EP0777733A1 - Verfaren zur behandlung von vögeln mit myelomonozytenwachstumsfaktor - Google Patents

Verfaren zur behandlung von vögeln mit myelomonozytenwachstumsfaktor

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Publication number
EP0777733A1
EP0777733A1 EP95925611A EP95925611A EP0777733A1 EP 0777733 A1 EP0777733 A1 EP 0777733A1 EP 95925611 A EP95925611 A EP 95925611A EP 95925611 A EP95925611 A EP 95925611A EP 0777733 A1 EP0777733 A1 EP 0777733A1
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EP
European Patent Office
Prior art keywords
mgf
cmgf
bird
avian
ovo
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Legal status (The legal status 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 status listed.)
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EP95925611A
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English (en)
French (fr)
Inventor
Paul A. Johnston
Molly M. Bland
April E. Kemper
Hong Liu
Julius K. Tyczkowski
Timothy W. Harding
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Embrex LLC
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Embrex LLC
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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
    • C07K14/53—Colony-stimulating factor [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • 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 to the treatment of birds by the in ovo administration of an avian myelomonocytic growth factor such as chicken myelomonocytic growth factor (cMGF) .
  • an avian myelomonocytic growth factor such as chicken myelomonocytic growth factor (cMGF) .
  • Chicken myelomonocytic growth factor is an avian hematopoietic cytokine which stimulates bone marrow cells to proliferate and produce cells of the monocyte/macrophage lineage.
  • cMGF was originally identified in lectin stimulated spleen cultures and purified from conditioned media produced by an LPS stimulated chicken macrophage cell line (HD11) by Leutz et al., EMBO J. 3, 3191-3197 (1984) .
  • cMGF is a glycoprotein which stimulates the growth of virally transformed chicken myeloid cell lines and formation of macrophage and granulocyte colonies in normal bone marrow cultures in vi tro (Leutz et al .
  • cMGF has been cloned by Leutz et al. , EMBO J 8, 175-181 (1989) , but sufficient pure cMGF, either native or recombinant, has not been available to characterize possible uses thereof.
  • U.S. Patent No. 5,028,421 to Fredericksen discloses a method for increasing the weight of treated birds after hatch by introducing a T-cell growth factor into eggs on about the eighteenth day of incubation.
  • a method of treating birds is disclosed herein. The method carried out by administering to a bird in ovo a biologically active amount of avian myelomonocytic growth factor (MGF) .
  • MMF myelomonocytic growth factor
  • a second aspect of the present invention is a method of making purified recombinant avian myelomonocytic growth factor.
  • the method includes culturing host cells (e.g., Escherichia coli , Pichia pastoris , and the like) which contain and express a recombinant DNA construct encoding MGF in a culture media, collecting culture media containing recombinant MGF from the host cells, and isolating the MGF from the culture media.
  • host cells e.g., Escherichia coli , Pichia pastoris , and the like
  • a third aspect of the present invention is a method of enhancing the growth of birds. This method is carried out by administering avian MGF to a bird in ovo in an amount effective to enhance the growth of the bird after hatch.
  • a fourth aspect of the present invention is a method of enhancing bone marrow proliferation in birds.
  • a fifth aspect of the present invention is a method of inhibiting the progression of a bacterial infection such as an Escheri chia coli infection or Salmonella infection in a bird.
  • the method comprises administering to said bird in ovo a avian myelomonocytic growth factor (MGF) in an amount effective to inhibit the progression of the bacterial infection.
  • MGF myelomonocytic growth factor
  • a sixth aspect of the present invention is a method of inhibiting the progression of a viral, fungal, or protozoal infection in a bird by administering to the bird in ovo a avian myelomonocytic growth factor (MGF) in an amount effective to inhibit the progression of the viral, fungal, or protozoal infection.
  • MMF myelomonocytic growth factor
  • Figure 1 is an illustration of Coomassie blue stained proteins of IPTG-induced or non-induced transformed BL21(DE3) Escherichia coli lysates separated and analyzed by 12% SDS-PAGE.
  • Figure 2 is a graphical illustration of bone marrow proliferation induced by cMGF eluted from non- reducing SDS-PAGE gels.
  • Figure 3 is a graphical illustration of bone marrow proliferation induced by the growth media of pPIC9-cMGF and pHIL-SI-cMGF transformed Pichia pastoris versus the GS115/His*Mut ⁇ albumin secreting stain.
  • Figure 4 is a graphical illustration of Fc- receptor mediated phagocytosis of sheep red blood cells opsonized with rabbit anti-SRBC IgG by bone marrow cells cultured +/-20 ⁇ g/ml cMGF.
  • Figure 5 is a graphical illustration of IS- induced nitrite production by bone marrow cells cultured +/-20 g/ml cMGF.
  • Figure 6 is a graphical illustration of Phorbol ester induced superoxide production by bone marrow cells cultured +/-20 ⁇ g/ml cMGF.
  • Figure 7 is a graphical illustration of Fc- receptor phagocytosis of sheep red blood cells opsonized with rabbit anti-SRBC IgG by bone marrow cells cultured for 48 Hours with the indicated doses of cMGF.
  • Figure 8 is a graphical illustration of IS (PA-14A, 2.5 ug/ml) induced nitrite production by bone marrow cells cultured for 48 hours with the indicated doses of cMGF.
  • Figure 9 is a graphical illustration of PMA- inducible superoxide production by adherence purified peripheral blood mononuclear leukocytes isolated from the pooled blood of chicks bled on day 4 post hatch of day 18 injected eggs receiving 2.5 ug/egg yeast expressed albumin or 2.5, 0.25 and 0.025 ug/egg Pichia pas tor is expressed cMGF.
  • Figure 10 is a graphical illustration of total pooled bone marrow cell counts obtained from 12 femurs removed from day old chicks hatched from egg injected with 0.1 U of IS, 0.25 ug of yeast expressed albumin, and either 0.25ug or 0.05ug of cMGF.
  • Figure 11 is a graphical illustration of Phorbol ester induced superoxide production by adherence purified bone marrow cells obtained from 12 femurs removed from day old chicks hatched from egg injected with 0.1 U of IS, 0.25 ug of yeast expressed albumin, and either 0.25ug or 0.05ug of cMGF.
  • in ovo refers to birds contained within an egg prior to hatch.
  • the present invention may be conceived of as both a method of treating eggs and a method of treating birds.
  • the present invention may be practiced with any type of bird egg, including chicken, turkey, duck, goose, quail, and pheasant eggs.
  • Chicken eggs are preferred.
  • Eggs treated by the method of the present invention are fertile eggs.
  • the eggs may be treated at any point during incubation, although it is preferable to treat the eggs in the fourth quarter of incubation, and most preferably to treat the eggs on about the eighteenth day of incubation (i.e., the eighteenth day of embryonic development) .
  • avian MGF as used MGF corresponding to MGF produced by any avian species.
  • the term “avian” is intended to encompass all avian species, including, but not limited to, chickens, turkeys, ducks, geese, quail, and pheasant. Various species of avian MGF are known. This term is also intended to include active fragments and analogs thereof.
  • the MGF may be provided in any suitable pharmaceutically acceptable carrier, but is preferably provided in an aqueous carrier such as a phosphate- buffered saline solution.
  • the administration of MGF in ovo provides a variety of useful results.
  • the administration of MGF in ovo may be used to enhance growth of the hatched chick.
  • the administration of MGF in ovo may be used to enhance the proliferation of bone marrow cells, resulting in a more fully developed immune system at an earlier stage ( i . e . , accelerate the onset of immune competence in the bird) .
  • the administration of MGF in ovo provides, as noted above, a method of inhibiting the progression of bacterial infections in a bird, such as Escherichia coli infections (i.e., avian colisepticemia) or Salmonella infections.
  • the administration of MGF in ovo provides, as also noted above, a method of inhibiting the progression of viral infections in a bird (e.g., infectious bursal disease virus infections, Newcastle's disease virus, Marek's disease virus, etc.) , of inhibiting the progression of fungal infections, and of inhibiting the progression of protozoal infections ⁇ e . g. , Eimeria species such as Eimeria tenella in avian coccidiosis) .
  • the MGF may be administered concurrently with a vaccine (e . g. , a live vaccine or a nonreplicating immunogen) effective for protecting the bird against the aforesaid infection.
  • MGF may be administered to eggs by any means which transports the compound through the shell.
  • the preferred method of administration is, however, by injection.
  • MGF may be injected into the egg at any site.
  • the site of injection is within either the region defined by the amnion, including the amniotic fluid and the embryo itself, in the yolk sac, or in the air cell .
  • Dosages of MGF used to carry out the methods described herein are not critical and can be determined in a routine manner.
  • the dosages will vary with the species of bird being treated, the time and site of administration, and the desired effect.
  • the upper limit of the dosage can be routinely determined, but in general will be as much as 10 or 100 ⁇ g per subject ( i . e . , per in ovo injection; per egg) or more.
  • the lower limit of the dosage likewise can be routinely determined, but can be as little as 1000 or 1 ng per subject or less.
  • the mechanism of injection is also not critical.
  • the method employed does not unduly damage the tissues and organs of the embryo or the extraembryonic membranes surrounding it so that the treatment will not decrease hatch rate.
  • a hypodermic syringe fitted with a needle of about 18 to 22 gauge is suitable.
  • the needle need only be inserted into the egg by about two millimeters.
  • a one inch needle when fully inserted from the center of the large end of the egg, will penetrate the shell, the outer and inner shell membranes enclosing the air cell, and the amnion.
  • a needle of this length will terminate either in the fluid above the chick or in the chick itself.
  • a pilot hole may be punched or drilled through the shell prior to insertion of the needle to prevent damaging or dulling of the needle.
  • the egg can be sealed with a substantially bacteria-impermeable sealing material such as wax or the like to prevent subsequent entry of undesirable bacteria.
  • a high speed automated injection system for avian embryos will be particularly suitable for practicing the present invention.
  • Numerous such devices are available, exemplary being those disclosed in U.S. Patents Nos. 4,903,635 and 4,681,063 to Hebrank, U.S. Patent No. 5,056,464 to Lewis, and U.S. Patent Nos. 4,040,388, 4,469,047, and 4,593,646 to Miller, the disclosures of which are incorporated by reference herein in their entirety. All such devices, as adapted for practicing the present invention, include an injector containing avian MGF as described herein, with the injector positioned to inject an egg carried by the apparatus with the avian MGF. Other features of the apparatus are discussed above.
  • a sealing apparatus operatively associated with the injection apparatus may be provided for sealing the hole in the egg after injection thereof.
  • U.S. Patent No. 4,903,635 to Hebrank and U.S. Patent No. 5,056,464 to Lewis, the disclosures of which are incorporated herein by reference in their entirety.
  • These devices comprise an injection apparatus for delivering fluid substances into a plurality of eggs and apparatus for aligning the eggs in relation to the injection apparatus.
  • the features of these apparatus may be combined with the features of the apparatus described above for practicing the present invention.
  • injected eggs are incubated to hatch and the birds are raised to at least 2 weeks of age.
  • Chicken myelomonocytic growth factor (cMGF) and DNA encoding the same is known (Leutz et al . , EMBO J. 3, 3191-3197 (1984) ; Leutz et al . , EMBO J 8, 175-181 (1989) ) .
  • the MGF proteins used to carry out the present invention may accordingly be made with techniques known in the art, or by variations thereof which will be readily apparent to those skilled in the art.
  • the production of recombinant DNA, vectors, host cells, and proteins by genetic engineering techniques is well known. See, e . g. , U.S. Patent No. 4,761,371 to Bell et al . at Col. 6 line 3 to Col. 9 line 65; U.S.
  • DNA sequences encoding MGF proteins may be recovered by use of the polymerase chain reaction (PCR) procedure and splicing by overlap extension (SOE) , as is known in the art. See U.S. Patents Nos. 4,683,195 to Mullis et al. and 4,683,202 to Mullis.
  • PCR polymerase chain reaction
  • SOE overlap extension
  • the MGF proteins may be synthesized in host cells transformed with vectors containing DNA encoding the MGF proteins.
  • a vector is a replicable DNA construct. Vectors are used herein either to amplify DNA encoding the MGF protein and/or to express DNA which encodes the MGF protein.
  • An expression vector is a replicable DNA construct in which a DNA sequence encoding the MGF protein is operably linked to suitable control sequences capable of effecting the expression of the MGF protein in a suitable host. The need for such control sequences will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.
  • Vectors useful for practicing the present invention include plasmids, viruses (including phage) , retroviruses, and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination) .
  • the vector replicates and functions independently of the host genome, or may, in some instances, integrate into the genome itself. Suitable vectors will contain replicon and control sequences which are derived from species compatible with the intended expression host.
  • Transformed host cells are cells which have been transformed or transfected with the MGF protein vectors constructed using recombinant DNA techniques. Transformed host cells ordinarily express the MGF protein, but host cells transformed for purposes of cloning or amplifying the MGF protein DNA need not express the MGF protein.
  • DNA regions are operably linked when they are functionally related to each other.
  • a promoter is operably linked to a coding sequence if it controls the transcription of the sequence
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
  • operably linked means contiguous and, in the case of leader sequences, contiguous and in reading frame.
  • Suitable host cells include prokaryotes, yeast cells or higher eukaryotic cells.
  • Prokaryotes include gram negative or gram positive organisms, for example Escherichia coli ⁇ E. coli ) or Bacilli.
  • Higher eukaryotic cells include established cell lines of mammalian origin as described below.
  • Exemplary host cells are E. coli W3110 (ATCC 27,325) , E. coli B, E. coli X1776 (ATCC 31,537) , and E. coli 294 (ATCC 31,446) .
  • Pseudomonas species, Bacillus species, and Serra tia marcesans are also suitable.
  • a broad variety of suitable microbial vectors are available.
  • a microbial vector will contain an origin of replication recognized by the intended host, a promoter which will function in the host and a phenotypic selection gene such as a gene encoding proteins conferring antibiotic resistance or supplying an auxotrophic requirement. Similar constructs will be manufactured for other hosts. E. coli is typically transformed using pBR322. See Bolivar et al . , Gene 2 , 95
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • Expression vectors should contain a promoter which is recognized by the host organism. This generally means a promoter obtained from the intended host. Promoters most commonly used in recombinant microbial expression vectors include the beta-lactamase
  • trp tryptophan promoter system
  • trp tryptophan promoter system
  • the tac promoter H. De Boer et al . , Proc . Na tl . Acad . Sci . USA 80, 21 (1983)
  • these are commonly used, other microbial promoters are suitable. Details concerning nucleotide sequences of many have been published, enabling a skilled worker to operably ligate them to DNA encoding the MGF protein in plasmid or viral vectors (Siebenlist et al . , Cell 20, 269
  • the promoter and Shine-Dalgarno sequence are operably linked to the DNA encoding the MGF protein, i.e., they are positioned so as to promote transcription of the MGF protein messenger RNA from the DNA.
  • Eukaryotic microbes such as yeast cultures may be transformed with suitable MGF protein-encoding vectors. See, e . g. , U.S. Patent No. 4,745,057. Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available.
  • Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS) , a promoter, DNA encoding the MGF protein, sequences for polyadenylation and transcription termination, and a selection gene.
  • An exemplary plasmid is YRp7, (Stinchcomb et al . , Na ture 282, 39 (1979) ; Kingsman et al . , Gene 7, 141 (1979) ; Tschemper et al . , Gene 10, 157 (1980)) .
  • This plasmid contains the trpl gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No.
  • yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al . , J. Biol . Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al . , J. Adv. Enzyme Reg. 7, 149 (1968) ; and Holland et al .
  • yeast host cell such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase , phosphofructokinase , glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al . , EPO Publn. No. 73,657.
  • a particularly preferred yeast host cell is
  • Pichia pastoris which is described along with suitable transformation vectors in U.S. Patents Nos. 4,683,293; 4,808,537; 4,812,405; 4,818,700; 4,837,148 4,855,231 4,857,467; 4,879,231; 4,882,279; 4,885,242 4,895, 800 4,929,555; 5,002,876; 5,004,688; 5,032,516 5,122,465 5,135,868; and 5,166,329, the disclosures of which applicant specifically intends to be incorporated herein by reference.
  • Cultures of cells derived from multicellular organisms are a desirable host for recombinant MGF protein synthesis.
  • any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture, including insect cells.
  • mammalian cells are preferred, as illustrated in the Examples. Propagation of such cells in cell culture has become a routine procedure. See Tissue Culture, Academic Press, Kruse and Patterson, editors (1973) .
  • useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI138, BHK, COS-7, CV, and MDCK cell lines.
  • Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the gene to be expressed, along with a ribosome binding site, RNA splice site (if intron-containing genomic DNA is used) , a polyadenylation site, and a transcriptional termination sequence.
  • the transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells are often provided by viral sources.
  • commonly used promoters are derived from polyoma, Adenovirus 2, and Simian Virus 40 (SV40) . See, e . g. , U.S. Patent No. 4,599,308.
  • the early and late promoters are useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. See Fiers et al. , Na ture 273, 113 (1978) .
  • the vaccinia virus may be used as a vector, as described in the Examples. Further, the MGF protein promoter, control and/or signal sequences, ay also be used, provided such control sequences are compatible with the host cell chosen.
  • An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV) , or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient. Rather than using vectors which contain viral origins of replication, one can transform mammalian cells by the method of cotransformation with a selectable marker and the MGF protein DNA.
  • DHFR dihydrofolate reductase
  • thymidine kinase See U.S. Pat. No. 4,399,216.
  • Such markers are proteins, generally enzymes, that enable the identification of transformant cells, i.e., cells which are competent to take up exogenous DNA. Generally, identification is by survival of transformants in culture medium that is toxic, or from which the cells cannot obtain critical nutrition without having taken up the marker protein.
  • Host cells such as insect cells (e.g., cultured Spodoptera frugiperda cells) and expression vectors such as the baculovirus expression vector (e.g., vectors derived from Autogrrapha calif ornica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed in carrying out the present invention, as described in U.S. Patents Nos. 4,745,051 and 4,879,236 to Smith et al .
  • a baculovirus expression vector comprises a baculovirus genome containing the gene to be expressed inserted into the polyhedron gene at a position ranging from the polyhedron transcriptional start signal to the ATG start site and under the transcriptional control of a baculovirus polyhedron promoter.
  • bp means base pairs
  • hr means hour
  • kDa mean kilodaltons
  • M means molar
  • ⁇ g means micrograms
  • ml means milliliters
  • SDS means sodium dodecyl sulfate
  • IPTG means Isopropyl- ⁇ - D-thiopyranoside
  • RPMI Rosewell Park Memorial Institute
  • FBS means fetal bovine serum
  • HPLC high pressure liquid chromatography
  • RP-HPLC means reverse phase high pressure liquid chromatography
  • ng means nanograms
  • YEA means yeast expressed albumin
  • U means units
  • IM means intramuscular
  • ⁇ L means microliters
  • mg means milligrams
  • cfu means colony forming units
  • °F means degrees Fahrenheit .
  • EXAMPLE 1 Cloning of Chicken Myelomonocytic Growth Factor This example describes the isolation of the DNA encoding cMGF described in Leutz et al . , EMBO J. 8, 175- 181 (1989) , except that a C was found at nucleotide 114 rather than a T as reported by Leutz.
  • RNA was prepared from guanadinium isothiocyanate lysed Concanavalin A (Con A) -stimulated splenocytes which had been isolated from 6 week leghorns .
  • PCR reverse transcription and polymerase chain reaction
  • cMGF-specific oligonucleotide primers gave a band of the expected size of -560 bp and a larger -800 bp band. Both bands were specific to splenocytes stimulated with Con A for 48 hrs and were not amplified in the controls or in splenocytes stimulated for only 24 hrs.
  • the 560 base pair PCR product encoding the cDNA for the mature cMGF protein was subcloned in the Escherichia coli (E. coli) expression vectors pET3A, B & C. Isopropyl- ⁇ -D-thiopyranoside (IPTG) was added to a growing culture of the BL21(DE3) E. coli host strain, to induce T7 polymerase, which in turn transcribes the target DNA in the plasmid. None of the cMGF recombinants cloned into pET3A, B & C using the Ndel and BamHI sites exhibited any IPTG inducible cMGF expression.
  • BamHI site allows the translation of the cMGF protein as a fusion with the first 11 amino acids of the gene-10 protein, which may translate more efficiently and confer stability on the expressed protein.
  • the cMGF clone was reamplified with cMGF primers 1+6 to engineer the BamHI sites and subclone it into pCRII.
  • cMGF cDNA was subcloned into the BamHI site into the pET3A vector. Recombinants, in the correct orientation, were then identified by Aval digestion. An IPTG inducible band ⁇ 21 kDa in BL21(DE3) cells transformed with the pET3A- cMGF-BamHI plasmid was observed.
  • a precursor of 26.5 kDa is synthesized whose 23 amino acid signal sequence is cleaved off and, in the presence of membranes, cMGF becomes glycosylated at two N- glycosylation sites to generate a 28 kDa mature cMGF form.
  • the calculated molecular weight of the mature cMGF protein is ⁇ 20.014 kDa, which is about 4 kDa smaller than that determined by SDS-PAGE.
  • the cells were broken open by sonication or mild detergent lysis with deoxycholate, followed by centrifugation to separate soluble and insoluble fractions. Samples were then analyzed by SDS-PAGE to determine whether the putative cMGF protein is expressed in the soluble or insoluble fraction. The results from both cell lysis methods indicated that cMGF is preferentially contained in the insoluble fraction, presumably in the inclusion bodies. Natural cMGF and the rcMGF-trpE eluted from non-reducing SDS-PAGE gels stimulate growth of hematopoietic colonies in chick bone marrow cells, indicating that biological activity is not destroyed by SDS but was found to be sensitive to exposure to reducing reagents.
  • rcMGF-glO targeted to inclusion bodies was recovered by washing inclusion bodies with 50% glycerol and solubilizing the inclusion bodies in 5% SDS.
  • Several methods were attempted to refold putative cMGF-glO protein after removing SDS and contaminating bacterial LPS by HPLC and DETOXI-GELTM columns from Pierce
  • Triton X-100 washed inclusion bodies containing the 21 kDa cMGF protein were solubilized in 5% SDS, 8 M urea and 6 M guanidinium-HCl and analyzed by RP-HPLC. Identical chromatography profiles were obtained for all solubilization methods and the 21 kDa cMGF protein was identified by SDS-PAGE. The dried RP-HPLC purified cMGF protein was solubilized by different techniques including SDS, Triton X-100, dipotassium phosphate and octyl- ⁇ -glucoside . Detergent was determined to be the most effective method of solubilization.
  • Pichia pastoris is methylotropic, and therefore capable of metabolizing methanol as a sole carbon source, and producing protein products with glycosylation similar to mammalian systems. Plasmids are available which direct protein products to be intracellular or secreted. Heterologous protein expression in this system is tightly regulated and induced to very high levels by the addition of methanol to the growth media.
  • the Pichia pastoris expression kit available from INVITROGEN ® was used (telephone number 602-748-4400) .
  • the cMGF clone was reamplfied with primers 7+6 to engineer in BamHI and EcoRI sites, and subcloned into two Pichia expression vectors, pPIC9 and pHIL-Sl both of which produce secreted fusion protein products with signal peptides from the ⁇ -mating factor or acid phosphatase genes respectively. Initially the PCR product was subcloned into the pCRTMII vector. Digestion with EcoRI/BamHI or EcoRI alone resulted in the correct size inserts.
  • the cMGF inserts were isolated from a low melting point agarose gel using the phenol/chloroform method and ligated into the EcoRI site of pPIC9 vector or the EcoRI/BamHI sites of pHIL-SI. The ligation was transformed into DH5 ⁇ f' and plated onto selective media. Potential cMGF subclones were verified by restriction digests of the DNA. One clone of each type was selected for further analysis. The correct orientation of cMGF insert in the pPIC9-cMGF and the pHIL-SI-cMGF subclones were checked by digestion with the enzymes Aval and Bgl II. DNA sequencing of the 5' end of the clones confirmed that the clones were subcloned correctly and the cMGF cDNA is in-frame with the Pichia vector signal sequences for secretion.
  • Spheroplasts were prepared from the GS115 (His-) strain by treatment with zymolase.
  • the Pichia spheroplasts were transformed with Bgll l digests of cMGF subcloned into pPIC9 and pHILSl .
  • the transformed yeast were plated onto selective media along with positive controls for transformation and secretion provided by Invitrogen.
  • the Pichia GS115 (His-) strain had a defect in the histidinal dehydrogenase activity coded for the gene HIS4. This defect enabled the strain to grow on complex media or minimal media supplemented with histidine.
  • GS115 His-
  • HIS4 histidine
  • cMGF recombinant clones Seven potential cMGF recombinant clones were selected from the transformation with the pHILSl-cMGF and pPIC9-cMGF vectors. These clones were grown in liquid culture in the presence of methanol to induce expression and secretion of the recombinant protein into the media. The culture media supernatant from each recombinant clone was collected and analyzed by SDS-PAGE. The results revealed five potential cMGF clones expressing two predominant proteins of -25 and 29 kDa, the size of the glycosylated forms of native cMGF.
  • the five potential positive samples were tested for in vi tro activity in the bone marrow proliferation assay and were found to be biologically active. Protein expression was scaled-up to a 40 mL, 2 day-induced culture with four of the five previously identified cMGF- expressing clones and the albumin-expressing transformant included as a negative control. Since the yeast methanol growth media inhibited bone marrow cell proliferation, the samples were processed using 10 k and 100 k CENTRIPREPTM ultrafilters from AMICONTM to exchange the yeast media with RPMI media and concentrate the samples. The results of the bone marrow proliferation assay are reported in Figure 3.
  • This new procedure produced material containing the 25 and 29 kDa putative cMGF bands when analyzed by SDS-PAGE, and can be resolved on RP-HPLC.
  • One of the fractions collected after RP-HPLC was active in the bone marrow proliferation assay and contained the two cMGF bands.
  • the Southern blotting technique was used to determine the cMGF gene copy number in each positive clone. The results indicated that all of the cMGF expressing clones contained one copy of the gene cassette.
  • Focus was also directed towards optimizing cell culture conditions to increase production of the cMGF product and one of the pPIC9-cMGF expressing clones was chosen for scale up to a 40.0 mL culture. Gentamicin was added to prevent bacterial contamination. The results obtained appeared to indicate that a 2-day methanol induction was optimal.
  • cMGF induced acquisition of Fc-receptor mediated phagocytosis and IS-induced nitrite production in BM cells after 48 hours of culture was dose dependent, beginning in the 10-100 ng/ml range and approaching maximal stimulation in the 1-2 micro-gram/ml range of cMGF concentration.
  • cMGF effects on cultured BM cell PMA-induced superoxide production were once again more complex with stimulation in the 10-100 ng/ml range and inhibition at doses of 500 ng/ml or greater.
  • cMGF induced proliferation of BM cells is also initiated in the nanogra range (data not shown) .
  • Pichia pastoris produced recombinant cMGF therefore stimulates both the proliferation of bone marrow cells and the acquisition of mature macrophage functional responses.
  • Concanavalin A stimulated splenocyte conditioned media (IS) which contains cMGF in addition to other cytokines, also stimulates the proliferation of bone marrow cells.
  • IS splenocyte conditioned media
  • cMGF was the most efficient at enhancing Fc- receptor mediated phagocytosis 37% versus 16% for Albumin, 5% for media alone and 2.5% for IS (data not shown) .
  • Both cMGF and IS treatment significantly stimulated IS-induced nitrite production relative to albumin and media cultured cells (data not shown) .
  • cMGF, IS, and albumin treatment all enhanced the PMA-induced superoxide response relative to media cultured cells (data not shown) .
  • Pichia pastoris produced recombinant cMGF therefore stimulates both the proliferation of bone marrow cells and the acquisition of mature macrophage functional responses.
  • IS which contains cMGF in addition to other cytokines (IL-2 & IFN, etc.), also stimulates both the proliferation of bone marrow cells and the acquisition of mature macrophage functional responses.
  • treatment of BM with IS had either no effect or inhibited acquisition of Fc-receptor mediated phagocytosis, an observation made previously with peritoneal elicited macrophages cultured with IS. It is therefore likely that in ovo delivery of cMGF will result in chicks hatching with greater numbers of functionally activated macrophages that are better able to cope with bacterial infections and perform their central role in the modulation of the immune response.
  • Each mean represents 5 chicks within one trial .
  • One hundred cells were counted for each chick sampled.
  • PBL's Peripheral blood mononuclear leukocytes
  • Non-adherent cells were removed by vigorous washing and the resulting adherent population of leukocytes assayed for their ability to perform Fc-receptor mediated phagocytosis, IS- induced nitrite production and phorbol ester induced superoxide production. There was no detectable IS or LPS inducible nitrite production and Fc-receptor mediated phagocytosis (opsonized-SRBC s) at any of the time points examined.
  • leukocytes were able to phagocytose non-opsonized SRBC's, which may be a reflection of the normal homeostatic functions of blood monocytes, that is the recognition and phagocytosis of senescent, damaged or foreign RBC's or other cells.
  • SRBC's normal homeostatic functions of blood monocytes
  • PBL's from the 0.25 & 0.025 ⁇ g cMGF/egg groups produced more 0 2 in response to PMA than albumin controls on days 1, 4, and 7.
  • PBL's from the 2.5 ⁇ g cMGF/egg group produced more 0 2 in response to PMA than albumin controls on day 4 post hatch but not on days 1 or 7.
  • bone marrow cells were isolated from the femurs of day old chicks hatched from eggs injected with yeast expressed cMGF or albumin and
  • Hatchability means are based on 9 replicates of 108 live embryonated eggs (n 972).* Treatment tested positive for bacterial contamination postinjection but not prior to injection, suggesting that the injectable may have became contaminated during the filling of syringes.
  • Hatcher Contamination E. coli Challenge Screening Models The hatcher contamination model involves placing eggs (typically 36 eggs) which have been injected into the aircell with the VPI E. coli strain (typically 10 4 CFU's) in the top tray of the hatcher. The chicks that hatch from these infected eggs then transmit the pathogen to the experimental chicks hatching from eggs incubated in the same hatcher. The various treatment groups are represented in two separate locations within the hatcher, and the locations are randomly varied from trial to trial. On the day of hatch, chicks are pulled, wingbanded, commingled, placed in floor pens and monitored for mortality twice daily for two weeks. At two weeks, chicks are weighed and counted. A series of three separate E. coli hatcher contamination challenge models were performed (Table 3) .
  • ovo cMGF consistently produces greater increases in both the number of bone marrow cells in chick femurs at hatch and the PMA inducible superoxide production from peripheral blood lymphocytes collected from Day 4 chicks when compared to YEA injected controls.
  • YEA does not stimulate bone marrow cells to proliferate in vi tro .
  • the YEA is dosed at an equivalent protein concentration to the 800 unit cMGF dose.
  • Table 4 Efficacy of cMGF and IM with or without .05 mg gentamicin in the hatcher contamination model.
  • the gentamicin treatment used as a positive control failed to reduce mortality below PBS injected controls, contrary to our previous data (Tables 3 and 4, above) .
  • the reason for this is unknown, although the chicks were held in the hatcher an additional day in an attempt to elevate early chick mortality so that differences between the efficacy of various YEA doses could be better discriminated. It may be that this additional stress may have overwhelmed the efficacy of the articles tested.
  • Treatment means based on approximately 105 chicks.
  • chicks are orally gavaged with approximately IO 9 CFU's of E. coli VPI strain immediately prior to placement in floor pens on the day of hatch.
  • the challenge culture is maintained on ice during the challenge procedure. Equal numbers of wingbanded chicks from each treatment group are commingled and randomly gavaged with 100 ⁇ L of the challenge culture prior to placement into floor pens. Chicks are monitored for mortality twice daily for 10 days.
  • Table 8 Efficacy of in ovo cMGF and IM with or without gentamicin against an oral E. coli challenge at hatch.
  • YEA fails to exhibit any of the other in vitro or in vivo effects of cMGF on bone marrow and immune cell populations
  • YEA has consistently demonstrated efficacy in the hatcher contamination model when it was dosed on an equivalent protein basis as 800 U cMGF.
  • the two preparations were tested in an oral challenge model to further evaluate and compare the efficacy of YEA to cMGF and also to determine if the protection could be attributed to in ovo delivery of protein (BSA) or products produced by the yeast expression system (YEM is the media derived from a transformed Pichia pastoris strain expressing intracellular 3-galactosidase) .
  • BSA protein
  • YEM is the media derived from a transformed Pichia pastoris strain expressing intracellular 3-galactosidase
  • Table 9 Efficacy of YEA, YEM, cMGF and BSA in the oral Day of Hatch Gavage E. coli challenge model.
  • the model is based on Leitner and Heller's finding (1992: Avian Diseases 36:211-220) that stress, (specifically 36 hour food and water deprivation) resulted in E. coli colonization of the liver, spleen, and blood of young turkeys.
  • the turkeys were orally gavaged with E. coli on hatch day, withdrawn from feed 5 days later and then organs harvested to evaluate colonization 36 hours later.
  • broiler chicks were administered either PBS, YEA (yeast expressed albumin) , 5, 50 or 500 U of cMGF in ovo, orally gavaged with IO 8 CFU E. coli (VPI 1990 strain) at hatch, removed from feed and water for 36 hours beginning on Day 4 posthatch and then either evaluated for tissue (liver and blood) colonization or Day 12 mortality.
  • the results are presented in Table 10.
  • Table 10 Efficacy of cMGF administration in ovo against E. coli organ invasion and early chick mortality.
  • cMGF was injected into the albumin at either 0.25, 2.5, 25 or 250 U on Day 0 of incubation. Eggs were removed directly from the incubator (99°F) and dipped in an E. coli broth at 40°F for four minutes on Day 13 of incubation. The temperature differential assists in the transport of the bacteria across the shell and shell membranes. A similar E. coli challenge on Day 18 of incubation resulted in a 7% decrease in hatchability and a 7% increase in two week mortality (Reid et al . , 1961) . A separate group of cMGF injected eggs were not exposed to E. coli to evaluate the effects of Day 0 cMGF injection on hatchability. The results are presented in Tables 12 and 13.
  • the hatcher contamination model is very similar to the E. coli hatcher contamination model described previously (see Example 9 above) except that the contaminated eggs (typically 15 eggs) placed in the top hatcher basket and used to deliver the challenge were injected with Salmonella typhimurium.
  • the posthatch seeder challenge model involves the commingling of Salmonella exposed and unexposed birds in floor pens simulating the horizontal transmission of pathogens from chick to chick.
  • cMGF and IM were all tested in both a Salmonella typhimurium hatcher contamination model and a posthatch seeder model. These models were conducted to evaluate the efficacy of these cytokine products against a different bacterial pathogen that is intracellular in nature.
  • the Day 10 mortality results for the Salmonella typhimurium Hatcher contamination model and Posthatch Seeder Model are presented below (Table 14) . Results are presented as percent of controls.
  • Table 14 Efficacy of cMGF and IM against Salmonella typhimuri urn .
  • PBS 100 100 100 100 100 100 100 100 100 (62) (62) (47) (43)
  • the challenge was very robust and produced higher levels of mortality than previously conducted trials of these models.
  • the only treatment that demonstrated efficacy was 0.5 mg Baytril in the hatcher contamination model, and even this exhibited a lower level of efficacy than in previous trials. It may be that the level of challenge may have overwhelmed the efficacy of the products tested.
  • EXAMPLE 15 Coccidiosis Challenge Screening Model
  • the efficacy of cMGF and IM were evaluated in a coccidiosis E. tenella oocyst challenge model (Table 15) .
  • the model measures fecal oocyst output after a low level oral challenge and evaluates the ability of the parasite to either infect or replicate within the host.
  • Efficacy measured by a reduction in the number of oocysts shed, suggests that either the parasite was unable to invade or infect cells within the subepithelial layer of the ceca, or that the parasite was unable to replicate once inside the cells and sexual reproduction or production of oocysts was inhibited.
  • Table 15 Efficacy of cMGF and IM against an -Eimeria tenella challenge.
  • Eimeria tenella oocysts Mean +/- S.D.
  • Birds were challenged either 1 or 7 days posthatch.
  • the oocyst output following challenge on Day 1 posthatch was unaffected by in ovo administration of cMGF or IM.
  • the mean oocyst production was numerically reduced but not significantly, and IM actually increased the number of oocysts produced.
  • the oocyst output following challenge on Day 7 posthatch was significantly reduced by in ovo administration of 40 U of cMGF. 800 U cMGF numerically reduced the oocyst output, while IM numerically increased oocyst output .
  • Eimeria tenella oocyst production was numerically reduced after in ovo cMGF administration at
  • Yeast expressed albumin (YEA) , at an equivalent protein concentration to cMGF at 800 U, exhibits comparable efficacy to 800 units of cMGF.
  • YEA Yeast expressed albumin
  • YEA does not stimulate bone marrow cells to proliferate in vi tro . It is therefore likely that the YEA efficacy in the E. coli hatcher contamination model is due to some alternate, as yet unknown, mechanism.

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