RECOMBINANT PIGEON POX VIRUS VACCINE

Abstract: The present invention provides a recombinant pigeon pox virus synthetically modified by the presence, in a non-essential region of the pigeon pox genome, of DNA not naturally occurring in pigeon pox virus. In a preferred embodiment of the invention, pigeon pox virus is modified by at least a portion of the Newcastle Disease virus F gene wherein the F gene is inserted within the thymidine kinase locus in the pigeon pox genome.

Description: RECOMBINANT PIGEON POX VIRUS VACCINE Field of the Invention The present invention relates to the field of treatments for avian Newcastle Disease. More particularly, the present invention relates to recombinant viral vaccine treatment of Newcastle Disease. Background of the Invention Newcastle disease is the most important viral disease of poultry. Newcastle disease is a highly contagious and destructive infection which attacks chickens, turkeys, pigeons and numerous species of wild and captive birds. Mortality due to Newcastle disease depends both on the immune status of the birds and on the virulence of the Newcastle disease virus. Mortality due to Newcastle disease can reach 100%. Chickens appear to the be most susceptible species. Newcastle disease virus, the avian paramyxovirus type 1 is the etiological agent of Newcastle disease. Newcastle disease virus strains are classified in three pathotypes: velogenic, mesogenic and lentogenic. Velogenic and mesogenic Newcastle disease virus strains are the pathogenic strains whilst lentogenic strains cause no mortality in birds of any age and are currently used as live vaccine strains against Newcastle disease virus. Currently used live vaccine strains include Hitchner, LaSota and Ulster strains. SUBSTITUTE SHEET Currently used live vaccines, however, have several disadvantages including thermal sensitivity, residual pathogenicity, possible reversion to virulence and neutralization by homologous antibodies. In addition, disease eradication programs are hindered by the inability to distinguish vaccinated from infected chickens on a serological basis. Genetically engineered vaccines are expected to overcome these problems. Recombinant vaccines have the advantage of providing immunity against the infectious agent without the possibility of reversion to the virulence of an attenuated strain. U.S.patent 4,603,112 issued July 29, 1986 to Paoletti and Panicali discloses a recombinant Vaccinia virus synthetically modified by the presence, in a non-essential region of the Vaccinia genome , of DNA not naturally occurring in the Vaccinia virus. Vaccinia virus was modified at the thymidine kinase locus of Vaccinia to express genes from herpes virus and influenza virus. Meulemans et al. (1988) Avian Pathology 12: 821-827 reports Newcastle disease virus F glycoprotein expressed from a recombinant Vaccinia virus vector protected chickens against live-virus challenge. Although the Vaccinia virus vaccine could protect chicks against infection with Newcastle disease virus, the researchers were unable to show seroconversion against the Vaccinia component of the recombinant virus in the inoculated chicks. Inability of Vaccinia virus to grow in chickens has been reported. Although adaptation of Vaccinia virus to the chicken is possible, nevertheless, the use of Vaccinia recombinant viruses in chickens is not reasonable since Vaccinia infects humans and its use as a vaccine could be hazardous for human health. There have been several reports of the use of fowl pox virus in a recombinant vaccine. PCT application publication number WO 88/02022 discloses recombinant fowl pox virus or SUBSTITUTE SHEET related avian pox virus modified by the insertion of foreign DNA into a non-essential region of the fowl pox virus. EPO publication number 0 314 569 discloses recombinant fowl pox virus wherein the fowl pox virus is modified in a non-coding region between genes to contain DNA coding for a heterologous protein. Although fowl pox virus has a more restricted range than Vaccinia virus, and does not infect humans, it nonetheless is generally more virulent in some avian species, particularly turkeys and chickens, two economically important species. Accordingly, it is an object of the invention to provide recombinant pigeon pox vaccines and pharmaceutical preparations for the inoculation of avian species against Newcastle disease infection that are less pathogenic for chickens and turkeys than recombinant fowl pox vaccines. It is another object of the invention to provide a pigeon pox vector that can be modified to express genes of choice for use as a vaccine. It is a further object of the invention to provide vaccines for inoculation of chickens against infectious agents. Summary of the Invention The present invention provides recombinant pigeon pox virus synthetically modified by the presence, in a non-essential region of the pigeon pox virus genome, of DNA not naturally occurring in pigeon pox virus. In preferred embodiments of the invention pigeon pox virus is modified within the thymidine kinase gene to contain DNA not naturally occurring in pigeon pox virus. A preferred DNA sequence for insertion within the thymidine kinase gene is DNA coding for the Newcastle disease virus F protein. This invention is more particularly pointed out in the appended claims and is described in its preferred embodiments in the following description. BSTITUTE SHEET Brief Description of the Drawings Figure 1 shows the nucleotide sequence of the insert of clone 11.14, in the mRNA sense, that codes for Newcastle disease F gene. Detailed Description of the Invention The present invention provides recombinant pigeon pox virus synthetically modified by the presence, in a non-essential region of the pigeon pox virus genome, of DNA not naturally occurring in pigeon pox virus. Using the recombinant virus of the invention, it will be possible to vaccinate avian species simultaneously against at least two different diseases. The recombinant virus of the invention will provide protection against both pigeon pox virus and a second virus, such as Newcastle disease virus, or other organism. In preferred embodiments of the invention, the recombinant pigeon pox virus contains the Newcastle disease virus F gene. When birds are inoculated with live attenuated Newcastle disease virus, it is not possible to determine when subsequent infection occurs which birds have been vaccinated and which birds are merely infected with the virus. Recombinant pigeon pox virus containing Newcastle disease F gene will differentiate Newcastle disease virus antibodies produced in response to the recombinant pigeon pox virus from antibodies due to infection since birds inoculated with the recombinant virus will not produce antibodies to all antigenic epitopes of Newcastle disease virus unless they are challenged by the virus itself. Except for pigeons in which the pigeon pox virus vaccine can cause severe reaction if used improperly, pigeon pox virus is less pathogenic for chickens and turkeys. Thus the chances of adverse reactions to the virus vector are reduced with the use of pigeon pox virus as the vector virus. The SUBSTITUTE SHEET recombinant pigeon pox virus is useful for inoculation of species capable of being infected with pigeon pox virus including chickens, turkeys and pigeons, as well as species capable of being infected with the organism whose characteristic DNA has been inserted into the pigeon pox virus. The recombinant pigeon pox virus of the invention may be administered as a pharmaceutical preparation according to established protocols for the administration of vaccines to birds, such as those disclosed in Tripathy and Cunningham Avian Pox in Diseases of Poultry, eighth edition, Iowa State University Press, Ames, Iowa, USA, 1984, Chapter 23 Avian Pox, including the wing web method and the feather follicle method, or according to the methods employed when administering commercially available pigeon pox or fowl pox vaccines. As a pharmaceutical preparation, the recombinant virus of the invention is administered in combination with a pharmaceutically acceptable carrier or diluent, such as water, saline, or other buffer. The pharmaceutical preparation may also contain additional ingredients such as an adjuvant or stabilizer. The particular route of administration, the age of birds at administration and the dosage will depend on such factors as the species, age and weight of the bird. The route of administration, the age of the birds, and the dosage of recombinant virus, vaccine or pharmaceutical preparation may also vary according to the type of DNA not naturally occurring in pigeon pox virus expressed by the recombinant virus. In preliminary tests using recombinant pigeon pox virus expressing the F gene of Newcastle disease virus, it has been found that vaccination with a virus produced in chicken embryo skin cells having a viral titer of ιo*5-3 TCID50 per milliliter was effective to protect SPF chickens when challenged with Newcastle disease virus strain Italian. UBSTITUTE SHEET Commercially available pigeon pox virus useful as a vaccine or research strains of pigeon pox virus are suitable for use as the viral vector in the invention. Pigeon pox virus used in a preferred embodiment of the invention was obtained from Rit SA, Genval, Belgium, and has registration number 6100. The recombinant pigeon pox virus of the invention is produced by synthetically inserting into the genome of the virus DNA not normally occurring in pigeon pox virus. In preferred embodiments of the invention, the DNA not normally occurring in pigeon pox virus, i.e. foreign DNA or heterologous DNA is inserted into a non-essential portion of the genome of the virus, i.e. within the thymidine kinase gene. In other embodiments of the invention, the foreign DNA may be inserted into intergenic regions of the pigeon pox virus. Foreign DNA coding for a desired gene product that will be inserted to the pigeon pox virus vector is selected according to the type of organism it is desired to protect against, or other purpose. Preferably the DNA not normally occurring in pigeon pox virus is characteristic of, or codes for a protein against which neutralizing antibodies can be formed in the inoculated host, particularly proteins involved with the fusion of a virus to a host cell. A preferred DNA sequence is the Newcastle disease virus F gene. Recombinant pigeon pox virus containing this gene will protect inoculated birds against infection by both pigeon pox and Newcastle disease viruses. DNA coding for antigenic proteins from other viruses or other organisms may be substituted for the Newcastle disease F gene to provide protection against the respective virus or other organism. The foreign DNA is preferably inserted into the pigeon pox vector operatively linked with transcription control elements to ensure proper transcription of the foreign gene. In a preferred embodiment of the invention, the foreign DNA is operatively linked with SUBSTITUTE SHEET the Vaccinia pll K promoter. The Vaccinia pll K promoter is derived from the Vaccinia virus gene that encodes a late 11,000-dalton polypeptide (UK) , see Bertholet, C.R. e__ .al. (1985) Proc. Natl. Acad. Sci. USA £2: 2096-2100; and Coupar, B.E.H. fit al * (1986) Eur. J. Immunol. 1£: 1479-1487, the disclosures of which are hereby incorporated as if fully set forth herein. Other transcription control elements that are compatible with pigeon pox virus, such as pigeon pox virus promoters may also be used. To facilitate the insertion of the foreign gene into pigeon pox, plasmid vectors may be constructed that contain defined Vaccinia virus promoters, restriction sites for the insertion of foreign coding sequences, and flanking sequences of the pigeon pox TK gene. In a preferred embodiment of the invention, the Newcastle disease F gene is operatively linked with Vaccinia promoter upstream, and the Newcastle disease F gene and Vaccinia promoter are linked upstream and downstream with flanking sequences of the pigeon pox thymidine kinase gene (thymidine kinase gene sequence, promoter, Newcastle disease F gene, thymidine kinase gene sequence, respectively) . The plasmid DNA is then transfected into cells infected with pigeon pox virus, wild-type or modified, where homologous recombination occurs. Transfection may be done by standard methods such as calcium phosphate coprecipitation or electroporation. The host cells are then tested for the presence of recombinant virus expressing the foreign gene. Insertion of the foreign DNA into the thymidine kinase gene renders recombinant virus TK-. TK ~ recombinants may be isolated by a plaque assay in the presence of bromodeoxyuridine (BUdR) a nucleoside analogous to thymidine, but highly mutagenic and poisonous to organisms such as a cell or virus when present in DNA contained therein. Other methods of selecting recombinant virus may be found in Boyle and Coupar (1988) Virus Research M: 343-356. SUBSTITUTE SHEET Although the recombinant virus of the invention has been cultured in chicken cells, cells of other species capable of infection with pigeon pox virus are also suitable for culture of the recombinant virus. In a preferred embodiment of the invention, two recombinant viruses expressing the F gene have been isolated. PPV/F 22 recombinant virus expressing the F gene was derived from high passaged pigeon pox virus. PPV/F 26 recombinant virus expressing the F gene and was derived from low passaged pigeon pox virus. Experimental The viral strain developed as vector virus is the pigeon pox virus available from Rit SA (Genval, Belgium) registration number 6100 which is for vaccinating chickens against fowl pox by the wing web procedure. All cell cultures used for the adaptation and multiplication of pigeon pox virus were primary cultures made from SPF chicken embryos (Lohman, Cuxhaven, Germany) . Primary chicken embryo skin cells were obtained by trypsinization of whole 12-day old embryos and grown in Opti-MEM (Gibco) containing 10% fetal calf serum. For maintenance of the cells, fetal calf serum was omitted from the medium. Primary chicken embryo fibroblast cells were prepared by trypsinization of minced 9-to 10-day old chicken embryos and grown in Opti-MEM. For maintenance of the cells, fetal calf serum was omitted from the medium. Primary chicken hepatocyte cells were prepared from the liver of 14 day old embryos in Opti-MEM containing 10% fetal calf serum as growth medium. Maintenance medium was ELY (Gibco) supplemented with 12% fetal calf serum. All cell cultures were incubated at 38.5° C in the presence of 5% carbon dioxide. One vial containing 1,000 doses of pigeon pox virus (10 7 -^ EID 50) was rehydrated in 10 ml Opti-MEM and inoculated on SUBSTITUTE SHEET primary chicken hepatocyte cells. After 6 days incubation, the progeny virus was recovered by three cycles of freezing and thawing and was further adapted to cell cultures by successive passages in chicken hepatocyte cells, chicken embryo skin cells and chicken embryo fibroblast cells. Two different adapted viruses were obtained: low passaged pigeon pox virus and high passaged pigeon pox virus. Low passaged pigeon pox virus was prepared by two passages in chicken hepatocyte cells, followed by two passages in chicken embryo skin cells, followed by two passages in chicken embryo fibroblast cells (6 passages in total) . The last passage (6th passage) was titrated in chicken embryo fibroblast cells and had a titer of 10 6 - 20 TCID50 per milliliter. High passaged cells were prepared by subjecting low passaged pigeon pox virus to an additional 25 passages in chicken embryo fibroblasts (31 passages in total) . High passaged pigeon pox virus was titrated in chicken embryo fibroblasts and had a titer of 10 7 - 00 TCID 50 . Determinat.ion of EID 50 and TCID 50 EID50 is the dose of virus that will infect fifty per cent of the test group of embryos. TCID5 Q is the dose of virus that will infect 50% of the test group of cell cultures. The EID50 and TCID50 may be calculated according to standard methods, such as the method in Villegas and Purchase, "Titration of Biological Suspensions" in Isolation and identification __£ uzla n Pathogens r second edition, The American Association of Avian Pathologists, Creative Printing Company, 2011 East Main Street, Endwell, New York 13760. Construction of recombinant pi smids Unless otherwise noted, recombinant DNA techniques were performed according to standard procedures such as those in Sambrook, et al. Molecular cloning. a Laboratory Manual, second edition,. Cold Spring Harbor Laboratory Press, Cold SUBSTITUTE SHEET Spring Harbor, Massachusetts, 1989. r.lon cr of Newcastle D sease F Gene The Newcastle disease F gene as disclosed in copending, commonly assigned U.S. patent application serial number 07/441,182, filed November 22, 1989, the disclosures of which are hereby incorporated by reference as if fully set forth herein, was used in constructing the recombinant pigeon pox virus. The method for cloning this gene is set forth below. A. MATF.RTA S AND METHODS VIRUS. Newcastle Disease Virus, strain Italian, was obtained from the International Reference Laboratory Weybridge, United Kingdom. It was grown in 9 to 11 day-old chick embryos, and used as typical virus source. The virus was purified as described by Le Long et al. (1986) J. Virol. 51: 1198-1202. EXTRACTION OF poly(A)+ -mRNA's FROM NDV-INFECTED CELLS. BHK-21 cells were infected at a multiplicity of 5 to 10 PFU (Plaque Forming Units) /cell. After an absorption period of thirty minutes, the cells were incubated at 37° C in Eagle minimal essential medium (MEM) . At 2.30 hour post-infection, actinomycin D was added at a final concentration of 2 ug/ l, and four hours later, the cells were washed twice with TNE buffer (0.01M Tris, pH 8.3, 0.15M NaCl, O.OOIM EDTA) and lysed in TNE-SDS buffer (0.01M Tris pH 8.3, 0.15M NaCl, O.OOIM EDTA, 0.5% SDS) containing 400 ug/ml of proteinase K. After addition of one volume of phenol: chloroform; isoamyl alcohol (25:24:1) the DNA was broken in a mixer and the mixture extracted twice with phenol:chloroform;isoamyl alcohol (1 volume) . After ethanol precipitation, the nucleic acids were pelleted and dissolved in elution buffer (0.1M Tris pH 7.5, O.OOIM EDTA, 0.5% SDS) and the mixture was made 2M in LiCl and incubated at 4° C for 12 hours. After centrifugation, the pellet was dissolved in 2ml of hybridization buffer (0.1M Tris, pH 7.5, SUBSTITUTE SHEET 0.35M NaCl, O.OOIM EDTA, 0.5% SDS) and passed through a 1.5 ml oligo(dT)-cellulose column. Poly(A)-containing NSs were eluted with elution buffer. Before use, the poly(A) + -mRNAs were tested for stimulation of protein synthesis in rabbit reticulocyte lysates (Palham, H.R.B. et al. Eur. J. Biochem. £1:247-256 (1976)); the synthesized viral proteins were detected by immunoprecipitation with specific anti-NDV antibodies, as described by Le Long et al. (1986) J. Virol, hi: 1198-1202. SYNTHESIS AND CLONING OF cDNAs. Using poly(A)+ -mRNAs from infected cells as templates and an oligo (dT)ιo-12 primer, the first strand cDNA copies were synthesized by reverse transcriptase. After heat/chill denaturation of the mRNA-cDNA hybrids, the 3' end loop from the single-stranded cDNAs were used as primers by DNA polymerase I for second strand synthesis. The hairpin loops from the resulting double- stranded cDNAs were then digested away by SI nuclease and oligo (dC) residues were added to the 3' ends of the cDNAs to form oligo(dC) tails. The cDNAs were then inserted into the PstI site of oligo(dG)-tailed pBR322 plasmid DNA by (dG) n - (dC) n hybridization, and the ligation mixture was used to transform E. coli strain MM294 (2ι≤dR17, i.e., r ~ κ m + κ) to tetracycline resistance. DETECTION OF VIRAL CLONES. Clones containing viral cDNAs as inserts (199 out of 1277 tetracycline-resistant and ampicillin-sensitive colonies) were detected by in situ colony hybridization according to the method of Grunstein, M. and Hogness, D.S. (1975) Pro. Natl. Acad. Sci. USA 22.: 3961, the disclosures of which are hereby incorporated by reference as if fully set forth herein, by using as a probe 32 _ a ] De ι ec j 50s viral RNA (isolated on a sucrose gradient (15-30% wt/wt) . Viral clones were then classified in several groups by performing other in situ colony hybridization experiments, using as probes randomly chosen plasmid DNAs containing cDNA SUBSTITUTE SHEET insert and 32p-labeled by nick-translation. IDENTIFICATION OF THE cDNAs. Taking advantage of the known coding assignments of the mRNAs of NDV (Collins, P.L. et al., J. Virol. 4^:1024-1031 (1982)), separation of mRNAs was performed by electrophoresis of poly(A) + -mRNAs from infected cells using 1.5% agarose gels in 0.025M sodium citrate, pH 3.5, containing 6M urea, and transfer of RNA was carried out on nitro-cellulose. Then, the nitrocellulose sheet was cut in several bands and each of them hybridized with one cDNA 32p_ labeled by nick-translation. Complementary identification of the cDNAs was performed by the hybridization-selection method, as described by Riezman, H. et al. Embo J. 12:2161-2168 (1983). Each mRNA selected by this method was translated in Xenopus oocytes (Gurdon, J.B. et al. , loc. cit.) , and the proteins obtained were immunoprecipitated with anti-NDV antibodies (le Long et al., loc. cit.) . DNA SEQUENCE ANALYSIS. DNA sequence was determined by the chemical method of Maxam & Gilbert and by the Sanger dideoxy sequencing method. (2) NU LEOTIDE SEQUENCE OF THE FUSTON GF.NF, AND ΑMTNO SEQUENCE OF PROTEIN F. The recombinant cDNA clone (11.14) was chosen for DNA sequence analysis because it possesses the largest insert among the clones containing F-specific sequences. More than 94% of the sequence of both strands of the F protein coding region was determined. The entire nucleotide sequence of the insert of clone 11.14, in the mRNA sense, is shown in Figure 1. It contains 1764 base pairs excluding poly(A) tract and G- C tails. There is only a single long open reading frame, starting at the ATG (nucleotides 68-70) , and ending with an core termination codon (nucleotides 1646-1648) . This end codon is followed by four additional in-frame terminators. SUBSTITUTE SHEET The coding region encodes a protein of 526 a ino acids (M r 56,464, not including any carbohydrate composition), which contains three characteristic hydrophobic regions. F2 probably extends from residue 19 to residue 98 and consists of 890 amino acids (M r 8,304 without any carbohydrate composition) ; F-^ is a polypeptide of 423 amino acids (residues 104-526; M r 45,541 when unglycoslyated) . F contains four potential glycoslyation sites: one on F2 and three on F-^. Cloninσ of the thvmidine kinase σene of piσeon pox virus into plasmid pSF65. High passaged pigeon pox virus was purified by sucrose gradient ultracentrifugation and viral DNA was extracted using the phenol/chloroform procedure. The DNA was recovered by ethanol precipitation and digested by EcoR I before ligation into lambda gt 10 vector (Promega, Madison, Wisconsin) . Clones containing the pigeon pox virus thymidine kinase gene were identified by hybridization using a 32p oligonucleotide made on the sequence of the thymidine kinase gene of fowl pox virus published by Boyle and Coupar (1988) Virus Research 1Q.: 343-356. The sequence of the oligonucleotide probe used is 5'GAT AAA TAC ATA GCC GTG TGT AGG 3'. A positive clone was multiplied and the insert was digested with restriction enzymes to give a Cla I - BamH I fragment of 2,540 base pairs containing the entire pigeon pox virus thymidine kinase gene. This fragment was cloned into the Ace I - Sma I site of plasmid p SP65 (Promega, Madison, Wisconsin) to create plasmid p SP65 TK. The EcoR I site of p SP65 TK was then deleted using the Klenow fragment of DNA polymerase I, and an EcoR I site was introduced into the unique Nco I site of the pigeon pox virus thymidine kinase gene. SUBSTITUTE SHEET An EcoR I fragment of 383 base pairs containing the p7.5 and pll K promoters from Vaccinia virus was obtained by enzymatic digestion of plasmid p SC 11, Chakrabarti <_____, al. (1985) Molecular and Cellular Biology 12.: 3403-3409 the disclosures of which are hereby incorporated by reference as if fully set forth herein. The F gene was obtained from pBR322 Clone (11.14) by enzymatic digestion using Bam HI and F sp I restriction enzymes. The Bam HI end of the F gene was made blunt using the Klenow fragment of DNA poly erase I. The blunt ended F gene was then cloned into the PvuII site of plasmid YEP36 (Butt et al. (1984) Proc. Natl. Acad. Sci USA £1: 3332-3336), which was modified by deletion of the galactokinase coding gene to create plasmid YEP36/F. An EcoRI fragment containing the Newcastle disease virus F gene was obtained by enzymatic digestion of plasmid YEP36/F, which was then ligated into the Eco RI site of pSP64 (Promega, Madison, Wisconsin) . The F gene from Newcastle disease virus was removed from plasmid p SP 64 by EcoR I digestion. The ECO RI fragment represents most of the F coding sequence (1721 nucleotides) and still contains an ATG codon, the second one in the F sequence. The p 7.5 - p 11 K and F fragments were ligated together into the unique EcoR I site (ERI) of p SP 65 TK. The recombinant plasmid was characterized by restriction analysis for the presence of the F sequence in the correct orientation, downstream of the p 11 K promoter. The sequence at the junction of the promoter and the Newcastle disease F gene was determined and was as follows: TAA ATG AAT TC|C AGG ATC CCG GTA CCT CTA ATG ERI p UK SUBSTITUTE SHEET Transaction and isolation of recombinant pigeon pox virus Primary chicken embryo fibroblast cells were infected either with low passaged pigeon pox virus or high passaged pigeon pox virus at 0.05 PFU/cell and transfected with 5 micrograms of plasmid using Lipofectin Reagent (Bethesda Research Laboratories, Gaithersburg, Maryland) according to the manufacturer's instructions. Recombination between plasmid and viral genome generated thymidine kinase negative pigeon pox virus recombinants which were isolated by plaque assay in primary chicken embryo skin cells in the presence of bromodeoxyuridine (BUdR) at a concentration of 25 micrograms per milliliter. Two recombinant viruses expressing the F gene were isolated. PPV/F 22 recombinant virus expressing the F gene was derived from high passaged pigeon pox virus. PPV/F 26 recombinant virus expressing the F gene was derived from low passaged pigeon pox virus. After four successive rounds of cloning in chicken embryo skin cells, the presence of the F sequence in the viral DNA was ascertained by polymerase chain reaction according to the method in Sambrook et al. supra, using primers made on the thymidine kinase flanking regions of the F gene. The sequences of the primers used are 5'CTA TGT TTT CCG GTA AAA CAT CGG 3 1 and 3•CTA TTT ATG TAT CGG CAC ACA TCC 5 • . Expression of the F gene in the infected cells was demonstrated by immunofluorescence with both anti-Newcastle disease virus polyclonal antibody and F protein monoclonal antibodies, according to the method of LeLong et al. (1986) J. Virol. 51: 1198-1202, and Espion et al. (1987) Arch. Virol. 95: 79-95, the disclosures of which are hereby incprporated by reference as if fully set forth herein. Purity of the pigeon pox virus recombinant viruses was checked after the third clonage by in situ staining of plaques obtained on primary chicken embryo skin cells using TITUTE SHEET biotinylated monoclonal antibodies directed against the F protein, according to the method of Kousoulas et al. (1984) 135: 379-394, the disclosures of which are hereby incorporated by reference as if fully set forth herein. All plaques were shown to express the Newcastle disease virus F protein. Stability of the recombinant viruses was assessed by making four successive multiplications in chicken embryo skin cells of the fourth clonage of PPV/F 22 and PPV/F 26 and testing the progeny virus by immunofluorescence and radio immunoprecipitation studies using a polyclonal antiserum to Newcastle disease virus or the F protein according to the methods of LeLong et al. supra, and Espion et al. supra. Both tests were positive. Immunization Studies The fourth chicken embryo skin cell passage of PPV/F 26 was used for vaccinating SPF chickens. The viral titer of the virus was 10^* 3 TCID 50 per milliliter. eighteen six week old sPF chickens were vaccinated by the feather follicle method. Challenge was performed 20 days later by intramuscular injection of 10^ EID 50 of the velogenic Newcastle disease virus strain Italian. Ten four week old SPF chickens were included as challenge controls. All the birds were observed for one month after challenge. All control chickens died within 6 days after challenge. Only one of the vaccinated chickens died during the total observation period. Serological testing was performed by ELISA and he agglutinin inhibition reaction according to the method in Meulemans et al. (1988) Avian Path. 12: 821-827, the disclosures of which are hereby incorporated by reference as if fully set forth herein. Serological testing demonstrated the presence of F protein antibodies in 17 of 18 vaccinated chickens and the absence of hemagglutinin antibodies twenty days after vaccination. One month after challenge, hemagglutinin and F protein antibodies were present in all survivors. SUBSTITUTE SHEET The above description and examples fully disclose the invention including preferred embodiments thereof. Modification of the methods described that are obvious to those of ordinary skill in moleculat genetics and related sciences are intended to be within the scope of the following claims. SUBSTITUTE SHEET

Claims: Claims 1. A recombinant pigeon pox virus synthetically modified by the presence, in a non-essential region of the pigeon pox virus genome, of DNA not naturally occurring in pigeon pox virus. 2. The pigeon pox virus of claim 1 wherein said non- essential region is within the thymidine kinase gene. 3. The pigeon pox virus of claim 1 wherein said DNA not naturally occurring in pigeon pox virus is at least a portion of the Newcastle disease virus F gene. 4. The pigeon pox virus of claim 2 wherein said DNA not naturally occurring in pigeon pox virus is at least a portion of the Newcastle disease virus F gene. 5. A vaccine comprising a recombinant pigeon pox virus synthetically modified by the presence, in a non-essential region of the pigeon pox virus genome, of DNA not naturally occurring in pigeon pox virus. 6. The vaccine of claim 5 wherein the non-essential region of the pigeon pox genome is within the thymidine kinase gene. 7. The vaccine of claim 5 wherein the DNA not naturally occurring in pigeon pox virus is at least a portion of the Newcastle disease virus F gene. SUBSTITUTE SHEET 8. A method of producing a vaccine for prevention of avian disease comprising (a) providing foreign DNA coding for a desired gene product under control of at least one Vaccinia virus promoter within flanking sequences of the pigeon pox virus thymidine kinase gene; and (b) introducing the DNA of step (a) into the thymidine kinase locus of pigeon pox virus. 9. The method of claim 8 wherein the foreign DNA is at least a portion of the Newcastle disease virus F gene. 10. A pharmaceutical preparation comprising the pigeon pox virus of claim 1 and a pharmaceutically acceptable carrier. 11. A pharmaceutical preparation comprising the vaccine of claim 4 and a pharmaceutically acceptable carrier. 12. A method of inoculating birds susceptible to infection by Newcastle disease virus comprising administering to such a bird an amount of the vaccine of claim 4 effective to inhibit infection of the bird by Newcastle disease virus . 13. A eukaryotic cell culture infected with the virus of claim 1. 14. The eukaryotic cell culture of claim 13 wherein said cell culture is a chicken cell culture. 15. The eukaryotic cell culture of claim 14 wherein said, cell culture is a chicken embryo skin cell culture. SUBSTITUTE SHEET

Inventors: LETELLIER CARINE, MEULEMANS GUY, LETELLIER, CARINE, MEULEMANS, GUY
Applicants: SMITHKLINE BEECHAM CORP, SMITHKLINE BEECHAM CORPORATION
Application year: 1991
Main classification: C12N7
IPC codes: A61K35, A61K39, A61P31, C07K14, C12N5, C12N7, C12N15