to improve vaccines.
In mammals, a group of composite hetero-dimeric cytokines has been identified based on complexes of a p40 protein subunit. The mammalian p40 element comprises a 40 kD protein which links covalently, by di-sulfide binding, with a p35 subunit to form interleukin-12 (IL-12) p70 (Gubler U, et al., PNAS USA 1991, 88: 4143-4147; Wolf S F, et al., J. Immunol. 1991, 146: 3047-3081; Trinchieri G., Blood 1994, 12: 4008-4027). In addition, p40 may form the composite cytokine IL-23, after combining with p19 (Wiekowski M T, et al., J. Immunol. 2001 166: 7563-7570), a molecule structurally related to IL-6, p35 and granulocyte-colony stimulating factor (G-CSF) (Oppmann B, et al., Immunity 2000, 13: 715-725). Moreover, p40 may form homodimers that have been shown to either compete for binding with IL-12 p70 to the IL-12 high affinity receptor and inhibit IL-12 bioactivity (Heinzel F P, et al., J. Immunol., 1997, 158: 4381-4388), or to enhance, rather than to decrease, IFN-γ production by CD8+ T cells and Th1 development (Piccotti J R, et al., J. Immunol. 1997, 158: 643-648).
The significance of p40 in vivo in various mammalian species has been demonstrated using recombinant p40. IL-12 antagonistic features of 80 KDa homodimeric (P40)2 have been clearly demonstrated in lipopolysaccharide (LPS)-induced IFN-γ-dependent lethal shock models (Mattner F, et al., Infect. Immun. 1997, 11: 4734-4737). The production of human p40, in the absence of bioactive IL-12 p70, has been demonstrated for brain microglial cells (De Goer-de Herve M G, et al., Cytokine 2001, 14: 88-96). In addition, the physiological role of mammalian p40 composite cytokines has been delineated in detail using in vivo gene-targeting approaches. It proved that following infection by Salmonella enteritidis mice genetically deficient for the p40 protein (p40−/−) showed a higher mortality rate and higher bacterial organ burden than mice capable of producing p40, but lacking the p35 gene (IL-12 p35−/−) (Lehmann J, et al., J. Immunol. 2001, 167: 5304-5315). Normal (wild-type) and IL-12 p35−/− mice cleared an infection with Mycobacterium bovis Calmette-Guerin (BCG) or pulmonary tuberculosis infection, while double-deficient IL-12 (p35−/−+p40−/−) mice showed high susceptibility to M. bovis BCG and tuberculosis infection (Holscher C, et al., J. Immunol. 2001, 167: 6957-6566). Susceptibility was associated with reduced antigen-specific Th1 and cytotoxic T cell responses. Interestingly, in vivo therapy with recombinant p40 homodimers reverted M. bovis BCG infected double-depleted (p35−/−+p40−/−) mice into a resistant phenotype. This demonstrates a protective and agonistic role of endogenous and exogenous p40 in mycobacterial infection, which is independent of IL-12 p70 (Holscher et al., 2001 supra). Similarly, Cryptococcus neoformans infected p40−/− mice died earlier and developed higher organ burdens than p35−/− mice, which suggests again a protective role for the p40 subunit independent of the IL-12 heterodimer (Decken K., et al., Infect. Immunity 1998, 66: 4994-5000). Also, p40−/− mice survived large doses of the intracellular bacterium Franscisella tularensis (LVS), but never cleared bacteria and developed chronic infection. In sharp contrast, p35−/− mice readily survived large doses of sub lethal LVS infection. This study suggests that clearance of LVS is dependent on p40 but not on IL-12 p70 (Elkins K L, et al., Infect. Immun. 2002, 70: 1936-1946). Also during murine cytomegalovirus (MCMV) infection p35−/− mice showed an altered phenotype compared to p40−/− mice, indicating that p40 may have an activity independent of and additional to IL-12 antagonism in vivo (Carr J A, et al., J. Interferon Cytokine Res. 1999, 19: 1145-1152).
Taken together, these experimental studies illustrate the crucial role in mammals of p40 based cytokines such as IL-12, IL-23 and (p40)2 in regulation of IFN-γ characteristic T helper-1 type immune responses essential in the control of mostly intracellular infections of bacterial, parasitic, fungal or viral nature.
The present invention concerns avian equivalents of the mammalian p40 based cytokines.
The cloning and sequencing of avian cytokines lags behind similar work done in mammals. Only a few avian cytokines have been identified so far. IFN-γ and IL-18 as well as a number of pro-inflammatory cytokines have been cloned, demonstrating the existence of a Th1-like cytokine network in chickens. Because of the low sequence homology to mammalian cytokines, usually somewhere around 30 to 50%, classical approaches to identify avian homologues of mammalian cytokines are usually not successful. The identification by PCR amplification using primers based on mammalian sequences is very difficult and unpredictive. (Hilton L. S. et al. Vet. Immunol. and Immunopathol. 2002, 85: 119-128; Staehi P. et al., J. Interferon Cytokine Res. 2001, 21: 993-1010) When some avian cytokines became available, work started to investigate their potential use as immune modulators or as immune adjuvants to enhance the efficiency of vaccines.
Most chickens produced in developed countries, both for consumption and egg-laying, are vaccinated. They are vaccinated against Marek's disease, and against Newcastle Disease Virus, Infectious Bursal Disease Virus, Infectious Bronchitis Virus, Fowlpox Virus, and Coccidial vaccines. Vaccination can be performed either before or after hatching. The immune systems of embryos and newly hatched birds is not yet fully developed and cannot give rise to an immune response that is as effective as 2-3 weeks after hatching. For the development of vaccines used pre-hatching or at-hatching, therefore a need exists for agents that enhance the immune response in birds after vaccination.
The present inventors have succeeded in identifying and determining both the amino acid- and the encoding gene sequence for novel avian cytokines. These proteins are useful for the above-mentioned purposes known for the mammalian counterparts, especially to enhance the effectiveness of avian vaccines.
SUMMARY OF THE INVENTION
The present invention provides a protein comprising at least one of the following polypeptide subunits:

    
    
        a subunit having an amino acid sequence showing at least 80% similarity with the amino acid sequence as depicted in SEQ ID NO 1,
        a subunit having an amino acid sequence showing at least 80% similarity with the amino acid sequence as depicted in SEQ ID NO 2.
    
    


DETAILED DESCRIPTION OF THE INVENTION
The sequence depicted in SEQ ID NO 1 represents a polypeptide having a molecular weight of approximately 40 kD. The sequence depicted in SEQ ID NO 2 represents a polypeptide having a molecular weight of approximately 35 kD.
The polypeptide subunit having a molecular weight of 40 kD will be referred to as “p40”, while the 35 kD subunit will be referred to as “p35”. Both sequences as depicted in SEQ ID NO 1 and 2 are derived from chicken DNA (chicken p40 and chicken p35).
As explained above for the mammalian cytokines, various p40 containing complexes may exist. p40 can appear as monomeric molecule, as homodimers, or as hetero-dimeric molecules. The p40 subunit, may be linked covalently, by di-sulfide binding, with the p35 subunit to form interleukin-12 (IL-12). In addition, p40 may form the composite cytokine IL-23, after combining with p19. The promiscuous binding of p40 to other cytokine peptide chains suggests the existence of hitherto other un-identified p40-complexing cytokines.
The proteins according to the invention may therefore be proteins consisting of one copy of one of the subunits, it may be homo-dimers of one of the subunits, especially p40, or a hetero-dimers consisting of one of the subunits (p40 or p35) together with another peptide subunit, or it may comprise both (p40 and p35) subunits. The invention also encompasses chimeric proteins comprising, for example, a chicken p40 or p35 subunit in combination with a p35 or p40 subunit derived from another species. Chimeras may be, for example, proteins wherein the chicken p35 is combined with a p40 derived from another avian or even non-avian species.
Together the p40 and p35, when linked by, for example, disulfide bonds, will form an avian Interleukin-12 (IL-12) which is likewise part of the present invention.
The proteins of the invention are in principle avian cytokines that can be used for different purposes, analogous to the mammalian counterparts. The cytokines according to the invention, especially the avian IL-12, more in particular the chicken IL-12, may be used as an adjuvant in avian vaccines to enhance the immune response.
Since it is obvious that minor modifications in the sequence of the protein are equally useful, the invention also provides for a protein comprising a polypeptide sub-unit having an amino acid sequence which has at least 80%, or preferably at least 90%, more preferably 95%, more preferably at least 99%, even most preferably 100% similarity to the sequence in SEQ ID NO 1 or SEQ ID NO 2.
The term “similarity” refers to a degree of similarity between proteins in view of differences in amino acids, but which different amino acids are functionally similar in view of almost equal size, lipophilicity, acidity etc. A percent similarity can be calculated by optimal alignment of the sequences using a similarity scoring matrix such as the Blosum62 matrix described in Henikoff S. and Henikoff J G., P. N. A. S. USA 1992, 89: 10915-10919. Calculation of the percentage similarity and optimal alignment of two sequences using the Blosum62 similarity matrix and the algorithm of Needleman and Wunsch (J. Mol. Biol. 1970, 48: 443-453) can be performed using the GAP program of the Genetics Computer Group (GCG, Madison, Wis., USA) using the default parameters of the program.
It is a further aspect of the invention to provide a protein which comprises a naturally occurring variant of one or both of the sub-units having the sequence as in SEQ ID NO 1 and SEQ ID NO 2. Such proteins, comprising a sub-unit having an amino acid sequence which has at least 80% similarity, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% similarity to the polypeptides defined in SEQ ID NO 1 or SEQ ID NO 2, are derived from avian species such as chicken, duck, goose turkey and pigeon.
Such sequences are presented in SEQ ID NO 5 and 7, which represent the duck and turkey equivalent respectively of the chicken p40 amino acid sequence depicted in SEQ ID NO 1.
Such polymorphic forms and avian species homologues are included in the class of proteins made available by this invention. Variants of the proteins that are likewise part of the present invention can be natural variants that may contain variations in the amino acid sequence due to deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. Amino acid substitutions that are expected not to essentially alter biological and immunological activities have been described. Amino acid replacements between related amino acids or replacements which have occurred frequently in evolution are, inter alia Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, 5: suppl. 3). Based on this information Lipman and Pearson developed a method for rapid and sensitive protein comparison and determination of the functional similarity between homologous polypeptides (Science 1985, 227: 1435-1441).
Other variants can be, for example, functional variants like salts, amides, esters, and specifically C-terminal esters, and N-acyl derivatives. Also included are peptides which are modified in vivo or in vitro, for example by glycosylation, amidation, carboxylation or phosphorylation.
Proteins comprising only a functional fragment of the p40 or p35 sub-unit (or both) are likewise considered as part of the present invention. A functional fragment of the polypeptide is a fragment that at least represents the part(s) of the polypeptide sub-unit(s), which is/are essential for the protein to be able to serve as a cytokine, and can fulfill this function, for example, when used alone or fused to heterologous sequences. Thus, such functional fragments, may be polypeptides that are functional per se, or the fragments may be functional when linked to other polypeptides, to obtain chimeric proteins. These functional fragments are understood to fall within the definition of the subunits.
Fragments can inter alia be produced by enzymatic cleavage of precursor molecules, using restriction endonucleases for the DNA and proteases for the polypeptides. Other methods include chemical synthesis of the fragments or the expression of peptide fragments by DNA fragments.
The polypeptide subunits have an apparent molecular weight of 40 or 35 kD respectively, based on the length of the amino acid (aa) sequence. The exact molecular weight can be determined in SDS-PAGE using reducing conditions.
Preferred proteins according to the invention are those proteins that comprise a polypeptide subunit having an amino acid sequence showing at least 80% similarity, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% similarity to the protein defined in SEQ ID NO 1. Such preferred proteins according to the invention may comprise one or two p40 subunits (i.e. be a mono- or dimer of p40). Within this preferred embodiment most preferred are the proteins comprising a p40 subunit derived from chicken. Examples of subunits having over 99% similarity to the chicken sequence of SEQ ID NO 1 are the duck and turkey p40 sequences depicted in SEQ ID NO 5 and 7 respectively.
Especially useful are those proteins wherein the p40 subunit is linked to a p35 subunit (by a disulfide linkage) such that an avian IL-12 is obtained.
In an especially preferred embodiment the invention provides the chicken IL-12, consisting of a p40 subunit having the amino acid sequence of SEQ ID NO 1 and a p35 subunit having the amino acid sequence depicted in SEQ ID NO 2, linked together, for example, by a disulfide bond.
The linkage of the p35 and p40 subunits can be established in various ways. Chicken IL-12 can be generated via expression vectors containing both the p35 and p40 cDNAs separated by, for example, an IRES (internal ribosome entry segment) element or directly linked via a Glycine/Serine-rich coding region (“hinge”) to form a single open reading frame. In addition, expression vectors containing either the p35 or p40 cDNA sequence under control of separate promoters can be used to generate chicken IL-12.
The preparation of the proteins, sub units or functional fragments thereof according to the invention is effected by means of one of the known organic chemical methods for peptide synthesis or with the aid of recombinant DNA techniques. This latter method involves the preparation of the desired peptide by means of expression using a recombinant polynucleotide with a nucleotide sequence, which is coding for one or more of the peptides in question in a suitable micro-organism as host.
These polynucleotides are likewise part of the present invention.
Thus the present invention furthermore provides a polynucleotide encoding at least one of the following polypeptide subunits:

    
    
        a subunit having an amino acid sequence having at least 80% similarity to the amino acid sequence as depicted in SEQ ID NO 1, and
        a subunit having an amino acid sequence having at least 80% similarity to the amino acid sequence as depicted in SEQ ID NO 2.
    
    


A polynucleotide encoding an avian IL-12 may comprise both sequences, for example, linked by a sequence encoding a hinge.
Fragments of the provided nucleic acid (na) sequence that encode a functional fragment of the polypeptide are likewise part of the present invention.
For example, a polynucleotide encoding such a functional fragment of the polypeptide may be fused to polynucleotides encoding transmembrane regions and/or signal sequences.
Polynucleotides as defined with the present invention also include polynucleotides having variations in the nucleic acid sequence when compared to the identified nucleic acid sequence or having polymorphic sites. With “variants” polynucleotides are meant that differ from the identified nucleic acid sequence but still encode a polypeptide that has a biological, e.g. cytokine, activity similar to the activity of a polypeptide having an amino acid sequence as depicted in SEQ ID NO 1 and/or 2.
Variants may be natural or non-natural variants. Natural variants will include homologues in various avian species. Non-naturally occurring variant may be introduced by mutagenesis. Natural variants may also be allelic variants. An allelic variant is one of several alternate forms of a gene occupying a locus on a chromosome of an organism. Sometimes, a gene is expressed in a certain tissue as a splicing variant, resulting in an altered 5′ or 3′ mRNA or the inclusion or exclusion of one or more exon sequences. These sequences, as well as the proteins encoded by these sequences, all are expected to perform the same or similar functions and form also part of the invention.
An isolated cDNA sequence may be incomplete due to incomplete transcription from the corresponding mRNA, or clones may be obtained containing fragments of the complete cDNA. Various techniques are known in the art to complete said cDNA sequences, such as RACE (Rapid Amplification of cDNA ends).
Polynucleotides that have a nucleic acid sequence that is a variant of the identified nucleic acid sequence may be isolated by a method comprising the steps of: a) hybridizing a DNA comprising all or part of the identified sequence as reflected in SEQ ID NO 3 or 4, under stringent conditions against nucleic acids being (genomic) DNA or cDNA isolated from avian cells which highly express the polynucleotide of interest; and b) isolating said nucleic acids by methods known to a person skilled in the art.
The hybridization conditions are preferably highly stringent.
According to the present invention the term “stringent” means washing conditions of 1×SSC, 0.1% SDS at a temperature of 65° C.; highly stringent conditions refer to a reduction in SSC towards 0.3×SSC, more preferably to 0.1×SSC. Preferably the first two washings are subsequently carried out twice each during 15-30 minutes. If there is a need to wash under highly stringent conditions an additional wash with 0.1×SSC is performed once during 15 minutes. Hybridization can be performed e.g. overnight in 0.5 M phosphate buffer pH 7.5 with 7% SDS at 65° C. Such hybridization methods are disclosed in any standard textbook on molecular cloning, for example: Molecular Cloning: a laboratory manual, 3rd ed.; eds: Sambrook et al., CSHL press, 2001.
As an alternative the isolation method might comprise nucleic acid amplification methodology using primers and/or probes derived from the nucleic acid sequence provided with the present invention. Such primers and/or probes are oligonucleotides that are at least 15 nucleotides in length; preferred oligo's have about 25-50 nucleotides.
Variants or other avian homologues of the sequences depicted in SEQ ID NO 3 and 4 may also be identified by comparing the sequence in silico to other avian sequences that may be comprised in a computer database. Sequences may be compared with sequences in databases using a BLAST program (BLASTF 2.1.2 [Oct. 19, 2000]) (Altschul, S F, T L Madden, A A Schaffer, J Zhang, Z Zhang, W Miller, and D J. Lipman, “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 1997, 25: 3389-3402).
The bioactivity of proteins according to the invention can be measured in vitro using a proliferation assay, in the following way:
COS-7 cells or chicken cells, e.g. CEF, HD-11, DT40 etc., may be seeded in 35-mm-diameter dishes at 5×105 cells/well. After a culture period of 16 hours cells can be transfected with 1 μg of plasmid DNA encoding chicken IL-12 using Lipofectamin PlusTM (Gibco BRL) according to the manufacturer's instructions. Culture media containing chicken IL-12 can be collected 72 hours after transfection. To check for chicken IL-12 bio-activity a bioassay based on the proliferation of peripheral blood lymphocytes (PBLs) needs to be developed. For this, the cytokine activity, encoded by the transfected plasmid(s), that is released into the culture medium may be analyzed using an adapted protocol of a previously described bioassay (Gately, M. K., Chizzonite, R. & Presky, D. H., “Measurement of human and mouse interleukin-12”, in: Current Protocols in Immunology, 1997, pp. 6.16.1-6.16.15, edited by J. E. Coligan et al., ed: John Wiley & Sons.)
In this bioassay that was developed specifically for mouse and human IL-12, human PBLs, isolated using LymphoprepTM (Nycomed), are cultured for 2 days in Iscoves medium containing 5 μg/ml Concanavalin A (ConA). To stimulate formation of blasts recombinant human interleukin-2 is added (50 units/ml) and cells are cultured for 3 additional days. Cells are washed, seeded in 96 well plates (2×104 cell/well), and cultured in the presence of the culture media of transfected cells. After 48 hours 3H-Thymidine (Amersham) is added and the incubation will be continued for 4 hours, whereafter the cells are harvested by an automated cell harvester. The incorporated radioactivity, which is a measure for cell proliferation and therefore IL-12 bio-activity, will be quantified by liquid scintillation counting.
In a further aspect the present invention provides polynucleotides comprising a nucleic acid sequence encoding a polypeptide subunit comprising an amino acid sequence having at least 80% similarity with the amino acid sequence as depicted in SEQ ID NO 1 or 2 respectively. Preferred are polynucleotides encoding polypeptides having at least 95% identity with SEQ ID NO 1 or 2 and more preferred are those polynucleotides encoding polyproteins having at least 97% identity with SEQ ID NO 1 or 2 wherein those encoding polypeptides having at least 98 or 99% are more preferred. Most preferred are polynucleotides encoding the polypeptide of SEQ ID NO 1 or 2. Due to the degeneracy of the genetic code, polynucleotides encoding an identical or substantially identical amino acid sequence may utilize different specific codons. All polynucleotides encoding the polypeptides as defined above are considered to be part of the invention.
In particular preferred polynucleotides according to the invention are isolated polynucleotides having at least 80% identity with the nucleic acid sequence of SEQ ID NO 3 or 4. More preferred are those polynucleotides having at least 90% identity, and yet more preferred at least 95, preferably 99% identity, most preferred 100% identity to the entire sequence of SEQ ID NO 3 or 4.
Such polynucleotides include polynucleotides comprising the nucleic acid sequence depicted in SEQ ID NO 3 and/or 4. A polynucleotide encoding a polypeptide with a sequence as depicted in SEQ ID NO 1 and/or 2 may comprise the nucleic acid sequence as depicted in SEQ ID NO 3 and/or 4. In a further preferred embodiment of the invention the polynucleotide consists of the nucleic acid sequence as depicted in SEQ ID NO 3 and/or 4.
Examples of polynucleotides showing over 99% homology with the nucleotide sequence depicted in SEQ ID NO 3 are the sequences depicted in SEQ ID NO 6 and 8, which are the coding sequences for the duck and turkey p40 respectively.
The polynucleotides according to the invention may be DNA or RNA, preferably DNA. DNA according to the invention may be obtained from cDNA. Alternatively, the coding sequence might be genomic DNA, or prepared using DNA synthesis techniques. If the polynucleotide is DNA, it may be in single stranded or double stranded form. The single strand might be the coding strand or the non-coding (anti-sense) strand.
Also included within the definition of polynucleotides are modified RNAs or DNAs. Modifications in the bases of the nucleic acid may be made, and bases such as Inosine may be incorporated. Other modifications may involve, for example, modifications of the backbone.
With “isolated” is meant that the polynucleotide is isolated from the natural state, i.e. it has been changed or moved from its natural environment or both. The molecule is separate and discrete from the whole organism with which the molecule is found in nature.
“% Identity” defines the relation between two or more polynucleotides or polypeptides on the basis of a comparison between their aligned sequences.
Identity can be calculated by known methods. Identity, or homology, percentages as mentioned herein are those that can be calculated with the GAP program, running under GCG (Genetics Computer Group Inc., Madison, Wis., USA).
Parameters for polypeptide sequence comparison included the following:
Algorithm: Needleman and Wunsch, J. Mol. Biol. 1970, 48: 443-453.
As a comparison matrix for amino acid alignments the Blosum62 matrix is used (Henikoff and Henikoff, supra) using the following parameters:
Gap penalty: 8
Gap length penalty: 2
No penalty for end gaps.
Parameters for nucleotide comparison that may be used:
Algorithm: Needleman and Wunsch (supra).
Comparison matrix: matches=+10, mismatch=0.
Gap penalty: 50.
Gap length penalty: 3.
The DNA according to the invention will be very useful for in vivo or in vitro expression of the encoded polypeptide in sufficient quantities and in substantially pure form. When the polynucleotides according to the invention are used for expression of the encoded polypeptide, the polynucleotides may include, in addition to the coding sequence for the polypeptide or functional fragment thereof, other coding sequences, for example, leader sequences or fusion portions, such as marker sequences and the like.
Application of p40-based composite cytokines, such as IL-12, IL-23, and (p40)2, may augment micro-organism-induced ongoing immune responses or vaccination-induced immune responses, based on both cellular and humoral immunity. In addition, intervention in the immunological cascade triggered by these cytokines, using e.g. antagonistic doses of (p40)2 may prevent unwanted pathological immune reactions following deregulated overproduction of the p40-based molecules.
The polynucleotides according to the invention may be used in the production of recombinant proteins according to the invention. The polynucleotides may also be used in DNA- or vector vaccines, together with other nucleic acid sequences encoding, for example, immunogenic proteins derived from avian pathogens.
The polynucleotides according to the invention will be very useful for in vivo or in vitro expression of the encoded polypeptide in sufficient quantities and in substantially pure form. When the polynucleotides according to the invention are used for expression of the encoded polypeptide, the polynucleotides may include, in addition to the coding sequence for the polypeptide or functional fragment thereof, other coding sequences, for example, leader sequences or fusion portions, such as marker sequences and the like.
A wide variety of host cell and cloning vehicle combinations may be usefully employed in cloning the nucleic acid sequence according to the invention. A polynucleotide according to the invention may be cloned into an appropriate expression system, such as a bacterial expression system (e.g. Escherichia coli DH5α), a viral expression system (e.g. Baculovirus), a yeast system (e.g. Sacharomyces cerevisiae, Pichia) or eukaryotic cells (e.g. Cos, BHK, HeLa, HD-11, DT40 or CEF cells). In all systems the polynucleotide is first cloned into an appropriate vector under control of a suitable constitutive or inducible promoter.
In another aspect the present invention therefore relates to a recombinant vector comprising a polynucleotide according to the invention. Suitable vectors are for example cosmids, bacterial or yeast plasmids, wide host range plasmids and vectors derived from combinations of plasmid and phage or virus DNA. Vectors derived from chromosomal DNA are also included. Furthermore an origin of replication and/or a dominant selection marker can be present in the vector according to the invention. The vectors according to the invention are suitable for transforming a host cell. Examples of suitable cloning vectors are plasmid vectors such as pBR322, the various pUC, pEMBL and Bluescript plasmids, or viral vectors such as HVT (Herpes virus of Turkeys), MDV (Marek disease virus), ILT (infectious laryngotracheitis virus), FAV (fowl adenovirus), FPV (Fowlpox virus), or NDV (Newcastle disease virus).
When used in the expression of the polypeptide or functional fragments thereof, a recombinant vector according to the present invention, may further comprise an expression control sequence operably linked to the nucleic acid sequence coding for the protein.
“Operably linked” refers to an arrangement wherein the control sequences are configured so as to perform their usual function, in effecting the expression of the polynucleotide.
Such expression control sequences generally comprise a promoter sequence and sequences which regulate transcription and translation and/or enhance expression levels. Not all of these control sequences need to be present in a recombinant vector as long as the desired polynucleotide is capable of being transcribed and translated. Of course expression control- and other sequences can vary depending on the host cell selected or can be made inducible. Such expression control sequences are well known in the art and extend to any eukaryotic, prokaryotic, or viral promoter or poly-A signal capable of directing gene transcription. Examples of useful promoters are the SV-40 promoter (Science 1983, 222: 524-527), the metallothionein promoter (Nature 1982, 296: 39-42), the heat shock promoter (Voellmy et al., P.N.A.S. USA 1985, 82: 4949-4953), the PRV gX promoter (Mettenleiter and Rauh, J. Virol. Methods 1990, 30: 55-66), the human CMV IE promoter (U.S. Pat. No. 5,168,062), the Rous Sarcoma virus LTR promoter (Gorman et al., P.N.A.S. USA 1982, 79: 6777-6781) or human elongation factor 1 alpha or ubiquitin promoter etc.
After the polynucleotide has been cloned into an appropriate vector, the construct may be transferred into the cell, bacteria, or yeast alone by means of an appropriate method, such as electroporation, CaCl2 transfection or lipofectins. When a baculovirus expression system is used, the transfer vector containing the polynucleotide may be transfected together with a complete baculo genome.
All these techniques are well known in the art and extensively described in protocols provided by manufactures of molecular biological materials (such as Promega, Stratagene, Clontech, and/or Invitrogen) and in literature or reference text books, for instance in Rodriguez, R. L. and D. T. Denhardt, ed., “Vectors: A survey of molecular cloning vectors and their uses”, Butterworths, 1988; Current protocols in Molecular Biology, eds.: F. M. Ausubel et al., Wiley N.Y., 1995; Molecular Cloning: a laboratory manual, supra; and DNA Cloning, Vol. 1-4, 2nd edition 1995, eds.: Glover and Hames, Oxford University Press).
The cells transformed with a polynucleotide or a vector according to the invention are likewise part of the present invention. Thus, in another aspect, the present invention provides a cell capable of expressing a recombinant polypeptide, characterized in that the cell comprises a polynucleotide according to the invention encoding the expressed recombinant polypeptide.
The term “recombinant” in this context refers to a polypeptide that is not expressed in the cell in nature. Thus, a host cell which comprises the DNA or expression vector according to the invention is also within the scope of the invention. The engineered host cells can be cultured in conventional nutrient media which can be modified e.g. for appropriate selection, amplification or induction of transcription. The culture conditions such as temperature, pH, nutrients etc. are well known to those ordinarily skilled in the art.
Cells that are transformed with a vector according to the invention may be of prokaryotic or eukaryotic origin, preferably the cells are of eukaryotic origin. Eukaryotic cells according to the invention may be of avian or non-avian origin. Cells that are of non-avian origin may be for example, BHK cells, insect cells, HeLa or COS cells. Preferably the cells are avian cells such as CEF, HD-11 or DT-40 cells.
A transformed cell according to the invention may comprise a polynucleotide according to the invention stably integrated into the genomic material or as part of an autonomously replicating vector.
A cell culture comprising a multitude of cells according to the invention is likewise part of the present invention. Cells according to the invention can be used to express the polypeptide subunits or the complete protein and can be isolated from the cell culture.
The cloning of the nucleotide sequences encoding the p40 and p35 subunits respectively, enables the production of pure proteins, free from other cytokines. This is especially useful in case of the production of antibodies specific for the proteins of the invention. These specific antibodies can be generated via techniques generally available. Preferably the specific antibodies are monoclonal antibodies. Thus the present invention furthermore provides for antibodies specific for the p40 and/or p35 subunits or for the chicken IL-12. The antibodies according to the invention are suitable for use in diagnostics or for isolation and purification of proteins such as avian chicken IL-12 from crude preparations. Moreover, the antibodies can be used to develop assays for quantitative analysis of protein production in vitro or for quantitative measurements of protein levels in vivo.
As already stated above, the proteins and polynucleotides according to the invention are especially useful to enhance the immune response to avian vaccines (i.e. they may be used as or in adjuvants).
Vaccination against an infectious disease aims to elicit an immune response that limits clinical symptoms associated with infection by a pathogen. It is important that the correct type of immune reaction is triggered, since many types of immune mechanisms that can be activated are inadequate for control of the particular pathogen. Low responsiveness to vaccine antigens can be overcome by administering the antigens in combination with adjuvants. Adjuvants are defined as those components of a vaccine formulation other than the antigen which contribute to enhanced immune responsiveness to the antigen, e.g. aluminum salts, oil emulsions, derivatives of muramyl peptide, monophosphoryl lipid A, liposomes, QS21TM, MF-59TM, IscomsTM, and the like.
The cellular and molecular mechanisms that are activated following vaccination are strongly influenced by the choice of adjuvant that is administered together with the vaccine antigen. Hence the selection of adjuvants may be as critical as the choice of vaccine antigens themselves in providing optimal efficacy.
Proteins according to the invention, in particular the chicken IL-12, may have a potent adjuvant effect on the immune response of a subject to a vaccine. Thus in another embodiment the invention provides for an adjuvant composition comprising an effective adjuvant amount of a protein according to the invention, in particular chicken IL-12. The adjuvant composition can be administered concomitantly or sequentially with a vaccine formulation.
The protein(s) according to the invention can be included in the vaccine formulation. Thus in another embodiment the present invention provides for a vaccine comprising at least one active agent, an effective adjuvant amount of a protein according to the invention, preferably chicken IL-12, and a pharmaceutically acceptable carrier or diluent.
A protein according to the present invention can be a molecule comprising the whole of the p40 and/or p35 sub-unit(s) or fragments thereof, provided said fragments have retained their ability to act as a cytokine (for example, when used in a vaccine, to retain their adjuvanting ability).
An adjuvant composition according to the present invention comprises a protein according to the invention, preferably chicken IL-12, and a pharmaceutically acceptable carrier. Suitable pharmaceutical carriers are water, saline, and the like. Additionally, the adjuvant composition may comprises one or more other adjuvants such as oil emulsions, aluminum salts, derivatives of muramyl dipeptide, monophosphoryl lipid A, liposomes, QS21 TM, MF-59TM, IscomsTM, and the like. The proteins according to the invention may also be used in conjunction with other cytokines.
The adjuvant composition according to the invention may, in the alternative, comprise a DNA plasmid capable of expressing a protein according to the invention. Said DNA plasmid comprises DNA sequences encoding a protein according to the invention, preferably chicken IL-12, operably linked to transcriptional regulatory sequences. Nucleotide sequences encoding for other cytokines that are used in conjunction with a protein according to the invention can be present on the same DNA plasmid or on a separate plasmid. Upon administration of such a DNA adjuvant composition to a subject, host cells take up and express encoded genes on the inoculated DNA, resulting in in vivo expression of the proteins according to the invention, for example, chicken IL-12.
A vaccine according to the invention comprises at least one active agent and an effective adjuvant amount of a protein according to the invention, i.e. in an amount which will cause the vaccinated subject to produce an enhanced immunological response as compared to the vaccine without said protein.
The required effective amount in an adjuvant composition or vaccine according to the invention is dependent on the type of active agent used, the type of pathogen immunized against, as well as the type of vaccinated subject. Determination of the effective amount is well within the routine skills of the practitioner, and will generally be in the amount of 0.001 to 500 μg/dose. Preferably the amount will be between 0.01 and 50 μg/dose, more preferably 0.1 to 5 μg/dose.
The active agent for use in a vaccine according to the invention can be of viral, bacterial or parasitic origin. The active agent may either be the whole pathogen which causes the disease, or may consist of components derived from said pathogen. In the event the active agent is a whole pathogen, said pathogen may be a live pathogen or an inactivated pathogen. Live pathogens are considered to be either attenuated or naturally occurring mild strains of said pathogen. Inactivated pathogens are pathogens killed by chemical or physical means, that is, the inactivated or “killed” pathogen is no longer capable of replication. Suitable means for chemical inactivation are formaldehyde, glutaraldehyde, β-propiolactone, ethyleneimine and derivatives, and the like. Suitable means for physical inactivation are UV radiation, γ-radiation, “heat-shock”, X-radiation, and the like. Alternatively, the active agent may constitute one or more components derived from said disease causing pathogen, e.g. purified protein, protein-polysaccharide, protein-lipopolysaccharides, lipopolysaccharides, and the like.
The active agent may be a DNA plasmid capable of in vivo expression of a pathogen or selected components derived from said pathogen. In addition, the vaccine may comprise a DNA plasmid capable of expressing a protein according to the invention in vivo. The DNA encoding said protein adjuvant and the DNA encoding said pathogen or selected components may be present on one and the same plasmid, or may be present on separate plasmids. Upon administration of the DNA vaccine to a subject, host cells will take up and express in vivo said active agent as well as said protein according to the invention. DNA vaccines are for example described in U.S. Pat. No. 5,580,859.
Pharmaceutically acceptable carriers or diluents that can be used to formulate an adjuvant composition or a vaccine composition according to the invention are sterile and physiologically compatible such as for example an aqueous buffer, a saline solution and the like. In addition stabilizers, preservatives and the like may be added to these compositions.
The compositions of the present invention may take any form that is suitable for oral or parenteral administration. For oral use, the adjuvant or vaccine compositions according to the invention may be formulated as solutions, syrups, suspensions, tablets, capsules and the like. For parenteral use, the compositions according to the present invention may be formulated in a form suitable for injection such as suspensions, solutions, dispersions, emulsions, and the like. Preparation of the compositions according to the present invention is carried out by means conventional for the skilled person.
Preferred administration routes are parenteral routes, e.g. intramuscular injection, intravenous injection, intradermal injection, subcutaneous injection, and mucosal routes, e.g. nasal drops, eye drops, (aerosol) sprays, and the like.
The following examples will illustrate the invention without limiting the invention thereto.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: DNA and protein sequence of clone pat.pk0055.c11 (=chicken ChIL-12 p35) (SEQ ID NO 4 and 2, respectively).
FIG. 2: Sequence of clone chicken ChEST582p2; 5′ and 3′ primers (SEQ ID NO 18 and 17, respectively)
FIG. 3: DNA and protein sequence of clone pND89 (=chicken ChIL-12 p40) (SEQ ID NO 3 and 1, respectively).
FIG. 4: DNA and protein sequence of clone pND115 (=Duck IL-12 p40) (SEQ ID NO 6 and 5, respectively).
FIG. 5: DNA and protein sequence of clone pND117 (Turkey IL-12 p40) (SEQ ID NO 8 and 7, respectively).
FIG. 6: DNA homology of chicken p40 to p40 sequences of duck and turkey. (SEQ ID NO 3, 6 and 8, respectively
FIG. 7: Protein homology of chicken p40 to p40 sequences of duck and turkey. (SEQ ID No 1, 5 and 7, respectively).
FIG. 8: Western blot analysis of COS-7 cell culture supernatants after transfection with chicken IL-12 and other cDNA molecules (mock=empty vector control).
FIG. 9: Chicken heterodimeric IL-12 dependent induction of IFN-γ (Fe=feline; mock=empty vector control).
FIG. 10: Chicken heterodimeric IL-12 dependent proliferation of chicken spleen cells (Fe=feline; mock=empty vector control).
FIG. 11: Chicken Flexi-IL-12 dependent induction of IFN-γ (Fe=feline; mock=empty vector control).
FIG. 12: Chicken Flexi-IL-12 dependent proliferation of chicken spleen cells (Fe=feline; mock=empty vector control).



EXAMPLES
Materials and Methods for All Examples
Cell Culture and LPS Treatment.
The chicken cell lines HD-11 (macrophage origin) and DT-40 (B-cell origin) were grown as cell suspensions in RPMI (supplemented with 10% fetal calf serum, 100 μg/ml streptomycin, 100 units/ml penicillin, 2 mM glutamin and 1 mM pyruvate) and DMEM (supplemented with 8% fetal calf serum, 2% chicken serum, 2 mM glutamin, 100 μg/ml streptomycin, 100 units/ml penicillin and 1 mM pyruvate), respectively. Lipopolysaccharide (LPS) treatment included the incubation of cells with 5 μg/ml LPS for 5 h. After the treatment, cells were washed 2-times with phosphate buffered saline and subsequently used for RNA isolation or stored at −70° C. Cells were maintained in a humidified atmosphere of 5% CO2 at 37° C.
The mammalian cell line COS-7 (African green monkey kidney cells) was grown in DMEM (supplemented with 10% FCS, 2 mM glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin and 1 mM pyruvate)
Chicken, Duck and Turkey Organs and LPS Treatment.
Three weeks old Normal White Leghorn specific pathogen free (SPF) chickens were derived from the Intervet animal facilities and housed under SPF conditions. The animals received water and food ad libitum.
Freshly isolated chicken, duck or turkey organs, i.e. spleen and kidney, were mashed using a sieve and Hanks buffer. Cells were pelleted at 1500 rpm for 3 minutes at room temperature and washed 2-times using the same procedure. Duck and turkey cells were subsequently used for RNA isolation. Chicken cells were resuspended in RPMI supplemented with 10% chicken serum, 100 μg/ml streptomycin and 100 units/ml penicillin, and grown for 16 h. Some cells were incubated with 5 μg/ml LPS for 5 h. After the treatment, cells were washed 2-times with phosphate buffered saline and subsequently used for RNA isolation or stored at −70° C. Cells were maintained in a humidified atmosphere of 5% CO2 at 41° C.
RNA Isolation.
Total RNA was isolated using Trizol ReagentTM (Gibco-Invitrogen) as described by the manufacturer. RNA quality was checked on a 1% agarose gel.
RT-PCR, PCR and Sequence Reactions.
Two μg of total RNA were reverse-transcribed into cDNA using the Superscript II RTTM protocol (Gibco-Invitrogen). This newly made cDNA was subsequently used as template for PCR amplification. For this, 1 μl of RT-cDNA reaction (10-20 ng of plasmid cDNA template) was mixed with 0.5 μl of 1 unit/μl SupertaqTM (HT Biotechnology Ltd.), 1 μl of 10 ng/μl of each primer, 1.6 μl of 2 mM dNTPs, and 2 μl of 10×ST PCR buffer (HT Biotechnology Ltd.) in a final volume of 20 μl. The reaction cycling conditions were 94° C. for 2 min, then 30 cycles of 94° C. for 30 s, 55° C. for 1 min, 72° C. for 1 min 30 s, then 72° C. for 2 min for a final extension. The PCR products were gel-purified using the Qiaquick Gel Extraction KitTM (Qiagen), cloned into the pDriveTM vector (Qiagen PCR Cloning kit, Qiagen) or into the pCR2.1-TOPTM vector (TA Cloning kit, Invitrogen).
Plasmid DNA was purified using the Qiagen Plasmid Midi KitTM (Qiagen). For general PCR-reactions, in which 10 ng of a plasmid template is being used, the same cycling conditions are used as described above.
All clones were extensively sequenced in both 5′ and 3′ directions using a DNA sequencing kit (BigDye Terminator v3.0 Cycle Sequencing Ready ReactionTM, Applied Biosystems). Sequences were analyzed with the SequencherTM 4.0 software (Gene Codes Corporation).
Sequence Analysis.
Sequence analysis, included use of Blast searches (through an internal server, InterBLAST, from Intervet Innovation, Schwabenheim, Germany), the Wisconsin PackageTM Version 10.2 (Genetics Computer Group; GCG), SequencherTM 4.0 (Gene Codes Corporation), OMIGATM 2.0 (Oxford Molecular Ltd.) and of GeneDocTM 2.6.
Chicken (Ch) IL-12 p35, ChIL-12 p40, ChFlexi-IL-12 and Feline (Fe) Flexi-IL-12 Eukaryotic Expression Constructs
ChIL-12 p35. Full length ChIL-12 p35 (clone pat.pk0055.c11), originally cloned in pcDNA3TM (Invitrogen) (Tirunagaru, V G et al., Genomics 2000, 66:144), was excised from pcDNA3 using EcoRI and NotI and cloned into the corresponding restriction sites of the eukaryotic expression vector pcDNA3.1(+)TM (Invitrogen). The EcoRI/NotI ChIL-12 p35 fragment was also cloned into the corresponding restriction sites of pcDNA3.1(−)TM (Invitrogen) to obtain an anti-sense control construct.
ChIL-12 p40. Full length ChIL-12 p40, present in a cDNA library constructed from pooled T and B cells isolated from vaccinated chickens and recloned into pDriveTM (Qiagen), was excised from pDrive using NotI and HindIII and cloned into the corresponding restriction sites of the eukaryotic expression vector pcDNA3.1(−) (Invitrogen).
ChFlexi-IL-12. A single chain chicken IL-12 molecule was generated by a strategy described by McMonagle et al. (Equine Vet. J. 2001, 33: 693).
The following primers were used to amplify ChIL-12 p35 without the putative 35 amino acid signal peptide sequence (as determined by the SPScan program from the Wisconsin Package (supra) and that introduced a 5′-BamHI and a 3′-HindIII restriction site:
5′-TTGGATCCGGTGGCGGCGGATCTCTGCCACCTCCTGCCCA-3′ (SEQ ID NO 9), and
5′-CCAAGCTTTTACATCTCTGCAGTGAGGGCACTCAGGTAGC-3′ (SEQ ID NO 10).
For ChIL-12 p40 the following primers were used that introduced a 5′-NotI and a 3′-BamHI restriction site:
5′-TTGCGGCCGCCATGTCTCACCTGCTATTTGCCTTACTTTC-3′ (SEQ ID NO 11) and
5′-TGGATCCACCACCGCCCGAGCCACCGCCACCTCTGCAAAGCGTGG-3′ (SEQ ID NO 12).
Both PCR fragments were separately cloned into pCR2.1-TOPOTM (Invitrogen) and extensively sequenced. ChIL-12 p40 was excised from pCR2.1-TOPO as a NotI/BamHI fragment and cloned into the corresponding restriction sites of the pcDNA3.1(−) vector (Invitrogen). The ChIL-12 p35 was excised from pCR2.1-TOPO as a BamHI/HindIII fragment and cloned into the corresponding restriction sites of the [ChIL-12 p40]-[pcDNA3.1(−)] construct downstream of the p40 fragment. This resulted in a single chain p40-p35 heterodimeric construct in which the p40 chain is linked to the p35 chain by an in-frame (Gly4Ser)3-linker; this molecule was designated ChFlexi-IL-12. FeFlexi-IL-12. Feline IL-12 was cloned into the eukaryotic pCI-neoTM vector (Promega) (Dr. L. Nicolson, Univ. of Glasgow Veterinary School, UK) producing a construct similar to the ChFlexi-IL-12.
Transient Expression of cDNA Clones in COS-7 Cells
COS-7 cells were transfected with 1.5 μg of each cDNA construct using the Invitrogen Life Technologies LipofectamineTM reagent (as described by the manufacturer) and cultured in 3-cm dishes with DMEM (without FCS and penicillin/streptomycin). After 8 h, transfected cells were washed and cultured in DMEM with penicillin/streptomycin and 10% FCS. After 72 h incubation at 37° C./5% CO2 the cell culture supernatants were harvested and centrifuged at 13,000 rpm for 10 min at 4° C. to remove cell debris. The supernatants were analyzed via Western blotting and used immediately or stored at −70° C.
Western Blot Analysis
Cell culture supernatants from transfected COS-7 cells were size fractionated using 4-12% Nu-PAGE T (Invitrogen) and blotted onto nitrocellulose filters (Schleicher & Schuell). Western blots were blocked in 3% skimmed milk (MPBS) in PBS, and subsequently incubated with a polyclonal antibody that was raised against a FeIL-12 p40 peptide diluted 1:300 in MPBS. Blocking and antibody incubation were each performed for 1 h at room temperature. After extensive washing (3 times 5 min), blots were incubated with alkaline peroxidase (AP)-conjugated goat anti-rabbit IgG antibodies (Sanbio) diluted 1:1000 in MPBS for 1 h at room temperature. After washing (3 times 5 min), bound AP-labeled secondary antibodies were visualized via staining.
Bioactivity Assays for Chicken (Ch) IL-12
NO-assay for the induction of splenic ChIFN-γ by ChIL-12. Chicken spleen cells were freshly isolated and seeded in triplicate in a 96-well plate at a density of 0.5×106 cells/well in 100 μl and incubated with 50 μl of serial dilutions of cell culture supernatants from COS-7 cells transfected with cDNA clones encoding ChIL-12 p40, ChIL-12 p40 mixed with ChIL-12 p35, ChFlexi-IL-12, FeFlexi-IL-12 or with an empty pcDNA3.1 plasmid (mock). Forty-eight hours after the addition of proteins, supernatants (75 μl) were collected and analyzed for the presence of biologically active ChIFN-γ. For this, 100 μl of 1.5×106/ml HD-11 cells were incubated with 75 μl of the collected supernatants for 24 h at 37° C./5% CO2 in 96-well plates. Activation of HD-11 cells by ChIFN-γ was measured as a function of nitrite accumulation in the culture supernatants using the Griess assay (Ding, A H et al., J. Immunol. 1988, 141: 2407; Stuehr, DJ, and CF Nathan, J. Exp. Med. 1989, 169:1543).
Assay for spleen cell proliferation by ChIL-12. After removing 75 μl of the supernatants (see NO-assay for the induction of splenic ChIFN-γ by ChIL-12 section) 50 μl medium and 18.5 kBq methyl-3H-Thymidine (25 μl per well) were added to the remaining 75 μl in the 96-well plate and incubated for 18-20 h at 41° C./5% CO2. After incubation the incorporated radioactivity was counted using an LKB BetaplateTM β-counter.
Statistical Analysis
The significance of the differences between the means of NO-production or between the means of cell proliferation was analyzed using the Student's t-test. Differences were considered significant at a confidence level of 95% (P<0.05).
Example 1
Isolation and Sequence Analysis of Clone pat.pk0055.c11 Encoding the Chicken IL-12 p35 Subunit (ChIL-12 p35)
Analysis of the open reading frame (nucleotides 1-618) of cDNA clone pat.pk0055.c11 (see FIG. 1), which was isolated from a high throughput sequencing project of the chicken Con A stimulated T cell cDNA library (Tirunagaru et al., supra) showed that it is homologous to the IL-12 p35 cDNA sequences of human (43% overall homology, M65271 in EMBL/Genbank), sheep (45% overall homology, AF173557 in EMBL/Genbank), horse (48% overall homology, Y11130 in EMBL/Genbank), cat (43% overall homology, Y07761 in EMBL/Genbank), bovine (45% overall homology, U14416 in EMBL/Genbank), mouse (42% overall homology; M86672 in EMBL/Genbank) and woodchuck (45% overall homology, X970189 in EMBL/Genbank) A multiple alignment of the Ch IL-12 p35 protein encoded by pat.pk0055.c11 to human, sheep, horse, cat, bovine, mouse and woodchuck IL-12 p35 proteins yields an overall amino acid homology of 27%, 25%, 30%, 24%, 25%, 29% and 21%, respectively. When removing the first 64 aa (residues 1-64) of pat.pk0055.c11 the homologies to human, sheep, horse, cat, bovine, mouse and woodchuck IL-12 p35 proteins increases to 33%, 32%, 37%, 25%, 32%, 34% and 28%, respectively, indicating that the N-terminal fragment of pat.pk0055.c11 is not as highly conserved as the rest of the protein. Based on these sequence homologies we conclude that clone pat.pk0055.c11 encodes the chicken IL-12 p35 subunit.
Example 2
Isolation of Clone pND89 Encoding the Chicken IL-12 p40 Subunit (ChIL-12 p40)
The coding sequence, i.e. nucleotides 35-1042, of the mouse Interleukin 12 p40 subunit (MuIL-12 p40; EMBL/Genbank Accession number M86671) was used to search the UMIST/Nottingham/Dundee Chicken EST Repository Database using the tBlastX program. A chicken EST sequence (clone ID: ChEST582p2; EST name 603603708F1; derived from adult kidney) was retrieved that showed 51% identity with aa 251-279 and 66% identity with aa 310-327 of the MuIL-12 p40 sequence. No Genbank Accession number has been assigned to this ChEST582p2 clone and no annotations pointing in the direction of IL-12 were made by the owners of this Chicken EST database. A database search in the same chicken EST database with this ChEST582p2 clone did not result in a longer or full length clone. A similar database search in the U.D. Chick EST database with the coding sequence, i.e. nucleotides 35-1042, of MuIL-12 p40 (EMBL/Genbank Accession number M86671) did not result in a valid hit using the BlastN program nor did a search with the ChEST582p2 clone result in a longer or full length clone.
The identified ChEST582p2 clone is 848 nucleotides long of which nucleotides 3-233 (which includes a stopcodon) encode a 76 aa long polypeptide (see FIG. 2). A multiple alignment of the predicted ChEST582p2 protein sequence showed that it aligns to the most C-terminal part of MuIL-12 p40 (35% overall homology; M86671 in EMBL/Genbank), and to the C-terminal part of IL-12 p40 of several other species including human (43% overall homology, M65272 in EMBL/Genbank), sheep (43% overall homology, AF004024 in EMBL/Genbank), horse (43% overall homology, Y11129 in EMBL/Genbank), cat (43% overall homology, Y07762 in EMBL/Genbank), bovine (42% overall homology, U11815 in EMBL/Genbank) and woodchuck (51% overall homology, X97019 in EMBL/Genbank).
To clone the full length chicken IL-12 p40 protein subunit (ChIL-12 p40), three approaches have been used.
In the first approach 3 degenerated primers:
5′-ATGTGTCACCAGYRGTTGGTCMTCTCYTG-3′ (SEQ ID NO 13),
5′-ATGTGTCYTCAGMAGYTRRYCATCTCCTG-3′ (SEQ ID NO 14), and
5′-ATGTGTCWYCAGYRGTTGGTCMTCTCCTG-3′) (SEQ ID NO 15),
and 1 specific 5′-end primer
5′-ATGCACCCTCAGCAGTTGGTCGTTTCCTG-3′ (SEQ ID NO 16),
based on the 5′-end of human (M65272 in EMBL/Genbank), reddeer (US7752 in EMBL/Genbank), horse (Y11129 in EMBL/Genbank), sheep (AF004024 in EMBL/Genbank), mouse (M86671 in EMBL/Genbank) and woodchuck (X97019 in EMBL/Genbank) were designed. In combination with a 3′-ChESTp582p2 primer

5′-TTATCTGCAAAGCGTGGACCACTCACTCCAGGAT-3′ (SEQ ID NO 17)

(complementary to nucleotide positions 233-200 in FIG. 2) an RT-PCR reaction was performed on total RNA isolated from chicken HD-11 (macrophage) cells, chicken DT-40 (B) cells, chicken kidney cells and chicken spleen cells treated with or without 5 μg/ml lipopolysaccharide (LPS). Surprisingly, none of the primer combinations resulted in a PCR-product. As a control, an RT-PCR reaction was performed simultaneously using a 5′-end ChESTp582p2 primer.

5′-ACCTGGACATATCCCAAGACCTGGAGCACA-3′ (SEQ ID NO 18)

(nucleotide positions 12-41 in FIG. 2) and the 3′-end ChESTp582p2 primer (SEQ ID NO 17, supra). This primer combination resulted in a PCR-fragment of 200 nucleotides. These results indicate that it is not possible to obtain a full length chicken IL-12 p40 molecule by using an RT-PCR approach based on 5′-end IL-12 p40 sequences from human, reddeer, horse, mouse or woodchuck with degenerated primers in combination with a specific 3′-end primer.

In the second approach we used plasmid pools isolated from a HD-11 cDNA library that was constructed from chicken HD-11 (macrophage) cells stimulated for 5 h with 5 μg/ml of LPS (Sick C, Schneider K, Staeheli P, Weining K C. Novel chicken CXC and CC chemokines. Cytokine 2000, 12:181-186) In this library cDNA molecules are unidirectionally cloned between the EcoRI and XhoI sites of the eukaryotic expression vector pcDNA1. In a PCR-reaction with a 5′-end pcDNA1 vector primer 116 nt upstream of the EcoRI restriction site
5′-CTGGCTAACTAGAGAACCCACTGCTTACTGGCTT-3′ (SEQ ID NO 19)
(nucleotide positions 2918-2951 of vector pcDNA1) and the 3′ChEST582p2 primer (SEQ ID NO 17, supra) a PCR-fragment of  ̃1000 nucleotides was obtained that was cloned into pDriveTM (Qiagen).
In the third approach we used plasmid pools isolated from a cDNA library that was constructed from pooled T and B cells isolated from vaccinated chickens. In this library cDNA molecules are unidirectionally cloned between the NotI and EcoRI sites of the eukaryotic expression vector pBlueScriptTM (Stratagene). In a PCR-reaction with a 5′-end pBlueScript vector primer approximately 120 nucleotides upstream of the NotI restriction site and the 3′ ChEST582p2 primer (SEQ ID NO 17, supra) a PCR-fragment of  ̃1000 nucleotides was obtained that was cloned into pDrive.
To investigate whether this clone, designated pND89, contains the  ̃200 nt IL-12 p40 fragment at the 3′-end, a PCR reaction using pND89 as template in combination with the 5′ChESTp582p2 (SEQ ID NO 18, supra) and the 3′ ChESTp582p2 primer (SEQ ID NO 17, supra) was performed. The results showed a PCR-fragment of  ̃200 nucleotides indicating that the  ̃1000 nt long pND89 cDNA clone contains the 222 nt long IL-12 p40 fragment.
Example 3
Sequence Analysis of cDNA Clone pND89
Clone pND89 was extensively sequenced which revealed that the cDNA clone (from start to stop) is 948 nucleotides long and encodes a protein of 315 aa (see FIG. 3. Analysis of the pND89 cDNA sequence containing the open reading frame (nucleotides 1-948) showed that it is homologous to the IL-12 p40 cDNA sequences of human (57% overall homology, M65272 in EMBL/Genbank), sheep (56% overall homology, AF004024 in EMBL/Genbank), horse (57% overall homology, Y11129 in EMBL/Genbank), cat (55% overall homology, Y07762 in EMBL/Genbank), bovine (56% overall homology, U11815 in EMBL/Genbank), mouse (55% overall homology; M86671 in EMBL/Genbank) and woodchuck (57% overall homology, X97019 in EMBL/Genbank). A multiple alignment of the Ch IL-12 p40 protein encoded by pND89 to human, sheep, horse, cat, bovine, mouse and woodchuck IL-12 p40 proteins yields an amino acid homology of 41%, 40%, 40%, 42%, 39%, 36% and 41%, respectively. Sequence analysis further revealed the presence of a signalpeptide with the cleavage site between aa 20-21 resulting in a signal peptide of 20 aa and a mature protein of 295 aa. The presence of a WSXWS box (aa 305-311), an Ig-like C2-type domain (aa residues 41-94) and a fibronectin type-III domain (aa residues 228-308), which are both characteristic for IL-12 p40, were confirmed by similarity. These sequence homologies prove that clone pND89 encodes the chicken IL-12 p40 subunit.
Example 4
Isolation and Sequence Analysis of Clones pND115 and pND117 Encoding the Duck and Turkey IL-12 p40 Subunits, Respectively
Using the chicken IL-12 p40 sequence and the existing high homology between chicken and duck/turkey, we tried to clone the duck and turkey IL-12 p40 subunits. In combination with a 5′-end chicken IL-12 p40 primer
5′-ATGTCTCACCTGCTATTTGC-3′ (SEQ ID NO 20)
(nucleotide positions 1-20 in FIG. 3) and a 3′-end chicken IL-12 p40 primer
5′-TTATCTGCAAAGCGTGGACCACT-3′ (SEQ ID NO 21)(complementary to nucleotide positions 948-926 in FIG. 3) an RT-PCR reaction was performed on total RNA isolated from either duck or turkey spleen and kidney cells. From the RT-PCR reactions PCR fragments of  ̃000 nt were obtained that were subsequently cloned into the pCR2.1TM vector (Invitrogen). The duck clone was designated pND115 and the turkey clone pND117. Clones pND115 and pND117 were extensively sequenced which revealed that both cDNA clones (from start to stop) are 948 nucleotides long and encode proteins of 315 aa (see FIGS. 4 and 5). Analysis of the pND115 and pND117 cDNA sequences containing the open reading frame (nucleotides 1-948) showed that both are >99% identical to the IL-12 p40 cDNA sequence of chicken (see FIG. 6). A multiple alignment of the predicted pND115 and pND117 proteins showed that pND115 is identical to chicken IL-12 p40 and that pND117 is >99% identical to the chicken IL-12 p40 protein (see FIG. 7). The small differences in homology between pND115, pND117 and chicken IL-12 p40 are the result of small substitutions in the cDNA sequence which results in silent amino acid residue mutations for both pND115 and pND117, and in 1 amino acid residue change for pND117 (see FIG. 7). Based on these high sequence homologies we conclude that clone pND115 encodes the duck IL-12 p40 subunit and that clone pND117 encodes the turkey IL-12 p40 subunit.
Example 5
Characterization of Recombinant Chicken (Ch) IL-12
To detect secreted ChIL-12 p40 and ChIL-12 p35 subunits after (co-) transfection of COS-7 cells non-denaturing Western blot analysis was applied. For this, a polyclonal antibody which was raised against a peptide of the Feline (Fe) IL-12 p40 subunit was used. With this anti-FeIL-12 p40-peptide antibody, ChIL-12 p40 and the ChIL-12 p40 homodimer: ChIL-12 p80 (Ch (p40)2) could be detected in the supernatants of transfected COS-7 cells (FIG. 8, lane 1). In the supernatants from COS-7 cells transfected with both ChIL-12 p35 and ChIL-12 p40 the heterodimeric ChIL-12 p70 protein (ChIL-12) (FIG. 8, lane 4) could be detected indicating that the p40 and p35 chain interact with each other into the heterodimeric IL-12 p70 molecule. The formation of ChIL-12 p70 was more efficient than the formation of homodimeric ChIL-12 p80 as no or only very small amounts of ChIL-12 p80 could be detected. Also, formation of ChIL-12 p70 is specific as co-transfection of ChIL-12 p40 cDNA with anti-sense ChIL-12 p35 cDNA or with a cDNA construct encoding an irrelevant viral protein (IBV-N) did not result in heterodimerization (FIG. 8, lanes 5-6).
Example 6
Bioactivity of Chicken IL-12
[1] ChIL-12 Dependent Induction of ChIFN-γ
A hallmark of IL-12 activity in mammals is its induction of IFN-γ by T lymphocytes. Therefore, the IFN-γ levels were assessed in the culture medium of freshly isolated chicken spleen cells incubated with dilutions of various proteins isolated after transient (co-)expression in COS-7 cells. ChIFN-γ was measured as a function of nitrite accumulation using HD-11 cells and the Griess assay. As shown in FIG. 9, only heterodimeric ChIL-12 p70 (a co-transfection of ChIL-12 p40 with ChIL-12 p35) is able to induce production of IFN-γ in chicken spleen cells in a concentration dependent manner. Transfection of mock vector or of ChIL-12 p40 alone, could not induce ChIFN-γ secretion to a level anywhere comparable to the heterodimeric ChIL-12 p70. Next to this it is clear that only species specific IL-12 induces IFN-γ in chicken spleen cells as the FeFlexi-IL-12, induced no significant amounts of ChIFN-γ. The differences in NO-production between ChIL-12 p40 and ChIL-12 p70 (a co-transfection of ChIL-12 p40 with ChIL-12 p35), and between ChIL-12 p70 and FeFlexi-IL-12 were significant (P<0.05). Taken together, these results indicate that ChIL-12 is bioactive and that the induction of IFN-γ via chicken spleen cells is ChIL-12 dependent.
Example 7
Bioactivity of Chicken IL-12
[2] ChIL-12-Dependent Proliferation of Chicken Spleen Cells
Another characteristic of IL-12, shared with several other cytokines, is its induction of T cell proliferation. The growth response of freshly isolated chicken splenocytes to various proteins, isolated after transient (co-) expression in COS-7 cells, was measured by a cell proliferation assay. Only heterodimeric ChIL-12 p70 (a co-transfection of ChIL-12 p40 with ChIL-12 p35) was able to induce the proliferation of chicken spleen cells (FIG. 10). The relatively low proliferation data observed for the first dilutions are possibly explained by overdose effects for this parameter. Neither ChIL-12 p40 alone nor FeFlexi-IL-12 were able to induce similar proliferative responses. From 1⁄6 dilution on the differences in proliferation between ChIL-12 p40 and ChIL-12 p70 (a co-transfection of ChIL-12 p40 with ChIL-12 p35), and between ChIL-12 p70 and FeFlexi-IL-12 were significant (P<0.05). Taken together, these results prove that ChIL-12 is bioactive and that the molecule is able to induce proliferation of chicken spleen cells.
Example 8
Bioactivity of Chicken IL-12
[3] Bioactivity of Single Chain ChFlexi-IL-12
After showing that co-transfection of single chain ChIL-12 p40 with single chain ChIL-12 p35 resulted in the formation of a bioactive ChIL-12 heterodimer (FIGS. 9 and 10), a single chain IL-12 molecule (ChFlexi-IL-12) was constructed. ChFlexi-IL-12 is a single chain p40-p35 heterodimeric construct in which the ChIL-12 p40 chain is linked to the ChIL-12 p35 chain by an in-frame (Gly4Ser)3-linker, also called a “hinge” region. By Western blot analysis it could be shown that the expression profile of the ChFlexi-IL-12 after transfection in COS-7 cells is comparable to that of FeFlexi-IL-12. Following incubation of freshly isolated chicken spleen cells with ChFlexi-IL-12 the release of both IFN-γ as well as cell proliferation were observed (FIG. 11 and FIG. 12). The results of these experiments prove that the chicken IL-12 flexi construct is also bioactive.

## Claims

1. An isolated protein comprising:
a subunit comprising an amino acid sequence showing at least 99% similarity with the amino acid sequence as depicted as SEQ ID NO: 1.

2. The protein according to claim 1, comprising a subunit having an apparent molecular weight of approximately 40 kD and having an amino acid sequence as depicted in SEQ ID NO: 1.

3. The protein according to claim 1, comprising
a subunit having an apparent molecular weight of approximately 40 kD and having an amino acid sequence as depicted in SEQ ID NO: 1 linked to another subunit with a molecular weight of approximately 35 kD).

4. An adjuvant composition comprising a protein according to claim 1, an adjuvant, and a pharmaceutically acceptable carrier.