WO1996003436A2

WO1996003436A2 - EQUINE η-INTERFERON

Info

Publication number: WO1996003436A2
Application number: PCT/GB1995/001732
Authority: WO
Inventors: David Edward Onions; Lesley Nicholson
Original assignee: Q-One Biotech Limited
Priority date: 1994-07-21
Filing date: 1995-07-21
Publication date: 1996-02-08
Also published as: GB2291645A; GB9414753D0; WO1996003436A3; AU3083295A

Abstract

The present invention provides a polynucleotide fragment encoding equine η-interferon, a recombinant vector comprising such a polynucleotide fragment, a host cell containing said polynucleotide fragment or recombinant vector, a recombinant equine η-interferon polypeptide, and pharmaceutical compositions comprising recombinant η-interferon for use as a prophylactic and/or therapeutic agent and also as an adjuvant.

Description

EQUINE -v-INTERFERON

The present invention relates to equine 7-interferon, a polynucleotide fragment encoding equine 7-interferon, a recombinant vector comprising such a polynucleotide fragment, a host cell containing said polynucleotide fragment, or recombinant vector comprising said polynucleotide fragment, an equine γ-interferon polypeptide, antibodies immuno-reactive with said polypeptide and pharmaceutical compositions comprising recombinant equine 7-interferon for use as a prophylactic and/or therapeutic agent and also as an adjuvant in horses.

Cyto ines are low molecular weight secreted proteins with immunomodulatory activity. The term applies to interferons, interleukins, lymphokines, monokines, colony stimulating factors and a number of growth factors (Balkwill and Burke 1989) . Interferons are a class of cytokines which exhibit antiviral activity. Three types exist, alph (α) , beta (β) and gamma (7) (Farrar and Schreiber 1993). The type I interferons, a and β are related and bind to a common cell structure surface receptor, although the multiple forms of α and the single β form may have different biological effects. Interferon gamma (IFN7) , also known as type II or immune interferon, is distinct from a and β interferons at a genetic level. Like β interferon it is encoded by a single gene. IFN7 is produced by natural killer (NK) cells and by T lymphocytes

(TH1 subset) in response to stimulation with antigen or mitogens. It possesses a range of biological effects of immunomodulatory or antiviral nature including enhancement of MHC class I and class II expression, suppression of the TH2 T lymphocyte response, activation of macrophages, enhancement of natural killer cell activity, and modulation of the synthesis and effect of a number of cytokines (Gray 1992). IFN7 has also exhibited adjuvanticity properties in some animal models (reviewed in Health and Playfair 1992) . Although the bovine and ovine IFN7S, which exhibit 93% amino acid identity, are cross-reactive (Radford et al. 1991) , IFN7S tend to be highly species-specific in their biological activity. Thus investigation of the potential applications of IFN7 in a particular species generally requires production of recombinant IFN7 of that species. Since the goals of most interferon cloning operations are deduction of the amino acid sequence and production of recombinant IFN, most IFN7 sequences have been derived from mRNA rather than chromosomal DNA. The advent of the polymerase chain reaction has, however, facilitated the cloning of cytokine cDNAs and the nucleotide sequences of the genes or cDNAs encoding a number of mammalian cytokines to been determined.

The most extensive structural studies of IFN7 have been performed on the human cytokine. The human IFN7 gene is 6kbp in size and possesses 3 introns (Taya et al. 1982) .

The human IFN7 gene encodes a 166 amino acid precursor possessing a 23 amino acid N-terminal signal sequence and two N-linked glycosylation sites. Functional IFN7 is homodimeric with the polypeptides associated in a non- covalent manner (Scahill et al. 1983) . Several forms of human IFN7 exist due to post-translational C-terminal proteolytic cleavage and differential glycosylation (Rinderknecht et al. 1984) . Regions of N-terminal and C- terminal sequence have been determined as being important in maintaining biological activity (Farrar and Schreiber, 1993) .

Cytokine and anti-cytokine therapies such as synthesis of inhibitors, soluble cytokine receptors, receptor antagonists or anti-cytokine antibodies, are finding increased clinical application in human medicine (Mire-Sluis, 1993) . Due to the lack of species cross- reactivity of many cytokines and the potential for antibody production against heterologous cytokines (Holmes, 1993), however, the ideal reagents in the horse will be substantially equine-specific.

The present invention provides a polynucleotide fragment, such as a DNA fragment, encoding equine 7- interferon.

The invention further provides a recombinant equine 7- interferon polypeptide.

"Polynucleotide fragment" as used herein refers to a chain of nucleotides such as deoxyribose nucleic acid (DNA) sequences and transcription products thereof, such as RNA, capable of giving rise to an equine 7-interferon protein or physiologically active fragment thereof. The term excludes the whole naturally ocurring genome.

Generally the polynucleotide will be in isolated form substantially free of biological material with which the whole genome is normally associated in vivo. Thus, this term includes double and single stranded DNA, and RNA sequences derived therefrom.

Jn general, the term "polypeptide" refers to a chain or sequence of amino acids displaying a biological activity substantially similar to the biological activity of equine 7-interferon and does not refer to a specific length of the product as such. The polypeptide if required, can be modified in vivo and in vitro. for example by glycosylation, amidation, carboxylation, phosphorylation and/or part translational cleavage; thus inter alia peptides, oligopeptides and proteins are encompassed thereby.

A polynucleotide fragment encoding equine 7-interferon can be amplified from equine 7-interferon cDNA, obtained by way of reverse transcription of mRNA, by polymerase chain reaction (PCR) , using primers designed against conserved regions of 7-interferon coding sequences from a single species, or a number of other species. An amplified fragment containing the equine 7-interferon is depicted in Figure 1.

The DNA fragment of Figure 1 was shown to encode an open reading frame (ORF) of 166 amino acids. A comparison of the amino acid sequence against previously sequenced 7- interferons revealed identities ranging from 45% with murine 7-interferon to 78% with bovine 7-interferon. The two potential N-linked glycosylation sites exist at positions conserved in bovine and pig 7-inteferon precursors at amino acids 39-41 and 106-108 in the equine precursor (data not shown) . In common with the human 7- interferon, equine 7-interferon has a predicted N-terminal signal sequence and possesses no cysteine residues.

The present invention includes polynucleotide and polypeptide sequences having at least 80 %, particularly at least 90 % and especially at least 95 % similarity with the sequences of Figure 1. "Similarity" refers to both identical and conservative replacement of nucleotides or amino acids, provided that the functionality of feline 7- interferon is substantially unimpaired.

The skilled man will appreciate that it is possible to genetically manipulate the gene or derivatives thereof, for example to clone the gene by recombinant DNA techniques generally known in the art and to express the polypeptides encoded thereby in vitro or in vivo. DNA fragments having the nucleotide sequence depicted in Figure 1 or derivatives thereof are preferably used for the expression of the equine 7-interferon.

It will be understood that for the particular equine 7-interferon polypeptide embraced herein, natural variations can exist between individuals or between species of the equis genus. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. All such derivatives showing active equine 7-interferon physiological activity are included within the scope of this invention. For example, for the purpose of the present invention conservative replacements

may be made between amino acids within the following groups:

(i) Alanine, Serine, Threonine;

(ii) Glutamic acid and Aspartic acid;

(iii) Arginine and Lysine;

(iv) Asparagine and Glutamine;

(v) Isoleuine, Leucine, and Valine;

(vi) Phenylalanine, Tyrosine and Tryptophan.

Moreover, recombinant DNA technology may be used to prepare nucleic acid sequences encoding the various derivatives outlined above.

As is well known in the art, the degeneracy of the genetic code permits substitution of bases in a codon resulting in a different codon which is still capable of coding for the same amino acid, e.g. the codon for the amino acid glutamic acid is both GAT and GAA. Consequently, it is clear that for the expression of a polypeptide with the amino acid sequence shown in Figure 1 or fragment thereof use can be made of a derivative nucleic acid sequence with such an alternative codon composition different from the nucleic acid sequence shown in said Figure 1.

Furthermore, fragments derived from the equine 7-interferon polypeptide or from the amino acid sequence depicted in Figure 1 which still display equine 7-interferon properties, or fragments derived from the nucleotide sequence encoding the equine 7-interferon polypeptide or derived from the nucleotide sequence depicted in Figure 1 encoding fragments of said equine 7-interferon polypeptides are also included in the present invention.

All such modifications mentioned above resulting in such derivatives of the equine 7-interferon polypeptide or gene are covered by the present invention so long as the characteristic equine 7-interferon biological properties remain substantially unaffected.

The equine 7-interferon polynucleotide fragment of the present invention is preferably linked to regulatory control sequences. Such control sequences may comprise promoters, operators, inducers, ribosome binding sites terminators etc. Suitable control sequences for a given host may be selected by those of ordinary skill in the art. In particular, an equine 7-interferon control sequence can be employed in a mammalian host.

A polynucleotide fragment according to the present invention can be ligated to various expression controlling sequences, resulting in a so-called recombinant nucleic acid molecule. Thus the present invention also includes an expression vector containing an expressible nucleic acid molecule. Said recombinant nucleic acid molecule can then be used for transformation of a suitable host. Such hybrid molecules are preferably derived from for example plasmids, or from nucleic acid sequence present in bacteriophages or viruses and are termed vector molecules.

Specific vectors which can be used to clone nucleic acid sequences according to the invention are known in the art (e.g. Rodriguez, R.L. and D.T. Denhardt, edit., Vectors: A survey of molecular cloning vectors and their uses, Butterworths , 1988).

A specific bacterial expression vector pHEX has been adapted for 7-interferon production (Reid, submitted for publication) . The pHEX vector has a stretch of 6 histidine residues and a thrombin site downstream of the vector- specified ATG initiation codon. The 6 histidine residues provide crude purification of recombinant proteins using affinity chromatography. The DNA insertion site is positioned 3' to the thrombin site so that should biological activity of the equine 7-interferon produced be compromised by the vector-specified N-terminus, cleavage of extraneous amino-acid sequence from the interferon moiety is possible.

The methods to be used for the construction of a recombinant nucleic acid molecule according to the invention are known to those of ordinary skill in the art and are inter alia set forth in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1989) .

The present invention also relates to a transformed cell containing the equine 7-interferon polynucleotide fragment in expressible form. "Transformation", as used herein, refers to the introduction of a heterologous polynucleotide fragment into a host cell, irrespective of the method used, for example direct uptake, transfection or transduction. The heterologous polynucleotide fragment may be maintained through autonomous replication or alternatively, may be integrated into the host genome. The recombinant nucleic acid molecules preferably are provided with appropriate control sequences compatible with the designated host which can regulate the expression of the inserted polynucleotide fragment e.g. tetracycline responsive promoter, thymidine kinase promoter and SV-40 promoter.

Suitable hosts for the expression of recombinant nucleic acid molecules can be prokaryotic or eukaryotic in origin. The most widely used hosts for expression of recombinant nucleic acid molecules may be selected from bacteria, yeast, insect cells and mammalian cells.

Since the biological half-life and the degree of glycosylation of recombinant equine 7-interferon may be important for use in vivo, yeast and baculovirus systems are preferred. The yeast strain Pichia pastoris exhibits potential for high level expression of recombinant proteins (Clare et al.1991) . The baculovirus system has been used successfully in the production of type 1 interferons (Smith et al. 1983).

The cloning and expression of recombinant equine 7-interferon also facilitates in producing reagents for the production of, for example, probes for in situ expression studies, production of anti-equine 7-interferon antibodies (particularly monoclonal antibodies) and evaluation of in vitro and in vivo biological activity of recombinant equine 7-interferon. The antibodies may be employed in diagnostic tests for 7-interferon.

The present invention further provides recombinant equine 7-interferon for the manufacture of reagents for use as prophylactic and/or therapeutic agents and also as an adjuvant in horses. In particular the invention provides pharmaceutical compositions comprising the recombinant 7- interferon together with a pharmaceutically acceptable carrier therefor.

A wide range of equine disorders, such as disease states attributable to equine 7-interferon deficiencies or over abundance and physiological or immunological abnormalities may benefit from equine 7-interferon therapy and/or prophylaxis. Such disease states include cancer, endotoxaemia, parasitic and bacterial infections, wound therapy, auto-immune and inflammatory diseases, allergies and viral infections. Equine 7-interferon may also be applied for vaccine adjuvantation.

Cytokines such as interferons , β and 7 and tumour necrosis factor, TNF, are reported as being capable of exhibiting direct antiviral activity in addition to their immunomodulatory properties (Ramsay et al. 1993) . The recombinant equine 7-interferon can prove beneficial as a prophylactic agent to modulate recrudescence of viruses, such as equine herpesvirus (EHV) , under conditions of stress and to reduce the clinical consequence of horizontal transmission of viral agents to in-contact animals. Herpes viruses and other viruses with complex genomes play an immunomodulatory role contributing to general or specific immunosuppression in the host. Administration of the equine 7-interferon can also serve to redirect the immune response such that more efficient virus clearance occurs. By way of example, administration of bovine IFN7 prior to experimental infection of cattle with bovine herpesvirus-1 (BHV-l) has been shown to reduce virus-induced suppression of cell-mediated cytotoxicity (Bielefeldt Ohmann and Babiuk, 1984).

As a consequence of the pleiotropic nature of the cytokine system, administration of the recombinant equine 7-interferon may have consequences on the expression and biological effect of other cytokines. Thus equine 7- interferon can be used as a tool to study these effects.

Non-specific stimulation of the immune response by adjuvant formulations is often beneficial in cases of prophylactic or therapeutic vaccination. However, in particular instances a more defined stimulation of the immune response using equine 7-interferon as an adjuvant or co- adjuvant can be more effective. Such a stimulation could take the form of administration of recombinant equine 7-interferon or expression of equine 7-interferon within live or disabled virus vectors. 7-interferon has previously been reported as being associated with adjuvancy in animals (Heath and Playfair, 1992). IFN7 upregulate MHC class II expression enhancing antigen presentation, and suppresses TH2 lymphocyte with resultant inhibition of humoral immunity. Through such actions an IFN7 adjuvant can drive a vaccine-protective immunity against viruses (Ra shaw et al. 1992) . Equine 7- interferon may also ameliorate clinical response to live virus vaccination which, providing protective immunity is not compromised, could be a desirable safety feature (Andrew et al. 1991; Ramshaw et al. 1992) . Candidate live virus vectors include poxviruses, adenovirus and herpesvirus, though the latent capacity of the latter coupled with potential immunopathology of cytokines may preclude cytokine insertion in this virus.

Administration of equine 7-interferon with live equine herpesvirus vaccines may be used to overcome mechanisms of immune evasion or diversion specified by the virus to provide a longer lasting protective immune response.

An embodiment of the invention will now be described by way of example only.

EXAMPLE 1

Molecular cloning of the equine 7-Interferon cDNA mRNA was isolated from mitogen-stimulated equine lymphocytes using the RNAgents (Trademark) Total RNA isolation system (supplied by Promega Corp.) 5μg of the mRNA was converted to cDNA using the Superscript (Trademark) system (supplied by Life Technologies) .

Two oligonucleotides (see below) were designed against interspecies conserved non-coding regions of 7- interferon mRNA.

Upstream primer (I):- 5'- ATGAAATATAC-3' Downstream primer (II):- 5'- TATTGCAGGCAG-3'

These oligonucleotides were then used as primers in a polymerase chain reaction (PCR) experiment to amplify the equine 7-interferon cDNA.

The PCR was performed as described by Saiki et al (1987) . Ten μl of lOOng/μl template cDNA from the reverse transcribed mRNA was added to a 40μl reaction mixture containing 200μM of dATP, dCTP, dGTP, dTTP, 50pmol of both primers (I) and (II) , 1 unit of DNA Polymerase and 5μl of lOx reaction buffer. The reaction buffer contained lOOmM Tris-HCl, 500mM potassium chloride, 0.01 per cent (w/v) gelatin and 1.5mM magnesium chloride, ultrapure water, TE (pH8.0). The solution was overlaid with two drops of mineral oil to prevent evaporation. To eliminate the possibility of false positives from the contamination of genomic samples with preparation of cDNA, amplification and analysis of the products were carried out. Thirty five cylces of amplification were performed using a Perkin Elmer Cetus thermal cycler. Each cycle consisted of 1 min. at 95°C to denature the DNA, 1 min. at 50°C to anneal the primers to the template and 1 min. at 72°C for primer extension. After the last cycle a further incubation for lOmins. at 72°C was performed to allow extension of any partially completed product. On completion of the amplification, lOμl of the reaction mixture was electrophoresed through a 1.5 per cent agarose gel. The DNA was visualised by staining with ethidium bromide and exposure to ultraviolet light (320nm) .

An amplified reaction product of approximately 600 bp was observed and the DNA purified and cloned into the plasmid vector pBluescript (Trademark) (supplied by Stratagene) .

EXAMPLE 2

Sequencing of the cloned equine 7-interferon cDNA

The pBluescript (Trademark) derived vector, containing the cloned equine 7-interferon DNA was prepared and purified. Double stranded DNA sequencing was carried out on this DNA using the T7 polymerase sequenase (Trademark) kit (supplied by USB Corp.) as per the manufacturers instructions.

The DNA sequence obtained is shown in Figure 1. The two underlined regions at the ends of the sequence correspond to the two primers (I) and (II) used to amplify the DNA. The two internal underlined regions represent potential glycosylation sites.

A translation of the DNA sequence into its corresponding amino acid sequence (one-letter code) is shown below the DNA sequence

EXAMPLE 3

Expression of recombinant equine 7-interferon in

Escherichia coli.

The open reading frame (ORF) encoding the equine 7- interferon was excised from the pBluescript derived vector and sub-cloned into the plasmid vector pHEX (Reid, 1994 submitted) for expression.

The pHEX vector was constructed from the vector pGEX- 2T (Smith and Johnson, 1988) . The sequence of the glutathione S-transferase gene was deleted from pGEX-2T and a sequence which contained a consensus to the 5' coding regions of bacterial mRNA and also six histidine residues was inserted 3' to the initiating AUG (see below)

pHEX BamHI EcoRI

Met Ala Lys lie Asn His His His His His His Gly Ser Glu Phe taOATG GCT AAA ATA AAT CAT CAC CAT CAC CAT CAC GGA TCC GAA TTC

On expression of the cloned equine 7-interferon DNA, a polypeptide of approximately 20 kilodaltons was observed, when crude lysates of IPTG induced cultures were analysed on a 12% acrylamide gel (Laemmli, 1970), followed by staining with coomasie brilliant blue dye. REFERENCES

1. Andrew ME, Karupiah G, Boyle DB, Blanden RV, Mullbacher A, Ramshaw IA and Couper BEH. (1991) . Effects of vaccinia virus-expressed interleukin 2 on the immune system of sublethally irradiated mice. Microbial Pathooenesis. 10, 363-371.

2. Balkwill FR and Burke F. (1989) . The cytokine network. Immunol. Today. 10, 299-304.

3. Bielefeldt Ohmann HB and Babiuk LA. In vitro and systemic effects of recombinant bovine interferons on natural cell-mediated cytotoxicity in healthy and herpes-infected calves. J.Leukocyte Biol.. 36, 451- 456.

4. Clare JJ, Rayment FB, Ballantine SP, Sreekrishna K and Romanos MA. (1991) . High-level expression of tetanus toxin fragment C in Pichis pastoris strains containing multiple tandem integrations of the gene. Biotechnology. 9, 455-460.

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Claims

1. A polynucleotide fragment encoding equine 7- interferon.

2. A polynucleotide fragment according to claim 1 characterised in that said DNA fragment encodes a polypeptide having an amino acid sequence shown in Figure 1 or a derivative thereof.

3. A polynucleotide fragment according to claim 1 characterised in that it is a DNA fragment which is substantially the same as the DNA fragment shown in Figure 1 or a derivative thereof.

4. A recombinant nucleic acid molecule comprising a polynucleotide fragment according to any one of claims 1 to 3.

5. A recombinant nucleic acid molecule according to claim 4 characterised in that the recombinant nucleic acid molecule comprises regulatory control sequences operably linked to said polynucleotide fragment for controlling expression of said polynucleotide fragment.

6. A recombinant nucleic acid molecule according to any one of claims 4 or 5 wherein the recombinant nucleic acid molecule is a plasmid.

7. A recombinant nucleic acid molecule according to any one of claims 4 or 5 wherein the recombinant nucleic acid molecule is derived from a viral vector.

8. A prokaryotic or eukaryotic host cell transformed, by a polynucleotide fragment or recombinant molecule according to any one of the preceding claims.

9. A recombinant equine 7-interferon polypeptide or derivative thereof displaying substantially the same biological activity as naturally occurring equine 7- interferon.

10. A recombinant equine 7-interferon polypeptide as shown in Figure 1 or derivatives thereof.

11. An antibody immuno-reactive with a polypeptide or fragment according to claims 9 or 10.

12. A polynucleotide fragment according to any one of claims 1 to 3 for use in therapy.

13. A recoir inant nucleic acid molecule according to any of claims 4 -o 6 for use in therapy.

14. A recombinant polypeptide or derivative thereof according to claims 9 or 10 for use in therapy.

15. A pharmaceutical composition comprising a polynucleotide fragment according to any of claims 1 to 3.

16. A pharmaceutical composition comprising a polypeptide or derivative thereof according to claims 9 or 10.