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WO1997000321A1 - Modulateurs de la reponse immunitaire et leur utilisation - Google Patents

Modulateurs de la reponse immunitaire et leur utilisation Download PDF

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Publication number
WO1997000321A1
WO1997000321A1 PCT/AU1996/000360 AU9600360W WO9700321A1 WO 1997000321 A1 WO1997000321 A1 WO 1997000321A1 AU 9600360 W AU9600360 W AU 9600360W WO 9700321 A1 WO9700321 A1 WO 9700321A1
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WO
WIPO (PCT)
Prior art keywords
ovine
seq
leu
nucleic acid
sequence
Prior art date
Application number
PCT/AU1996/000360
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English (en)
Inventor
Heng-Fong Seow
Paul Wood
Original Assignee
Commonwealth Scientific And Industrial Research Organisation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AUPN3502A external-priority patent/AUPN350295A0/en
Priority claimed from AUPN6244A external-priority patent/AUPN624495A0/en
Application filed by Commonwealth Scientific And Industrial Research Organisation filed Critical Commonwealth Scientific And Industrial Research Organisation
Priority to AU59917/96A priority Critical patent/AU5991796A/en
Publication of WO1997000321A1 publication Critical patent/WO1997000321A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5409—IL-5
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates generally to recombinant polypeptides having ovine cytokine properties and to genetic sequences encoding same. More particularly, the present invention is directed to recombinant ovine interleukins and specifically interleukin-5 (IL-5) and interleukin- 12 (LL-12) and their use as immune response modulators, especially in vaccine compositions.
  • IL-5 interleukin-5
  • LL-12 interleukin- 12
  • Cytokine research is of particular importance, especially as these molecules regulate the proUferation, differentiation and function of a variety of cells such as cells involved in mediating an immune response.
  • Administration of recombinant cytokines or regulating cytokine function and/or synthesis is becoming, increasingly, the focus of medical research into the treatment of a range of disease conditions in humans and animals.
  • Cytokines are the hormones of the immune system which control and determine the nature of the immune response (Balkwill and Burke, 1989).
  • Examples of cytokines include interleukins which primarily effect the functional activity of the lymphocytes involved in specific cell-mediated and antibody responses; colony stimulating factors which regulate the maturation of precursor cells into macrophages, granulocytes, mast cells and lymphocytes which are involved in innate resistance to many pathogens (Metealf, 1987); the interferons, which, in addition to their direct anti-viral action, also stimulate antibody synthesis, the activity of natural killer cells and the antimicrobial activity of macrophages and neutrophils (Bielefeldt Ohmann et al, 1987). All these molecules have the potential to alter the disease resistance and immune responsiveness of animals to a wide variety of infectious diseases and vaccines.
  • cytokines Much research has been undertaken into the use of cytokines to augment the immune response and to enhance the immunocompetence of the host to eliminate foreign pathogens.
  • cytokines comparatively few cytokines are used directly or targeted in therapeutic regimens, especially in animals.
  • cytokines are used directly or targeted in therapeutic regimens, especially in animals.
  • One reason for this is the pleiotropic nature of many cytokines.
  • interleukin- 11 EL-11
  • EL-11 is a functionally pleiotropic molecule capable of inducing multipotential haemopoietin progenitor cell proliferation, enhancing megakaiyocyte and platelet formation, stimulating acute phase protein synthesis and inhibiting adipocyte lipoprotein lipase activity.
  • IL-5 is a potent growth promoter of early haemopoietic progenitor cells. It also promotes the generation of cytotoxic cells from thymocytes and murine EL-5 stimulates the production and secretion of IgM and IgA by B cells in synergism with bacterial endotoxins (Sonada et al, 1992). Secretory IgA antibodies directed against specific virulence determinants of infecting organisms play an important role in overall mucosal immunity. IL-5 is also a specific stimulator of eosinophil differentiation as well as a selective chemoattractant and eosinophil activation factor.
  • IL-12 is a heterodimeric cytokine composed of a 40-kDa subunit (p40) disulfide-linked to a 35-kDa subunit (p35) (Kobayashi et al 1989); (Stern et al 1990). It induces the production of IFN- ⁇ by T and NK cells, stimulates the proliferation of activated T and NK cells and enhances the specific and non-specific cytolytic lymphocyte responses. Accumulating evidence suggests that the lack of effective protection against infectious pathogens may result from the selective activation of T cells with an aberrant cytokine profile. Generally, protection against intracellular bacteria and viruses requires a Thl-type response. IL-12 is the critical cytokine that drives differentiation of naive cells to the Thl subset resulting in the Thl-type immune response. Thus, IL-12 plays a vital role in inducing protective effector mechanisms against bacterial and viral infections.
  • p40 40-kDa subunit
  • p35 35-kDa sub
  • one aspect of the present - vention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding, or complementary to a nucleotide sequence encoding, an ovine cytokine or a functional or immunologically interactive homologue, analogue or derivative thereof, wherein said ovine cytokine is IL-5 or IL-12 or a polypeptide subunit of IL-12, or is a fusion cytokine between different subunits of IL-12.
  • references to "IL-12” or “ovine IL-12” shall be taken to include all possible 5 monomeric, dimeric or other multimeric forms comprising the 35 kDa or 40 kDa polypeptide subunits, including heterodimers and homodimers comprising same.
  • References herein to "IL- 12” shall also be taken to include all possible fusion cytokines between the 35 kDa and the 40 kDa polypeptide subunits of ovine IL-12. In a particularly preferred embodiment however, references contained herein to "IL-12” indicates a heterodimer formed between the 35 kDa and 10 40 kDa polypeptide subunits.
  • nucleotide sequence of the cloned cytokines will most preferably include the sequences set forth in SEQ ID No: 1 or SEQ ID No: 3 for IL-5, SEQ ID No: 5 or SEQ ID No:
  • (ii) is capable of hybridising under at least medium stringency conditions to one or more of the nucleotide sequences set forth in SEQ ID Nos: 1,3,5,7, or 9 or a complementary sequence or a homologue, analogue or derivative thereof; and (iii) wherein said interleukin comprises an amino acid sequence corresponding to all
  • the present invention provides an isolated DNA molecule which: 30 (i) encodes a molecule having IL-5 activity;
  • interleukin comprises an amino acid sequence corresponding to SEQ ID No: 2 or SEQ ID No: 4 or having 70% or greater similarity thereto.
  • (i) encodes a molecule having EL- 12 activity; (ii) is capable of hybridising under at least medium stringency conditions to one or more ofthe nucleotide sequences set forth in SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ
  • ID NO: 9 or a complementary form or a homologue, analogue or derivative thereof.
  • interleukin comprises an amino acid sequence corresponding to all or a part of one or more ofthe amino acid sequences set forth in SEQ ID No: 6 or SEQ
  • a high stringency wash is defmed herein to be 0. l-0.2xSSC, 0.1% w/v SDS at 55-65 °C for 20 minutes and a medium level of stringency is considered herein to be 2xSSC, 0.1% w/v SDS at ⁇ 45°C for 20 minutes.
  • the alternative conditions are applicable depending on concentration, purity and source of nucleic acid molecules.
  • the present invention provides an isolated nucleic acid molecule which encodes or is complementary to a nucleic acid molecule which encodes an ovine IL-5 and having a nucleotide sequence substantially as set forth in SEQ ID NO: 1 or SEQ ID NO: 3, or a homologue, analogue or derivative thereof.
  • the present invention provides an isolated nucleic acid molecule which encodes, or is complementary to a nucleic acid molecule which encodes a 35 kDa subunit of ovine IL-12 and having a nucleotide sequence substantially as set forth in SEQ ID NO: 5 or SEQ ID NO: 7, or a homologue, analogue or derivative thereof.
  • the present invention provides an isolated nucleic acid molecule which encodes or is complementary to a nucleic acid molecule which encodes a 40 kDa subunit of ovine EL- 12 and having a nucleotide sequence substantially as set forth in SEQ ID NO: 9 or a homologue, analogue or derivative thereof.
  • homologue in relation to a variant genetic sequence, refers to a gene which encodes a polypeptide which retains its function as an interleukin molecule or subunit of same, although said polypeptide may contain amino acid substitutions, deletions and/or additions.
  • homologue in relation to a variant polypeptide refers to a polypeptide containing amino acid substitutions, amino acid deletions and/or amino acid additions which do not affect the function ofthe polypeptide.
  • amino acids may be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment or antigenicity, and so on.
  • the present invention clearly extends to homologues of ovine IL-5 or IL-12 genetic sequences.
  • analogue as used hereinafter in reference to a nucleic acid molecule, shall be taken to refer to a variant genetic sequence which is functionally equivalent to a genetic sequence which encodes or is complementary to a genetic sequence which encodes an ovine IL- 5 or ovine IL-12 polypeptide, but which contains certain non-naturally occurring or modified residues.
  • analogue when used in relation to a polypeptide molecule shall be taken to refer to a variant polypeptide which is functionally equivalent to an ovine IL-5 or an ovine IL-12 polypeptide, but which contains certain non-naturally occurring or modified residues.
  • a "derivative" of a nucleic acid molecule or a polypeptide molecule may not have the same physiological activity as the genetic sequence or polypeptide from which it was derived, however it is useful in the isolation of related genetic sequences or polypeptides, or in modifying gene expression, for example by antisense or ribozyme technology, or in the production of useful immunoreactive molecules, for example the production of useful subunit vaccines.
  • the present invention provides a genetic construct comprising at least one of the nucleotide sequences set forth in SEQ ID Nos: 1,3,5,7 or 9 or a homologue, analogue or derivative thereof.
  • the genetic constructs ofthe present invention are particularly useful for the production of recombinant cytokine molecules encoded therein, when introduced into a cell line and under conditions suitable for gene expression to occur. Such conditions will depend upon the cell line and the expression vector used in each case and would be well-known to the person skilled in the art.
  • any number of expression vectors can be employed depending on whether expression is required in a eukaryotic or prokaryotic cell.
  • the promoter sequence used in the expression vector will also vary depending upon the level of expression required and whether expression is intended to be constitutive or regulated.
  • Examples of eukaryotic cells contemplated herein include mammalian, yeast, insect or plant cells and examples of prokaryotes include Escherichia coli, Bacillus sp. and Pseudomonas sp.
  • Typical promoters suitable for expression in bacterial cells such as E. coli include, but are not limited to, tac promoter, the lac ⁇ promoter, or the phage lambda ⁇ L or ⁇ R promoters.
  • a still further aspect ofthe present invention provides a recombinant isolated ovine IL-5 or IL-12 polypeptide or a homologue, analogue or derivative thereof.
  • recombinant cytokine or related term “recombinant molecule” is meant a glycosylated or unglycosylated polypeptide molecule, with or without other associated molecules (eg.
  • lipids produced by recombinant means such as presence of a DNA molecule in an expression vector in the correct reading frame relative to a promoter and introducing the resultant recombinant expression vector into a suitable host and growing said host under conditions appropriate for expression and, if necessary, transportation ofthe recombinant protein or its derivative from said host and then purifying the recombinant molecule.
  • a recombinant polypeptide comprising a sequence of amino acids which is substantially the same as the amino acid sequence set forth in any one ir more of SEQ ID Nos: 2,4,6,8, or 10, or is at least 70% identical to same.
  • the present invention extends to any derivatives of ovine IL-5 or IL-12 polypeptides set forth in SEQ ID NOS. 2, 4, 6, 8 or 10. Derivatives of ovine IL-5 and IL-12 include single or multiple amino acid substitutions, deletions and/or additions to the molecule.
  • these are prepared by first making single or multiple nucleotide substitutions, deletions and/or additions to the nucleic acid molecule encoding the ovine cytokine.
  • amino acids can be chemically added by established techniques and in any sequence required to give the desired mutant. All such derivatives are encompassed by the present invention.
  • Amino acid insertional derivatives of the ovine cytokines of the present invention include amino and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening ofthe resulting product.
  • Deletional variants are characterised by the removal of one or more amino acids from the sequence.
  • Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Typical substitutions are those made in accordance with Table 1.
  • amino acids are generally replaced by other amino acids having like properties, such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and the like.
  • Amino acid substitutions are typically of single residues.
  • Amino acid insertions will usually be in the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues.
  • deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues and a corresponding insertion of two residues. TABLE 1 Suitable residues for amino acid substitutions
  • ovine cytokine IL-5 or IL-12 includes reference to any derivatives thereof as contemplated above.
  • the amino acid variants referred to above may be readily made using synthetic peptide techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having known or partially known sequence are well known and include, for example, Ml 3 mutagenesis. The manipulation of DNA sequence to produce variant proteins which manifest as substitutional, insertional or deletional variants are conveniently described, for example, in Sambrook et al (1989).
  • recombinant or synthetic mutants and derivatives of the ovine cytokines of the present invention include single or multiple substitutions, deletions and/or additions of any molecule associated with the enzyme such as carbohydrates, lipids and/or proteins or polypeptides.
  • ovine IL-5 and IL-12 molecules contemplated herein will find particular appUcation in the intensive livestock industries such as the live animal export trade, feed-lots and intensive rearing industries. Animals in close containment are subjected to greater environmental challenge with infectious diseases, particularly respiratory infections and are more prone to the immunodepressive effects of stress leading to higher susceptibility to opportunistic pathogens.
  • a method for the treatment and/or prophylaxis of a Uvestock animal exposed to or infected with a pathogenic organism comprising administering to said animal an immunoresponsive effective amount of ovine EL-5 and/or ovine IL-12 or a homologue, analogue or derivative thereof for a time and under conditions sufficient to maintain, stimulate or enhance the immunoresponsiveness of said animal.
  • the ovine cytokine is a recombinant molecule.
  • livestock animal extends to sheep, horses, pigs, cows, donkeys, emus, ostriches, alpacas, camels, deer, goats, amongst other animals, provided that the ovine cytokines are effective in those animals.
  • Particularly preferred livestock animals are sheep and cows.
  • cytokines of the present invention are used as natural adjuvants for vaccines, particularly for subunit vaccines produced by recombinant DNA technology.
  • IL-5 and/or IL-12 are used in vaccines to enhance the immunogenicity of antigens, particularly in subunit vaccines. Advances in slow- release technology and the development of live non-pathogenic bacteria and viruses as delivery vectors for these molecules will ensure their cost-effectiveness in sheep and cattle. They may also be used as in nucleic acid vaccination.
  • the present invention extends to a method of enhancing and/or stimulating an immune response to one or more antigens in an animal, said method comprising administering to said animal an immunoresponsive effective amount of IL-5 and/or EL- 12.
  • a vaccine comprising an antigen and recombinant ovine IL-5 and/or IL-12 or their derivatives.
  • the vaccine may also comprise one or more pharmaceutically acceptable carriers and/or diluents.
  • the carriers and/or diluents are also required to be acceptable for veterinary use.
  • the ovine IL-5 and/or IL-12 may also be deUvered by genetic means.
  • the recombinant ovine EL-5 and/or IL-12 may be encoded by a genetic construct present in a delivery system such as a virus, yeast, bacterium, protozoan, insect or mammalian cell. The presence of such a delivery system in a target animal will enable delivery of the recombinant ovine cytokine.
  • a genetic construct comprising a first nucleotide sequence encoding ovine IL-5 or ovine IL-12 or their derivatives and a second nucleotide sequence defining a deUvery vehicle.
  • the delivery vehicle is capable of replication in a delivery cell such as a bacterial, yeast, insect, a protozoan animal or a mammalian cell. Generally, the delivery cells would not in normal use be harmful or pathogenic to the target animal.
  • attenuated deUvery cells are employed. Particularly useful delivery cells are bacterial cells, attenuated viruses and particularly suitable delivery vectors are recombinant viral and bacterial vectors.
  • an attenuated infectious virus is used as a live vaccine.
  • the genetic sequence encoding ovine IL-5 and/or IL-12 or their derivatives is cloned into the viral sequence and the recombinant virus used to infect target animals.
  • the recombinant virus causes infection and replicates in the animal cells resulting in production of the recombinant cytokine.
  • the infecting recombinant virus may subsequently be eliminated after production of an immunomodulating effective amount of the cytokine.
  • a similar protocol is adopted with live bacterial carriers.
  • a recombinant viral vector may be used.
  • a viral vector provides a modified virus capable of infecting a cell but not replicating therein.
  • a viral vector provides a means of introducing a genetic sequence which is transiently capable of expression into the desired cytokine.
  • An "immunomodulating effective amount” is an amount of cytokine sufficient to effect immunomodulation in the target animal, i.e. to enhance the ability of the immune system to develop an effective immune response or to enhance the immunocompetence of the animal or immunogenicity of an antigen which may also be expressed in the genetic vector.
  • the present invention extends to a veterinary pharmaceutical composition for use in Uvestock animals such as to enhance the immune system or accelerate its maturation or improve its immunocompetence or to facilitate immunomodulation in said animals, said composition comprising recombinant ovine EL-5 and/or IL-12 or their derivatives, recombinant ovine IL-5 and/or IL-12 fused to an antigen or to each other with or without antigen or genetic sequences encoding same in suitable delivery vehicles.
  • the composition comprises a recombinant cytokine
  • the composition is injected or orally administered.
  • the composition comprises genetic material, it is administered as part of a viral vector, live viral vector, live bacterial vector or nucleic acid vaccine.
  • Conditions in Uvestock animals for which treatment might be required include infectious disease, cancer, immunosuppression, allergy and to enhance or suppress reproductive systems. Conditions would also include situations where animals are in an immunocompromised state such as during or following stress, due to overcrowding and transport process, changes in climate and early weaning.
  • the administration of the cytokine molecules may also promote growth and or early maturation.
  • the animal to be treated and the cytokine in the composition might be "homologous" in the sense that both are ofthe same species, i.e. both ovine species or may be "heterologous" where the ovine cytokine is effective in another animal.
  • the compositions may also contain other active molecules such as antibiotics or antigen molecules. Combinations of cytokine molecules with antigen molecules may increase the efficacy of vaccines.
  • the present invention therefore, provides a veterinary pharmaceutical composition
  • a veterinary pharmaceutical composition comprising an immunomodulatingly effective amount of ovine IL-5 and/or IL-12 or their derivatives or genetic sequences capable of expressing same and one or more carriers and/or diluents acceptable for veterinary use.
  • the active ingredient(s) of the pharmaceutical composition is/are contemplated to exhibit exceUent activity in stimulating, enhancing or otherwise faciUtating an immune response in an animal species and in particular a Uvestock animal when administered in an amount which depends on the particular case.
  • the variation depends, for example, on the cytokine and, in some cases, the antigen involved in stimulating the immune response. For example, from about 0.5 ⁇ g to about 100 ⁇ g of a particular cytokine which may be combined with other cytokines, per kilogram of body weight per day may be required. Dosage regimen may be adjusted to provide the optimum therapeutic response.
  • the active compound may be administered by injection or by oral ingestion in any convenient manner or may be administered via a genetic sequence such as in a viral or bacterial vector or a nucleic acid vaccine.
  • the active compounds may also be administered in dispersions prepared in glycerol,
  • the pharmaceutical forms suitable for parenteral administration include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants.
  • the prevention ofthe action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, tJ irmerosal and the like. In many cases, it wiU be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example.
  • Carriers and/or diluents suitable for veterinary use include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the Uke.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the composition is contemplated.
  • Supplementary active ingredients can also be incorporated into the compositions. The latter is particularly contemplated as far as the present invention extends to multivalent vaccines or multi-component cytokine molecules.
  • compositions of the present invention may comprise in addition to IL-5 and/or IL-12 or their derivatives, one or more other active compounds such as antigens and/or immune stimulating compounds.
  • the cytokine may also be delivered by a live delivery system such as using a bacterial expression system to express the cytokine protein in bacteria which can be incorporated into gut flora.
  • a viral expression system can be employed or incorporated into a BCG vaccine.
  • one form of viral expression is the administration of a live vector generally by spray, feed or water where an infecting effective amount of the live vector (e.g. virus or bacterium) is provided to the animal.
  • Another form of viral expression system is a non-repUcating virus vector which is capable of infecting a cell but not replicating therein.
  • the non-replicating viral vector provides a means of introducing genetic material for transient expression into a cytokine.
  • the mode of administering such a vector is the same as a live viral vector.
  • the present invention extends to antibodies raised against ovine IL-5 or IL-12.
  • the antibodies may be monoclonal or polyclonal and may be used for developing enzyme- immunosorbent assays for the rapid diagnosis of infectious diseases of livestock animals.
  • an antibody preparation comprising antibodies or derivatives thereof, immuno interactive with either IL-5 or IL-12 or derivatives thereof.
  • Immunoassays are useful in detecting the presence of EL-5 and/or D -12 in a target animal.
  • a second IL-5/IL-12 antibody labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing sufficient time for the formation of a tertiary complex of antibody-EL-5/IL-12-antibody. Any unreacted material is washed away, and the presence ofthe tertiary complex is determined by observation of a signal produced by the reporter molecule. The results may either be quaUtative, by simple observation of the visible signal or may be quantitated by comparison with a control sample containing known amounts of hapten.
  • the sohd substrate is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing the molecule to the insoluble carrier.
  • reporter molecule is meant a molecule which, by its chemical nature, produces an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecule in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes).
  • an enzyme immunoassay an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist which are readily available to one skilled in the art.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, ⁇ -galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. It is also possible to employ fluorogenic substrates, which yield a fluorescent product.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescent compounds may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by iUumination with Ught of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the Ught at a characteristic colour visually detectable with a light microscope.
  • the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the hapten of interest.
  • Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method.
  • reporter molecules such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed. It will be readily apparent to the skilled techmcian how to vary the procedure to suit the required purpose.
  • Figure 1 is a schematic representation showing the nucleotide sequence of the exons and the intron/exon splice junctions of the ovine IL-5 gene [SEQ ID NO: 1].
  • Figure 2 is a schematic representation showing the ahgnment ofthe deduced amino acid sequence of ovine IL-5 [SEQ ID NO:2] with human and mouse IL-5 polypeptides.
  • Figure 3 is a schematic representation showing an alignment of the deduced partial amino acid sequence of ovine IL-12 35kDa subunit [SEQ ID NO: 6] with human and mouse IL- 12 polypeptide molecules.
  • the symbol (•) indicates that the amino acid is missing.
  • Figure 4 is a schematic representation showing the construction ofthe expression vector pCI-neo/p35, which expresses ovine IL-5 as a fusion protein with a polyhistidine (6xHis) polypeptide.
  • Figure 5 is a schematic representation showing the construction of a vector which expresses ovine EL-5 as a fusion protein with glutathione-S-transferase in a pGEX bacterial expression vector.
  • Figure 6 is a schematic representation showing the construction of a vector which expresses 35 kDa ovine EL- 12 as a fusion protein with glutathione-S-transferase in a pGEX bacterial expression vector.
  • Figure 7 is a schematic representation showing the construction of a vector which expresses 40 kDa ovine IL-12 as a fusion protein with glutathione-S-transferase in a pGEX bacterial expression vector.
  • Figure 8 is a schematic representation showing the expression vector pCI-neo/IL-12 which co-expresses the 35 kDa and 40 kDa ovine IL-12 subunits under the control of the CMV I.E promoter/enhancer sequence.
  • Figure 9 is a graphical representation showing the biological activity of recombinant ovine IL-5 (rOvIL-5) in a murine BAF cell (IL-5 dependent cell line) proliferation assay.
  • An ovine genomic library constructed in EMBL3 vector was obtained from Clontech,
  • Phage were used to infect Escherichia coli strain K802. One hundred thousand plaques were transferred onto nylon filters and screened with a ( 32 P ⁇ -labelled fragment of human IL-5 cDNA. The filters were washed at 2xSSC, 0.1% (w/v) SDS, 65°C for 20 minutes. Five positive plaques were purified after three rounds of plating and screening. When the phage DNA was used as the template for PCR, all five preparations of DNA gave a strong intense band of approximately 2 kb in size. The phage DNA was used as template in the PCR using the primers as follows:
  • the conditions for PCR were 30 cycles of 94°C for one min, 55 ⁇ C for two min and 72 °C for 2 min.
  • the amplified PCR product was cloned into the Smal site of pUC18 vector and 3 clones were sequenced by dideoxy sequencing using an automatic DNA sequencer.
  • RNA from peripheral lymph node cells stimulated for 24 hours with Concanavalin (5 ⁇ g/ml) was isolated using Tri ol (Gibco, BRL) according to the manufacturer's instructions.
  • RNA (5 ⁇ g) was reverse-transcribed to produce single-stranded cDNA, using Superscript RNase H-reverse transcriptase (Gibco- BRL).
  • Ovine EL-5 cDNA sequences were then amplified in a polymerase chain reaction using Taq polymerase (Gibco- BRL) and the following primers: CGCGGATCCATGCATCTGCGTTTGACCTTG [SEQ ID No: 13] forward primer and
  • the primers were based on the genomic sequence of ovine IL-5 gene set forth in SEQ ID NO: 1.
  • the conditions for PCR were 30 cycles of amplification as follows: 94°C for 30 seconds;
  • the ampUfied DNA was cloned into the Sfrl site of pCRSCRIPTSK + (Stratagene, USA). Four clones were sequenced in both directions using the M13 forward primer and the reverse primer using the AppUed Biosystem 373 A DNA sequencer.
  • the complete nucleotide sequence of the IL-5 cDNA clone is set forth in SEQ ID NO: 3.
  • the predicted amino acid sequence of full-length ovine IL-5 is set forth in SEQ ID NO: 4.
  • the cells were washed twice in Dulbecco's modified Eagle's medium (Flow, AustraUa) supplemented with 20 mM Hepes, 9 mM sodium bicarbonate, 2 mM glutamine, 50 ⁇ M 2-mercaptoethanol, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 10% (v/v) heat- inactivated foetal bovine serum.
  • the cells were resuspended in culture medium and viable cells enumerated by trypan blue exclusion. Cells were found to be greater than 95% macrophages as estimated by microscopic examination.
  • the macrophages were cultured for 5 h at 37° C in 90 mm plastic tissue culture petri dishes (1 x IO 7 cells/dish) containing 12 ml of culture media and 20 ⁇ g/ml of lipopolysaccharide (Sigma, USA). The adhered macrophages were washed with PBS and then lysed in 1 ml Trizol (Gibco-BRL).
  • RNA from LPS-stimulate ovine alveolar macrophages lysed in Trizol were isolated according to manufacturer's instruction. An amount of 5 ⁇ g of RNA was used for first strand complementary DNA synthesis using Superscript RNase H- reverse transcriptase (Gibco-BRL) and PCR performed with Taq polymerase (Gibco-BRL) and the following primers:
  • the amplified DNA fragment was subcloned into the BamHl site plasmid pUC18. DNA sequencing by the dideoxy termination method was performed on both strands using the universal and reverse primers.
  • Figure 3 shows the aUgnment ofthe deduced partial amino acid sequence [SEQ ID NO: 6] of the 35 kDa subunit of ovine IL-12 compared to the bovine, human and mouse IL-12 cytokines.
  • the level of amino acid homology with human and mouse equivalents are 79 and 61%, respectively.
  • EXAMPLE 4 CLONING A FULL-LENGTH cDNA ENCODING THE 35 kDa SUBUNIT OF
  • Ovine alveolar macrophages were lipopolysaccaride (LPS)-stimulated as described in the preceding Examples for 4 hours. Macrophages were subsequently lysed in Trizol (Gibco BRL) and RNA was isolated according to the manufacturer's instructions. RNA (5 ⁇ g) was used as a template for first-strand cDNA synthesis using Superscript RNase H- reverse transcriptase (Gibco-BRL). Ovine IL-12 sequences were amplified using the cDNA as a template and the following primers:
  • the ampUfied DNA fragment was subcloned into the Smal site of plasmid pUC 18.
  • DNA sequencing was performed using the Applied Biosystem 373A DNA sequencer. Both strands were sequenced using the universal and reverse sequencing primers.
  • IL-12 is set forth in SEQ ID NO: 7.
  • the predicted amino acid sequence of the 35 kDa subunit of ovine EL-12 is set forth in SEQ ID NO: 8.
  • Ovine peripheral lymph node cells were cultured with the phorbol ester, phorbol myristate acetate (PMA) at 10 ng/ml and calcium ionophore A23187 (0.5 ⁇ g/ml) for 24 hours and the cells were lysed in TRizol (Gibco BRL) according to the manufacturer's instructions.
  • RNA was isolated and PCR performed with the following primers:
  • the PCR product was cloned into the Smal site of the plasmid pUC18 and four clones were sequenced with the Ml 3 universal and reverse sequencing primers using the Applied Biosystem 373 A DNA sequencer.
  • the sequencing reactions were performed using the PRISMTM Dye DeoxyTM Terminator Cycle Sequencing Kit (Applied Biosystems). Using these primers approximately 350-400 nucleotide sequence could be determined from the 5' and 3' ends of the cDNA.
  • Subcloning step One of the fuU-length cDNAs encoding the 40 kDa subunit of ovine EL- 12 was digested with Sau3 AI and EcoRI and a DNA fragment of approximately 400bp was subcloned into the Ba HI/EcoKl site of pUCl 18. This subclone was sequenced using the universal and reverse primers as described above.
  • the complete nucleotide sequence ofthe fuU-length cDNA encoding the 40 kDa subunit of ovine IL-12 is set forth in S ⁇ Q ID NO: 9.
  • the predicted amino acid sequence of the full- length 40 kDa subunit of ovine IL-12 is set forth in S ⁇ Q ID NO: 10.
  • IL-5 dependent murine BAF cells were grown in the presence of murine X63 cell line supernatant [5%(v/v)] as an IL-5 source, in DMEM /10%(w/v)FCS.
  • a well-grown cell culture, grown in a 75cm 3 flask was decanted into a 50 ml centrifuge tube and centrifuged at 1200 rpm for 10 mins. Cells were then resuspended in 10 % (v/v) DMEM and recentrifuged at 1200rpm for 10 mins. Cell washes were repeated twice to remove exogenous EL-5 and finally resuspended to a concentration of 5 X IO 4 cells /ml.
  • Recombinant ovine IL-5 protein generated from the expression system was titrated in triplicate across a 96 well tissue culture plate then 100 ⁇ L of the washed BAF cell suspension was added to a final concentration of 5 X IO 3 cells/well .
  • Murine IL-5 was used as a positive control for cell proliferation.
  • the cell cultures were incubated in 5%(v/v) CO 2 at 37°C for 2 days then pulsed for 8-18 hr with tritiated thymidine, harvested and counted to determine the amount of radioactivity incorporated.
  • Recombinant ovine EL- 5 and IL-12 are prepared basically as described for the preparation of recombinant ovine IL-l ⁇ in International Patent Application No. PCT/ AU91/00419. Briefly, IL-5 and IL-12 are prepared as follows:
  • the ovine IL-5 cDNA encoding the mature IL-5 protein was obtained as described in Example 2.
  • the coding sequence for the mature form of the EL-5 gene was PCR-amplified using a thermostable polymerase and introduced into the polylinker of an expression vector belonging to the pQE-30 series of vectors, wherein the vector was selected such that the reading frame of IL-5 mature form coding sequence was in-frame with the reading frame of polyhistidine (6xHis) contained therein.
  • the resultant expression construct was designated pQE-30-IL-5.
  • Figure 4 is a schematic illustration of the expression construct pQE-30-IL-5.
  • the pQE-30-IL-5 expression construct was introduced into E.coli strain DH5 ⁇ , where induction of the P5 promoter results in high level expression of a fusion protein comprising the polyHis and IL-5 polypeptides.
  • the cDNA insert is ligated into the multiple cloning site of an expression vector belonging to the pGEX series of vectors, wherein the vector is selected such that the reading frame of IL-5 is in-frame with the reading frame of glutathione-s-tranferase contained therein (Smith and Johnson, 1988).
  • the IL-5 open reading frame is cloned immediately downstream of the thrombin cleavage site, to produce an in-frame fusion.
  • the recombinant plasmid is designated pGEX-IL-5.
  • Figure 5 is a schematic illustration ofthe expression plasmid pGEX-IL-5 showing the site of thrombin cleavage of the fusion protein. Transformants o ⁇ Escherichia coli strain JMl 09 are then produced.
  • bacterial colonies transformed with pQE-30-IL-5 were picked and cultured overnight at 37°C in LB growth medium [l%(w/v) tryptone, 0.5%(w/v) yeast extract, 1% (w/v) NaCl] supplemented with ampicillin (50 ⁇ g/ml). Flasks containing IL of LB growth medium and ampicillin (50 ⁇ g/ml) and a 1 :50 inoculum of overnight cultures were shaken at 37°C. After 2 hours, the P5 promoter of the expression construct pQE-30-EL-5 was induced with IPTG to a final concentration of ImM and incubated for a further for two hours.
  • IL-5 When IL-5 is expressed under the control of the tac promoter, overnight cultures of the pGEX-IL-5 plasmid are diluted in 250ml of Luria Broth (lOg/L bacto-tryptone, 5g/L yeast extract, lOg/L NaCl) or Terrific Broth (16.43g/L K 2 HPO 4 .3H 2 0, 2.3 lg/L KH 2 PO 4 , tryptone 12g/L, yeast 24g/L, glycerol 4ml/L) containing 100/ ⁇ g/ml ampicillin.
  • Luria Broth lOg/L bacto-tryptone, 5g/L yeast extract, lOg/L NaCl
  • Terrific Broth (16.43g/L K 2 HPO 4 .3H 2 0, 2.3 lg/L KH 2 PO 4 , tryptone 12g/L, yeast 24g/L, glycerol 4ml/L) containing 100/ ⁇
  • the cultures are grown for 2h at 37 °C before adding IPTG (isopropyl- ⁇ -tbiogalactopyranoside) to 0.2mM (or as indicated in the legend). Induction of the tac promoter results in high-level expression of a fusion protein between GST and EL-5. After 4h, the cultures are harvested and centrifuged. The pellets are weighed and resuspended in the appropriate volume of buffer (50mM Tris/HCl, pH7.5; lOml/g of wet weight of pellet). The cells are lysed on ice by sonication and then centrifuged.
  • buffer 50mM Tris/HCl, pH7.5; lOml/g of wet weight of pellet.
  • the supernatant is loaded onto a 5ml giutathione Sepharose column (sulphur-linkage, Sigma). The flow through is retained and the column in then washed thoroughly with at least 5 bed volumes of 50mM Tris/HCl, pH7.5.
  • the recombinant IL-5 protein is eluted either as a fusion product with 5mM giutathione or as free IL-5 by cleavage with human thrombin (lOU/ml; ICN) at room temperature for lh.
  • the eluted proteins are analysed by electrophoresis on a 15% (w/v) SDS/polyacrylamide gel and visualised by staining with 0.05% (w/v) Coomassie Brilliant Blue R
  • the cDNAs encoding the mature form of the 35 kDa and 40 kDa subunits of ovine IL-l ⁇ are cloned into the multiple cloning site of an expression vector belonging to the pGEX series of vectors, wherein the vector is selected such that the reading frame of each IL-12 cDNA sequence is in-frame with the reading frame of glutathione-s-tranf erase contained therein (Smith and Johnson, 1988), to produce the expression plasmids pGEX- EL12a and pGEX-IL12b, respectively.
  • Figures 6 and 7 are schematic illustrations of the expression plasmids pGEX-IL12a and pGEX-IL12b respectively, showing the sites of thrombin cleavage of the fusion protein. Induction of the lac promoter ofthe expression plasmids pGEX-lL12a and pGEX-IL12b results in high level expression of a fusion protein in each case.
  • elution from the column with giutathione yields GST-IL12 fusion proteins with approximate molecular weights of 61 kDa for the 35 kDa IL-12 subunit) and 66 kDa (for the 40 kDa JL-12 subunit).
  • the cDNA encoding the 35kDa subunit of ovine IL-12 is cloned into the multiple cloning site of the mammalian expression vector pCI-neo, between the CMV I.E enhancer/promoter/ intron sequence and the S V40 late polyadenylation sequence, to produce the intermediate expression vector pCI-neo/p35.
  • Expression of the 35 kDa EL- 12 polypeptide in pCI-neo/p35 is under the control of the CMV I.E enhancer/promoter and chimeric intron sequences
  • FIG. 8 is a schematic representation of the dual expression construct pCI-neo/IL-12.
  • the construct pCI-neo/TL-12 is introduced into a mammalian cell line for transient or stable expression. Expression is constitutive for both genes.
  • the EL- 12 heterodimer forms from the subunits and is secreted into the culture medium.
  • Protein assays Protein concentrations are estimated by the Bradford dye assay (Biorad) using bovine serum albumin as standard.
  • Recombinant IL-5 (rOvIL-5) was prepared and purified from cells transformed with the expression construct pQE-30-IL-5 as described in Example 7 and subsequently assayed for biological activity using the BAF mouse cell line bioassay described in Example 6. As shown in Figure 9, significant biological activity above that observed for control samples, was detected for rOvIL-5 produced using the expression vector pQE-30-IL-5 ( Figure 4), when the recombinant polypeptide was present in the assay samples at a concentration greater than 1/32 dilution.
  • the recombinant Taenia ovis fusion protein GST-45W (Johnson et al., 1989) is used as the model antigen in all studies.
  • Vaccines are formulated with 50 ⁇ g/dose of GST-45W and 0, 10 or lOO ⁇ g/dose of recombinant DL-5 and/or 35 kDa IL-12 and/or 40 kDa IL-12 polypeptides in either phosphate buffered saline (PBS) or the conventional adjuvants Quil A (1 or 5mg/ml), incomplete Freund's adjuvant (IFA; 1 : 1, oil:water) and aluminium hydroxide 5 (6 mg/ml). Sheep are injected intramuscularly (i/m) (lml) into the left hind leg for the primary inoculation and 4 weeks later boosted with an i/m injection ofthe same vaccine preparation into the right hind leg.
  • PBS phosphate buffered saline
  • Quil A (1 or 5m
  • Sera are collected from all animals before the primary inoculations and then at weekly intervals until 4 weeks post secondary inoculation. Sera are stored at -20 °C until
  • EIA enzyme immunoassay
  • Sheep are randomly allocated into 12 groups of 5 animals. Serum samples were collected prior to first vaccination and then at weekly intervals until 4 weeks post secondary vaccination. Serum antibody levels to purified 45W are quantified by EIA. All vaccine formulations are standardised to contain 50 ⁇ g of GST-45W per dose. The addition of at least 10-100 ⁇ g of recombinant DL-5 to aqueous and aluminium hydroxide (A1OH) vaccine formulations results in significant increases in serum IgG anti-45W.
  • A1OH aluminium hydroxide
  • Example 7 An experiment similar to that described in Example 11 for recombinant IL-5, is conducted for the co-expressed recombinant IL-12 35 kDa and 40 kDa polypeptide subunits, produced from the plasmid pCI-neo/IL-12 (Example 7.4), to ascertain the adjuvant potential of recombinant IL-12. Serum antibody levels are quantified by EIA using GST-45W antigen. As in Example 11, adjuvant effects are seen in the PBS and A1OH vaccine groups when approximately at least 10-100 ⁇ g of recombinant IL-12 is incorporated.
  • the combination of both IL-5 and IL-12 is studied in A1OH vaccine formulations.
  • Table 4 shows the vaccine formulations used for this experiment. Animals (five per group) are injected i/m in the rear leg on day 0 and receive a second i/m injection in the opposing rear leg on day 28.
  • the cytokines exert synergistic co-adjuvant effects when administered with A1OH.
  • Antibody titres are elevated significantly compared to titres obtained when A1OH alone is used as adjuvant.
  • the level of antibody obtained with the AlOH-cytokine combination is commensurate with that obtained with Quil A.
  • Vaccine formulations comprising combinations of recombinant IL-5 and DL-12 polypeptides
  • GAC CTC CAA AAA AGG AAG TGT GGA GGA GAA AGA TGG AGA GTG AAA CAA 336 A ⁇ p Leu Gin Ly ⁇ Arg Lys Cys Gly Gly Glu Arg Trp Arg Val Lys Gin 100 105 110
  • Trp Glu Tyr Pro A ⁇ p Thr Trp Ser Thr Pro Hi ⁇ Ser Tyr Phe Ser Leu 260 265 270

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Abstract

La présente invention concerne une molécule d'acide nucléique qui comprend une séquence de nucléotides codant une molécule de cytokine IL-5 ou IL-12 d'ovins ou qui est complémentaire d'une telle séquence codant une telle molécule de cytokine. L'invention traite aussi de polypeptides IL-5 et IL-12 isolés et de recombinaison, d'ovins, qui peuvent être utilisés comme modulateurs de la réponse immunitaire pour le bétail.
PCT/AU1996/000360 1995-06-14 1996-06-14 Modulateurs de la reponse immunitaire et leur utilisation WO1997000321A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998017689A3 (fr) * 1996-10-18 1998-08-20 Genemedicine Inc Expression du gene il-12, systemes d'apport et utilisations
WO1998017814A3 (fr) * 1996-10-18 1998-08-27 Genemedicine Inc Expression de genes, systemes d'apport et utilisations
WO1998041229A1 (fr) * 1997-03-19 1998-09-24 F. Hoffmann-La Roche Ag Utilisation du il-12p40 comme immunostimulant
WO1999047678A3 (fr) * 1998-03-19 1999-12-09 Valentis Inc Plasmides d'interferon alpha, systemes d'administration et procedes de fabrication desdits plasmides
WO2000065058A1 (fr) * 1999-04-23 2000-11-02 Pharmexa A/S Procede de regulation negative de l'interleukine 5 (il-5)
WO2000067761A1 (fr) * 1999-05-06 2000-11-16 Wake Forest University Compositions et procedes d'identification d'antigenes provoquant une reponse immunitaire
US6537781B1 (en) * 1999-08-10 2003-03-25 Idexx Laboratories, Inc. Methods and compositions concerning canine interleukin 5
US6929794B1 (en) 1996-05-31 2005-08-16 National University Of Ireland IL-12 as an adjuvant for Bordetella pertussis vaccines
DE102004015762A1 (de) 2004-03-31 2005-11-03 Braun Gmbh Epilationsgerät und Epilationsverfahren
US7115712B1 (en) 1999-12-02 2006-10-03 Maxygen, Inc. Cytokine polypeptides
WO2014093622A2 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Délivrance, fabrication et optimisation de systèmes, de procédés et de compositions pour la manipulation de séquences et applications thérapeutiques

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AU8527891A (en) * 1990-09-13 1992-04-15 Commonwealth Scientific And Industrial Research Organisation Ovine cytokine genes
EP0621341A2 (fr) * 1993-04-22 1994-10-26 American Cyanamid Company Anticorps monoclonaux contre des cytokines bovines

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6929794B1 (en) 1996-05-31 2005-08-16 National University Of Ireland IL-12 as an adjuvant for Bordetella pertussis vaccines
WO1998017689A3 (fr) * 1996-10-18 1998-08-20 Genemedicine Inc Expression du gene il-12, systemes d'apport et utilisations
WO1998017814A3 (fr) * 1996-10-18 1998-08-27 Genemedicine Inc Expression de genes, systemes d'apport et utilisations
WO1998041229A1 (fr) * 1997-03-19 1998-09-24 F. Hoffmann-La Roche Ag Utilisation du il-12p40 comme immunostimulant
WO1999047678A3 (fr) * 1998-03-19 1999-12-09 Valentis Inc Plasmides d'interferon alpha, systemes d'administration et procedes de fabrication desdits plasmides
WO2000065058A1 (fr) * 1999-04-23 2000-11-02 Pharmexa A/S Procede de regulation negative de l'interleukine 5 (il-5)
US7285273B1 (en) 1999-04-23 2007-10-23 Pharmexa A/S Method for down-regulating IL5 activity
US6746669B1 (en) 1999-04-23 2004-06-08 Pharmexa A/S Method for down-regulating IL5 activity
US6716623B2 (en) 1999-05-06 2004-04-06 Wake Forest University School Of Medicine Compositions and methods for identifying antigens which elicit an immune response
US6500641B1 (en) 1999-05-06 2002-12-31 Wake Forest University School Of Medicine Compositions and methods for identifying antigens which elicit an immune response
US7273752B2 (en) 1999-05-06 2007-09-25 Wake Forest University Health Sciences Compositions and methods for identifying antigens which elicit an immune response
WO2000067761A1 (fr) * 1999-05-06 2000-11-16 Wake Forest University Compositions et procedes d'identification d'antigenes provoquant une reponse immunitaire
US7749752B2 (en) 1999-05-06 2010-07-06 Wake Forest University Health Sciences Compositions and methods for identifying antigens which elicit an immune response
US6537781B1 (en) * 1999-08-10 2003-03-25 Idexx Laboratories, Inc. Methods and compositions concerning canine interleukin 5
US7115712B1 (en) 1999-12-02 2006-10-03 Maxygen, Inc. Cytokine polypeptides
DE102004015762A1 (de) 2004-03-31 2005-11-03 Braun Gmbh Epilationsgerät und Epilationsverfahren
WO2014093622A2 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Délivrance, fabrication et optimisation de systèmes, de procédés et de compositions pour la manipulation de séquences et applications thérapeutiques

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