AU729518B2 - Recombinant fowlpox viruses and uses thereof - Google Patents

Recombinant fowlpox viruses and uses thereof Download PDF

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AU729518B2
AU729518B2 AU64819/96A AU6481996A AU729518B2 AU 729518 B2 AU729518 B2 AU 729518B2 AU 64819/96 A AU64819/96 A AU 64819/96A AU 6481996 A AU6481996 A AU 6481996A AU 729518 B2 AU729518 B2 AU 729518B2
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Mark D. Cochran
David E. Junker
Philip A Singer
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Syntro Corp
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Description

WO 96/40880 PCT/US96/11187 RECOMBINANT FOWLPOX VIRUSES AND USES THEREOF Within this application several publications are referenced by arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION The present invention relates to recombinant fowlpox virus useful in live vaccine to protect fowl against Newcastle disease virus and fowlpox virus.
The ability to isolate DNA and clone this isolated DNA into bacterial plasmids has greatly expanded the approaches available to make viral vaccines. The method used to make the present invention involve modifying cloned DNA sequences by insertions, deletions and single or multiple base changes. The modified DNA is then inserted into a viral genome, and the resulting virus may then be used in a vaccine to elicit an immune response in a host animal and provide protection to the animal against disease.
Fowlpox virus (FPV) is a member of the poxviridiae family of viruses. There are two subfamilies in this classification, and they are differentiated based upon WO 96/40880 PCT/US96/11187 -2the host range (vertebrate or invertebrate) of the virus.
Among the vertebrate poxviruses, there is serological cross reactivity to group specific antigens that has aided in classification of the viruses into six genera, and FPV has been placed in the avipoxvirus genera along with seven additional poxviruses that primarily infect birds. In general, poxviruses are the largest of the animal viruses and can be visualized with the light microscope. Under the electron microscope, the virus takes on a biscuit like or oval shaped appearance. The principal chemical components of the poxviruses are protein (90% by weight), deoxyribonucleic acid (DNA) and lipid but in FPV the lipid component is -1/3 of the dry weight. Polyacrylamide gel electrophoresis (PAGE) of solubilized virions indicates that there are >100 different proteins associated with the viruses that include: structural polypeptides, enzymes associated with translation of messenger ribonucleic acid (mRNA), enzymes involved in RNA synthesis, and enzymes associated with DNA replication. The genome of poxviruses consists double-stranded DNA that varies in base composition (32% G+C to 64% G+C) and length (140 kilobasepairs [kb] to 280 kb for FPV) depending upon individual virus. The complete nucleotide sequence of the vaccina virus (VV) genome has recently been determined, and most of the essential genes have been found to lie within the highly conserved middle region of the genome while nonessential functions seem to map nearer to the termini of the DNA.
The poxviruses are unique in their propensity to replicate within the cytoplasmic space of the infected cell, and in the case of VV, mature virus particles are moved out of the assembly areas and into the periphery of the cell where additional membrane encapsulation occurs.
With FPV, the assembled viral particles become associated with a dense viral-derived protein matrix that occludes the virus in the form of cellular inclusions that may help protect the virion from lytic activities. Depending WO 96/40880 PCT/US96/11187 -3upon the specific poxvirus and strain (from 1% to 30% of different mature VV strains) varying levels of mature virus can be found extracellularly, but the majority of the virus population remains associated with the cell at the end of the growth cycle.
Fowlpox is unique throughout the world, but because its host-range is limited to birds it is not considered to be a public health hazard. All chickens can be infected by the virus with a resulting decline in the growth rate of the bird and temporary decreases in egg production.
Usually, transmission of FPV occurs through physical contact of injured skin, but there are reports that the virus is also transmitted via arthropod vectors. After an incubation period of four to ten days, the disease is typically manifested in the following ways: skin lesions in non-feathered areas, lesions of the nasal passages, and lesions of the mouth. A normal FPV infection usually lasts three to four weeks, and afterward the bird is conferred life-long immunity to the disease.
Currently, conventionally derived FPV vaccines are being used in commercial settings to provide protection to chickens and turkeys. Typically, the vaccine viruses are attenuated by serial passage in cell culture selecting for strains that have altered growth and/or virulence properties. The modified live vaccine is prepared by growth in vitro in chicken embryo fibroblast cells or by growth on the chorioallantoic membrane of the chicken embryo. The vaccine virus is given to birds subcutaneously.
The present invention concerns the use of FPV as a vector for the delivery of specific vaccine antigens to poultry.
The idea of using live viruses as delivery systems for antigens (vectoring) has a long history that is associated with introduction of the first live viral WO96/40880 PCTIUS96/11187 -4vaccines. The antigens that were delivered were not foreign but were naturally expressed by the live virus in the vaccine. The use of viruses to deliver foreign antigens in the modern sense became obvious with the recombinant DNA studies. The vaccinia virus was the vector and various antigens from other disease causing pathogens were the foreign antigens, and the vaccine was created by genetic engineering. While the concept became obvious with these disclosures, what was not obvious were the answers to more practical questions concerning what makes the best candidate viral vector and what constitutes the best foreign gene or gene to deliver. In answering these questions, details of the pathogenicity, site of replication or growth, the kind of elicited immune response, expression levels for the virus and foreign gene of interest (GOI), its suitability for genetic engineering, its probability of being licensed by regulatory agencies, etc. are all factors in the configuration. The prior art does not teach these questions of utility.
The presently preferred method for creating recombinant poxviruses uses a plasmid of bacterial origin that contains at least one cassette consisting of a poxvirus promoter followed by the gene of interest. The cassette(s) is flanked by poxvirus genomic DNA sequences that direct the gene of interest to the corresponding homologous nonessential region of the viral genome by homologous recombination. Cells are initially infected with the wild-type virus, and shortly thereafter the plasmid DNA is introduced into the infected cells. Since poxviruses have their own RNA polymerase and transcriptional apparatus, it is necessary that the gene of interest be regulated by a promoter of poxvirus origin. There are three characteristic poxvirus promoters that are differentiated based upon their temporal regulation of gene expression relative to the WO96/40880 PCT/US96/11187 infective cycle of the virus: early, intermediate and late expression. Each promoter type can be identified by a typical consensus sequence that is -30 bp in length and specific to each promoter type. In vaccinia virus, some viral genes are regulated by tandem early/late promoters that can be used by the virus to continually express the downstream gene throughout the infective cycle.
It is generally agreed that poxviruses contain nonessential regions of DNA in various parts of the genome, and that modifications of these regions can either attenuate the virus, leading to a non-pathogenic strain from which a vaccine may be derived, or give rise to genomic instabilities that yield mixed populations of virus. The degree of attenuation of the virus is important to the utility of the virus as a vaccine.
Insertions or deletions which cause too much attenuation or genetic deletions which cause too much attenuation or genetic instability of the virus will result in a vaccine that fails to elicit an adequate immune response.
Although several examples of deletions/insertions are known for poxviruses, the appropriate configuration is not readily apparent.
Thus far, gene expression from foreign genes of interest have been inserted into the genome of poxviruses has been obtained for five different pox viruses: vaccinia, canary pox, pigeon pox, raccoon pox and fowlpox. Vaccinia virus is the classically studied poxvirus, and it has been used extensively to vector foreign genes of interest; it is the subject of U.S. Patents 4,603,112 and 4,722,848.
Raccoon pox (Esposito, et al., 1988) and Canary pox (Taylor, et al., 1991) have bene used to express antigens from the rabies virus. More recently, FPV has been used to vector a number of different foreign gene of interest, and is the subject of patent applications (EPA 0 284 416, PCT WO 89/03429, PCT WO 89/12684, PCT WO 91/02072, PCT WO WO 96/40880 PCT/US96/11187 -6- 89/03879, PCT etc.). However, these publications do not teach the vectored antigen configuration, the FPV insertion sites, or the promoter sequences and the arrangement of the present invention.
A foreign gene of interest targeted for insertion into the genome of FPV can be obtained from any pathogenic organism of interest. Typically, the gene of interest will be derived from pathogens that cause diseases in poultry that have an economic impact on the poultry industry. The genes can be derived from organisms for which there are existing vaccines, and because of the novel advantages of the vectoring technology the FPV derived vaccines will be superior. Also, the gene of interest may be derived from pathogens for which thee is currently no vaccine but where there is a requirement for control of the disease. Typically, the gene of interest encodes immunogenic polypeptides of the pathogen, and may represent surface proteins, secreted proteins and structural proteins.
One relevant avian pathogen that is a target for FPV vectoring in the present invention is Infectious Laryngotracheitis virus (ILT). ILT is a member of the herpesviridiae family, and this pathogen causes an acute disease of chickens which is characterized by respiratory depression, gasping and expectoration of bloody exudate.
Viral replication is limited to cells of the respiratory tract, where in the trachea the infection gives rise to tissue erosion and hemorrhage. In chickens, no drug has been effective in reducing the degree of lesion formation or in decreasing clinical signs. Vaccination of birds with various modified forms of the ILT virus derived by cell passage and/or tedious regimes of administration have conferred acceptable protection in susceptible chickens. Because of the degree of attenuation of current ILT vaccines, care must be taken to assure that WO 96/40880 PCT/US96/11187 -7the correct level of virus is maintained; enough to provide protection, but not enough to cause disease in the flock.
An additional target for the FPV vectoring approach is Newcastle disease, an infectious, highly contagious and debilitating disease that is caused by the Newcastle disease virus (NDV), a single-stranded RNA virus of the paramyxovirus family. The various pathotypes of NDV (velongic, mesogenic, lentogenic) differ with regard to the severity of the disease, the specificity and symptoms, but most types seem to infect the respiratory system and the nervous system. NDV primarily infects chickens, turkeys and other avian species. Historically, vaccination has been used to prevent disease, but because of maternal antibody interference, life-span of the bird and route of administration, the producer needs to adapt immunization protocols to fit specific needs.
Marek's disease of poultry is a lymphoproliferative tumor producing disease of poultry that primarily affects the peripheral nervous system and other visceral tissues and organs. Marek's disease exists in poultry producing countries throughout the world, and is an additional target described by the present invention for a FPV-based vectored vaccine. The causative agent of Marek's disease is a cell associated gammaherpesvirus that has been designated as Marek's disease virus (MDV). Three classes of viruses have been developed as conventional vaccines for protecting chickens against Marek's disease: attenuated serotype 1 MDV, herpesvirus of turkeys (HVT), and naturally avirulent serotype 2 isolates of MDV.
Protection obtained with these vaccines is principally directed toward the tumorigenic aspect of the disease.
The occurrence of excessive Marek's disease losses in such conventionally vaccinated flocks has led to the requirement for forming admixtures of the various vaccine WO 96/40880 PCT/US96/11187 -8types. Such polyvalent vaccines while generally ore effective in disease control, complicate the vaccine regime.
1 WO 96/40880 PCT/US96/11187 -9- SUMMARY OF THE INVENTION This invention provides a recombinant fowlpox virus comprising a foreign DNA sequence inserted into the fowlpox virus genomic DNA, wherein the foreign DNA sequence is inserted within a non-essential region of the fowlpox virus genomic DNA and is capable of being expressed in a fowlpox virus infected host cell.
The invention further provides homology vectors, vaccines and methods of immunization.
WO 96/40880 PCT/US96/11187 BRIEF DESCRIPTION OF THE FIGURES Ficures 1A-1C: Detailed description of the Sfil fragment insert in Homology Vector 502-26.22. The diagram shows the orientation of DNA fragments assembled in the cassette. The origin of each fragment is described in the Materials and Methods section. The sequences located at the junctions between each fragment and at the ends of the marker gene are shown, including junction A (SEQ ID NO: 15), junction B (SEQ ID NO: 16), junction C (SEQ ID NO: 17), and junction D (SEQ ID NO: 18). The restriction sites used to generate each fragment are indicated at the appropriate junction. The location of the NDV F and HN genes is shown.
Numbers in parenthesis refer to amino acids, and restriction sites in brackets indicate the remnants of sites which were destroyed during construction.
Figures 2A-2D: Detailed description of fowlpox virus S-FPV-099 and S-FPV-101 and the DNA insertion in Homology Vector 751-07.D1. Diagram showing the orientation of DNA fragments assembled in plasmid 751-07.D1. The origin of each fragment is indicated in the table.
The sequences located at each of the junctions between fragments is also shown. Figures 2A-2D'show the sequences located at Junction A (SEQ ID NO: (SEQ ID NO: C (SEQ ID NO: D (SEQ ID NO: and E (SEQ ID NO: between fragments and the sequences located at the junctions. The restriction sites used to generate each fragment as well as synthetic linker sequences which are used to join the fragments are described for each junction. The WO 96/40880 PCTIUS96/11187 -11location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, refer to amino acids, and restrictions sites in brackets, indicate the remnants of sites which are destroyed during construction. The following abbreviations are used: fowlpox virus (FPV), chicken interferon (cIFN), Escherichia coli coli), pox synthetic late promoter 2 early promoter 2 (LP2EP2), pox synthetic late promoter 1 (LP1), base pairs polymerase chain reaction (PCR).
Figures 3A-3D: Detailed description of fowlpox virus S-FPV-100 and the DNA insertion in Homology Vector 751-56.C1.
Diagram showing the orientation of DNA fragments assembled in plasmid 751-56.Cl. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments is also shown. Figures 3A-3D show the sequences located at Junction A (SEQ ID NOS: (SEQ ID NO: C (SEQ ID NO: D (SEQ ID NO: and E (SEQ ID NO: between fragments and the sequences located at the junctions. The restriction sites used to generate each fragment as well as synthetic linker sequences which are used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. The following two conventions are used: numbers in parentheses, refer to amino acids, and restrictions sites in brackets, indicate the remnants of sites which are destroyed during construction. The following abbreviations are used: fowlpox virus (FPV), chicken myelomoncytic growth factor (cMGF), Escherichia coli coli), pox synthetic late promoter 2 early promoter 2 (LP2EP2), pox synthetic late promoter 1 (LP1), base pairs o~ WO 96/40880 PCT/US96/1 1187 -12polymerase chain reaction (PCR).
SWO 96/40880 PCT/US96/11187 -13- DETAILED DESCRIPTION OF THE INVENTION This invention provides a recombinant fowlpox virus comprising a foreign DNA sequence inserted into the fowlpox virus genomic DNA, wherein the foreign DNA sequence is inserted within a 2.8 kB EcoRI fragment of the fowlpox virus genomic DNA and is capable of being expressed in a fowlpox virus infected host cell.
In one embodiment the foreign DNA sequence is inserted within a SnaBI restriction endonuclease site within the approximately 2.8 kB EcoRI fragment of the fowlpox virus genomic DNA.
This invention provides a recombinant fowlpox virus comprising a foreign DNA sequence inserted into the fowlpox virus genomic DNA, wherein the foreign DNA sequence is inserted within a 3.5 kB EcoRI fragment of the fowlpox virus genomic DNA and is capable of being expressed in a fowlpox virus infected host cell.
In one embodiment the recombinant fowlpox virus the foreign DNA sequence is inserted within a HpaI restriction endonuclease site within the approximately 3.5 kB EcoRI fragment of the fowlpox virus genomic DNA.
The present invention provides a recombinant fowlpox virus comprising a foreign DNA sequence inserted into the fowlpox virus genomic DNA, wherein the foreign DNA sequence is inserted within a 4.2 kB EcoRI fragment of the fowlpox virus genomic DNA and is capable of being expressed in a fowlpox virus infected host cell.
In one embodiment of the recombinant fowlpox virus foreign DNA sequence is inserted within a MluI restriction endonuclease site within the approximately 4.2 kB EcoRI fragment of the fowlpox virus genomic DNA.
1 WO 96/40880 PCT/US96/11187 -14- The invention provides a recombinant fowlpox virus comprising a foreign DNA sequence inserted into the fowlpox virus genomic DNA, wherein the foreign DNA sequence is inserted within a non-essential region of the fowlpox virus genomic DNA and is capable of being expressed in a fowlpox virus infected host cell.
In one embodiment this invention provides a recombinant fowlpox virus wherein the foreign DNA sequence is inserted into an open reading frame within the nonessential region the fowlpox virus genomic DNA.
For purposes of this invention, "a recombinant fowlpox virus capable of replication" is a live fowlpox virus which has been generated by the recombinant methods well known to those of skill in the art, the methods set forth in HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV in Materials and Methods and has not had genetic material essential for the replication of the recombinant fowlpox virus deleted.
The invention further provides a foreign DNA sequence or foreign RNA which encodes a polypeptide. Preferably, the polypeptide is antigenic in the animal. Preferably, this antigenic polypeptide is a linear polymer of more than amino acids linked by peptide bonds which stimulates the animal to produce antibodies.
The invention further provides a recombinant fowlpox virus capable of replication which contains a foreign DNA encoding a polypeptide which is a detectable marker.
Preferably the detectable marker is the polypeptide E.
coli 0-galactosidase or E. coli beta-glucuronidase.
In one embodiment of the recombinant fowlpox virus the foreign DNA sequence encodes a cytokine. In another "WO 96/40880 PCT/US96/11187 embodiment the cytokine is chicken myelomonocytic growth factor (cMGF) or chicken interferon (cIFN). Cytokines include, but are not limited to: transforming growth factor beta, epidermal growth factor family, fibroblast growth factors, hepatocyte growth factor, insulin-like growth factor, vascular endothelial growth factor, interleukin 1, IL-1 receptor antagonist, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin- 6, IL-6 soluble receptor, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, interleukin-13, angiogenin, chemokines, colony stimulating factors, granulocyte-macrophage colony stimulating factors, erythropoietin, interferon, interferon gamma, c-kit ligand, leukemia inhibitory factor, oncostatin M, pleiotrophin, secretory leukocyte protease inhibitor, stem cell factor, tumor necrosis factors, and soluble TNF receptors. These cytokines are from humans, bovine, equine, feline, canine, porcine or avian.
This invention provides a recombinant fowlpox virus further comprising a newcastle disease virus hemagglutinin (NDV HN), or a newcastle disease virus fusion (NDV F).
Antigenic polypeptide of a human pathogen which are derived from human herpesvirus include, but are not limited to: hepatitis B virus and hepatitis C virus hepatitis B virus surface and core antigens, hepatitis C virus, human immunodeficiency virus, herpes simplex virus-1, herpes simplex virus-2, human cytomegalovirus, Epstein-Barr virus, Varicella-Zoster virus, human herpesvirus-6, human herpesvirus-7, human influenza, measles virus, hantaan virus, pneumonia virus, rhinovirus, poliovirus, human respiratory syncytial virus, retrovirus, human T-cell leukemia virus, rabies virus, mumps virus, malaria (Plasmodium falciparum), i, WO 96/40880 PCT/US96/11187 -16- Bordetella pertussis, Diptheria, Rickettsia prowazekii, Borrelia berfdorferi, Tetanus toxoid, malignant tumor antigens.
The antigenic polypeptide of an equine pathogen can derived from equine influenza virus, or equine herpesvirus. In one embodiment the antigenic polypeptide is equine influenza neuraminidase or hemagglutinin.
Examples of such antigenic polypeptide are equine influenza virus type A/Alaska 91 neuraminidase, equine influenza virus type A/Prague 56 neuraminidase, equine influenza virus type A/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81 neuraminidase, equine influenza virus type A/Kentucky 92 neuraminidase equine herpesvirus type 1 glycoprotein B, equine herpesvirus type 1 glycoprotein D, Streptococcus equi, equine infectious anemia virus, equine encephalitis virus, equine rhinovirus and equine rotavirus.
The present invention further provides an antigenic polypeptide which includes, but is not limited to: hog cholera virus gEl, hog cholera virus gE2, swine influenza virus hemagglutinin, neuromanidase, matrix and nucleoprotein, pseudorabies virus gB, gC and gD, and PRRS virus ORF7.
For example, the antigenic polypeptide of derived from infectious bovine rhinotracheitis virus gE, bovine respiratory syncytial virus equine pathogen can derived from equine influenza virus is bovine respiratory syncytial virus attachment protein (BRSV bovine respiratory syncytial virus fusion protein (BRSV F), bovine respiratory syncytial virus nucleocapsid protein (BRSV bovine parainfluenza virus type 3 fusion protein, and the bovine parainfluenza virus type 3 hemagglutinin neuraminidase WO 96/40880 PCT/US96/11187 -17- The present invention provides a recombinant fowlpox virus wherein the foreign DNA sequence encodes an antigenic polypeptide which is derived or derivable from a group consisting of: feline immunodeficiency virus gag, feline immunodeficiency virus env, infectious laryngotracheitis virus glycoprotein B, infectious laryngotracheitis virus gI, infectious laryngotracheitis virus gD, infectious bovine rhinotracheitis virus glycoprotein G, infectious bovine rhinotracheitis virus glycoprotein E, pseudorabies virus glycoprotein pseudorabies virus II glycoprotein B, pseudorabies virus III glycoprotein C, pseudorabies virus glycoprotein E, pseudorabies virus glycoprotein H, marek's disease virus glycoprotein A, marek's disease virus glycoprotein B, marek's disease virus glycoprotein D, newcastle disease virus hemagglutinin or neuraminadase, newcastle disease virus fusion, infectious bursal disease virus VP2, infectious bursal disease virus VP3, infectious bursal disease virus VP4, infectious bursal disease virus polyprotein, infectious bronchitis virus spike, infectious bronchitis virus matrix, and chick anemia virus.
The present invention provides a recombinant fowlpox virus wherein the foreign DNA sequence is under control of a promoter. In one embodiment the foreign DNA sequence is under control of an endogenous upstream poxvirus promoter. In another embodiment the foreign DNA sequence is under control of a heterologous upstream promoter. In another embodiment the promoter is selected from a group consisting of: synthetic pox viral promoter, pox synthetic late promoter 1, pox synthetic late promoter 2 early promoter 2, pox OIL promoter, pox I4L promoter, pox I3L promoter, pox I2L promoter, pox IlL promoter, pox EO1R promoter, HCMV immediate early, BHV-1.1 VP8, marek's disease virus glycoprotein A, marek's disease virus glycoprotein B, marek's disease virus glycoprotein D, WO 96/40880 PCT/US96/11187 -18laryngotracheitis virus glycoprotein I, infectious laryngotracheitis virus glycoprotein B, and infectious laryngotracheitis virus gD.
The present invention also provides a recombinant fowlpox virus designated S-FPV-097. The S-FPV-097 has been deposited on February 25, 1994 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR 2446.
The present invention also provides a vaccine which comprises an effective immunizing amount of the recombinant virus designated S-FPV-097 and a suitable carrier. The vaccine may contain either inactivated or live fowlpox virus S-FPV-097, although live virus is presently preferred. The present invention also provides a method of immunizing an animal, particularly poultry, against disease caused by fowlpox virus, Newcastle disease virus and infectious laryngotracheitis virus.
This method comprises administering to the animal an effective immunizing dose of the vaccine of the present invention. The vaccine may be administered by any of the methods well known to those skilled in the art, for example, by intramuscular, intraperitoneal, intravenous or intradermal injection. Alternatively, the vaccine may be administered intranasally, orally, or ocularly.
The present invention also provides a recombinant fowlpox virus designated S-FPV-095. The present invention also provides a vaccine which comprises an effective immunizing amount of the recombinant virus designated S- FPV-095 and a suitable carrier. The vaccine may contain either inactivated or live fowlpox virus S-FPV-095, although live virus is presently preferred. The present WO 96/40880 PCT/US96/11187 -19invention also provides a method of immunizing an animal, particularly poultry, against disease caused by fowlpox virus, Newcastle disease virus and infectious laryngotracheitis virus. This method comprises administering to the animal an effective immunizing dose of the vaccine of the present invention. The vaccine may be administered by any of the methods well known to those skilled in the art, for example, by intramuscular, intraperitoneal, intravenous or intradermal injection.
Alternatively, the vaccine may be administered intranasally, orally, or ocularly.
The present invention also provides a recombinant fowlpox virus designated S-FPV-074. The present invention also provides a vaccine which comprises an effective immunizing amount of the recombinant virus designated S- FPV-074 and a suitable carrier. The vaccine may contain either inactivated or live fowlpox virus S-FPV-074, although live virus is presently preferred. The present invention also provides a method of immunizing an animal, particularly poultry, against disease caused by fowlpox virus and Newcastle disease virus. This method comprises administering to the animal an effective immunizing dose of the vaccine of the present invention. The vaccine may be administered by any of the methods well known to those skilled in the art, for example, by intramuscular, intraperitoneal, intravenous or intradermal injection.
Alternatively, the vaccine may be administered intranasally, orally, or ocularly.
The present invention also provides a recombinant fowlpox virus designated S-FPV-081. The present invention also provides a vaccine which comprises an effective immunizing amount of the recombinant virus designated S- FPV-081 and a suitable carrier. The vaccine may contain either inactivated or live fowlpox virus S-FPV-081, although live virus is presently preferred. The present v WO 96/40880 PCT/US96/11187 invention also provides a method of immunizing an animal, particularly poultry, against disease caused by fowlpox virus and Marek's disease virus. This method comprises administering to the animal an effective immunizing dose of the vaccine of the present invention. The vaccine may be administered by any of the methods well known to those skilled in the art, for example, by intramuscular, intraperitoneal, intravenous or intradermal injection.
Alternatively, the vaccine may be administered intranasally, orally, or ocularly.
The present invention also provides a recombinant fowlpox virus designated S-FPV-085. The present invention also provides a vaccine which comprises an effective immunizing amount of the recombinant virus designated S- FPV-085 and a suitable carrier. The vaccine may contain either inactivated or live fowlpox virus S-FPV-085, although live virus is presently preferred. The present invention also provides a method of immunizing an animal, particularly poultry, against disease caused by fowlpox virus, Newcastle disease virus, infectious laryngotracheitis virus and Marek's disease virus. This method comprises administering to the animal an effective immunizing dose of the vaccine of the present invention.
The vaccine may be administered by any of the methods well known to those skilled in the art, for example, by intramuscular, intraperitoneal, intravenous or intradermal injection. Alternatively, the vaccine may be administered intranasally, orally, or ocularly.
The present invention also provides a recombinant fowlpox virus designated S-FPV-082, S-FPV-083, S-FPV-099, S-FPV- 100, and S-FPV-101.
Suitable carriers for use with the recombinant fowlpox virus vaccines of the present invention are those well WO 96/40880 PCT/US96/11187 -21known in the art and include proteins, sugars, etc. One example of such a suitable carrier is a physiologically balanced culture medium containing one or more stabilizing agents such as stabilized, hydrolyzed proteins, lactose, etc.
An "effective immunizing amount" of the recombinant viruses of the present invention is an amount within the range of 102-109 PFU/dose. Preferably, the effective immunizing amount is from about 1 0 3-10S PFU/dose for the live virus vaccine. Preferable, the live vaccine is created by taking tissue culture fluids and adding stabilizing agents such as stabilized, hydrolyzed proteins.
WO 96/40880 PCT/US96/11187 -22- MATERIAL AND METHODS PREPARATION OF FOWLPOX VIRUS STOCK SAMPLES. Fowlpox virus samples were prepared by infecting chicken embryo fibroblast (CEF) cells at a multiplicity of infection of 0.01 PFU/cell in a 1:1 mixture of HAM's F10 medium and Medium 199 (F10/199) containing 2 mM glutamine and antibiotics (referred to as CEF negative medium). Prior to infection, the cell monolayers were washed once with CEF negative medium to remove fetal bovine serum. The FPV contained in the initial inoculum (0.5 ml for 10 cm plate; 10 ml for T175 cm flask) was allowed to absorb onto the cell monolayer for two hours, being redistributed every half hour. After this period, the original inoculum was brought up to an appropriate final volume by the addition of complete CEF medium (CEF negative medium plus 2% fetal bovine serum). The plates were incubated at 37 0 C in 5% CO 2 until cytopathic effect was complete. The medium and cells were harvested, frozen at -70 0 C, thawed and dispensed into 1.0 ml vials and refrozen at -70 0 C. Virus titers typically range between 10' and 107 PFU/ml.
PREPARATION OF FPV DNA. For fowlpox virus DNA isolation, a confluent monolayer of CEF cells in a T175 cm 2 flask was infected at a multiplicity of 0.1 and incubated 4-6 days until the cells were showing 100% cytopathic effect. The infected cells were harvested by scraping into the medium and centrifuging at 3000 rpm for 5 minutes in a clinical centrifuge. The medium was decanted, and the cell pellet was gently resuspended in 1.0 ml PBS (per T175) and subjected to two successive freeze-thaws (-70 0 C to 37°C).
After the last thaw, the cells (on ice) were sonicated two times for 30 seconds each with 45 seconds cooling time in between. Cellular debris was removed by centrifuging (Sorvall RC-5B Superspeed Centrifuge) at 3000 rpm for 5 minutes in an HB4 rotor at 4°C. FPV WO 96/40880 PCT/US96/11187 -23virions, present in the supernatant, were pelleted by centrifugation at 15,000 rpm for 20 minutes at 4°C in a SS34 rotor (Sorvall) and resuspended in 10mM Tris (pH This fraction was then layered onto a 36% sucrose gradient (w/v in 10 mM Tris pH 7.5) and centrifuged (Beckman L8-70M Ultracentrifuge) at 18,000 rpm for minutes in a SW41 rotor at 4°C. The virion pellet was resuspended in 1.0 ml of 10 mM Tris pH 7.5 and sonicated on ice for 30 seconds. This fraction was layered onto a 20% to 50% continuous sucrose gradient and centrifuged at 16,000 rpm for 60 minutes in a SW41 rotor at 4°C. The FPV virion band located about three quarters down the gradient was harvested, diluted with 20% sucrose and pelleted by centrifugation at 18,000 rpm for 60 minutes in a SW41 rotor at 4°C. The resultant pellet was then washed once with 10 mM Tris pH 7.5 to remove traces of sucrose and finally resuspended in 10mM Tris pH 7.5. FPV DNA was then extracted from the purified virions by lysis (four hours at 60°C) following the addition of EDTA, SDS, and proteinase K to final concentrations of 20 mM, and 0.5 mf/ml, respectively. After digestion, three phenol-chloroform extractions were conducted and the sample precipitated by the addition of two volumes of absolute ethanol and incubated at -20 0 C for 30 minutes.
The sample was then centrifuged in an Eppendorf minifuge for five minutes at full speed. The supernatant was decanted, and the pellet air dried and rehydrated in 0.01 M Tris pH 7.5, 1mM EDTA at 4 0
C.
MOLECULAR BIOLOGICAL TECHNIQUES. Techniques for the manipulation of bacteria and DNA, including such procedures as digestion with restriction endonucleases, gel electrophoresis, extraction of DNA from gels, ligation, phosphorylation with kinase, treatment with phosphatase, growth of bacterial cultures, transformation of bacteria with DNA, and other molecular biological methods are described by Maniatis et al (1982) and I- WO 96/40880 PCT/US96/11187 -24- Sambrook et al (1989). Except as noted, these were used with minor variation.
DNA SEQUENCING. Sequencing was performed using the BRL Sequenase Kit and 3 "S-dATP (NEN). Reactions using both the dGTP mixes and the dITP mixes were performed to clarify areas of compression. Alternatively, compressed areas were resolved on formamide gels. Templates were double-stranded plasmid subclones or single stranded M13 subclones, and primers were either made to the vector just outside the insert to be sequenced, or to previously obtained sequence. Sequence obtained was assembled and compared using Dnastar software. Manipulation and comparison of sequences obtained was performed with Superclone and Supersee programs from Coral Software.
STRATEGY FOR THE CONSTRUCTION OF SYNTHETIC POX VIRAL PROMOTERS. For recombinant fowlpox vectors synthetic pox promoters offer several advantages including the ability to control the strength and timing of foreign gene expression. We chose to design four promoter cassettes EP1 (SEQ ID NO:8, LP1 (SEQ ID NO:9), EP2 (SEQ ID and LP2 (SEQ ID NO:11) based on promoters that have been defined in the vaccinia virus (Bertholet et al. 1986, Davidson and Moss, 1989a, and Davidson and Moss, 1989b).
Each cassette was designed to contain the DNA sequences defined in vaccina flanked by restriction sites which could be used to combine the cassettes in any order or combination. Initiator methionines were also designed into each cassette such that inframe fusions could be made at either EcoRI or BamHi sites. A set of translational stop codons in all three reading frames and an early transcriptional termination signal (Earl, et al., 1990) was also engineered downstream of the inframe fusion site. DNA encoding each cassette was synthesized according to standard techniques and cloned into the appropriate homology vectors.
1. WO 96/40880 PCT/US96/11187 cDNA CLONING PROCEDURE. cDNA cloning refers to the methods used to convert RNA molecules into DNA molecules following state of the art procedures. Applicants' methods are described in (Gubler and Hoffman, 1983).
Bethesda Research Laboratories (Gaithersburg, MD) have designed a cDNA Cloning Kit that is very similar to the procedures used by applicants, and contains a set of reagents and protocols that may be used to duplicate our results.
For cloning virus mRNA species, a host cell line sensitive to infection by the virus was infected at 5-10 plaque forming units per cell. When cytopathic effect was evident, but before total destruction, the medium was removed and the cells were lysed in 10 mls lysis buffer (4 M guanidine thiocyanate, 0.1% antifoam A, 25 mM sodium citrate pH 7.0, 0.5% N-lauroyl sarcosine, 0.1 M betamercaptoethanol). The cell lysate was poured into a sterilized Dounce homogenizer and homogenized on ice 8-10 times until the solution was homogenous. For RNA purification, 8mls of cell lysate were gently layered over 3.5 mls of CsCl solution (5.7 M CsCl, 25 mM sodium citrate pH 7.0) in a Beckman SW41 centrifuge tube. The samples were centrifuged for 18 hrs at 20 0 C at 36000 rpm in a Beckman SW41 rotor. The tubes were put on ice and the supernatants from the tubes were carefully removed by aspiration to leave the RNA pellet undisturbed. The pellet was resuspended in 400 pl glass distilled water, and 2.6 mls of guanidine solution (7.5 M guanidine-HCl, 25 mM sodium citrate pH 7.0, 5 mM dithiothreitol) were added. Then 0.37 volumes of 1 M acetic acid were added, followed by 0.75 volumes of cold ethanol and the sample was put at -20 0 C for 18 hrs to precipitate RNA. The precipitate was collected by centrifugation in a Sorvall centrifuge for 10 min at 4 0 C at 10000 rpm in an SS34 rotor. The pellet was dissolved in 1.0 ml distilled water, recentrifuged at 13000 rpm, and the supernatant WO 96/40880 PCT/US96/11187 -26saved. RNA was re-extracted from the pellet 2 more times as above with 0.5 ml distilled water, and the supernatants were pooled. A 0.1 volume of 2 M potassium acetate solution was added to the sample followed by 2 volumes of cold ethanol and the sample was put at for 18 hrs. The precipitated RNA was collected by centrifugation in the SS34 rotor at 4°C for 10 min at 10000 rpm. The pellet was dissolved in 1 ml distilled water and the concentration taken by adsorption at A260/280. The RNA was stored at -70 0
C.
mRNA containing polyadenylate tails (poly-A) was selected using oligo-dT cellulose (Pharmacia #27 5543-0). Three mg of total RNA was boiled and chilled and applied to a 100 mg oligo-dT cellulose column in binding buffer (0.1 M Tris pH 7.5, 0.5 M LiCl, 5 mM EDTA pH 8.0, 0.1% lithium dodecyl sulfate). The retained poly-A+ RNA was eluted from the column with elution buffer (5 mM Tris pH 7.5, 1 mM EDTA pH 8.0, 0.1% sodium dodecyl sulfate). This mRNA was reapplied to an oligo-dT column in binding buffer and eluted again in elution buffer. The sample was precipitated with 200 mM sodium acetate and 2 volumes cold ethanol at -20 0 C for 18 hrs. The RNA was resuspended in 50 pl distilled water.
Ten Ag poly-A+ RNA was denatured in 20 mM methyl mercury hydroxide for 6 min at 22 0 C. 3-mercaptoethanol was added to 75 mM and the sample was incubated for 5 min at 22°C.
The reaction mixture for first strand cDNA synthesis in 0.25 ml contained 1 Ag oligo-dT primer (P-L Biochemicals) or 1 fg synthetic primer, 28 units placental ribonuclease inhibitor (Bethesda Research Labs #5518SA), 100 mM Tris pH 8.3, 140 mM KC1, 10 mM MgC12, 0.8 mM dATP, dCTP, dGTP, and dTTP (Pharmacia), 100 microcuries 32Plabeled dCTP (New England Nuclear #NEG-013H), and 180 units AMV reverse transcriptase (Molecular Genetics Resources #MG 101). The reaction was incubated at 42°C WO 96/40880 PCT/US96/11187 -27for 90 min, and then was terminated with 20 mM EDTA pH The sample was extracted with an equal volume of phenol/chloroform and precipitated with 2 M ammonium acetate and 2 volumes of cold ethanol -20 0 C for 3 hrs. After precipitation and centrifugation, the pellet was dissolved in 100 jil distilled water. The sample was loaded onto a 15 ml G-100 Sephadex column (Pharmacia) in buffer (100 mM Tris pH 7.5, 1 mM EDTA pH 100 mM NaCI). The leading edge of the eluted DNA fractions were pooled, and DNA was concentrated by lyophilization until the volume was about 100 il, then the DNA was precipitated with ammonium acetate plus ethanol as above.
The entire first strand sample was used for second strand reaction which followed the Gubler and Hoffman (1983) method except that 50 ig/ml dNTP's, 5.4 units DNA polymerase I (Boerhinger Mannheim #642-711), and 100 units/ml E. coli DNA ligase (New England Biolabs #205) in a total volume of 50 microliters were used. After second strand synthesis, the cDNA was phenol/chloroform extracted and precipitated. The DNA was resuspended in pl distilled water, treated with 1 yg RNase A for min at 22 0 C, and electrophoresed through a 1% agarose gel (Sigma Type II agarose) in 40 mM Tris-acetate buffer pH 6.85. The gel was strained with ethidium bromide, and DNA in the expected size range was excised from the gel and electroeluted in 8 mM Tris-acetate pH 6.85.
Electroeluted DNA was lyophilized to about 100 microliters, and precipitated with ammonium acetate and ethanol as above. The DNA was resuspended in 20 Al water.
Oligo-dC tails were added to the DNA to facilitate cloning. The reaction contained the DNA, 100 mM potassium cacodylate pH 7.2, 0.2 mM dithiothreitol, 2 mM CaCl 2 80 jimoles dCTP, and 25 units terminal 1. WO 96/40880 PCT/US96/11187 -28deoxynucleotidyl transferase (Molecular Genetic Resources #S1001) in 50 Al. After 30 min at 37 0 C, the reaction was terminated with 10 mM EDTA, and the sample was phenol/chloroform extracted and precipitated as above.
The dC-tailed DNA sample was annealed to 200 ng plasmid vector pBR322 that contained oligo-dG tails (Bethesda Research Labs #5355 SA/SB) in 200 Al of 0.01 M Tris pH 0.1 M NaCl, 1 mM EDTA pH 8.0 at 65 0 C for 2 min and then 57 0 C for 2 hrs. Fresh competent E. coli DH-1 cells were prepared and transformed as described by Hanahan (1983) using half the annealed cDNA sample in twenty 200 pl aliquots of cells. Transformed cells were plated on L-broth agar plates plus 10 Ag/ml tetracycline. Colonies were screened for the presence of inserts into the ampicillin gene using Ampscreen\ (Bethesda Research Labs #5537 UA), and the positive colonies were picked for analysis.
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. This method relies upon the homologous recombination between FPV DNA and the plasmid homology vector DNA which occurs in the tissue culture cells containing both FPV DNA and transfected plasmid homology vector. For homologous recombination to occur, monolayers of CEF cells are infected with S-FPV-001 (A mild fowlpox vaccine strain available as Bio-Pox' from Agri-Bio Corporation, Gainsville, Georgia) at a multiplicity of infection of 0.01 PFU/cell to introduce replicating FPV DNA synthesis) into the cells. The plasmid homology vector DNA is then transfected into these cells according to the "Infection-Transfection Procedure".
INFECTION-TRANSFECTION PROCEDURE. CEF cells in 6 cm plates (about 80% confluent) were infected with S-FPV-001 at a multiplicity of infection of 0.01 PFU/cell in CEF 1, o WO 96/40880 PCT/US96/11187 -29negative medium and incubated at 370C in a humidified
CO
2 incubator for five hours. The transfection procedure used is essentially that recommended for Lipofectin T M Reagent (BRL). Briefly, for each 6 cm plate, micrograms of plasmid DNA were diluted up to 100 microliters with H 2 0. Separately, 50 micrograms of Lipofectin M Reagent were diluted to 100 microliters with
H
2 0. The 100 microliters of diluted Lipofectin™ Reagent were added dropwise to the diluted plasmid DNA contained in a polystyrene, 5 ml, snap cap tube and mixed gently.
The mixture was then incubated for 15-20 minutes at room temperature. During this time, the virus inoculum was removed from the 6 cm plates and the cell monolayers washed once with CEF negative medium. Three mls of CEF negative medium were added to the plasmid DNA/lipofectin mixture and the contents pipetted onto the cell monolayer. Following overnight (about 16 hours) incubation at 37 0 C in a humidified 5% CO 2 incubator, the medium was removed and replaced with 5 ml CEF complete medium. The cells were incubated at 37°C in 5% CO 2 for 3- 7 days until cytopathic effect from the virus was 100%. Virus was harvested as described above for the preparation of virus stocks. This stock was referred to as a transfection stock and was subsequently screened for recombinant virus by the "Plaque Hybridization Procedure For Purifying Recombinant FPV".
PLAQUE HYBRIDIZATION PROCEDURE FOR PURIFYING RECOMBINANT FPV. CEF cell monolayers were infected with various dilutions of the infection/transfection viral stocks, overlaid with nutrient agarose media (equal volumes of agarose and 2X M199) and incubated 6-7 days for plaque development to occur. The agarose overlay and plate were marked with the same three asymmetrical dots (India ink) to aid in positioning the Nitrocellulose (NC) membrane (cell monolayer) and agarose overlay. The agarose overlay was transferred to the lid of the 10 cm 1, WO 96/40880 PCT/US96/11187 dish and stored at 4*C. The CEF monolayer was overlaid with a pre-wetted (PBS) NC membrane and pressure applied to transfer the monolayer to the NC membrane. Cells contained on the NC membrane were then lysed by placing the membranes in 1.5 ml of 1.5 M NaCI and 0.5 M NaOH for five minutes. The membranes were placed in 1.5 ml of 3 M sodium acetate (pH 5.2) for five minutes. DNA from the lysed cells was bound to the NC membrane by baking at 0 C for one hour. After this period the membranes were prehybridized with a solution containing 6X SSC, 3% skim milk, 0.5% SDS, salmon sperm DNA (50 gg/ml) and incubated at 65°C for one hour. Radio-labeled probe DNA (alpha 32
P-
dCTP) was added and incubated at 65 0 C overnight (12 hours). After hybridization the NC membranes were washed two times (30 minutes each) with 2X SSC at 65 0 C, followed by two additional washes at 65 0 C with 0.5X SSC. The NC membranes were dried and exposed to X-ray film (Kodak X- OMAT, AR) at -70 0 C for 12 hours. Plaques corresponding to positive signals seen on the autoradiogram were picked from the agarose overlay, using a pasteur pipette, and were resuspended into 1 ml of CEF media and stored at 0 C. Typically, 5-6 rounds of plaque purification were required to ensure purity of the recombinant virus.
SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FPV USING BLACK PLAQUE ASSAYS. To analyze expression of foreign antigens expressed by recombinant fowlpox viruses, monolayers of CEF cells were infected with recombinant FPV, overlaid with nutrient agarose media and incubated for 6-7 days at 37 0 C for plaque development to occur. The agarose overlay was removed from the dish, the cells fixed with 100% methanol for 10 minutes at room temperature and air dried. The primary antibody was diluted to an appropriate concentration with PBS and incubated on the cell monolayer for two hours at room temperature. Unbound antibody was removed from the cells by washing three times with PBS at room temperature. A WO 96/40880 PCT/US96/11187 -31horseradish peroxidase conjugated secondary antibody was diluted with PBS and incubated on the cell monolayer for two hours at room temperature. Unbound secondary antibody was then removed by washing the cells three times with PBS at room temperature. The cells were incubated 15-30 minutes at room temperature with freshly prepared substrate solution (100 Ag/ml 4-chloro-1naphthol, 0.003% H 2 0 2 in PBS). Plaques expressing the correct antigen stain black.
SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. When the E. coli 0-galactosidase (lacZ) or 3glucuronidase (uidA) marker gene was incorporated into a recombinant virus the plaques containing recombinants were visualized by a simple assay. The enzymatic substrate was incorporated (300 gg/ml) into the agarose overlay during the plaque assay. For the lacZ marker gene the substrates Bluogal M (halogenated indolyl-o-Dgalactosidase, Bethesda Research Labs) for blue plaques or CPRG (chlorophenol Red Galactopyranoside, Boehringer mannheim) for red plaques were used. For the uidA marker gene the substrate X-Glucuro Chx (5-bromo-4chloro-3-indolyl-o-D-glucuronic acid Cyclohexylammonium salt, Biosynth AG) was used. Plaques that expressed active marker enzyme turned either red or blue. The plaques were then picked onto fresh cells and purified by further plaque isolation.
RNA ISOLATED FROM CONCANAVALIN A STIMULATED CHICKEN SPLEEN CELLS. Chicken spleens were dissected from 3 week old SPAFAS hatched chicks, washed, and disrupted through a syringe/needle to release cells. After allowing stroma and debri to settle out, the cells were pelleted and washed twice with PBS. The cell pellet was treated with a hypotonic lysis buffer to lyse red blood cells, and splenocytes were recovered and washed twice with PBS.
Splenocytes were resuspended at 5 x 106 cells/ml in RPMI p WO 96/40880 PCT/US96/11187 -32containing 5% FBS and 5 Ag/ml Concanavalin A and incubated at 390 for 48 hours. Total RNA was isolated from the cells using guanidine isothionate lysis reagents and protocols from the Promega RNA isolation kit (Promega Corporation, Madison WI). 4pg of total RNA was used in each 1st strand reaction containing the appropriate antisense primers and AMV reverse transcriptase (Promega Corporation, Madison WI). cDNA synthesis was performed in the same tube following the reverse transcriptase reaction, using the appropriate sense primers and Vent® DNA polymerase (Life Technologies, Inc. Bethesda, MD).
HOMOLOGY VECTOR 451-79.95. The plasmid 451-79.95 was constructed for the purpose of inserting the NDV HN gene into FPV. A lacZ marker gene followed by the NDV HN gene was inserted as a cassette into the homology vector 443- 88.14 at the unique Sfil site. The cassette may be constructed utilizing standard recombinant DNA techniques (Maniatis et al., 1982 and Sambrook et al., 1989), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated. The first fragment is the synthetic late promoter LP1 (SEQ ID NO:9). The second fragment contains the coding region of E. coli lacZ and is derived from plasmid pJF751 (Ferrari et al., 1985). Note that the promoter and lacZ gene are fused so as to express a hybrid protein consisting of 4 amino acids derived from the synthetic promoter followed by amino acids 10 to 1024 of the lacZ gene. The third fragment is another copy of the synthetic late promoter LP1. the fourth fragment contains the coding region of the NDV HN gene and was derived from the full length HN cDNA clone. Note that the promoter and HN gene are fused so as to express a hybrid protein consisting of 4 amino acids derived from the synthetic promoter followed by amino acids 2 to 577 of the HN gene. Both genes are in the opposite transcriptional orientation relative to the WO 96/40880 PCT/US96/11187 -33- ORF1 gene in the parental homology vector.
HOMOLOGY VECTOR 489-21.1. The plasmid 489-21.1 was constructed for the purpose of inserting the NDV HN gene into FPV. The NDV HN gene was inserted as a cassette into the homology vector 443-88.8 at the unique Sfil site. The cassette may be constructed utilizing standard recombinant DNA techniques (Maniatis et al., 1982 and Sambrook et al., 1989), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated. The first fragment is the synthetic early/late promoter EP1LP2 (SEQ ID NO:8/SEQ ID NO:11).
The second fragment contains the coding region of the NDV HN gene and was derived from the full length HN cDNA clone. Note that the promoter and HN gene are fused so as to express a hybrid protein consisting of 4 amino acids derived from the synthetic promoter followed by amino acids 2 to 577 of the HN gene. The HN gene is in the opposite transcriptional orientation relative to the ORF in the parental homology vector.
HOMOLOGY VECTORS 502-26.22. The plasmid 502-26.22 was constructed for the purpose of inserting the NDV HN and F genes into FPV. The NDV HN and F genes were inserted as a SfiI fragment (SEQ ID NO:12) into the homology vector 443-88.8 at the unique Sfil site. The NDV HN and F genes were inserted in the same transcriptional orientation as the ORF in the parental homology vector.
A detailed description of the Sfil is shown in Figures 1A-1C. The inserted Sfil fragment may be constructed utilizing standard recombinant DNA techniques (Maniatis et al. and Sambrook et al., 1989), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figures 1A-1C. Fragment 1 is approximately 1811 base pair Avail to NaeI restriction fragment of the full length NDV HN cDNA clone (B1 strain). Fragment 2 is an approximately 1812 base pair WO 96/40880 PCT/US96/11187 -34- BamHI to PstI restriction fragment of the full length NDV F cDNA (Bl strain). Fragment 3 is an approximately 235 base pair PstI and Scal restriction fragment of the plasmid pBR322.
HOMOLOGY VECTOR 502-27.5. The plasmid 502-27.5 was constructed for the purpose of inserting the NDV F gene into FPV. A LacZ marker gene followed by the NDV F gene was inserted as a cassette into the homology vector 443- 88.14 at the unique SfiI site. The cassette may be constructed utilizing standard recombinant DNA techniques (Maniatis et al., 1982 and Sambrook et al., 1989), joining restriction fragments from the following sources with the synthetic DNA sequences indicated. The first fragment is the synthetic late promoter LP1 (SEQ ID NO:9). The second fragment contains the coding region of E. coli LacZ and is derived from plasmid pJF751 (Ferrari et al., 1985). Note that the promoter and LacZ gene are fused so as to express a hybrid protein consisting of 4 amino acids derived from the synthetic promoter followed by amino acids 10 to 1024 of the LacZ gene. The third fragment is the synthetic early/late promoter EP1LP2 (SEQ ID NO:8/SEQ ID NO:11). The fourth fragment contains the coding region of the NDV F gene and was derived from the full length F cDNA clone. Note that the promoter and F gene are fused so as to express a hybrid protein consisting of 4 amino acids dervied from the synthetic promoter followed by 10 amino acids derivied from the F gene 5' untranslated region followed by amino acid 1 to 544 of the F gene. Both genes are in the opposite transcriptional orientation relative to the ORF in the parental homology vector.
HOMOLOGY VECTOR 586-36.6. The plasmid 586-36.6 was constructed for the purpose of inserting the infectious laryngotracheitis virus (ILT) gB and gD genes into the FPV. An E. coli 9-glucuronidase uidA marker gene WO 96/40880 PCT/US96/11187 preceeded by the ILT gB and gD genes was inserted as a cassette into the homology vector 451-08.22 at the unique Sfil site. The cassette may be constructed utilizing standard recombinant DNA techniques (Maniatis et al., 1982 and Sambrook et al., 1989), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated. The first fragment is the synthetic early/late promoter EP1LP2 (SEQ ID NO:8/SEQ ID NO:11). The second fragment contains the coding region of ILT gB and is dervied from an approximately 3000 base pair ILT virus genomic EcoRI fragment. Note that the promoter and gB gene are fused so as to express the complete coding region of the gB gene (amino acids 1- 883). The third fragment is the synthetic early/late promoter EP1LP2 (SEQ ID NO:8/SEQ ID NO:11). The fourth fragment contains the coding region of the ILT gD gene (SEQ ID NO:19) and was derived from an approximately 2060 base pair EcoRI to BclI restriction sub-fragment of the ILT KpnI genomic restriction fragment #8 (10.6 KB). Note that the promoter and gD gene are fused so as to express a hybrid protein consisting of 3 amino acids dervied from the synthetic promoter followed by amino acids 3 to 434 of the gD gene. The fifth fragment is the synthetic late promoter LP1 (SEQ ID NO:9). The last fragment contains the coding region of E. coli uidA and is derived from plasmid pRAJ260 (Clonetech). Note that the promoter and uidA gene are fused so as to express a hybrid protein consisting of 3 amino acids derived from the synthetic promoter followed by amino acids 1 to 602 of the uidA gene. All three genes are in the opposite transcriptional orientation relative to ORFI in the parental homology vector.
HOMOLOGY VECTOR 608-10.3. The plasmid 608-10.3 was constructed for the purpose of inserting the Marek's Disease virus (MDV) gD and gB genes into FPV. A LacZ marker gene preceeded by the MDV gD and gB genes was WO 96/40880 PCT/US96/11187 -36inserted as a cassette into the homology vector 443-88.14 at the unique Sfil site. The cassette may be constructed utilizing standard recombinant DNA techniques (Maniatis et al., 1982 and Sambrook et al., 1989), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated. The first fragment is the synthetic late/early promoter LP2EP2 (SEQ ID NO:11/SEQ ID NO:10). The second fragment contains the coding region of MDV gD and is derived from an approximately 2177 base pair NcoI to SalI sub-fragment of the MDV BglII 4.2 KB genomic restriction fragment (Ross, et al., 1991). Note that the promoter and gD are fused so as to express a hybrid protein consisting of 3 amino acids derived from the synthetic promoter followed by amino acids 3 to 403 of the gD gene. The third fragment is the synthetic early/late promoter EP1LP2 (SEQ ID NO:8/SEQ ID NO:11). The fourth fragment contains the coding region of the MDV gB gene and was derived from an approximately 3898 base pair SalI to EcoRI genomic MDV fragment (Ross, et al., 1989). Note that the promoter and gB gene are fused so as to express a hybrid protein consisting of 3 amino acids derived from the synthetic promoter followed by amino acids 3 to 865 of the gB gene.
The fifth fragment is the synthetic late promoter LP1 (SEQ ID NO:9). The sixth fragment contains the coding region of E. coli LacZ and is derived from plasmid pJF751 (Ferrari, et al., 1985). Note that the promoter and LacZ gene are fused so as to express a hybrid protein consisting of 4 amino acids derived from the synthetic promoter followed by amino acids 10 to 1024 of the LacZ gene. All three genes are in the opposite transcriptional orientation relative to ORF1 in the parental homology vector.
HOMOLOGY VECTOR 538-51.27. The plasmid 538-51.27 was constructed for the purpose of inserting the genes for Infectious Bronchitis virus (IBV) Massachusetts Spike WO 96/40880 PCT/US96/11187 -37protein (Mass Spike) and Massachusetts Matrix protein (Mass Matrix) into FPV. A lacZ maiker gene and the genes for IBV Mass Spike and Mass Matrix.were inserted as a cassette into the homology vector 443-88.14 at the unique Sfil site. The inserted Sfil fragment is constructed utilizing standard recombinant DNA techniques (Maniatis et al., 1982 and Sambrook et al., 1989), by joining restriction fragments from the following sources. The first fragment is the synthetic early/late promoter EP1LP2 (SEQ ID NO: 8/ SEQ ID NO: 11). The second fragment contains the coding region for the IBV Mass Spike gene and (amino acids 3-1162) is derived from an approximately 3500 base pair BsmI to PvuI IBV cDNA fragment. The third fragment is the synthetic early/late promoter EP1LP2 (SEQ ID NO: 8/ SEQ ID NO: 11). The fourth fragment contains the coding region for the IBV Mass Matrix gene (amino acids 1-232) and is derived from an approximately 1500 base pair XbaI to Spel IBV cDNA fragment. The fifth fragment is the synthetic late promoter LP1 (SEQ ID NO: The sixth fragment contains the coding region of E. coli lacZ and is derived from plasmid pJF751 (Ferrari, et al. 1985).
HOMOLOGY VECTOR 622-49.1. The plasmid 622-49.1 was constructed for the purpose of inserting the IBV Massachusetts (Mass) Nucleocapsid gene into FPV. A uidA marker gene and the IBV Mass Nucleocapsid gene was inserted as a cassette into the homology vector 451-08.22 at the unique Sfil site. The inserted Sfil fragment was constructed utilizing standard recombinant DNA techniques (Maniatis et al., 1982 and Sambrook et al., 1989), by joining restriction fragments from the following sources.
The first fragment is the synthetic early/late promoter EP1LP2 (SEQ ID NO: 8/ SEQ ID NO: 11). The second fragment contains the coding region for the IBV Mass Nucleocapsid gene and is derived from an approximately 3800 base pair PstI to IBV cDNA fragment. The third fragment is the synthetic late promoter LP1 (SEQ ID NO: i- WO 96/40880 PCT/US96/11187 -38- The fourth fragment contains the coding region of E.
coli uidA and is derived from plasmid pRAJ260 (Clonetech).
HOMOLOGY VECTORS 584-36.12. The plasmid 584-36.12 was constructed for the purpose of inserting the NDV HN and F genes into FPV. The NDV HN and F genes were inserted as a Sfil fragment into the homology vector 443-88.14 (see example IB) at the unique Sfil site. The NDV HN and F genes were inserted in the same transcriptional orientation as the ORF in the parental homology vector.
A detailed description of the Sfil fragment is shown in Figures 1A-1C. The inserted Sfil fragment was constructed utilizing standard recombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figures 1A-1C.
Fragment 1 is an approximately 1811 base pair Avail to NaeI restriction fragment of the full length NDV HN cDNA clone (BI strain). Fragment 2 is an approximately 1812 base pair BamHI to PstI restriction fragment of the full length NDV F cDNA (BI strain). Fragment 3 is an approximately 235 base pair PstI to Scal restriction fragment of the plasmid pBR322.
HOMOLOGY VECTOR 694-10.4. The plasmid 694-10.4 was constructed for the purpose of inserting the infectious laryngotracheitis virus (ILTV) gB and gD genes into FPV.
An E.coli 0-glucuronidase uidA marker gene preceded by the ILTV gB and gD genes was inserted as a cassette into the homology vector 451-08.22 at the unique Sfil site.
The cassette was constructed utilizing standard recombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joining restriction fragments from the following sources with the synthetic DNA sequences indicated. The first fragment is the synthetic early/late promoter EP1LP2 (SEQ ID NO:8/SEQ ID NO:11).
WO 96/40880 PCT/US96/11187 -39- The second fragment contains the coding region of ILTV gB and is derived from an approximately 3000 base pair ILT virus genomic EcoRI fragment. Note that the promoter and gB gene are fused so as to express the complete coding region of the gB gene (animo acids 1-883). The third fragment is the synthetic early/late promoter EP1LP2 (SEQ ID NO:8/SEQ ID NO:11). The fourth fragment contains the coding region of the ILTV gD gene and was derived from an approximately 2060 base pair EcoRI to BclI restriction sub-fragment of the ILTV KpnI genomic restriction fragment #8 (10.6 KB). Note that the promoter and gD gene are fused so as to express a hybrid protein consisting of 3 amino acids derived from the synthetic promoter followed by amino acids 3 to 434 of the gD gene. The fifth fragment is the synthetic late promoter LP1 (SEQ ID The last fragment contains the coding region of E.coli uidA and is derived from plasmid pRAJ260 (Clonetech). Note that the promoter and uidA gene are fused so as to express a hybrid protein consisting of 3 amino acids derived from the synthetic promoter followed by amino acids 1 to 602 of the uidA gene.
HOMOLOGY VECTOR 749-75.82. The plasmid 749-75.82 was used to insert foreign DNA into FPV. It incorporates an E.
coli P-galactosidase (lacZ) marker gene and the infectious bursal disease virus (IBDV) polymerase gene flanked by FPV DNA. When this plasmid was used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV a virus containing DNA coding for the foreign genes results. Note that the -galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1) and the IBDV polymerase gene is under the control of a synthetic late/early pox promoter (LP2EP2). The homology vector was constructed utilizing standard recombinant DNA techniques (11 and 14), by joining restriction fragments from the following sources with the appropriate synthetic DNA sequences. The plasmid WO 96/40880 PCT/US96/11187 vector was derived from an approximately 2999 base pair EcoRI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1184 base pair EcoRI to SnaBI restriction sub-fragment of the 2.8 kb EcoRI FPV genomic fragment (SEQ ID NO. Fragment 2 is an approximately 2700 EcoRI to AscI restriction fragment synthesized by cDNA cloning and polymerase chain reaction (PCR) from an IBDV RNA template. cDNA and PCR primers CACGAATTCTGACATTTTCAACAGTCCACAGGCGC-3'; 12/93.4) (SEQ ID NO: and 5'-GCTGTTGGACATCACGGGCCAGG-3'; 9/93.28) (SEQ ID NO: were used to synthesize an approximately 1100 base pair EcoRI to BclI fragment at the 5' end of the IBDV polymerase gene. cDNA and PCR primers ACCCGGAACATATGGTCAGCTCCAT-3'; 12/93.2) (SEQ ID NO: and 5'-GGCGCGCCAGGCGAAGGCCGGGGATACGG-3'; 12/93.3) (SEQ ID NO: were used to synthesize an approximately 1700 base pair BclI to AscI fragment at the 3' end of the IBDV polymerase gene. The two fragments were ligated at the BclI site to form the approximately 2800 base pair EcoRI to BclI fragment. Fragment 3 is an approximately 3002 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 4 is an approximately 1626 base pair SnaBI to EcoRI restriction sub-fragment of the 2.8 kb EcoRI FPV genomic fragment (SEQ ID NO. HOMOLOGY VECTOR 751-07.Dl. The plasmid 751-07.Dl was used to insert foreign DNA into FPV. It incorporates an E.
coli P-galactosidase (lacZ) marker gene and the chicken interferon (cIFN) gene flanked by FPV DNA. When this plasmid was used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV a virus containing DNA coding for the foreign genes results. Note that the 0-galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1) and the cIFN gene is under the control of a synthetic late/early pox promoter (LP2EP2). The homology vector was constructed utilizing standard recombinant DNA I WO 96/40880 PCT/US96/11187 -41techniques by joining restriction fragmen ts from the following sources with the appropriate synthetic DNA sequences. The plasmid vector was derived from an approximately 2999 base pair EcoRI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1626 base pair EcoRI to SnaBI restriction sub-fragment of the 2.8 kb EcoRI FPV genomic fragment (SEQ ID NO. Fragment 2 is an approximately 577 base pair EcoRI to BglII fragment coding for the cIFN gene (17) derived by reverse transcription and polymerase chain reaction (PCR) (Sambrook, et al., 1989) of RNA ISOLATED FROM CONCANAVALIN A STIMULATED CHICKEN SPLEEN CELLS. The antisense primer (6/94.13) used for reverse transcription and PCR was CGACGGATCCGAGGTGCGTTTGGGGCTAAGTGC-3' (SEQ ID NO: The sense primer (6/94.12) used for PCR was CCACGGATCCAGCACAACGCGAGTCCCACCATGGCT-3' (SEQ ID NO: The BamHI fragment resulting from reverse transcription and PCR was gel purified and used as a template for a second PCR reaction to introduce a unique EcoRI site at the 5' end and a unique BglII site at the 3' end. The second PCR reaction used primer 6/94.22 CCACGAATTCGATGGCTGTGCCTGCAAGCCCACAG-3'; SEQ ID NO: at the 5' end and primer 6/94.34 CGAAGATCTGAGGTGCGTTTGGGGCTAAGTGC-3'; SEQ ID NO: at the 3' end to yield an approximately 577 base pair fragment.
The DNA fragment contains the coding sequence from amino acid 1 to amino acid 193 of the chicken interferon protein (17) which includes a 31 amino acid signal sequence at the amino terminus and 162 amino acids of the mature protein encoding chicken interferon. Fragment 3 is an approximately 3002 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 4 is an approximately 1184 base pair SnaBI to EcoRI restriction sub-fragment of the 2.8 kb EcoRI FPV genomic fragment (SEQ ID NO. WO 96/40880 PCT/US96/11187 -42- HOMOLOGY VECTOR 751-56.Cl. The plasmid 751-56.Cl was used to insert foreign DNA into FPV. It incorporates an E.
coli 0-galactosidase (lacZ) marker gene and the chicken myelomonocytic growth factor (cMGF) gene flanked by FPV DNA. When this plasmid was used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV a virus containing DNA coding for the foreign genes results. Note that the -galactosidase (lacZ) marker gene is under the control of a synthetic late pox promoter (LP1) and the cMGF gene is under the control of a synthetic late/early pox promoter (LP2EP2).
The homology vector was constructed utilizing standard recombinant DNA techniques (11 and 14), by joining restriction fragments from the following sources with the appropriate synthetic DNA sequences. The plasmid vector was derived from an approximately 2999 base pair EcoRI restriction fragment of pSP64 (Promega). Fragment 1 is an approximately 1184 base pair EcoRI to SnaBI restriction sub-fragment of the 2.8 kb EcoRI FPV genomic fragment (SEQ ID NO. Fragment 2 is an approximately 640 base pair EcoRI to BamHI fragment coding for the cMGF gene (16) derived by reverse transcription and polymerase chain reaction (PCR) (Sambrook, et al., 1989) of RNA ISOLATED FROM CONCANAVALIN A STIMULATED CHICKEN SPLEEN CELLS. The antisense primer (6/94.20) used for reverse transcription and PCR was CGCAGGATCCGGGGCGTCAGAGGCGGGCGAGGTG-3' (SEQ ID NO: The sense primer (5/94.5) used for PCR was GAGCGGATCCTGCAGGAGGAGACACAGAGCTG-3' (SEQ ID NO: The BamHI fragment derived from PCR was subcloned into a plasmid and used as a template for a second PCR reaction using primer 6/94.16 SEQ ID NO: at the 5' end and primer 6/94.20 CGCAGGATCCGGGGCGTCAGAGGCGGGCGAGGTG-3'; SEQ ID NO: at the 3' end to yield an approximately 640 base pair fragment. The DNA fragment contains the coding sequence from amino acid 1 to amino acid 201 of the cMGF protein 22-11-00:12:45 ;DAVIES COLLISON CAVE Pat. &Trad ;61 7 3368 2262 IS/ WO 96/40880 PCT/US96/11187 -43- (16) which includes a 23 amino acid signal sequence at the amino terminus and 178 amino acids of the mature protein encoding cMGF. Fragment 3 is an approximately 3002 base pair BamHI to PvuII restriction fragment of plasmid pJF751 Fragment 4 is an approximately 1626 base pair SnaBI to EcoRI restriction sub-fragment of the 2.8 kb EcoRI FPV genomic fragment (SEQ ID NO. Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.
so Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
so* 0.
0 g 22/11 '00 WED 13:49 [TX/RX NO 6922] o WO 96/40880 PCT/US96/11187 -44- Example 1 Sites for Insertion of Foreign DNA into FPV In order to define appropriate insertion sites, a library of FPV EcoRI restriction fragments was generated in the plasmid vector pSP64 (Promega). Several of these restriction fragments were subjected to restriction mapping analysis. Unique blunt cutting restriction endonuclease sites were identified and mapped within the cloned FPV DNA regions. The blunt restriction sites were converted to Not I and Sfi I sites through the use of synthetic DNA linkers (oligo 66.04; GGCGGCCGCGGCCCTCGAGGCCA-3' SEQ ID NO: 1 and oligo 66.05; 5' TGGCCTCGAGGGCCGCGGCCGCC 3' SEQ ID NO: A 0galactosidase (lacZ) marker gene was inserted in each of the potential sites. A plasmid containing such a foreign DNA insert may be used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV to construct a FPV containing the foreign DNA. For this procedure to be successful it is important that the insertion site be in a region non-essential to the replication of the FPV and that the site be flanked with FPV DNA appropriate for mediating homologous recombination between virus and plasmid DNAs. The plasmids containing the lacZ marker gene were utilized in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The generation of recombinant virus was determined by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. Three sites were successfully used to generate a recombinant viruses. In each case the resulting virus was easily purified to 100%, clearly defining an appropriate site for the insertion of foreign DNA. The three homology vectors used to define these sites are described below.
Example 1A WO 96/40880 PCT/US96/11187 Homolorv Vector 443-88.8 The homology vector 443-88.8 contains a 3.5 KB FPV genomic EcoRI fragment and is useful for the insertion of foreign DNA into FPV. This EcoRI fragment maps to the approximately 5.5 KB overlap of FPV genomic fragments SalI C and PstI F (Coupar et al., 1990). The Notl/SfiI linker described above was inserted into a unique HpaI site in this fragment. This site is designated the 680 insertion site.
The homology vector 443-88.8 was characterized by DNA sequence analysis. Approximately 1495 base pairs of DNA sequence flanking the HpaI site was determined (SEQ ID NO: This sequence indicates that the open reading frame of 383 amino acids spans the HpaI insertion site.
The HpaI site interrupts this ORF at amino acid 226.
This ORF shows no amino acid sequence homology to any known pox virus genes.
Example 1B Homolocry Vector 443-88.14 The homology vector 443-88.14 contains a 2.8 KB FPV genomic EcoRI fragment and is useful for the insertion of foreign DNA into FPV. The NotI/SfiI linker described above was inserted into a unique SnaBI site in this fragment. This site is designated the 681 insertion site.
The homology vector 443-88.14 was characterized by DNA sequence analysis. The entire sequence of the 2.8 KB fragment was determined (SEQ ID NO: This sequence indicates that the SnaBI site is flanked on one side by a complete ORF of 422 amino acids (ORF1) reading toward the restriction site and on the other side by an WO 96/40880 PCT/US96/11187 -46incomplete ORF of 387 amino acids (ORF2) also reading toward the restriction site. Both ORF1 and ORF2 share homology with the vaccinia virus MIL gene (ref). The MIL gene shares homology with the vaccinia virus KIL gene which has been shown to be involved in viral host-range functions.
Example IC Homology Vector 451-08.22 The homology vector 451-08.22 contains a 4.2 KB FPV genomic EcoRI fragment and is useful for the insertion of foreign DNA into FPV. The Notl/SfiI linker described above was inserted into a unique StuI site in this fragment. A unique MluI site is located approximately 500 base pairs away from the StuI insertion site. This site is designated the 540 insertion site.
Example 2 Bivalent Vaccines Against Newcastle Disease and Fowlpox Recombinant FPV expressing proteins from NDV make bivalent vaccines protecting against both Marek's Disease and Newcastle disease. We have constructed several recombinant FPV expressing NDV proteins: S-FPV-013 (example 2A), S-FPV-035 (example 2B), S-FPV-041 (example 2C), S-FPV-042 (example 2D), and S-FPV-043 (example 2E).
Example 2A S-FPV-013 S-FPV-013 is a recombinant fowlpox virus that expresses two foreign genes. The gene for E. coli f-galactosidase (lacZ gene) and the gene for Newcastle Disease virus WO 96/40880 PCT/US96/11187 -47hemagglutinin-neuraminidase (HN) protein were inserted into the 681 insertion site. The lacZ gene is under the control of a synthetic late promoter.LP1 and the HN gene is under the control of the synthetic late promoter LP2.
S-FPV-013 was derived from S-FPV-001. This was accomplished utilizing the homology vector 451-79.95 (see Materials and Methods) and virus S-FPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The final result of red plaque purification was the recombinant virus designated S-FPV- 013. This virus was assayed for P-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in the materials and methods. After the initial three rounds of purification all plaques observed were blue indicating that the virus was pure, stable and expressing the marker gene.
S-FPV-013 was assayed for expression of NDV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FPV. An NDV HN specific monoclonal antibody (3-1G-5) was shown to react specifically with S-FPV-013 plaques and not with S-FPV- 001 negative control plaques. All S-FPV-013 observed plaques reacted with the monoclonal antibody antiserum indicating that the virus was stably expressing the NDV foreign gene.
Example 2B S-FPV-035 S-FPV-035 is a recombinant fowlpox virus that express a foreign gene. The Newcastle Disease virus HN gene was inserted at the 680 insertion site (see example 1A) The WO 96/40880 PCT/US96/11187 -48- HN gene is under the control of the synthetic early/late promoter EP1LP2.
S-FPV-035 was derived from S-FPV-001. This was accomplished utilizing the homology vector 489-21.1 (see Materials and Methods) and virus S-FPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the PLAQUE HYBRIDIZATION PROCEDURE FOR PURIFYING RECOMBINANT FPV. The final result of plaque hybridization purification was the recombinant virus designated S-FPV-035.
S-FPV-035 was assayed for expression of NDV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FPV. An NDV HN specific monoclonal antibody (3-1G-5) was shown to react specifically with S-FPV-035 plaques and not with S-FPV- 001 negative control plaques. All S-FPV-035 observed plaques reacted with the monoclonal antibody indicating that the virus was stably expressing the NDV foreign gene.
Example 2C S-FPV-041 S-FPV-041 is a recombinant fowlpox virus that expresses two foreign genes. The gene for E. coli 3-galactosidase (lacZ gene) and the gene for Newcastle Disease virus fusion protein were inserted into the 681 insertion site. The lacZ gene is under the control of a synthetic late promoter LP1 and the F gene is under the control of the synthetic early/late promoter EP1LP2.
S-FPV-041 was derived from S-FPV-001. This was accomplished utilizing the homology vector 502-27.5 (see o WO 96/40880 PCT/US96/11187 -49- Materials and Methods) and virus S-FPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The final result of red plaque purification was the recombinant virus designated S-FPV-041. This virus was assayed for /-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in the materials and methods. After the initial three rounds of purification all plaques observed were blue indicating that the virus was pure, stable and expressing the marker gene.
S-FPV-041 was assayed for expression of NDV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FPV. An NDV F specific monoclonal antibody (5-3F-2) was shown to react specifically with S-FPV-041 plaques and not with S-FPV- 001 negative control plaques. All S-FPV-041 observed plaques reacted with the monoclonal antibody indicating that the virus was stably expressing the NDV foreign gene.
Example 2D S-FPV-042 S-FPV-042 is a recombinant fowlpox virus that expresses three foreign genes. The gene for E. coli 3galactosidase (lacZ gene) and the gene for Newcastle Disease virus fusion protein was inserted into the 681 insertion site. The lacZ gene is under the control of a synthetic late promoter LP1 and the F gene is under the control of the synthetic early/late promoter EP1LP2.
The Newcastle Disease virus hemagglutinin (HN) gene were inserted at the 680 insertion site. The HN gene is under the control of the synthetic early/late promoter EP1LP2.
WO 96/40880 PCT/US96/11187 S-FPV-042 was derived from S-FPV-035. This was accomplished utilizing the homology vector 502-27.5 (see Materials and Methods) and virus. S-FPV-035 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The final result of red plaque purification was the recombinant virus designated S-FPV-042. This virus was assayed for -galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in the materials and methods. After the initial three rounds of purification all plaques observed were blue indicating that the virus was pure, stable and expressing the marker gene.
S-FPV-042 was assayed for expression of NDV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FPV. Monoclonal antibodies specific for both HN (3-1G-5) and F (5-3F-2) were shown to react specifically with S-FPV-042 plaques and not with S-FPV-001 negative control plaques. All S-FPV-042 observed plaques reacted with the monoclonal antibodies indicating that the virus was stably expressing the NDV foreign genes.
Example 2E S-FPV-043 S-FPV-043 is a recombinant fowlpox virus that expresses two foreign genes. The genes for Newcastle Disease virus F protein and HN protein were inserted at the 680 insertion site. The F and HN genes are each under the control of a synthetic early/late promoter EP1LP2.
S-FPV-043 was derived from S-FPV-001. This was accomplished utilizing the homology vector 502-26.22 (see WO 96/40880 PCT/US96/11187 -51- Materials and Methods) and virus S-FPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the PLAQUE HYBRIDIZATION PROCEDURE FOR PURIFYING RECOMBINANT FPV. The final result of plaque hybridization purification was the recombinant virus designated S-FPV-043.. The S-FPV-043 has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. VR 2395.
S-FPV-043 was assayed for expression of NDV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FPV. Monoclonal antibodies specific for both HN (3-1G-5) and F (5-3F-2) were shown to react specifically with S-FPV-043 plaques and not with S-FPV-001 negative control plaques. All S-FPV-043 observed plaques reacted with the monoclonal antibodies antiserum indicating that the virus was stably expressing the NDV foreign genes.
TESTING OF RECOMBINANT FPV EXPRESSING NDV ANTIGENS Groups of one day old SPF chicks (HyVac Inc.) were immunized with recombinant fowlpox viruses S-FPV-035, S- FPV-041, or S-FPV-043. Non vaccinated controls were also included. Three weeks post-vaccination, the birds were challenged intramuscularly with either virulent NDV or virulent FPV (Table The challenged chicks were observed daily for 14 days for clinical signs and death due to NDV. Non vaccinated control birds showed 100% mortality. S-FPV-043 vaccinated birds showed 100% protection against FPV challenge. Birds vaccinated with S-FPV-035 showed 95% protection compared with 85% seen 1 WO 96/40880 PCT/US96/11187 -52with birds immunized with S-FPV-041. These results suggest that recombinants expressing HN or F alone provide only partial protection. When both NDV proteins are combined into the same virus S-FPV-043, an enhancement of protection against lethal NDV challenge is obtained, resulting in a lower protective dose. The chicks that were challenged with FPV were scored for pox lesions. Non vaccinated control birds showed no protection against FPV lesions. Birds vaccinated with S-FPV-043 were completely protected from FPV lesions.
The duration of immunity conferred by vaccination with S- FPV-043 was examined. A group of SPF chicks was immunized with S-FPV-043 at one day of age and then challenged six weeks post-vaccination with either NDV or FPV. Complete protection was observed against both NDV and FPV challenge in S-FPV-043 vaccinated birds, whereas non vaccinated controls were totally susceptible to both challenge viruses. These results suggest that the duration of immunity afforded by vaccination with S-FPV- 043 would span the life of a broiler bird 6 weeks).
The effect of vaccinating hens in lay with the recombinant S-FPV-043 was evaluated by assessing egg production post-vaccination. One group of 50 hens was vaccinated and a second group of 50 hens, housed under conditions identical to the vaccinated group, served as non vaccinated controls. Daily egg production was monitored for four weeks post-vaccination. No differences were observed in egg production between the two groups of hens, indicating this vaccine will not adversely affect egg production in laying hens.
A study was conducted to determine whether S-FPV-043 could actively immunize chicks in the presence of maternal antibodies to both NDV and FPV. Chicks obtained from NDV and FPV immunized flocks were vaccinated with S- WO 96/40880 PCT/US96/11187 -53- FPV-043 and three weeks after vaccination, they were challenged with either virulent NDV or virulent FPV.
Clinical responses were compared with non vaccinated chicks from the same flock and with non-vaccinated chicks from an antibody negative flock (Table Chicks derived from antibody negative flocks showed 100% mortality after NDV challenge. Protection against NDV challenge, in nonvaccinated chicks known to have maternally derived antibody against NDV, ranged from 30 to 60%. Protection levels increased, to a range of 75 to 85%, when the maternal antibody positive chicks were vaccinated with S- FPV-043 suggesting an active immunization. The increase in NDV protection from 30% to 75% (flock 1) and 55% to (flock 2) clearly demonstrate the ability of S-FPV- 043 to partially overcome maternal antibody to both NDV and FPV. A decrease in FPV protection was observed in flock 1, suggesting some inhibition of FPV replication.
"WO 96/40880 PCT/US96/11187 -54- Table 1. Immunity conferred by Fowlpox recombinant vaccines vectoring different genes from Newcastle disease virus.
Challengea VIRUS DOSEb NDV FPV
FPV/NDV-HN
FPV/NDV-F
FPV/NDV-HN+F
Controls 8 x 10 5 2 x 10 4 2 x 10 3 85 100
NTC
NT
100 none a Percent protection following challenge 3 vaccination b PFU/0.1 ml dose c Not tested weeks post- 4' WO 96/40880 "WO 9640880PCTIUS96/1 1187 Table 2. A~bility of recombinant vaccine FPV/NDV-HN+F (S- FPV-043) to vaccinate chicks with maternal antibody.
Challenge a History Flock Vaccination Hen Antibodyb
NDV
FPV
NDV-HIc NDV NDV FPV ELISA FPV-AGPd Vacc. Con. Vacc. Con.
NDN
NDN
Nc 1:36 1:1738 Neg 75 30 I+ FPV 1:64 1:2852 Neg 85 55 Ionly 1:92 1:4324 Neg 80 60 ~ne Neg Neg Neg 0 Percent protection following challenge 3 post-vaccination.
Every flock antibody.
HI -Hemagglutination Inhibition Assay AGP -Agar Gel Precipitation Assay 100 weeks SUBSTTUTE SHEET (RULE 26) WO 96/40880 PCT/US96/11187 -56- Example 2F S-FPV-074 S-FPV-074 is a recombinant fowlpox virus that expresses two foreign genes. The genes for Newcastle Disease virus F protein and HN protein were inserted at the 681 insertion site. The F and HN genes are each under the control of a synthetic late/early promoter LP2EP2.
S-FPV-074 was derived from S-FPV-001. This was accomplished utilizing the homology vector 584-36.12 (see Materials and Methods) and virus S-FPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the PLAQUE HYBRIDIZATION PROCEDURE FOR PURIFYING RECOMBINANT FPV. The final result of plaque hybridization purification was the recombinant virus designated S-FPV-074.
S-FPV-074 was assayed for expression of NDV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FPV. Monoclonal antibodies specific for NDV HN (3-1G-5) and F (5-3F-2) were shown to react specifically with S-FPV-074 plaques and not with S- FPV-001 negative control plaques. All S-FPV-074 observed plaques reacted with the monoclonal antibodies indicating that the virus was stably expressing the NDV foreign genes.
S-FPV-074 expresses foreign antigens from NDV. This virus is useful as a multi-valent vaccine against Newcastle Diseases and Fowlpox.
WO 96/40880 PCT/US96/11187 -57- Example 3 Recombinant fowlpox viruses expressing proteins from Marek's disease virus (MDV) make vaccines protecting against both fowlpox virus and Marek's disease virus. We have constructed several recombinant FPV expressing MDV proteins: S-FPV-081, S-FPV-082 and S-FPV-085. Of these S-FPV-082 and S-FPV-085 also express proteins from Newcastle disease virus. These viruses are useful for vaccinating against fowlpox virus, Marek's disease virus, and Newcastle disease virus.
S-FPV-085 further expresses proteins from infectious laryngotracheitis virus (ILTV), making them useful as vaccines against ILTV.
Example 3A S-FPV-081 S-FPV-081 is a recombinant fowlpox virus that expresses three foreign genes. The gene for E.coli 3-galactosidase (lacZ gene) and the genes for Marek's Disease virus (MDV) glycoprotein D (gD) and glycoprotein B (gB) were inserted into the 681 insertion site. The lac Z gene is under the control of a synthetic late promoter LP1 and the MDV gD and gB genes are under the control of the synthetic early/late promoters LP2EP2 and EP1LP2 respectively.
S-FPV-081 was derived from S-FPV-001. This was accomplished utilizing the homology vector 608-10.3 (see Materials and Methods) and virus S-FPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The final result of red plaque purification was the recombinant virus designated S-FPV-081. This "WO 96/40880 PCTIUS96/11187 -58virus was assayed for f-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in the materials and methods. After the initial three rounds of purification all plaques observed were blue indicating that the virus was pure, stable and expressing the marker gene.
S-FPV-081 was assayed for expression of MDV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FPV. Convalescent sera from MDV infected chickens was shown to react specifically with S-FPV-081 plaques and not with S-FPV-001 negative control plaques. All S-FPV-081 observed plaques reacted with the chicken antiserum indicating that the virus was stably expressing the MDV foreign genes. Western blot assays of infected cell lysates using convalescent sera from MDV-infected chickens indicated that S-FPV-081 was expressing a MDV glycoprotein B and MDV glycoprotein D.
S-FPV-081 expresses foreign antigens from MDV. This virus is useful as a multi-valent vaccine against Marek's Disease and Fowlpox.
Example 3B S-FPV-082 S-FPV-082 is a recombinant fowlpox virus that expresses five foreign genes. The genes for Newcastle Disease virus F protein and HN protein were inserted at the 680 insertion site. The F and HN genes are each under the control of a synthetic early/late promoter EP1LP2. The gene for E. coli P-galactosidase (lacZ gene) and the genes for Marek's Disease virus (MDV) gD and gB were inserted into the 681 insertion site. The lacZ gene is under the control of a synthetic late promoter LP1 and the MDV gD and gB genes are under the control of the WO 96/40880 PCTIUS96/11187 -59synthetic early/late promoters LP2EP2 and EP1LP2 respectively.
S-FPV-082 was derived from S-FPV-043. This was accomplished utilizing the homology vector 608-10.3 (see Materials and Methods) and virus S-FPV-043 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The final result of red plaque purification was the recombinant virus designated S-FPV-082. This virus was assayed for 0-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in the materials and methods. After the initial three rounds of purification all plaques observed were blue indicating that the virus was pure, stable and expressing the marker gene.
S-FPV-082 was assayed for expression of MDV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FPV. Convalescent sera from MDV infected chickens was shown to react specifically with S-FPV-082 plaques and not with S-FPV-001 negative control plaques. All S-FPV-082 observed plaques reacted with the chicken antiserum indicating that the virus was stably expressing the MDV foreign genes.
S-FPV-082 expresses foreign antigens from NDV and MDV.
This virus will be valuable as a multi-valent vaccine against Newcastle Disease, Marek's Disease and Fowlpox.
Example 3C S-FPV-085 S-FPV-085 is a recombinant fowlpox virus that expresses eight foreign genes. The genes for Newcastle Disease WO 96/40880 PCT/US96/11187 virus F protein and HN protein are inserted at the 680 insertion site. The F and HN genes are each under the control of a synthetic early/late promoter EP1LP2. The gene for E.coli 9-galactosidase (lacZ gene) and the genes for Marek's Disease virus (MDV) gD and gB are inserted into the 681 insertion site. The lac Z gene is under the control of a synthetic late promoter LP1 and the MDV gD and gB genes are under the control of the synthetic early/late promoters LP2EP2 and EP1LP2 respectively. The gene for E.coli /-glucuronidase (uidA gene) and the genes for Infectious Laryngotracheitis virus (ILTV) gD and gB are inserted into the 540 insertion site. The uidA gene is under the control of a synthetic late promoter LP1 and the ILTV gD and gB genes are each under the control of a synthetic early/late promoter EP1LP2.
S-FPV-085 is derived from S-FPV-082. This is accomplished utilizing the homology vector 586-36.6 (see Materials and Methods) and virus S-FPV-082 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV.
The transfection stock is screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque (0-glucuronidase) purification is the recombinant virus designated S-FPV- 085. This virus is assayed for f-glucuronidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in the materials and methods. After the initial three rounds of purification all plaques observed are blue indicating that the virus is pure, stable and expressing the marker gene.
S-FPV-085 is assayed for expression of MDV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FPV. S-FPV-085 expresses foreign antigens from NDV, MDV and ILTV. This virus is useful as a multi-valent vaccine against Newcastle WO 96/40880 PCT/US96/11187 -61- Disease, Marek's Disease, Infectious Laryngotracheitis and Fowlpox.
Example 4 Recombinant fowlpox virus (FPV) expressing proteins from infectious laryngotracheitis virus (ILTV) make vaccines protecting against both FPV and ILTV. We have constructed several recombinant FPV expressing ILTV proteins: S-FPV-095, S-FPV-083, and S-FPV-097. Of these, S-FPV-083 and S-FPV-097 also express proteins from Newcastle disease virus (NDV), making them useful as vaccines against NDV as well.
Example 4A S-FPV-095 S-FPV-095 is a recombinant fowlpox virus that expresses three foreign genes. The gene for E.coli 0-glucuronidase (uidA gene) and the genes for Infectious Laryngotracheitis virus (ILTV) glycoprotein D (gD) and glycoprotein B (gB) were inserted into the 540 insertion site. The uidA gene is under the control of a synthetic late promoter LP1 and the ILTV gD and gB genes are each under the control of a synthetic early/late promoter EP1LP2.
S-FPV-095 was derived from S-FPV-001. This was accomplished utilizing the homology vector 694-10.4 (see Materials and Methods) and virus S-FPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification (0-glucuronidase) was the recombinant virus designated S-FPV-095. This virus was assayed for 3- WO 96/40880 PCT/US96/11187 -62glucuronidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in the materials and methods. After the initial three rounds of purification all plaques observed were blue indicating that the virus was pure, stable and expressing the marker gene.
S-FPV-095 was assayed for expression of ILTV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FPV. Antibodies to ILTV gB and gD was shown to react specifically with S-FPV-095 plaques and not with S-FPV-001 negative control plaques. All S- FPV-095 observed plaques reacted with the antiserum indicating that the virus was stably expressing the ILTV foreign genes.
S-FPV-095 expresses foreign antigens from ILTV. This virus is useful as a multi-valent vaccine against Infectious Laryngotracheitis and Fowlpox.
Example 4B S-FPV-083 S-FPV-083 is a recombinant fowlpox virus that expresses five foreign genes. The genes for Newcastle Disease virus F protein and HN protein were inserted at the 680 insertion site. The F and HN genes are each under the control of a synthetic early/late promoter EP1LP2. The gene for E. coli /-glucuronidase (uidA gene) and the genes for Infectious Laryngotracheitis virus (ILT) gD and gB were inserted into the 540 insertion site. The uidA gene is under the control of a synthetic late promoter LP1 and the ILT gD and gB genes are each under the control of a synthetic early/late promoter (EP1LP2).
S-FPV-083 was derived from S-FPV-043. This was WO 96/40880 PCT/US96/11187 -63accomplished utilizing the homology vector 586-36.6 (see Materials and Methods) and virus S-FPV-043 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification was the recombinant virus designated S-FPV- 083. This virus was assayed for -glucuronidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in the materials and methods. After the initial three rounds of purification all plaques observed were blue indicating that the virus was pure, stable and expressing the marker gene.
S-FPV-083 was assayed for expression of ILTV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FPV. Convalescent sera from ILTV infected chickens was shown to react specifically with S-FPV-083 plaques and not with S-FPV-001 negative control plaques. All S-FPV-083 observed plaques reacted with the chicken antiserum indicating that the virus was stably expressing the ILTV foreign genes.
S-FPV-083 expresses foreign antigens from NDV and ILTV.
This virus will be valuable as a multi-valent vaccine against Newcastle Disease, Infectious Laryngotracheitis and Fowlpox.
Example 4C S-FPV-097 S-FPV-097 is a recombinant fowlpox virus that expresses five foreign genes. The genes for Newcastle Disease virus F protein and HN protein were inserted at the 680 insertion site. The F and HN genes are each under the WO 96/40880 PCT/US96/11187 -64control of a synthetic early/late promoter EP1LP2. The gene for E.coli 6-glucuronidase (uidA gene) and the genes for Infectious Laryngotracheitis virus (ILTV) glycoprotein D (gD) and glycoprotein B (gB) were inserted into the 540 insertion site. The uidA gene is under the control of a synthetic late promoter LP1 and the ILTV gD and gB genes are each under the control of a synthetic early/late promoter EP1LP2.
S-FPV-097 was derived from S-FPV-043. This was accomplished utilizing the homology vector 694-10.4 (see Materials and Methods) and virus S-FPV-043 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification was the recombinant virus designated S-FPV- 097. This virus was assayed for 0-glucuronidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in the materials and methods. After the initial three rounds of purification all plaques observed were blue indicating that the virus was pure, stable and expressing the marker gene.
S-FPV-097 was assayed for expression of ILTV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FPV. Antibodies to ILTV gB and gD was shown to react specifically with S-FPV-097 plaques and not with S-FPV-001 negative control plaques. All S- FPV-097 observed plaques reacted with the antiserum indicating that the virus was stably expressing the ILTV foreign genes. All S-FPV-097 observed plaques reacted with the chicken antiserum to ILTV indicating that the virus was stably expressing the ILTV foreign genes.
Monoclonal antibodies specific for NDV HN (3-1G-5) and F (5-3F-2) were shown to react specifically with S-FPV-097 *WO 96/40880 PCT/US96/11187 plaques and not with S-FPV-001 negative control plaques.
All S-FPV-097 observed plaques reacted with the monoclonal antibodies indicating that the virus was stably expressing the NDV foreign genes.
S-FPV-097 expresses foreign antigens from NDV and ILTV.
This virus is useful as a multi-valent vaccine against Newcastle Disease, Infectious Laryngotracheitis and Fowlpox.
WO 96/40880 PCT/US96/11187 -66- Example Recombinant fowlpox virus (FPV) expressing proteins from infectious bronchitis virus (IBV) make vaccines protecting against both FPV and IBV. We have constructed two recombinant FPV expressing IBV proteins: S-FPV-072 and S-FPV-079. Both of these viruses also express proteins from Newcastle disease virus (NDV), making them useful as vaccines against NDV.
Example S-FPV-072 S-FPV-072 is a recombinant fowlpox virus that expresses five foreign genes. The genes for Newcastle Disease virus F protein and HN protein were inserted at the 680 insertion site. The F and HN genes are each under the control of a synthetic early/late promoter EP1LP2. The gene for E.coli 1-galactosidase (lacZ gene) and the genes for Infectious Bronchitis virus (IBV) Massachusetts Spike protein (Mass Spike) and Massachusetts Matrix protein (Mass Matrix) were inserted into the 681 insertion site.
The lac Z gene is under the control of a synthetic late promoter LP1 and the IBV Mass Spike and Mass Matrix genes are each under the control of the synthetic early/late promoter EP1LP2.
S-FPV-072 was derived from S-FPV-043. This was accomplished utilizing the homology vector 538-51.27 (see Materials and Methods) and virus S-FPV-043 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The final result of red plaque purification was the recombinant virus designated S-FPV- 072. This virus was assayed for B-galactosidase expression, purity, and insert stability by multiple WO 96/40880 PCTIUS96/11187 -67passages monitored by the blue plaque assay as described in the materials and methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus was pure, stable and expressing the marker gene.
S-FPV-072 was assayed for expression of NDV and IBV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FPV. Monoclonal antibody 15-88 to the IBV Mass Spike protein was shown to react specifically with S-FPV-072 plaques and not with S- FPV-001 negative control plaques. All S-FPV-072 observed plaques reacted with the monoclonal antibodies indicating that the virus was stably expressing the IBV foreign gene. Western blot assays of infected cell lysates using monoclonal antibody 15-88 to the IBV Mass Spike protein indicated that S-FPV-072 was expressing a 90 kD IBV Mass Spike protein. Monoclonal antibodies specific for both HN (3-1G-5) and F (5-3F-2) were shown to react specifically with S-FPV-072 plaques and not with S-FPV- 001 negative control plaques. All S-FPV-072 observed plaques reacted with the monoclonal antibodies indicating that the virus was stably expressing the NDV foreign genes.
S-FPV-072 expresses foreign antigens from NDV and IBV.
This virus is useful as a multi-valent vaccine against Newcastle Diseases, Infectious Bronchitis, and Fowlpox.
Example S-FPV-079 is a recombinant fowlpox virus that expresses seven foreign genes. The genes for Newcastle Disease virus F protein and HN protein were inserted at the 680 insertion site. The F and HN genes are each under the control of a synthetic early/late promoter EP1LP2. The gene for E.coli 0-galactosidase (lacZ gene) and the genes for Infectious Bronchitis virus (IBV) Massachusetts Spike WO 96/40880 PCT/US96/11187 -68protein (Mass Spike) and Massachusetts Matrix protein (Mass Matrix) were inserted into the 681 insertion site.
The lac Z gene is under the control of a synthetic late promoter LP1 and the IBV Mass Spike and Mass Matrix genes are each under the control of the synthetic early/late promoter EP1LP2. The gene for the E. coli flglucuronidase (uidA) gene and the gene for the IBV Mass Nucleocapsid protein were inserted into the 540 insertion site. The uidA gene is under the control of the synthetic late/early promoter LP2EP2 and the IBV Mass Nucleocapsid gene is under the control of the synthetic early/late promoter EP1LP2.
S-FPV-079 was derived from S-FPV-072. This was accomplished utilizing the Homology Vector 611-49.1 (see Materials and Methods) and virus S-FPV-072 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The final result of red plaque purification was the recombinant virus designated S-FPV- 079. This virus was assayed for B-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in the materials and methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus was pure, stable and expressing the marker gene.
S-FPV-079 was assayed for expression of NDV and IBV specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FPV. Monoclonal antibody 15-88 to the IBV Mass Spike protein was shown to react specifically with S-FPV-072 plaques and not with S- FPV-001 negative control plaques. All S-FPV-079 observed plaques reacted with the monoclonal antibody antiserum to IBV indicating that the virus was stably expressing the WO 96/40880 PCT/US96/11187 -69- IBV foreign gene. Western blot assays of infected cell lysates using monoclonal antibody 15-88 to the IBV Mass Spike protein indicated that S-FPV-079 was expressing a kD IBV Mass Spike protein. Monoclonal antibodies specific for both HN (3-1G-5) and F (5-3F-2) were shown to react specifically with S-FPV-079 plaques and not with S-FPV-001 negative control plaques. All S-FPV-079 observed plaques reacted with the monoclonal antibodies indicating that the virus was stably expressing the NDV foreign genes.
S-FPV-079 expresses foreign antigens from NDV and IBV.
This virus is useful as a multi-valent vaccine against Newcastle Diseases, Infectious Bronchitis, and Fowlpox.
Example 6 Recombinant fowlpox virus, S-FPV-099 or S-FPV-101, expressing chicken interferon (cIFN) or S-FPV-100, expressing chicken myelomonocytic growth factor (cMGF), are useful to enhance the immune response when added to vaccines against diseases of poultry. Chicken myelomonocytic growth factor (cMGF) is homologous to mammalian interleukin-6 protein, and chicken interferon (cIFN) is homologous to mammalian interferon Type I. When used alone or in combination with vaccines against specific avian diseases, S-FPV-099, S-FPV-100 and S-FPV- 101 provide enhanced mucosal, humoral, or cell mediated immunity against avian disease-causing viruses including, but not limited to, Marek's disease virus, Newcastle disease virus, infectious laryngotracheitis virus, infectious bronchitis virus, infectious bursal disease virus.
S-FPV-099 S-FPV-099 is a recombinant fowlpox virus that expresses WO 96/40880 PCT/US96/11187 two foreign genes. The genes for chicken interferon (cIFN) and E. coli lacZ were inserted at the uniqe SnaBI restriction endonuclease site in the 2.8 kB EcoRI FPV genomic fragment (681 insertion site). The cIFN gene is under the control of a synthetic late/early promoter LP2EP2, and the E. coli lacZ gene is under the control of a synthetic late promoter LP1.
S-FPV-099 was derived from S-FPV-001. This was accomplished utilizing the homology vector 751-07.D1 (see Materials and Methods) and virus S-FPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The final result of red plaque purification was the recombinant virus designated S-FPV-099. This virus was assayed for A-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus S-FPV-099 was pure, stable, and expressing the foreign gene.
Supernatants from S-FPV-099 have interferon activity in cell culture. Addition of S-FPV-099 conditioned media to chicken embryo fibroblast (CEF) cell culture inhibits infection of the CEF cells by vesicular stomatitis virus or by herpesvirus of turkeys. S-FPV-099 is useful to enhance the immune response alone or when added to vaccines against diseases of poultry.
S-FPV-100 S-FPV-100 is a recombinant fowlpox virus that expresses two foreign genes. The genes for chicken myelomonocytic growth factor (cMGF) and E. coli lacZ were inserted at WO 96/40880 PCT/US96/11187 -71the uniqe SnaBI restriction endonuclease site in the 2.8 kB EcoRI FPV genomic fragment (681 insertion site). The cMGF gene is under the control of a synthetic late/early promoter LP2EP2, and the E. coli lacZ gene is under the control of a synthetic late promoter LP1.
S-FPV-100 was derived from S-FPV-001. This was accomplished utilizing the homology vector 751-56.Cl (see Materials and Methods) and virus S-FPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The final result of red plaque purification was the recombinant virus designated S-FPV-100. This virus was assayed for 0-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus S-FPV-100 was pure, stable, and expressing the foreign gene.
S-FPV-100 is useful to enhance the immune response alone or when added to vaccines against diseases of poultry.
S-FPV-101 S-FPV-101 is a recombinant fowlpox virus that expresses four foreign genes. The genes for chicken interferon (cIFN) and E. coli lacZ were inserted at the uniqe SnaBI restriction endonuclease site in the 2.8 kB EcoRI FPV genomic fragment (681 insertion site). The cIFN gene is under the control of a synthetic late/early promoter LP2EP2, and the E. coli lacZ gene is under the control of a synthetic late promoter LP1. The genes for Newcastle Disease virus F protein and HN protein were inserted at the 680 insertion site. The F and HN genes are each under 4 WO 96/40880 PCT/US96/11187 -72the control of a synthetic early/late promoter EP1LP2.
S-FPV-101 was derived from S-FPV-043. This was accomplished utilizing the homology vector 751-07.D1 (see Materials and Methods) and virus S-FPV-043 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The final result of red plaque purification was the recombinant virus designated S-FPV-101. This virus was assayed for f-galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus S-FPV-101 was pure, stable, and expressing the foreign gene.
Supernatants from S-FPV-101 have interferon activity in cell culture. Addition of S-FPV-101 conditioned media to chicken embryo fibroblast (CEF) cell culture inhibits infection of the CEF cells by vesicular stomatitis virus or by herpesvirus of turkeys. S-FPV-101 is useful to enhance the immune response alone or when added to vaccines against diseases of poultry. S-FPV-101 is useful as a multi-valent vaccine against Newcastle Diseases and Fowlpox.
Example 7 Recombinant fowlpox virus expressing Newcastle's disease virus HN and F proteins lacking the membrane anchor sequences is a superior vaccine against fowlpox and Newcastle's disease.
Day old chicks from hens which have been exposed to or vaccinated against Newcastle's disease virus carry WO 96/40880 PCT/US96/11187 -73antibodies to NDV which may neutralize a vaccine containing a recombinant fowlpox virus expressing the NDV HN and F proteins. In vitro virus neutralization
(VN)
assays using VN monoclonal antibodies specific for either NDV HN or F proteins have been shown to neutralize recombinant fowlpox virus expressing the NDV HN and F proteins. These results suggest that the NDV HN and F glycoproteins are incorporated into the fowlpox virus virion. To increase the efficacy of a vaccine in the presence on maternal antibodies against Newcastle's disease virus, a recombinant fowlpox virus is constructed which expresses the NDV HN and F proteins lacking the membrane anchor domains of each protein. The resulting recombinant virus produces NDV HN and F proteins secreted into the serum of the vaccinated animal producing a strong humoral and cell mediated immune response to the Newcastle's disease virus. The NDV HN and F proteins are not presented on the surface of the FPV particle and thus evade neutralization by maternal antibodies present in the vaccinated day old chicks.
The hemagglutinin-Neuraminidase (HN) and Fusion genes from the B1 strain of Newcastle Disease Virus (ATCC VR- 108) were isolated as cDNA clones, using oligo dT primed poly A selected mRNA.
The fusion protein mediates penetration of NDV into host cells by fusion of the viral envelope with the host cell plasma membrane. A posttranslational cleavage of inactive precursors Fo into two disulfide-bonded polypeptides, Fl and F2, is necessary to produce fusion active F protein and thereby yield infectious virions.
The new hydrophobic N-terminus of Fl generated after cleavage of Fo is responsible for the fusion characteristic of paramyxoviruses and thus determines virulence. The required proteolytic cleavage signal (paired basic residues) in the NDV Bl strain is altered, WO 96/40880 PCT/US96/11187 -74thereby preventing cleavage of Fo into Fl and F2, resulting in an attenuated NDV strain.
The addition of the NDV F signal sequence (aal-25) to VP2 (vFP147), resulted in the secretion of VP2 in the TC fluid, but abolished its protective response (Paoletti, et. al WO 93/03145). Three hydrophobic domains exist within the F glycoprotein which interact with the lipid bilayer The signal sequence at the N-terminus of the primary translation product Fo; the N-terminus of Fl; and the transmembrane anchor domain near the Cterminus of Fl. The F glycoprotein of the Bi strain of NDV is 544 amino acids in length with the transmembrane anchor domain spanning 27 amino acids from position 500 to 526 (LITYIVLTIISLVFGILSLILACYLMY). Amino acids 1-499 of the NDV F protein are expressed under the control of a synthetic promoter element which functions as both an early and late promoter, such as EP1LP2 or LP2EP2, directing expression throughout the reproduction cycle.
This results in the deletion of amino acids 527-544, the cytoplasmic tail, thought to interact with the inner membrane protein before or during virus assembly. A recombinant fowlpox virus is constructed which expresses the NDV F protein lacking the C-terminal membrane anchor domain from a synthetic early/late promoter.
The hemagglutinin-neuraminidase (HN) glycoprotein provides NDV with the ability to agglutinate and elute erythrocytes. The process consists of two stages: attachment of the virus to the receptor on the red blood cell surface (agglutination) and destruction of the receptor by the neuraminidase enzyme activity (elution).
The major hydrophobic anchor domain is present near the N-terminus of HN, supporting the view that the N-terminus is anchored to the lipid bilayer. The HN glycoprotein of the B1 strain of NDV is 577 amino acids in length with the transmembrane anchor domain spanning 28 amino acids 1, WO 96/40880 PCT/US96/11187 from position 27 to 54 (IAILFLTVVTLAISVASLLYSMGASTPS).
The extreme N-terminal amino acids (1 to 26) are relatively hydrophilic. Amino acids 55 to 577 of the HN protein are expressed under the control of a synthetic promoter element which functions as both an early and late promoter, such as EP1LP2 or LP2EP2, directing expression throughout the reproduction cycle. THE NDV HN polypeptide has a membrane transport signal sequence, such as the PRV gX signal sequence, at its amino terminus to direct the protein to be secreted into the serum of a vaccinated animal. A recombinant fowlpox virus is constructed which expresses the NDV HN protein lacking the N-terminal membrane anchor domain and containing an N-terminal PRV gX signal sequence from a synthetic early/late promoter. Alternatively the NDV HN polypeptide contains a deletion of the transmembrane anchor domain spanning 28 amino acids from position 27 to 54 and retains amino acids 1 to 26 and 55 to 577. A recombinant fowlpox virus is constructed which expresses the NDV HN protein lacking the membrane anchor domain (amino acids 27 to 54) from a synthetic early/late promoter.
A recombinant fowlpox virus is constructed which expresses both the NDV HN and F proteins lacking the membrane anchor domains of each protein from a synthetic early/late promoter. The resulting recombinant virus produces NDV HN and F proteins secreted into the serum of the vaccinated animal producing a strong humoral and cell mediated immune response to the Newcastle's disease virus. The NDV HN and F proteins are not presented on the surface of the FPV particle and thus evade neutralization by maternal antibodies present in the vaccinated day old chicks.
Example 8 Recombinant fowlpox virus expressing cell surface WO 96/40880 PCT/US96/11187 -76receptors on the surface of the FPV viral particle useful for targeting gene products to specific tissues or organs.
Serum from chickens carrying maternal antibodies to Newcastle's disease virus inhibits productive infection and plaque formation by S-FPV-043 on chicken embryo fibroblasts in cell culture. One explanation for this result is that the antigenic epitopes of the NDV HN and F proteins expressed in S-FPV-043 are displayed on the surface of the fowlpox viral particle. Display of proteins on the surface of the FPV particle is useful to target specific gene products to specific normal cell types or tumor cell types. Proteins which are displayed on the surface of the FPV particle include but are not limited to integrins which would target the virus to integrin receptors on the cell surface; erythropoetin which would target the virus to erythropoetin receptors on the surface of red blood cells; antibodies or other proteins which would target to specific proteins or receptors on the surface of normal or tumor cells. The fowlpox virus also delivers cytokines, interleukins, interferons, or colony stimulating factors which stimulate a strong humoral or cell mediated immune response against a tumor or disease causing organism. The proteins displayed on the surface of the fowlpox virus are expressed from the fowlpox genome as fusion proteins to the membrane anchor domains of the NDV HN or F proteins, or to other proteins containing membrane anchor domains. The cytokines, interleukins, interferons, or colony stimulating factors are expressed as fusion proteins to PRV gX, E. coli P-galactosidase or another protein in a soluble, not membrane bound, form. The fusion protein stabilizes the cytokine protein and allows it to diffuse in the serum of the animal to reach its cellular target.
WO 96/40880 PCT/US96/11187 -77- Example 9 S-FPV-098 S-FPV-098 is a recombinant fowlpox virus that expresses two foreign genes. The genes for infectious bursal disease virus (IBDV) polymerase gene and E. coli lacZ were inserted at the 681 insertion site. The IBDV polymerase gene is under the control of a synthetic late/early promoter LP2EP2, and the E. coli lacZ gene is under the control of a synthetic late promoter LP1.
S-FPV-098 was derived from S-FPV-001. This was accomplished utilizing the homology vector 749-75.82 (see Materials and Methods) and virus S-FPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The final result of red plaque purification was the recombinant virus designated S-FPV-098. This virus was assayed for -galactosidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in Materials and Methods. After the initial three rounds of purification, all plaques observed were blue indicating that the virus S-FPV-098 was pure, stable, and expressing the foreign gene.
S-FPV-098 is useful for expression of IBDV polymerase protein. S-FPV-098 is useful in an in vitro approach to a recombinant IBDV attenuated vaccine. RNA strands from the attenuated IBDV strain are synthesized in a bacterial expression system using T3 or T7 promoters (pBlueScript plasmid; Stratagene, Inc.) to synthesize double stranded short and long segments of the IBDV genome. The IBDV double stranded RNA segments and S-FPV-098 are transfected into Vero cells. The fowlpox virus expresses the IBDV polymerase but does not replicate in Vero cells.
11 "WO 96/40880 PCTIUS96/11187 -78- The IBDV polymerase produced from S-FPV-098 synthesizes infectious attenuated IBDV virus from the double stranded RNA genomic templates. The resulting attenuated IBDV virus is useful as a vaccine against infectious bursal disease in chickens.
As an alternative to the construction of a IBD vaccine using a viral vectored delivery system and/or subunit approaches, IBD virus RNA is directly manipulated reconstructing the virus using full length RNA derived from cDNA clones representing both the large (segment A) and small (segment B) double-stranded RNA subunits.
Generation of IBD virus is this manner offers several advantages over the first two approaches. First, if IBD virus is re-generated using RNA templates, one is able to manipulate the cloned cDNA copies of the viral genome prior to transcription (generation of RNA). Using this approach, it is possible to either attenuate a virulent IBD strain or replace the VP2 variable region of the attenuated vaccine backbone with that of virulent strains. In doing so, the present invention provides protection against the virulent IBDV strain while providing the safety and efficacy of the vaccine strain.
Furthermore, using this approach, the present invention constructs and tests temperature sensitive IBD viruses generated using the RNA polymerase derived from the related birnavirus infectious pancreatic necrosis virus (IPNV) and the polyprotein derived from IBDV. The IPNV polymerase has optimum activity at a temperature lower than that of IBDV. If the IPNV polymerase recognizes the regulatory signals present on IBDV, the hybrid virus is expected to be attenuated at the elevated temperature present in chickens. Alternatively, it is possible to construct and test IBD viruses generated using the RNA polymerase derived from IBDV serotype 2 viruse and the polyprotein derived from IBDVserotype 1 virus.
"WO 96/40880 PCTIUS96/11187 -79cDNA clones representing the complete genome of IBDV (double stranded RNA segments A and B) is constructed, initially using the BursaVac vaccine strain (Sterwin Labs). Once cDNA clones representing full length copies of segment A and B are constructed, template RNA is prepared. Since IBDV exists as a bisegmented doublestranded RNA virus, both the sense and anti-sense RNA strands of each segment are produced using the pBlueScript plasmid; Stratagene, Inc.). These vectors utilize the highly specific phage promoters SP6 or T7 to produce substrate amounts of RNA in vitro. A unique restriction endonuclease site is engineered into the 3' PCR primer to linearize the DNA for the generation of run-off transcripts during transcription.
The purified RNA transcripts (4 strands) are transfected into Vero cells to determine whether the RNA is infectious. If IBD virus is generated, as determined by black plaque assays using IBDV specific Mabs, no further manipulations are required and engineering of the vaccine strain can commence. The advantage of this method is that engineered IBD viruses generated in this manner will be pure and require little/no purification, greatly decreasing the time required to generate new vaccines.
If negative results are obtained using the purified RNA's, functional viral RNA polymerase is required by use of a helper virus. Birnaviruses replicate their nucleic acid by a strand displacement (semi-conservative) mechanism, with the RNA polymerase binding to the ends of the double-stranded RNA molecules forming circularized ring structures (Muller Nitschke, Virology 159, 174- 177, 1987). RNA polymerase open reading frame of about 878 amino acids in fowlpox virus is expressed and this recombinant virus (S-FPV-098) is used to provide functional IBDV RNA polymerase in trans. Fowlpox virus expressed immunologically recognizable foreign antigens in non-avian cells (Vero cells), where there are no signs WO 96/40880 PCT/US96/11187 of productive replication of the viral vector (Paoletti et al., Technological Advances in Vaccine Development, 321-334, 1988, Alan R. Liss, In the present invention the IBDV polymerase protein is expressed in the same cells as the transfected RNA using the fowlpox virus vector without contaminating the cells with FPV replication.
With the demonstration that IBD virus is generated in vitro using genomic RNA, an improved live attenuated virus vaccines against infectious bursal disease is developed. Using recombinant DNA technology along with the newly defined system of generating IBD virus, specific deletions within the viral genome, facilitating the construction of attenuated viruses are made. Using this technology, the region of IBDV responsible for virulence and generate attenuated, immunogenic IBDV vaccines are identified. The present invention provides a virulent IBD strain or replacement of the VP2 variable region of the attenuated vaccine backbone with that of a virulent strain, thus protecting against the virulent strain while providing the safety and efficacy of the vaccine strain.
Example The chicken interferon (cIFN) gene was cloned into wild type (FPV) viruses by homologous recombinant techniques.
Briefly, the entire coding region of cIFN was isolated from activated chicken spleen cell RNA by RT/PCR using primer sequences from the recently published cIFN sequence (Sekellick, et al., 1994). Recombinant FPV viruses containing cIFN, and FPV/cIFN (S-FPV-099), were engineered to contain the entire cIFN ORF under the control of a synthetic pox virus promoter (LP2EP2), which functions as both an early and late promoter, directing expression throughout the entire viral replication cycle.
WO 96/40880 PCT/US96/11187 -81- A third recombinant virus, FPV/cIFN+NDV, (S-FPV-101) was made in a similar manner, except that a FPV virus previously engineered to contain the Newcastle Disease (NDV) antigens HN and F was used as the parent virus during homologous recombination, thus yielding a recombinant fowlpox virus co-expressing the cIFN and NDV genes. All recombinant viruses contain the lac Z gene engineered in tandem with cIFN under the control of a synthetic late (LP1) pox promoter. All promoter/gene constructs were sequenced at the promoter/cIFN junction to confirm the integrity of the proper DNA coding frame.
Co-expression of 0-galactosidase facilitated the isolation and plaque purification of the recombinant viruses. Independent viral insertion sites were used for insertion of the cIFN gene and the NDV genes in the fowlpox virus. The insertion sites were found to interrupt nonessential virus genes in both SPV and FPV.
To confirm the presence of the cIFN gene, recombinant viral DNAs were analyzed by PCR, using cIFN specific primers flanking the coding region. All viral DNA's yielded the expected 600 bp amplified cIFN DNA product.
In addition, southern blot analysis on the viral DNA was performed using a non-radioactive labeled cIFN cDNA probe. Plasmid constructs containing the cIFN gene cassettes were sequenced across the transcriptional and translational initiation/termination signals, to confirm the integrity of the ORF.
Growth Properties of Recombinant Viruses in Cell Culture.
Recombinant FPV/cIFN and FPV/cIFN+NDV were found to be attenuated with respect to their growth in chicken embryo fibroblast (CEF) cells. Plaque size was decreased significantly and viral titers were 0.9-1.4 logs less when compared to wild type FPV. We suggest that fowlpox virus has anti-IFN mechanisms, similar to anti-IFN 1. WO 96/40880 PCT/US96/11187 -82mechanisms reported for other pox viruses, e.g. vaccinia, cowpox. And that these mechanisms help the virus to overcome the inhibitory effects of exogenously expressed cIFN. Therefore, fowlpox virus is able to infect, replicate and retain a productive infectious state.
In Vivo Properties of Recombinant FPV/cTFN Virus in Chicks.
10-day old chicks were inoculated, subcutaneously, with recombinant FPV/cIFN (S-FPV-099) virus at increasing dosages. At 10 days post inoculation, all chicks were inoculated with a mixture of sheep red blood cells (SRBC) and Brucella abortus At 15 days post FPV/cIFN virus inoculation, sera was collected, total body weights and antibody responses to SRBC's and BA were measured, and chicks were sacrificed for necropsy analysis. These data show that there were no significant differences in chick body weight, SRBC and BA antibody responses or gross pathologyc associated with inoculation of recombinant FPV/cIFN virus, as compared to chicks inoculated with PBS alone. Therefore, this virus appears to be safe in 10-day old chicks.
Table 3. Determination of safety of recombinant FPV/cIFN virus in 10-day old chicks.
FPV/cIFN Total body Antibody titers a,d (pfu/chick) weight (grams) a b BA SRBC 0 (PBS) 438 4.66 2.16 600 460 4.00 2.00 6,000 461 4.25 2.00 SUBSTITUTE SHEET (RULE 26) WO 96/40880 PCT/US96/11187 -83- 60,000 460 4.62 2.00 a. Measured 15 days post FPV/cIFN virus inoculation b. Mean body weight c. There were no detectable gross pathological changes in any of the groups.
Mean antibody titers were determined by agglutination assay and expressed as log 2 One-day old chicks were inoculated intranasally/intraocularly with NDV B1 (106 ELDso/chick) alone or in addition to subcutaneous inoculation with FPV/cIFN (10 3 pfu/chick). Chick mortality was recorded 2 weeks post vaccination. Chicks vaccinated with NDV B1l alone or with NDV B1l plus FPV wild-type virus showed mortality compared to chickens co-vaccinated with NDV-Bl and FPV/cIFN, in which group, all chicks remained alive. Subsequently, all chicks were challenged at 4 weeks post vaccination with a pathogenic strain of NDV (GB-TX). All chicks were protected, except for those in the "no treatment control group. These data show that NDV Bl vaccine induced mortality was reduced without affecting the vaccine's protective ability.
Table 4. Effect of recombinant FPV/cIFN virus on NVD B1l vaccine induced chick mortality and NDV Bl induced protection from NDV challenge.
SUBSTITUTE SHEET (RULE 26) WO 96/40880 PCT/US96/11187 -84- Treatment Vaccine Challenge Post vaccination antiinduced induced NDV antibody responses.
mortality., mortality.b 0 c Dead/Total Dead/Total 2 weekad 4 weeks No treatment 0/25 15/15 <1 <1 NDVB1 alone 7/30 0/12 1.87 (0.31) 2.15 (0.32) NDVB1 FPV 9/30 0/10 1.96 (0.54) 1.99 (0.35) NDVB1 0/30 0/19 2.00 (0.42) 2.15 (0.37) FPV/cIFN a. Mortality was measured 2 weeks post vaccination.
b. Chicks were challenged 4 weeks post vaccination, intramuscularly, with 10,000 ELD 5 oNDV GB-TX.
c. Mortality was measured 2 weeks post challenge d. Antibody titers were determined by NDV virus neutralization and expressed as group mean (log 0 o).
17-day-old chicken embryos were inoculated with 500 pfu/embryo with FPV/cIFN/NDV virus, FPV wild-type virus or PBS diluent (0.2 ml). Chicks were allowed to hatch and then placed in an isolation unit and observed for mortality for one week. These data show that inoculation of chicken embryos with FPV/cIFN+NDV or FPV wild-type does not interfere with normal hatching.
Table 5. Effect of FPV/cIFN/NDV virus in ovo.
Treatment Number of Eggs Mortality Hatched/Total (Dead/Total) a Diluent (PBS) 15/17 1/15 FPV (wild-type) 15/17 3/15 FPV/cIFN/NDV 14/18 0/14 a 1 week post hatch SUBSTITUTE SHEET (RULE 26) "WO 96/40880 PCT/US96/11187 Three week old SPF chicks were vaccinated, subcutaneously, with 500 pfu/chick of FPV/cIFN/NDV recombinant virus. Sera were collected 9 days and 28 days post vaccination to measure neutralizing antibody responses raised against NDV. All chickens were challenged 28 days post vaccination with a pathogenic strain of NDV and observed for NDV induced mortality for days. These data show that vaccinated chicks developed detectable anti-NDV antibody responses as little as 9 days post vaccination with FPV/NDV/cIFN recombinant virus. These antibody levels were maintained for at least 28 days. In addition, chickens vaccinated with FPV/cIFN/NDV recombinant virus were all protected against challenge with a virulent strain of
NDV.
Table 6. Protective efficacy of FPV/cIFN/NDV vaccine in 3-week-old-chickens.
Vaccine Post Post Vaccination Antibody Challenge Responses Mortality a Dead/Total 9 days 28 days None 19/19 b <1 c FPV-IFN-NDV 0/20 1.36 (0.12) 1.33 (0.31) a. Chicks were challenged intramuscularly, 28 days post vaccination, with 10,000 ELDsoNDV GB-TX.
b. Antibody responses were determined by VN test and expressed as geometric mean titer (log1O) of chickens c. Antibody responses were determined by VN test and expressed as geometric mean titer (log1O) of chickens SUBSTITUTE SHEET (RULE 26) "WO 96/40880 PCTIUS96/11187 -86- One day old SPF chicks were vaccinated, subcutaneously, with 500 pfu/chick of FPV/cIFN/NDV recombinant virus.
Chicks were challenged intranasally/intraocularly at 4 7 and 15 days post vaccination with virulent NDV (GB-TX), and observed for NDV induced mortality for 15 days in each case. These data show that vaccinated chicks.are resistant to virulent NDV when challenged at 7 days post vaccination, but not as early as 4 days post vaccination.
Thus, onset of immunity to NDV following vaccination with FPV/cIFN/NDV recombinant virus occurs between 4 and 7 days post vaccination.
Table 6. Protective efficacy of FPV/cIFN/NDV vaccine in one day old chicks.
Mortality following challenge at 4, 7, and 15 days post vaccination.
Experiment Vaccine 4-days 7-days No.
Dead/Total Dead/Total Dead/Total 1 None NDa 10/10 10/10 FPV-IFN-NDV ND 0/10 0/10 2 None 10/10 10/10 10/10 FPV-IFN-NDV 10/10 1/10 0/10 NDV-B1 4/10 0/10 0/10 a Not Done SUBSTITUTE SHEET (RULE 26) WO 96/40880 PCT/US96/11187 -87- Conclusions 1. Recombinant fowlpox viruses express biologically active chicken interferon into the supernatants of infected cells, as measured by protection of CEF cells from VSV infection.
2. Chicken interferon expressed in supernatants from recombinant SPV/cIFN infected cells has been shown to protect CEF cells against infection with HVT in a dose dependent manner.
3. Chicken interferon expressed from SPV/cIFN acted synergistically with LPS to activate chicken macrophages as detected by nitric oxide induction.
4. Recombinant FPV/cIFN virus was found to be safe in day old chicks at a dosage of 6 x 104 pfu/chick.
Recombinant FPV/cIFN virus was shown to reduce NDV B1 vaccine induced mortality without affecting the vaccine's ability to protect chicks against NDV infection.
6. Inoculation of recombinant FPV/cIFN/NDV virus in ovo does not appear to interfere with normal hatching.
7. Recombinant FPV/cIFN/NDV virus was shown to induce anti-NDV neutralizing antibody in 3-week-old chicks as early as 9 days post vaccination with sustained immunity thru 28 days post vaccination. Furthermore, three-weekold chicks were fully protected against virulent NDV challenge at 28 days post vaccination.
WO 96/40880 PCT/US96/11187 -88- 8. Recombinant FPV/cIFN/NDV virus was shown to protect one-day-old chicks from virulent NDV. challenge as early as 7 days post vaccination.
9. The foregoing data indicate that recombinant fowlpox viruses expressing chicken IFN may have beneficial applications as immune modulating agents in vitro, in vivo and in ovo.
WO 96/40880 PCT/US96/11187 -89- References 1. C. Bertholet, et al., EMBO Journal 5, 1951-1957, 1986.
2. B.H. Coupar, et al., Virology 179, 159-167, 1990.
3. A.J. Davidson and B. Moss, J. Mol. Biol. 210, 749- 769.
4. A.J. Davidson and B. Moss, J. Mol. Biol., 210, 771- 784.
P.L. Earl, et al., Journal of Virology 64, 2448- 2451, 1990.
6. J. Esposito, et al., Virology 165, 313.
7. F.A. Ferrari, et al., Journal of Bacteriology 161, 556-562, 1985.
8. U. Gubler and B.J. Hoffman, Gene 25, 263-269.
9. D. Hanahan, Molecular Biology 166, 557-580, 1983.
M.A. Innis, et al., PCR Protocols A Guide to Methods and Applications, 84-91, Academic Press, Inc., San Diego 1990.
11. Maniatis, et al., Molecular Cloning, Cold Spring Harbor Laboratory, New York 1982.
12. L.J.N. Ross, et al., Journal of General Virology, 1789-1804 (1989).
13. L.J.N. Ross, et al., Journal of General Virology, 72, 949-954 (1991).
,WO 96/40880 PCTIUS96/1 1187 14. J. Sambrook, et al., Molecular Cloning A Laboratory Manual Second Edition, Cold Spring Harbor Press, 1989.
15. J. Taylor, et al., Vaccine 9, 190-193, 1991.
16. A. Leutz, et al., EMBO Journal 8: 175-182 (1989).
17. M.J. Sekellick, et al., Journal of Interferon Reserch 14: 71-79 (1994).
WO 96/40880 PCT/US96/11187 -91- SEQUENCE LISTING GENERAL INFORMATION: APPLICANTS: Mark D. Cochran and David E. Junker (ii) TITLE OF INVENTION: Recombinant Fowlpox Viruses and Uses Thereof (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: John P. White STREET: 1185 Avenue of the Americas CITY: New York STATE: New York COUNTRY: USA ZIP: 10036 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: Not Yet Known FILING DATE: 07-JUN-1995
CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION: NAME: White, John P REGISTRATION NO: 28,678 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (212)278-0400 TELEFAX: (212)391-0526 TELEX: 422523 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CATAAGGCGG CCGCGGCCCT CGAGGCCA 28 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid o WO 96/40880 PCT/US96/11187 -92- STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CATAATGGCC TCGAGGGCCG CGGCCGCC 28 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1507 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 260..1411 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CTACTTCATA AAAAGTTTAA ACCTTCCGAA AGATTTTTGG ATAAAAGTAG AGAACTCGCA TTGCGATTAT GCTCTAGGAC AATCCTGTAA AGTGTCTCGA TCTTAGCATA TAGATAAATG 120 TTTGAACTAA TATCCTAAAG CCTGTATGTA ACAGTTGGTG CCTATTGAAA GATACTGATT 180 ATCAAGGAGA AGAATAATAT AAATCGTAAA AATAATACTT ATTATATAAT ATAATGTATA 240 ATAATATACA AAAACAGCC ATG ATA CGT ATT ATA ATA TTA TCG TTA TTA TTT 292 Met Ile Arg Ile Ile Ile Leu Ser Leu Leu Phe 1 5 ATT AAC GTA ACA ACA GAT AGT CAA GAA TCT TCA AAA AAT ATA CAA AAT 340 Ile Asn Val Thr Thr Asp Ser Gln Glu Ser Ser Lys Asn Ile Gln Asn 20 GTA TTG CAC GTT ACA GAA TAT AGT AGA ACT GGT GTA ACA GCT TGC TCG 388 Val Leu His Val Thr Glu Tyr Ser Arg Thr Gly Val Thr Ala Cys Ser 35 TTA CAT TGT TTT GAT CGT TCC AAA GGT TTA GAT CAA CCA AAA ACA TTT 436 Leu His Cys Phe Asp Arg Ser Lys Gly Leu Asp Gln Pro Lys Thr Phe 45 50 ATC CTG CCT GGT AAA TAT AGC AAT AAC AGT ATA AAA CTA GAA GTA GCT 484 Ile Leu Pro Gly Lys Tyr Ser Asn Asn Ser Ile Lys Leu Glu Val Ala 65 70 ATT GAT ACA TAT AAA AAA GAT AGC GAC TTC AGT TAT TCT CAC CCA TGT 532 Ile Asp Thr Tyr Lys Lys Asp Ser Asp Phe Ser Tyr Ser His Pro Cys "WO 96/40880 PCT/US96/11187 -93- CAA ATA TTC Gin Ile Phe CAG TTC TGT GTG TCT GGT AAT TTT AGT GGT AAA CGG TTC Gin Phe Cys Val Ser Gly Asn Phe Ser Gly Lys Arg Phe 100 105 GAT CAT TAT CTA TAT GGG TAT ACA ATT TCC GGA TTT ATA GAT ATT GCT Asp His Tyr Leu Tyr Gly Tyr Thr Ile Ser Gly Phe Ile Asp Ile Ala 110 115 120 CCA AAA TAT TAT AGC GGT ATG TCT ATA AGT ACT ATT ACT GTT ATG CCA Pro Lys Tyr Tyr Ser Gly Met Ser Ile Ser Thr Ile Thr Val Met Pro 125 130 135 580 628 676
TTA
Leu 140 CAA GAA GGA TCA TTA AAG CAT GAT GAT GCC GAT GAC TAT GAC TAC Gin Glu Gly Ser Leu Lys His Asp Asp Ala Asp Asp Tyr Asp Tyr 145 150 155 GAT GAT GAT TGT GTT CCT TAT AAA GAA ACC CAG CCT CGA CAT ATG CCA Asp Asp Asp Cys Val Pro Tyr Lys Glu Thr Gln Pro Arg His Met Pro 160 165 170 GAA TCG GTA ATA AAA GAA GGA TGT AAA CCC ATT CCA CTA CCA AGG TAT Glu Ser Val Ile Lys Glu Gly Cys Lys Pro Ile Pro Leu Pro Arg Tyr 175 180 185 GAT GAA AAT GAC GAT CCT ACT TGT ATT ATG TAT TGG GAT CAC TCG TGG Asp Glu Asn Asp Asp Pro Thr Cys Ile Met Tyr Trp Asp His Ser Trp 190 195 200 GAT AAT TAC TGT AAT GTT GGA TTT TTT AAT TCT CTA CAG AGT GAT CAC Asp Asn Tyr Cys Asn Val Gly Phe Phe Asn Ser Leu Gln Ser Asp His 205 210 215
AAT
Asn 220 CCT CTG GTT TTT CCG TTA ACA AGT TAT TCT GAT ATA AAC AAT GCA Pro Leu Val Phe Pro Leu Thr Ser Tyr Ser Asp Ile Asn Asn Ala 225 230 235 TTT CAT GCT TTT CAA TCA TCT TAT TGT AGA TCA CTA GGC TTT AAC CAA Phe His Ala Phe Gln Ser Ser Tyr Cys Arg Ser Leu Gly Phe Asn Gin 240 245 250 TCA TAC AGT GTA TGC GTA TCT ATA GGT GAT ACA CCA TTT GAG GTT ACG Ser Tyr Ser Val Cys Val Ser Ile Gly Asp Thr Pro Phe Glu Val Thr 255 260 265 TAT CAT AGT TAT GAA AGT GTT ACT GTT GAT CAG TTA TTA CAA GAA ATT Tyr His Ser Tyr Glu Ser Val Thr Val Asp Gln Leu Leu Gln Glu Ile 270 275 280 AAA ACA CTA TAT GGA GAA GAT GCT GTA TAT GGA TTA CCG TTT AGA AAT Lys Thr Leu Tyr Gly Glu Asp Ala Val Tyr Gly Leu Pro Phe Arg Asn 285 290 295 964 1012 1060 1108 1156 1204 1252 1300 1348
ATA
Ile 300 ACT ATA AGG GCG CGT ACA CGG ATT CAA AGT TTA CCT CTT ACT AAC Thr Ile Arg Ala Arg Thr Arg Ile Gln Ser Leu Pro Leu Thr Asn 305 310 315 AAT ACC TGT ATC CCT AAA CAA GAC GAT GCT GAT GAT GTT GAC GAT GCT Asn Thr Cys Ile Pro Lys Gln Asp Asp Ala Asp Asp Val Asp Asp Ala 320 325 330 GAT GAT GTT GAC GAT GCT GAT GAT GCT GAC GAT GAT GAT GAT TAC GAG Asp Asp Val Asp Asp Ala Asp Asp Ala Asp Asp Asp Asp Asp Tyr Glu 335 340 345 TTA TAT GTA GAA ACT ACA CCA AGA GTG CCA ACA GCG AGA AAA AAA CCC "WO 96/40880 PCT/US96/11187 -94- Leu Tyr Val Glu Thr Thr Pro Arg Val Pro Thr Ala Arg Lys Lys Pro 350 355 360 GTT ACA GAA GAA TAT AAT GAT ATA TTT AGT AGT TTT GAT AAT TTT GAC 1396 Val Thr Glu Glu Tyr Asn Asp Ile Phe Ser Ser Phe Asp Asn Phe Asp 365 370 375 ATG AAA AAG AAA TAAGACATAT TTTATTAAAT CAAAAAGTCT GTCGAACTTT 1448 Met Lys Lys Lys 380 TAGTGTTTAA CCTATATCGA TTTATGATTT TTCCATGATG ATCCAGGCTA TGACTGACT 1507 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 383 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Ile Arg Ile Ile Ile Leu Ser Leu Leu Phe Ile Asn Val Thr Thr 1 5 10 Asp Ser Gln Glu Ser Ser Lys Asn Ile Gln Asn Val Leu His Val Thr 20 25 Glu Tyr Ser Arg Thr Gly Val Thr Ala Cys Ser Leu His Cys Phe Asp 40 Arg Ser Lys Gly Leu Asp Gln Pro Lys Thr Phe Ile Leu Pro Gly Lys 55 Tyr Ser Asn Asn Ser Ile Lys Leu Glu Val Ala Ile Asp Thr Tyr Lys 70 75 Lys Asp Ser Asp Phe Ser Tyr Ser His Pro Cys Gln Ile Phe Gln Phe 90 Cys Val Ser Gly Asn Phe Ser Gly Lys Arg Phe Asp His Tyr Leu Tyr 100 105 110 Gly Tyr Thr Ile Ser Gly Phe Ile Asp Ile Ala Pro Lys Tyr Tyr Ser 115 120 125 Gly Met Ser Ile Ser Thr Ile Thr Val Met Pro Leu Gln Glu Gly Ser 130 135 140 Leu Lys His Asp Asp Ala Asp Asp Tyr Asp Tyr Asp Asp Asp Cys Val 145 150 155 160 Pro Tyr Lys Glu Thr Gln Pro Arg His Met Pro Glu Ser Val Ile Lys 165 170 175 Glu Gly Cys Lys Pro Ile Pro Leu Pro Arg Tyr Asp Glu Asn Asp Asp 180 185 190 Pro Thr Cys Ile Met Tyr Trp Asp His Ser Trp Asp Asn Tyr Cys Asn 195 200 205 Val Gly Phe Phe Asn Ser Leu Gln Ser Asp His Asn Pro Leu Val Phe 210 215 220 4'WO 96/40880 PCT/US96/11187 Pro Leu Thr Ser Tyr Ser Asp Ile Asn Asn Ala Phe His Ala Phe Gin 225 230 235 240 Ser Ser Tyr Cys Arg Ser Leu Gly Phe Asn Gln Ser Tyr Ser Val Cys 245 250 255 Val Ser Ile Gly Asp Thr Pro Phe Glu Val Thr Tyr His Ser Tyr Glu 260 265 270 Ser Val Thr Val Asp Gin Leu Leu Gln Glu Ile Lys Thr Leu Tyr Gly 275 280 285 Glu Asp Ala Val Tyr Gly Leu Pro Phe Arg Asn Ile Thr Ile Arg Ala 290 295 300 Arg Thr Arg Ile Gin Ser Leu Pro Leu Thr Asn Asn Thr Cys Ile Pro 305 310 315 320 Lys Gin Asp Asp Ala Asp Asp Val Asp Asp Ala Asp Asp Val Asp Asp 325 330 335 Ala Asp Asp Ala Asp Asp Asp Asp Asp Tyr Glu Leu Tyr Val Glu Thr 340 345 350 Thr Pro Arg Val Pro Thr Ala Arg Lys Lys Pro Val Thr Glu Glu Tyr 355 360 365 Asn Asp Ile Phe Ser Ser Phe Asp Asn Phe Asp Met Lys Lys Lys 370 375 380 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 2849 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 300..1568 (ix) FEATURE: NAME/KEY: CDS LOCATION: complement (1685..2848) (xi) SEQUENCE DESCRIPTION: SEQ ID AAGCCAGTTT GAATTCAATA TTCATCGCCG ATAGTTGGTA GAAATACTAT TCATGAAATT TACCTTCTTC CGTGGCTTAA AAACTTATTG TATGTACCAT TCATTATAAG ATCTGATACT 120 ATCGGCATCT TCTATTTTCC GAGTTTTTTA CATCTGGTTA CTAGTATCCA TGTTCGTCTA 180 ATAAGAGGGA AGGAATATAT CTATCTACAT AAACATCATA AGGTTCTTTG ATAGATTTAT 240 ATCGCTAATA AAATATAAAT AATAATTAAA GATTTTATGA TATATCGAGC TTTGCAAAA 299 WO 96/40880 PCT/US96/11187 -96- ATG TCT GTT GAT TGG CGT ACA GAA ATC TAT TCG GGT GAT ATA TCC CTA Met Ser Val Asp Trp Arg Thr Glu Ile Tyr Ser Gly Asp Ile Ser Leu 1 5 10 GTA GAA AAA CTT ATA AAG AAT AAA GGT AAT TGC ATC AAT ATA TCT GTA Val Glu Lys Leu Ile Lys Asn Lys Gly Asn Cys Ile Asn Ile Ser Val 25 GAG GAA ACA ACA ACT CCG TTA ATA GAC GCT ATA AGA ACC GGA AAT GCC Glu Glu Thr Thr Thr Pro Leu Ile Asp Ala Ile Arg Thr Gly Asn Ala 40 AAA ATA GTA GAA CTA TTT ATC AAG CAC GGA GCG CAA GTT AAT CAT GTA Lys Ile Val Glu Leu Phe Ile Lys His Gly Ala Gln Val Asn His Val 50 55 AAT ACT AAA ATT CCT AAT CCC TTG TTA ACA GCT ATC AAA ATA GGA TCA Asn Thr Lys Ile Pro Asn Pro Leu Leu Thr Ala Ile Lys Ile Gly Ser 70 75 CAC GAT ATA GTA AAA CTG CTG TTG ATT AAC GGA GTT GAT ACT TCT ATT His Asp Ile Val Lys Leu Leu Leu Ile Asn Gly Val Asp Thr Ser Ile 90 TTG CCA GTC CCC TGC ATA AAT AAA GAA ATG ATA AAA ACT ATA TTA GAT Leu Pro Val Pro Cys Ile Asn Lys Glu Met Ile Lys Thr Ile Leu Asp 100 105 110 AGT GGT GTG AAA GTA AAC ACA AAA AAT GCT AAA TCT AAA ACT TTC TTG Ser Gly Val Lys Val Asn Thr Lys Asn Ala Lys Ser Lys Thr Phe Leu 115 120 125 347 395 443 491 539 587 635 683
CAT
His
GAG
Glu 145
ATA
Ile
TTA
Leu
CCG
Pro
GTT
Val
ACA
Thr 225
CTG
Leu
CCA
Pro TAC GCG ATT AAG AAT AAT GAC TTA GAG GTT ATC AAA ATG CTT TTT Tyr Ala Ile Lys Asn Asn Asp Leu Glu Val Ile Lys Met Leu Phe 130 135 140 TAT GGA GCT GAT GTT AAT ATA AAA GAT GAT AAC ATA TGT TAT TCT Tyr Gly Ala Asp Val Asn Ile Lys Asp Asp Asn Ile Cys Tyr Ser 150 155 160 CAC ATA GCT ACT AGG AGT AAT TCA TAT GAA ATC ATA AAA TTA CTA His Ile Ala Thr Arg Ser Asn Ser Tyr Glu Ile Ile Lys Leu Leu 165 170 175 GAA AAA GGT GCT TAT GCA AAC GTA AAA GAC AAT TAT GGT AAT TCT Glu Lys Gly Ala Tyr Ala Asn Val Lys Asp Asn Tyr Gly Asn Ser 180 185 190 TTA CAT AAC GCG GCT AAA TAT GGC GAT TAT GCT TGT ATT AAA TTA Leu His Asn Ala Ala Lys Tyr Gly Asp Tyr Ala Cys Ile Lys Leu 195 200 205 TTA GAC CAT ACT AAT AAC ATA AGC AAT AAG TGC AAC AAC GGT GTT Leu Asp His Thr Asn Asn Ile Ser Asn Lys Cys Asn Asn Gly Val 210 215 220 CCG TTA CAT AAC GCT ATA CTA TAT AAT AGA TCT GCC GTA GAA TTA Pro Leu His Asn Ala Ile Leu Tyr Asn Arg Ser Ala Val Glu Leu 230 235 240 ATT AAC AAT CGA TCT ATT AAT GAT ACG GAT GTA GAC GGA TAT ACT Ile Asn Asn Arg Ser Ile Asn Asp Thr Asp Val Asp Gly Tyr Thr 245 250 255 CTA CAT TAT GCT TTG CAA CCT CCG TGT AGT ATA GAT ATT ATA GAT Leu His Tyr Ala Leu Gln Pro Pro Cys Ser Ile Asp Ile Ile Asp 260 265 270 731 779 827 875 923 971 1019 1067 1115 -WO 96/40880 WO 9640880PCT/US96/1 1187 -97- ATA CTA CTA TAT AAC AAC GCC GAT ATA TCT ATA AAA GAT AAT AAC GGA Ile Leu Leu Tyr Asn Asn Ala Asp Ile Ser Ile Lys Asp Asn Asn Giy 275 280 285 CGC AAT Arg Asn 290 ATA AAA Ile Lys 305 CCT ATC GAT ACG GCG TIT AAG TAT ATT AAC AGA GAT AGC GTT Pro Ile Asp Thr Ala Phe Lys Tyr Ile Asn Arg Asp Ser Val 295 300 GAA CTT CTC CGA AAC GCC GTG TTA ATT AAC GAG GTC GGT AAA Giu Leu Leu Arg Asn Aia Vai Leu Ile Asn Giu Vai Gly Lys 310 315 320 TTA AAA GAT ACT ACT ATC 'ITA GAA CAC AAA GAA ATA AAA GAC AAT ACC Leu Lys Asp Thr Thr Ile Leu Giu His Lys Giu Ile Lys Asp Asn Thr 325 330 335 GTG TTT TCA AAC TTT GTG TAC GAA TGT AAT GAA GAA ATT AAA AAA ATG Val Phe Ser Asn Phe Vai Tyr Giu Cys Asn Giu Giu Ile Lys Lys Met 340 345 350 AAG AAA ACT AAA TOT GTC GOT GAC TAT AGT ATG TTT GAC GTA TAC ATG Lys Lys Thr Lys Cys Vai Giy Asp Tyr Ser Met Phe Asp Vai Tyr Met 355 360 365 ATA AGO TAT AAA CAC AAA TAT GAC GGT AAT AAG GAT AGT ATT AAA GAC Ile Arg Tyr Lys His Lys Tyr Asp Gly Asn Lys Asp Ser Ile Lys Asp 370 375 380 TAT TTG Tyr Leu 385 GAT ATT Asp Ile TAT GAT Tyr Asp
GGTATAAA
GGCCAGTA
TTAGACCA
TTATGTC7
TAGTAAGT
TTTTTACT
TCTTTTAG
ATTTCTCT
ATATGTGC
CTACTATC
AATGCTA'I
TCTAATAA
GTAGATAA
CTCATATP
CGT TGT CTT GAT GAT AAT AGT ACT CGT ATG TTA AAA ACT ATA Arg Cys Leu Asp Asp Asn Ser Thr Arg Met Leu Lys Thr Ile 390 395 400 AAT GAA TIT CCT ATA TAT TCT ATG TAT CTC GTA AGA TGC CTA Asn Giu Phe Pro Ile Tyr Ser Met Tyr Leu Vai Arg Cys Leu 405 1410 415 ATG GTA ATA TAT TAAAAGAAAT GGGCTCTTGC ATACATAATC 1163 1211 1259 1307 1355 1403 1451 1499 1547 1595 1655 1715 1775 1835 1895 1955 2015 2075 2135 2195 2255 2315 2375 2435 Met Val Ile Tyr 420 AA ATAACGAAAT TATTAGCGGT .GC TCAGTATTTC CTATAAACTC AC CGCTAGAATC TAATATCA 'AT TACTGGCTAA GGATATGGAA 'AT TAA'TTCCTT TATTATAGGA AA TAACGAATAT ATCTAAAGAG TIT CTTCCTTACA ACTCAACCAA 'CA AGGGG'rITAC TTCAC'rrCTG ,TA TGATATATCT AAAAGAAAAC TC TAAGTTCAGC ACCATAATGT TA ATAAAGGATA TTGATTCCTT ,CA TTITATAAC 'ITCTGG'TI'A ,GT AITrTTTITT AGAATTAACA ,CG GATTTCC ATATAAGGCA TACATATCTT ACGGCGGCCG CGGCCCTCGA
TAATATTGAG
TCTAATTTTG
G'ITTTAAGAC
AGTTTGATAT
ATCTACTTTT
GATCTCCGTA
TTATTI-ACTA AGTGATGTAA TAAAACATAT TAATACGAAT ATACTTTTAA ACGTATCATC
ATATCGTGAC
ATATTACTGT
AATAATATAT
ATGTTAATAG
CAA'ITCTT'TT
ITAGCTCCTC
AAATGTAAAA
GTATAAAATC
TAAGGCTCTT
TTACTATACC
CATTTACATC
TACACGCATA
TATCTATGAG
CCGTTCCTAT
CCGGAGAAGT
TTCTAATATT
ATTATAGAAA
CAGGTTCATG
.TTTAGATATA
GCTTTGAATA
TTGTATACAT
ATCGATGACC
ATGATATTCT
CGCTCCGTTA
ATGCAACGGA
CGTTTTTACA
CTTCTGCGAT
t, WO 96/40880 'WO 9640880PCTIUS96/1 1187
AACGCTTCTA
ACAGCGGCAT
AATTCTAACA
GGAGTGATAT
ATGTTTTCTG
TI'TTAGCGT
GGTGCTTGTA
-98- TATCGGCCCC GTAATCTAAA AGAGTGTTTA TGATAACTAC ATTGTTTCTT AATGAATAGG CGTCTTGTCA CAATAATCTC TAGCATTTAC GTTCGCTCCC ACGTTATAAC TGTATC'ITTA TATCTATCTA GAGTAGAGGC TTGATGTAAT ACAGACTATC AGCGGCGTTA ACATCTGCAC CCCGCATTAT TA.AAGTTCTA TATCGTATCC A ITCTTAGCC ATGAGATACA GAGGAGTTTC TCCTTTAATG TAACATCTAT TCCTCT'ITCC AATAACTTGG GTACTAGTCT ACTTAACGAA CCGTGTAATG CAAAGGAGTA TI'CTATAAA CATCTATAGA ATTC 2495 2555 2615 2675 2735 2795 2849 INFORMATION FOR SEQ ID NO:6: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 422 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Ser Val Asp Val Glu Lys Asn His Leu Ser His Giu 145 Ile Leu Glu Glu Ile Thr Asp Pro Gly Tyr 130 Tyr His Glu Leu Thr Giu Ile Val Pro 100 Lys Ile Ala Ala Gly 180 Trp Ile Thr Leu Pro Lys 85 Cys Val Lys Asp Thr 165 Ala Lys Pro Phe Asn 70 Leu Ile Asn Asn Val 150 Arg Tyr Arg Thr Glu Ile Lys Ile Lys Leu Leu Lys Lys 120 Asp Ile Asn Asn Gly 25 Asp His Leu Ile Glu 105 Asn Leu Lys Ser Val 185 Tyr Asn Ala Gly Thr Asn Met Ala Giu Asp Tyr 170 Lys Ser Gly Asp Ile Ser Leu Cys Ile Ala Ala 75 Gly Ile Lys Val Asp 155 Glu Asp Ile Arg Gin Ile Val Lys Ser Ile 140 Asn Ile Asn Asn Thr Val Lys Asp Thr Lys 125 Lys Ile Ile Tyr Cys 205 Ile Gly Asn Ile Thr Ile 110 Thr Met Cys Lys Gly 190 Val Ala Val Ser s0 Ile Asp Leu Phe Ser 160 Leu Ser Pro Leu His Asn Ala Ala Lys Tyr Gly Asp Tyr Ala 195 200 Ile Lys Leu -WO 96/40880 PCT/US96/11187 -99- Val Thr 225 Leu Pro Ile Arg Ile 305 Leu Val Lys Ile Tyr 385 Asp Tyr Leu 210 Pro Ile Leu Leu Asn 290 Lys Lys Phe Lys Arg 370 Leu Ile Asp Asp Leu Asn His Leu 275 Pro Glu Asp Ser Thr 355 Tyr Arg Asn Met His His Asn Tyr 260 Tyr Ile Leu Thr Asn 340 Lys Lys Cys Glu Val 420 Thr Asn Arg 245 Ala Asn Asp Leu Thr 325 Phe Cys His Leu Phe 405 Ile Asn Ala 230 Ser Leu Asn Thr Arg 310 Ile Val Val Lys Asp 390 Pro Tyr Asn 215 Ile Ile Gin Ala Ala 295 Asn Leu Tyr Gly Tyr 375 Asp Ile lie Leu Asn Pro Asp 280 Phe Ala Glu Glu Asp 360 Asp Asn Tyr Ser Tyr Asp Pro 265 Ile Lys Val His Cys 345 Tyr Gly Ser Ser Asn Lys Cys 220 Asn Arg Ser 235 Thr Asp Val 250 Cys Ser Ile Ser Ile Lys Tyr Ile Asn 300 Leu Ile Asn 315 Lys Glu Ile 330 Asn Glu Glu Ser Met Phe Asn Lys Asp 380 Thr Arg Met 395 Met Tyr Leu 410 Asn Asn Gly Val Ala Asp Asp Asp 285 Arg Glu Lys Ile Asp 365 Ser Leu Val Glu Tyr 255 Ile Asn Ser Gly Asn 335 Lys Tyr Lys Thr Cys 415 Leu 240 Thr Asp Gly Val Lys 320 Thr Met Met Asp Ile 400 Leu INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 387 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Asn Ser Ile Asp Val Tyr Lys Asn Thr Pro Leu His Tyr Thr Val Gin 1 5 10 Ala Pro Ser Leu Ser Arg Leu Val Pro Lys Leu Leu Glu Arg Gly Ile 20 25 Asp Val Asn Ala Lys Asn Ile Lys Gly Glu Thr Pro Leu Tyr Leu Met 40 Ala Lys Asn Gly Tyr Asp Thr Glu Asn Ile Arg Thr Leu Ile Met Arg 55 WO 96/40880 PCT/US96/11187 -100- Gly Gin Glu Ile Leu Val 145 Thr Ser Ile Arg Asn 225 Asp His Asn Trp Asn 305 Leu Tyr Ala Glu Ala Ala Leu His Asp 130 Leu Leu Thr Lys Asn 210 Ile Lys Ile Pro Leu 290 Met Leu Tyr Ser Ile 370 Asp Ser Gly Tyr 115 Tyr His Ile Pro Met 195 Gin Leu Ser Cys Leu 275 Ser Phe His Phe Asn 355 Leu Val Thr Ala 100 Ala Gly Phe Asp Leu 180 Leu Tyr Leu Leu Ile 260 Arg Cys Tyr His Tyr 340 Arg Asp Asn Leu 85 Asn Ala Ala Ala Arg 165 His Leu Pro His Asn 245 Gin Glu Lys Ser Leu 325 Asn His Ser Ala 70 Asp Val Val Asp Leu 150 Gly Tyr Asp Leu Tyr 230 Ser Asp Ile Glu Leu 310 Val Tyr Lys Ser Ala Asp Ser Leu Arg Asn Arg Ile 135 Tyr Ala Ala Asn Leu 215 Gly Asn Phe Ile Glu 295 Asp Asn Gly Ile Gly 375 Tyr Ala Asn 120 Glu Gly Asn Cys Gly 200 Ile Ala Met Ile Gin 280 Leu Ile Asn Asp Leu 360 Trp Lys Arg 105 Asn Ala Thr Val Lys 185 Ala Ala Glu Phe Arg 265 Ser Lys Phe Pro Arg 345 Glu Ser Asp 90 Asp Val Leu Asn Asn 170 Lys Asp Leu Leu Ser 250 His Asp Asp Val Ile 330 Leu Lys Lys Tyr 75 Thr Tyr Val Ser Pro 155 Ser Asn Val Glu Arg 235 Phe Asp Asp Ile Ile 315 Ile Lys Ser Leu Ile Thr Pro Val Ile Thr Cys Asp Lys 110 Ile Ile Asn 125 Gin Lys Ile 140 Tyr Met Ser Lys Cys Asn Tyr 220 Asp Arg ile Thr Ser 300 Ser Lys Thr Arg Leu 380 Asn Lys Ala 205 His Ser Tyr Arg Phe 285 Lys Lys Glu Ser Ser 365 Arg Lys Pro 190 Ile Gly Arg Ile Ser 270 Lys Ile Asn Ile Ile 350 Lys Ile Leu Leu Thr Thr Gly Val Tyr 175 Glu Asn Ile Val Ile 255 Glu Ser Arg Met Asn 335 Ser Leu Ser His Leu Pro Leu Thr Lys 160 Leu Val Ile Val Ile 240 Ala Val Ile Ile Asn 320 Thr Leu Asp Asn Ser Gln Tyr 385 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs WO 96/40880 PCT/US96/11187 -101- TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AAAAATTGAA AAACTATTCT AATTTATTGC ACGGAGATCT INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: AATTTCATTT TGTTIrTTTC TATGCTATAA AT 32 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID GTATCCTAAA ATTGAATTGT AATTATCGAT AATAAAT 37 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO WO 96/40880 PCT/US96/11187 -102- (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: ±TTTTTTTTTT i-TTTTTTrrr GGCATATAAA TGAATTCGGA TC 42 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 4177 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 115..1860 (ix) FEATURE: NAME/KEY: CDS LOCATION: 2095..3756 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CATACTGGCC TCGAGGGCCG CGGCCGCCTG CAGGTCGACT CTAGAAAAAA TTGAAAAACT ATTCTAATTT ATTGCACGGA GATCTTTTTT TrTTT-ITIT TTTTTGGCAT ATAA ATG 117 Met 1 AAT TCG GAT CCG GAC CGC GCC GTT AGC CAA GTT GCG TTA GAG AAT GAT 165 Asn Ser Asp Pro Asp Arg Ala Val Ser Gin Val Ala Leu Glu Asn Asp 10 GAA AGA GAG GCA AAA AAT ACA TGG CGC TTG ATA TTC CGG ATT GCA ATC 213 Glu Arg Glu Ala Lys Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala Ile 25 TTA TTC TTA ACA GTA GTG ACC TTG GCT ATA TCT GTA GCC TCC CTT TTA 261 Leu Phe Leu Thr Val Val Thr Leu Ala Ile Ser Val Ala Ser Leu Leu 40 TAT AGC ATG GGG GCT AGC ACA CCT AGC GAT CTT GTA GGC ATA CCG ACT 309 Tyr Ser Met Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Ile Pro Thr 50 55 60 AGG ATT TCC AGG GCA GAA GAA AAG ATT ACA TCT ACA CTT GGT TCC AAT 357 Arg Ile Ser Arg Ala Glu Glu Lys Ile Thr Ser Thr Leu Gly Ser Asn 75 CAA GAT GTA GTA GAT AGG ATA TAT AAG CAA GTG GCC CTT GAG TCT CCA 405 Gln Asp Val Val Asp Arg Ile Tyr Lys Gin Val Ala Leu Glu Ser Pro 90 TTG GCA TTG TTA AAT ACT GAG ACC ACA ATT ATG AAC GCA ATA ACA TCT 453 Leu Ala Leu Leu Asn Thr Glu Thr Thr Ile Met Asn Ala Ile Thr Ser 100 105 110 -WO 96/40880 PCT/US96/11187 -103- CTC TCT TAT CAG ATT AAT GGA GCT GCA AAC AAC AGC GGG TGG GGG GCA 501 Leu Ser Tyr Gln Ile Asn Gly Ala Ala Asn Asn Ser Gly Trp Gly Ala 115 120 125 CCT ATT CAT GAC CCA GAT TAT ATA GGG GGG ATA GGC AAA GAA CTC ATT 549 Pro Ile His Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Glu Leu Ile 130 135 140 145 GTA GAT GAT GCT AGT GAT GTC ACA TCA TTC TAT CCC TCT GCA TTT CAA 597 Val Asp Asp Ala Ser Asp Val Thr Ser Phe Tyr Pro Ser Ala Phe Gin 150 155 160 GAA CAT CTG AAT TTT ATC CCG GCG CCT ACT ACA GGA TCA GGT TGC ACT 645 Glu His Leu Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr 165 170 175 CGA ATA CCC TCA TTT GAC ATG AGT GCT ACC CAT TAC TGC TAC ACC CAT 693 Arg Ile Pro Ser Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr His 180 185 190 AAT GTA ATA TTG TCT GGA TGC AGA GAT CAC TCA CAC TCA CAT CAG TAT 741 Asn Val Ile Leu Ser Gly Cys Arg Asp His Ser His Ser His Gln Tyr 195 200 205 TTA GCA CTT GGT GTG CTC CGG ACA TCT GCA ACA GGG AGG GTA TTC TTT 789 Leu Ala Leu Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Val Phe Phe 210 215 220 225 TCT ACT CTG CGT TCC ATC AAC CTG GAC GAC ACC CAA AAT CGG AAG TCT 837 Ser Thr Leu Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn Arg Lys Ser 230 235 240 TGC AGT GTG AGT GCA ACT CCC CTG GGT TGT GAT ATG CTG TGC TCG AAA 885 Cys Ser Val Ser Ala Thr Pro Leu Gly Cys Asp Met Leu Cys Ser Lys 245 250 255 GCC ACG GAG ACA GAG GAA GAA GAT TAT AAC TCA GCT GTC CCT ACG CGG 933 Ala Thr Glu Thr Glu Glu Glu Asp Tyr Asn Ser Ala Val Pro Thr Arg 260 265 270 ATG GTA CAT GGG AGG TTA GGG TTC GAC GGC CAA TAT CAC GAA AAG GAC 981 Met Val His Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Glu Lys Asp 275 280 285 CTA GAT GTC ACA ACA TTA TTC GGG GAC TGG GTG GCC AAC TAC CCA GGA 1029 Leu Asp Val Thr Thr Leu Phe Gly Asp Trp Val Ala Asn Tyr Pro Gly 290 295 300 305 GTA GGG GGT GGA TCT TTT ATT GAC AGC CGC GTG TGG TTC TCA GTC TAC 1077 Val Gly Gly Gly Ser Phe Ile Asp Ser Arg Val Trp Phe Ser Val Tyr 310 315 320 GGA GGG TTA AAA CCC AAT ACA CCC AGT GAC ACT GTA CAG GAA GGG AAA 1125 Gly Gly Leu Lys Pro Asn Thr Pro Ser Asp Thr Val Gln Glu Gly Lys 325 330 335 TAT GTG ATA TAC AAG CGA TAC AAT GAC ACA TGC CCA GAT GAG CAA GAC 1173 Tyr Val Ile Tyr Lys Arg Tyr Asn Asp Thr Cys Pro Asp Glu Gln Asp 340 345 350 TAC CAG ATT CGA ATG GCC AAG TCT TCG TAT AAG CCT GGA CGG TTT GGT 1221 Tyr Gln Ile Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Arg Phe Gly 355 360 365 GGG AAA CGC ATA CAG CAG GCT ATC TTA TCT ATC AAA GTG TCA ACA TCC 1269 Gly Lys Arg Ile Gln Gln Ala Ile Leu Ser Ile Lys Val Ser Thr Ser 370 375 380 385 WO 96/40880 WO 9640880PCTUS96/1 1187 -104- TTA GGC GAA GAC CCG GTA CTG ACT GTA CCG CCC AAC ACA GTC ACA CTC 1317 Leu Gly Glu Asp Pro Val Leu Thr Val Pro Pro Asn Thr Val Thr Leu 390 395 400 ATG GGG GCC GAA GGC AGA AIT CTC ACA GTA GGG ACA TCC CAT TTC TTG 1365 Met Gly Ala Giu Gly Arg Ile Leu Thr Val Gly Thr Ser His Phe Leu 405 410 415 TAT CAG CGA GGG TCA TCA TAC TrC TCT CCC GCG TTA TTA TAT CCT ATG 1413 Tyr Gin Arg Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met 420 425 430 ACA GTC AGC AAC AAA ACA GCC ACT CTT CAT AGT CCT TAT ACA TTC AAT 1461 Thr Val Ser Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Thr Phe Asn 435 440 445 GCC TTC ACT CGG CCA GGT AGT ATC CCT TGC CAG GCT TCA GCA AGA TGC 1509 Ala Phe Thr Arg Pro Gly Ser Ile Pro Cys Gin Ala Ser Ala Arg Cys 450 455 460 465 CCC AAC TCA TGT GTT ACT GGA GTC TAT ACA GAT CCA TAT CCC CTA ATC 1557 Pro Asn Ser Cys Val Thr Gly Val Tyr Thr Asp Pro Tyr Pro Leu Ile 470 475 480 ITC TAT AGA AAC CAC ACC TTG CGA GGG GTA TTC GGG ACA ATG CTT GAT 1605 Phe Tyr Arg Asn His Thr Leu Arg Gly Val Phe Gly Thr Met Leu Asp 485 490 495 GGT GAA CAA GCA AGA CTT AAC CCT GCG TCT GCA GTA TTC GAT AGC ACA 1653 Gly Glu Gin Ala Arg Leu Asn Pro Ala Ser Ala Val Phe Asp Ser Thr 500 505 510 TCC CGC AGT CGC ATA ACT CGA GTG AGT TCA AGC AGC ATC AAA GCA GCA 1701 Ser Arg'Ser Arg Ile Thr Arg Val Ser Ser Ser Ser Ile Lys Ala Ala 515 520 525 TAC ACA ACA TCA ACT TGT TTT AAA GTG GTC AAG ACC AAT AAG ACC TAT 1749 Tyr Thr Thr Ser Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr Tyr 530 535 540 545 TGT CTC AGC ATT GCT GAA ATA TCT AAT ACT CTC TTC GGA GAA TTC AGA 1797 Cys Leu Ser Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Giu Phe Arg 550 555 560 ATO GTC CCG TTA CTA G'IT GAG ATC CTC AAA CAT GAC GGG GTT AGA GAA 1845 Ile Val Pro Leu Leu Val Glu Ile Leu Lys Asp Asp Gly Val Arg Glu 565 570 575 GCC AGG TCT GGC TAGTTGAGTC AACTATGAAA GAGTTGGAAA GATGGCATTG 1897 Ala Arg Ser Gly 580 TATCACCTAT C'ITCTGCGAC ATCAAGAATC AAACCGAATG CCCGGATCCA TAATTAATTA 1957 ATTAATTTT ATCCCTCGAC TCTAGAAAAA ATTGAAAAAC TATTCTAATT TATTGCACGG 2017 AGATCTI 777ITTTT 7T1I~TTGGCA TATAAATGAA TTCGGATCGA TCCCGGTTGG 2077 60 CGCCCTCCAG GTGCAGG ATG GOC TCC AGA CCT TCT ACC AAG AAC CCA GCA 2127 Met Gly Ser Arg Pro Ser Thr Lys Asn Pro Ala 1 5 CCT ATG ATG CTG ACT ATC CGG GTC GCG CTG GTA CTG AGT TGC ATC TGT 2175 Pro Met Met Leu Thr Ile Arg Val Ala Leu Val Leu Ser Cys Ile Cys 20 -WO 96/40880 PCT/US96/11187
CCG
Pro
GTT
Val -105- GCA AAC TCC ATT GAT GGC AGG CCT CTT GCA GCT GCA GGA ATT GTG Ala Asn Ser Ile Asp Gly Arg Pro Leu Ala Ala Ala Gly Ile Val 35 ACA GGA GAC AAA GCA GTC AAC ATA TAC ACC TCA TCC CAG ACA GGA Thr Gly Asp Lys Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gly 50 TCA ATC ATA GTT AAG CTC CTC CCG AAT CTG CCA AAG GAT AAG GAG GCA Ser Ile Ile Val Lys Leu Leu Pro Asn Leu Pro Lys Asp Lys Glu Ala 65 70 TGT GCG AAA GCC CCC TTG GAT GCA TAC AAC AGG ACA TTG ACC ACT TTG Cys Ala Lys Ala Pro Leu Asp Ala Tyr Asn Arg Thr Leu Thr Thr Leu 80 85 CTC ACC CCC CTT GGT GAC TCT ATC CGT AGG ATA CAA GAG TCT GTG ACT Leu Thr Pro Leu Gly Asp Ser Ile Arg Arg Ile Gln Glu Ser Val Thr 100 105 ACA TCT GGA GGG GGG AGA CAG GGG CGC CTT ATA GGC GCC ATT ATT GGC Thr Ser Gly Gly Gly Arg Gln Gly Arg Leu Ile Gly Ala Ile Ile Gly 110 115 120 GGT GTG GCT CTT GGG GTT GCA ACT GCC GCA CAA ATA ACA GCG GCC GCA Gly Val Ala Leu Gly Val Ala Thr Ala Ala Gln Ile Thr Ala Ala Ala 125 130 135 GCT CTG ATA CAA GCC AAA CAA AAT GCT GCC AAC ATC CTC CGA CTT AAA Ala Leu Ile Gln Ala Lys Gln Asn Ala Ala Asn Ile Leu Arg Leu Lys 140 145 150 155 GAG AGC ATT GCC GCA ACC AAT GAG GCT GTG CAT GAG GTC ACT GAC GGA Glu Ser Ile Ala Ala Thr Asn Glu Ala Val His Glu Val Thr Asp Gly 160 165 170 TTA TCG CAA CTA GCA GTG GCA GTT GGG AAG ATG CAG CAG TTC GTT AAT Leu Ser Gln Leu Ala Val Ala Val Gly Lys Met Gln Gln Phe Val Asn 175 180 185 GAC CAA TTT AAT AAA ACA GCT CAG GAA TTA GAC TGC ATC AAA ATT GCA Asp Gln Phe Asn Lys Thr Ala Gln Glu Leu Asp Cys Ile Lys Ile Ala 190 195 200 CAG CAA GTT GGT GTA GAG CTC AAC CTG TAC CTA ACC GAA TCG ACT ACA Gin Gln Val Gly Val Glu Leu Asn Leu Tyr Leu Thr Glu Ser Thr Thr 205 210 215 GTA TTC GGA CCA CAA ATC ACT TCA CCT GCC TTA AAC AAG CTG ACT ATT Val Phe Gly Pro Gln Ile Thr Ser Pro Ala Leu Asn Lys Leu Thr Ile 220 225 230 235 CAG GCA CTT TAC AAT CTA GCT GGT GGG AAT ATG GAT TAC TTA TTG ACT Gin Ala Leu Tyr Asn Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Thr 240 245 250 AAG TTA GGT ATA GGG AAC AAT CAA CTC AGC TCA TTA ATC GGT AGC GGC Lys Leu Gly Ile Gly Asn Asn Gln Leu Ser Ser Leu Ile Gly Ser Gly 255 260 265 TTA ATC ACC GGT AAC CCT ATT CTA TAC GAC TCA CAG ACT CAA CTC TTG Leu Ile Thr Gly Asn Pro Ile Leu Tyr Asp Ser Gln Thr Gln Leu Leu 270 275 280 GGT ATA CAG GTA ACT CTA CCT TCA GTC GGG AAC CTA AAT AAT ATG CGT Gly Ile Gln Val Thr Leu Pro Ser Val Gly Asn Leu Asn Asn Met Arg 285 290 295 2223 2271 2319 2367 2415 2463 2511 2559 2607 2655 2703 2751 2799 2847 2895 2943 2991 ,WO 96/40880PCIS6118 PCTIUS96/11187 -106- GCC ACC TAC TrG GAA ACC T1'A TCC GTA AGC ACA ACC AGG GGA TTT GCC 3039 Ala Thr Tyr Leu Giu Thr Leu Ser Val Ser Thr Thr Arg Gly Phe Ala 300 305 310 315 TCG GCA CTT GTC CCA AAA GTG GTG ACA CGG GTC GGT TCT GTG ATA GAA 3087 Ser Ala Leu Val Pro Lys Val Val Thr Arg Val Gly Ser Val Ile Giu 320 325 330 GAA CTT GAC ACC TCA TAC TGT ATA GAA ACT GAC TTA GAT TTA TAT TGT 3135 Giu Leu Asp Thr Ser Tyr Cys Ile Glu Thr Asp Leu Asp Leu Tyr Cys 335 340 345 ACA AGA ATA GTA ACG TTC CCT ATG TCC CCT GGT ATT TAC TCC TGC TTG 3183 Thr Arg Ile Val Thr Phe Pro Met Ser Pro Gly Ile Tyr Ser Cys Leu 350 355 360 AGC GGC AAT ACA TCG GCC TGT ATG TAC TCA AAG ACC GAA GGC GCA CTT 3231 Ser Gly Asn Thr Ser Ala Cys Met Tyr Ser Lys Thr Glu Giy Ala Leu 365 370 375 ACT ACA CCA TAT ATG ACT ATC AAA GGC TCA GTC ATC GCT AAC TGC AAG 3279 Thr Thr Pro Tyr Met Thr Ile Lys Gly Ser Val Ile Ala Asn Cys Lys 380 385 390 395 ATG ACA ACA TGT AGA TGT GTA AAC CCC CCG GGT ATC ATA TCG CAA AAC 3327 Met Thr Thr Cys Arg Cys Val Asn Pro Pro Gly Ile Ile Ser Gin Asn 400 405 410 TAT GGA GAA GCC GTG TCT CTA ATA GAT AAA CAA TCA TGC AAT GTT TTA 3375 Tyr Gly Glu Ala Val Ser Leu Ile Asp Lys Gin Ser Cys Asn Val Leu 415 420 425 TCC TTA GGC GGG ATA ACT TTA AGG CTC AGT GGG GAA TTC GAT GTA ACT 3423 Ser Leu Gly Gly Ile Thr Leu Arg Leu Ser Gly Giu Phe Asp Val Thr 430 435 440 TAT CAG AAG AAT ATC TCA ATA CAA GAT TCT CAA GTA ATA ATA ACA GGC 3471 Tyr Gin Lys Asn Ile Ser Ile Gin Asp Ser Gin Vai Ile Ile Thr Gly 445 450 455 AAT CTT GAT ATC TCA ACT GAG CTT GGG AAT GTC AAC AAC TCG ATC AGT 3519 Asn Leu Asp Ile Ser Thr Giu Leu Giy Asn Val Asn Asn Ser Ile Ser 460 465 470 475 AAT GCC TTG AAT AAG TTA GAG GAA AGC AAC AGA AAA CTA GAC AAA GTC 3567 Asn Ala Leu Asn Lys Leu Giu Glu Ser Asn Arg Lys Leu Asp Lys Vai 480 485 490 AAT GTC AAA CTG ACC AGC ACA TCT GCT CTC A'IT ACC TAT ATC GTT TTG 3615 Asn Val Lys Leu Thr Ser Thr Ser Ala Leu Ile Thr Tyr Ile Val Leu 495 500 505 ACT ATC ATA TCT CTI' G'IT TTT GGT ATA CIT AGC CTG ATT CTA GCA TGC 3663 Thr Ile Ile Ser Leu Val Phe Gly Ile Leu Ser Leu Ile Leu Ala Cys 510 515 520 TAC CTA ATG TAC AAG CAA AAG GCG CAA CAA AAG ACC TTA TTA TGG CTT 3711 Tyr Leu Met Tyr Lys Gin Lys Ala Gin Gin Lys Thr Leu Leu Trp Leu 525 530 535 GGG AAT AAT ACC CTA GAT CAG ATG AGA GCC ACT ACA AAA ATG TGAACACAGA 3763 Giy Asn Asn Thr Leu Asp Gin Met Arg Ala Thr Thr Lys Met 540 545 550 TGAGGAACGA AGG'rITCCCT AATAGTAATT TGTGTGAAAG TTCTGGTAGT CTGTCAGTTC 3823 GGAGAGTTAA GAAAAAAAAA AAACCCCCCC CCCCCCCCCC CCCCCCCCCT GCAGGCATCG 3883 -WO 96/40880PCIS6118 PCTIUS96/11187 -107- TGGTGTCACG CTCGTCGTTT GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC 3943 GAGITACATG ATCCCCCATG TTGTGCAAAA AAGCGG'ITAG CTCCTTCGGT CCTCCGATCG 4003 TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT 4063 CTCTTACTGT CATGCCATCC GTAAGATGCT TITCTGTGAC TGGTGAGTGA TCCATAATTA 4123 ATTAATTAAT TITTATCCCG GGTCGACCTG CAGGCGGCCG CGGCCCTCGA GGCC 4177 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 581 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13: Met Asn Ser Asp Pro Asp Arg Ala Val Ser Gin Val Ala Leu Glu Asn 1 5 10 Asp Glu Arg Glu Ala Lys Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala 25 Ile Leu Phe Leu Thr Val Val Thr Leu Ala Ile Ser Val Ala Ser Leu 35 40 Leu Tyr Ser Met Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Ile Pro 55 Thr ArgIle Ser Arg Ala Glu Glu Lys Ile Thr Ser Thr Leu Gly Ser 70 75 Asn Gin Asp Val Val Asp Arg Ile Tyr Lys Gin Val Ala Leu Giu Ser 90 Pro Leu Ala Leu Leu Asn Thr Giu Thr Thr Ile Met Asn Ala Ile Thr 100 105 110 Ser Leu Ser Tyr Gin Ile Asn Gly Ala Ala Asn Asn Ser Gly Trp Gly 115 120 125 Ala Pro Ile His Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Giu Leu 130 135 140 Ile Val Asp Asp Ala Ser Asp Val Thr Ser Phe Tyr Pro Ser Ala Phe 145 150 155 160 Gin Glu His Leu Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys 165 170 175 Thr Arg Ile Pro Ser Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr 180 185 190 His Asn Val Ile Leu Ser Gly Cys Arg Asp His Ser His Ser His Gin 195 200 205 Tyr Leu Ala Leu Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Val Phe 210 215 220 Phe Ser Thr Leu Arg Ser Ile Asn Leu Asp Asp Thr Gin Asn Arg Lys 225 230 235 240 WO 96/40880 WO 9640880PCTIUS96/1 1187 -108- Ser Lys Arg Asp Gly 305 Tyr Lys Asp Gly Ser 385 Leu Leu Met Asn Cys 465 Ile Asp Thr Ala Tyr 545 Arg Cys Ala Met Leu 290 Val Gly Tyr Tyr Gly 370 Leu Met Tyr Thr Ala 450 Pro Phe Gly Ser Tyr 530 Cys Ile Val Ser Ala Thr Pro Leu Giu 260 His Val Gly Leu Ile 340 Ile Arg Giu Ala Arg 420 Ser Thr Ser Arg Gin 500 Ser Thr Ser Pro 245 Thr Gly Thr Gly Lys 325 Tyr Arg Ile Asp Giu 405 Gly Asn Arg Cys Asn 485 Ala Arg Ser Ile Leu 565 Giu Arg Thr Ser 310 Pro Lys Met Gin Pro 390 Gly Ser Lys Pro Val 470 His Arg Ile Thr Ala 550 Leu Giu Leu Leu 295 Phe Asn Arg Ala Gin 375 Val Arg Ser Thr Gly 455 Thr Thr Leu Thr Cys 535 Glu Val Giu Gly 280 Phe Ile Thr Tyr Lys 360 Ala Leu Ile Tyr Ala 440 Ser Gly Leu Asn Arg 520 Phe Ile Glu Asp 265 Phe Gly Asp Pro Asn 345 Ser Ile Thr Leu Phe 425 Thr Ile Val Arg Pro 505 Val Lys Ser Ile Gly 250 Tyr Asp Asp Ser Ser 330 Asp Ser Leu Val Thr 410 Ser Leu Pro Tyr Gly 490 Al a Ser Val Asn Leu 570 Cys Asp Asn Ser Gly Gin Trp Val 300 Arg Val 315 Asp Thr Thr Cys Tyr Lys Ser Ile 380 Pro Pro 395 Val Giy Pro Ala His Ser Cys Gin 460 Thr Asp 475.
Vai Phe Ser Ala Ser Ser Val Lys 540 Thr Leu 555 Lys Asp Met Ala Tyr 285 Ala Trp Val Pro Pro 365 Lys Asn Thr Leu Pro 445 Ala Pro Gly Val Ser 525 Thr Phe Asp Leu Val 270 His Asn Phe Gin Asp 350 Gly Val Thr Ser Leu 430 Tyr Ser Tyr Thr Phe Ile Asn Gly Gly Ser Thr Lys Pro Val 320 Giy Gin Phe Thr Thr 400 Phe Pro Phe Arg Leu 480 Leu Ser Ala Thr Phe 560 Arg Giu Ala Arg Ser Gly 580 ,'WO 96/40880 PCT/US96/11187 -109- INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 553 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14: Met Gly Ser Arg Pro Ser Thr Lys Asn Pro Ala Pro Met Met Leu Thr 1 5 10 Ile Arg Val Ala Leu Val Leu Ser Cys Ile Cys Pro Ala Asn Ser Ile 25 Asp Gly Arg Pro Leu Ala Ala Ala Gly Ile Val Val Thr Gly Asp Lys 40 Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gly Ser Ile Ile Val Lys 55 Leu Leu Pro Asn Leu Pro Lys Asp Lys Glu Ala Cys Ala Lys Ala Pro 65 70 75 Leu Asp Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly 90 Asp Ser Ile Arg Arg Ile Gln Glu Ser Val Thr Thr Ser Gly Gly Gly 100 105 110 Arg Gln Gly Arg Leu Ile Gly Ala Ile Ile Gly Gly Val Ala Leu Gly 115 120 125 Val Ala Thr Ala Ala Gln Ile Thr Ala Ala Ala Ala Leu Ile Gln Ala 130 135 140 Lys Gln Asn Ala Ala Asn Ile Leu Arg Leu Lys Glu Ser Ile Ala Ala 145 150 155 160 Thr Asn Glu Ala Val His Glu Val Thr Asp Gly Leu Ser Gln Leu Ala 165 170 175 Val Ala Val Gly Lys Met Gln Gln Phe Val Asn Asp Gln Phe Asn Lys 180 185 190 Thr Ala Gln Glu Leu Asp Cys Ile Lys Ile Ala Gln Gln Val Gly Val 195 200 205 Glu Leu Asn Leu Tyr Leu Thr Glu Ser Thr Thr Val Phe Gly Pro Gin 210 215 220 Ile Thr Ser Pro Ala Leu Asn Lys Leu Thr Ile Gln Ala Leu Tyr Asn 225 230 235 240 Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Thr Lys Leu Gly Ile Gly 245 250 255 Asn Asn Gln Leu Ser Ser Leu Ile Gly Ser Gly Leu Ile Thr Gly Asn 260 265 270 Pro Ile Leu Tyr Asp Ser Gln Thr Gln Leu Leu Gly Ile Gln Val Thr 275 280 285 Leu Pro Ser Val Gly Asn Leu Asn Asn Met Arg Ala Thr Tyr Leu Glu 290 295 300 j. -WO 96/40880 PCT/US96/11187 -110- Thr Leu Ser Val Ser Thr Thr Arg Gly Phe Ala Ser Ala Leu Val Pro 305 310 315 320 Lys Val Val Thr Arg Val Gly Ser Val Ile Glu Glu Leu Asp Thr Ser 325 330 335 Tyr Cys Ile Glu Thr Asp Leu Asp Leu Tyr Cys Thr Arg Ile Val Thr 340 345 350 Phe Pro Met Ser Pro Gly Ile Tyr Ser Cys Leu Ser Gly Asn Thr Ser 355 360 365 Ala Cys Met Tyr Ser Lys Thr Glu Gly Ala Leu Thr Thr Pro Tyr Met 370 375 380 Thr Ile Lys Gly Ser Val Ile Ala Asn Cys Lys Met Thr Thr Cys Arg 385 390 395 400 Cys Val Asn Pro Pro Gly Ile Ile Ser Gln Asn Tyr Gly Glu Ala Val 405 410 415 Ser Leu Ile Asp Lys Gin Ser Cys Asn Val Leu Ser Leu Gly Gly Ile 420 425 430 Thr Leu Arg Leu Ser Gly Glu Phe Asp Val Thr Tyr Gln Lys Asn Ile 435 440 445 Ser Ile Gln Asp Ser Gln Val Ile Ile Thr Gly Asn Leu Asp Ile Ser 450 455 460 Thr Glu Leu Gly Asn Val Asn Asn Ser Ile Ser Asn Ala Leu Asn Lys 465 470 475 480 Leu Glu Glu Ser Asn Arg Lys Leu Asp Lys Val Asn Val Lys Leu Thr 485 490 495 Ser Thr Ser Ala Leu Ile Thr Tyr Ile Val Leu Thr Ile Ile Ser Leu 500 505 510 Val Phe Gly Ile Leu Ser Leu Ile Leu Ala Cys Tyr Leu Met Tyr Lys 515 520 525 Gin Lys Ala Gln Gln Lys Thr Leu Leu Trp Leu Gly Asn Asn Thr Leu 530 535 540 Asp Gln Met Arg Ala Thr Thr Lys Met 545 550 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 182 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID i. -W0 96/40880 PCT/US96/11187 -111- GGCCTCGAGG GCCGCGGCCG CCTGCAGGTC GACTCTAGAA AAAATTGAAA AACTATTCTA ATTTATTGCA CGGAGATCTT TrITITrTTT rTTTrri-TG- GCATATAAAT GAATTCGGAT 120 CCGGACCGCG CCGTTAGCCA AGTTGCGTTA GAGAATGATG AAAGAGAGGC AAAAAATACA 180 TG 182 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 178 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: ATCTTCTGCG ACATCAAGAA TCAAACCGAA TGCCCGGATC CATAATTAAT TAATTAATTT TTATCCCTCG ACTCTAGAAA AAATTGAAAA ACTATTCTAA TTTATTGCAC GGAGATCTTT 120 TTrTTTTrTTT TTTTTTTTGG CATATAAATG AATTCGGATC GATCCCGGTT GGCGCCCT 178 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: AAAAACCCCC CCCCCCCCCC CCCCCCCCCC CTGCAGGCAT CGTGGTGTCA CGCTCGTCGT INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 120 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO *WO 96/40880 PCT/US96/11187 -112- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ATAATTCTCT TACTGTCATG CCATCCGTAA GATGCTTTTC TGTGACTGGT GAGTGATCCA TAATTAATTA ATTAATTTTT ATCCCGGGTC GACCTGCAGG CGGCCGCGGC CCTCGAGGCC 120 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 1305 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1305 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: ATG CAC CGT CCT CAT CTC AGA CGG CAC TCG CGT TAC TAC GCG AAA GGA 48 Met His Arg Pro His Leu Arg Arg His Ser Arg Tyr Tyr Ala Lys Gly 1 5 10 GAG GTG CTT AAC AAA CAC ATG GAT TGC GGT GGA AAA CGG TGC TGC TCA 96 Glu Val Leu Asn Lys His Met Asp Cys Gly Gly Lys Arg Cys Cys Ser 25 GGC GCA GCT GTA TTC ACT CTT TTC TGG ACT TGT GTC AGG ATT ATG CGG 144 Gly Ala Ala Val Phe Thr Leu Phe Trp Thr Cys Val Arg Ile Met Arg 40 GAG CAT ATC TGC TTT GTA CGC AAC GCT ATG GAC CGC CAT TTA TTT TTG 192 Glu His Ile Cys Phe Val Arg Asn Ala Met Asp Arg His Leu Phe Leu 50 55 AGG AAT GCT TTT TGG ACT ATC GTA CTG CTT TCT TCC TTC GCT AGC CAG 240 Arg Asn Ala Phe Trp Thr Ile Val Leu Leu Ser Ser Phe Ala Ser Gln 70 75 AGC ACC GCC GCC GTC ACG TAC GAC TAC ATT TTA GGC CGT CGC GCG CTC 288 Ser Thr Ala Ala Val Thr Tyr Asp Tyr Ile Leu Gly Arg Arg Ala Leu 90 GAC GCG CTA ACC ATA CCG GCG GTT GGC CCG TAT AAC AGA TAC CTC ACT 336 Asp Ala Leu Thr Ile Pro Ala Val Gly Pro Tyr Asn Arg Tyr Leu Thr 100 105 110 AGG GTA TCA AGA GGC TGC GAC GTT GTC GAG CTC AAC CCG ATT TCT AAC 384 Arg Val Ser Arg Gly Cys Asp Val Val Glu Leu Asn Pro Ile Ser Asn 115 120 125 GTG GAC GAC ATG ATA TCG GCG GCC AAA GAA AAA GAG AAG GGG GGC CCT Val
TTC
Asp Asp Met Ile Ser Ala Ala Lys Glu Lys Glu Lys Gly Gly Pro 130 135 140 GAG GCC TCC GTC GTC TGG TTC TAC GTG ATT AAG GGC GAC GAC GGC 432 480 *WO 96/40880 PCT/US96/11187 -113- Phe Glu Ala Ser Val Val Trp Phe Tyr Val Ile Lys Gly Asp Asp Gly 145 150 155 160 GAG GAC AAG TAC TGT CCA ATC TAT AGA AAA GAG TAC AGG GAA TGT GGC 528 Glu Asp Lys Tyr Cys Pro Ile Tyr Arg Lys Glu Tyr Arg Glu Cys Gly 165 170 175 GAC GTA CAA CTG CTA TCT GAA TGC GCC GTT CAA TCT GCA CAG ATG TGG 576 Asp Val Gln Leu Leu Ser Glu Cys Ala Val Gln Ser Ala Gln Met Trp 180 185 190 GCA GTG GAC TAT GTT CCT AGC ACC CTT GTA TCG CGA AAT GGC GCG GGA 624 Ala Val Asp Tyr Val Pro Ser Thr Leu Val Ser Arg Asn Gly Ala Gly 195 200 205 CTG ACT ATA TTC TCC CCC ACT GCT GCG CTC TCT GGC CAA TAC TTG CTG 672 Leu Thr Ile Phe Ser Pro Thr Ala Ala Leu Ser Gly Gln Tyr Leu Leu 210 215 220 ACC CTG AAA ATC GGG AGA TTT GCG CAA ACA GCT CTC GTA ACT CTA GAA 720 Thr Leu Lys Ile Gly Arg Phe Ala Gln Thr Ala Leu Val Thr Leu Glu 225 230 235 240 GTT AAC GAT CGC TGT TTA AAG ATC GGG TCG CAG CTT AAC TTT TTA CCG 768 Val Asn Asp Arg Cys Leu Lys Ile Gly Ser Gln Leu Asn Phe Leu Pro 245 250 255 TCG AAA TGC TGG ACA ACA GAA CAG TAT CAG ACT GGA TTT CAA GGC GAA 816 Ser Lys Cys Trp Thr Thr Glu Gln Tyr Gln Thr Gly Phe Gin Gly Glu 260 265 270 CAC CTT TAT CCG ATC GCA GAC ACC AAT ACA CGA CAC GCG GAC GAC GTA 864 His Leu Tyr Pro Ile Ala Asp Thr Asn Thr Arg His Ala Asp Asp Val 275 280 285 TAT CGG GGA TAC GAA GAT ATT CTG CAG CGC TGG AAT AAT TTG CTG AGG 912 Tyr Arg Gly Tyr Glu Asp Ile Leu Gln Arg Trp Asn Asn Leu Leu Arg 290 295 300 AAA AAG AAT CCT AGC GCG CCA GAC CCT CGT CCA GAT AGC GTC CCG CAA 960 Lys Lys Asn Pro Ser Ala Pro Asp Pro Arg Pro Asp Ser Val Pro Gin 305 310 315 320 GAA ATT CCC GCT GTA ACC AAG AAA GCG GAA GGG CGC ACC CCG GAC GCA 1008 Glu Ile Pro Ala Val Thr Lys Lys Ala Glu Gly Arg Thr Pro Asp Ala 325 330 335 GAA AGC AGC GAA AAG AAG GCC CCT CCA GAA GAC TCG GAG GAC GAC ATG 1056 Glu Ser Ser Glu Lys Lys Ala Pro Pro Glu Asp Ser Glu Asp Asp Met 340 345 350 CAG GCA GAG GCT TCT GGA GAA AAT CCT GCC GCC CTC CCC GAA GAC GAC 1104 Gin Ala Glu Ala Ser Gly Glu Asn Pro Ala Ala Leu Pro Glu Asp Asp 355 360 365 GAA GTC CCC GAG GAC ACC GAG CAC GAT GAT CCA AAC TCG GAT CCT GAC 1152 Glu Val Pro Glu Asp Thr Glu His Asp Asp Pro Asn Ser Asp Pro Asp 370 375 380 TAT TAC AAT GAC ATG CCC GCC GTG ATC CCG GTG GAG GAG ACT ACT AAA 1200 Tyr Tyr Asn Asp Met Pro Ala Val Ile Pro Val Glu Glu Thr Thr Lys 385 390 395 400 AGT TCT AAT GCC GTC TCC ATG CCC ATA TTC GCG GCG TTC GTA GCC TGC 1248 Ser Ser Asn Ala Val Ser Met Pro Ile Phe Ala Ala Phe Val Ala Cys 405 410 415 *WO 96/40880 PCT/US96/11187 -114- GCG GTC GCG CTC GTG GGG CTA CTG GTT TGG AGC ATC GTA AAA TGC GCG 1296 Ala Val Ala Leu Val Gly Leu Leu Val Trp Ser Ile Val Lys Cys Ala 420 425 430 CGT AGC TAA 1305 Arg Ser 435 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 434 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met His Arg Pro His Leu Arg Arg His Ser Arg Tyr Tyr Ala Lys Gly 1 5 10 Glu Val Leu Asn Lys His Met Asp Cys Gly Gly Lys Arg Cys Cys Ser 20 25 Gly Ala Ala Val Phe Thr Leu Phe Trp Thr Cys Val Arg Ile Met Arg 40 Glu His Ile Cys Phe Val Arg Asn Ala Met Asp Arg His Leu Phe Leu 55 Arg Asn Ala Phe Trp Thr Ile Val Leu Leu Ser Ser Phe Ala Ser Gin 70 75 Ser Thr Ala Ala Val Thr Tyr Asp Tyr Ile Leu Gly Arg Arg Ala Leu 90 Asp Ala Leu Thr Ile Pro Ala Val Gly Pro Tyr Asn Arg Tyr Leu Thr 100 105 110 Arg Val Ser Arg Gly Cys Asp Val Val Glu Leu Asn Pro Ile Ser Asn 115 120 125 Val Asp Asp Met Ile Ser Ala Ala Lys Glu Lys Glu Lys Gly Gly Pro 130 135 140 Phe Glu Ala Ser Val Val Trp Phe Tyr Val Ile Lys Gly Asp Asp Gly 145 150 155 160 Glu Asp Lys Tyr Cys Pro Ile Tyr Arg Lys Glu Tyr Arg Glu Cys Gly 165 170 175 Asp Val Gln Leu Leu Ser Glu Cys Ala Val Gln Ser Ala Gln Met Trp 180 185 190 Ala Val Asp Tyr Val Pro Ser Thr Leu Val Ser Arg Asn Gly Ala Gly 195 200 205 Leu Thr Ile Phe Ser Pro Thr Ala Ala Leu Ser Gly Gln Tyr Leu Leu 210 215 220 Thr Leu Lys Ile Gly Arg Phe Ala Gln Thr Ala Leu Val Thr Leu Glu 225 230 235 240 Val Asn Asp Arg Cys Leu Lys Ile Gly Ser Gln Leu Asn Phe Leu Pro 245 250 255 -WO 96/40880 *WO 9640880PCT/US961 1187 -115- Ser His Tyr Lys 305 Glu Giu Gin Giu Tyr 385 Ser Ala Arg Lys Leu Arg 290 Lys Ile Ser Ala Val 370 Tyr Ser Val Ser Trp 260 Pro Tyr Pro Ala Giu 340 Ala Giu Asp Ala Leu 420 Thr Ile Glu Ser Val 325 Lys Ser Asp Met Val 405 Val Thr Ala Asp Ala 310 Thr Lys Gly Thr Pro 390 Ser Gly Giu Asp Ile 295 Pro Lys Aia Giu Giu 375 Ala Met Leu Gin Thr 280 Leu Asp Lys Pro Asn 360 His Vai Pro Leu Tyr 265 Asn Gin Pro Ala Pro 345 Pro Asp Ile Ile Val 425 Gin Thr Gly Phe Gin Arg Trp Pro 315 Gly Asp Ala Pro Vai 395 Aia Ser Ala 285 Asn Ser Thr Giu Pro 365 Ser Giu Phe Val 270 Asp Leu Val Pro Asp 350 Giu Asp Thr Vai Lys 430 Giy Giu Asp Val Leu Arg Pro Gin 320 Asp Ala 335 Asp Met Asp Asp Pro Asp Thr Lys 400 Aia Cys 415 Cys Ala

Claims (2)

  1. 22-11-00:12:45 ;DAVIES COLLISON CAVE Pat.&Trad :61 7 336B 2262 16/ -116- The claims defining the invention are as follows:- 1. A recombinant fowlpox virus comprising a foreign DNA encoding an avian cytokine inserted into a fowlpox virus genome, wherein the foreign DNA is inserted within a region of the genome which corresponds to a 2.8 kb EcoRI fragment of the fowlpox virus genome and is capable of being expressed in a host cell into which the virus is introduced. 2. The recombinant fowlpox virus of claim 1, wherein the foreign DNA is inserted within a SnaBI site within the region of the genome which corresponds to the 2.8 kb EcoRI fragment. 3. A recombinant fowlpox virus comprising a foreign DNA encoding an avian cytokine inserted into a fowlpox virus genome, wherein the foreign DNA is inserted within a region of the :genome which corresponds to a 3.5 kb EcoRI fragment within a Sall C fragment and PstI F fragment of the fowlpox virus genome and is capable of being expressed in a host cell into which the virus is introduced. 4. The recombinant fowlpox virus of claim 3, wherein the foreign DNA is inserted within a Hpal site within the region of the genome which corresponds to the 3.5 kb EcoRI fragment- A recombinant fowlpox virus comprising a foreign DNA encoding an avian cytokine inserted into a fowlpox virus genome, wherein the foreign DNA is inserted within a region of the genome which corresponds to a 4.2 kb EcoRI fragment of the 22/11 '00 WED 13:49 [TX/RX NO 6922] 22-11-UO;12:45 ;DAVIES COLLISON CAVt Pat.&Irad ;51 ~Jbb ;Ze I 1 I 0 -117- fowlpox virus genome and is capable of being expressed in a host cell into which the virus is introduced. 6. The recombinant fowlpox virus of claim 5, wherein the foreign DNA is inserted within a Mlul site within the region of the genome which corresponds to the 4.2 kb EcoRI fragment. 7. The recombinant fowlpox virus of any one of claims 1 to 6, further comprising a second foreign DNA inserted within a non- essential region of the fowlpox virus genome which is capable of being expressed in a host cell into which the recombinant fowlpox virus has been introduced. 8. The recombinant fowlpox virus of claim 7, wherein the second foreign DNA encodes a polypeptide. 9. The recombinant fowlpox virus of claim 7, wherein the second foreign DNA encodes a detectable marker. 10. The recombinant fowlpox virus of claim 9, wherein the detectable marker is E. coli beta-galactosidase or E. coli beta-glucuronidase. 11. The recombinant fowlpox virus of claim 8, wherein the polypeptide is Newcastle Disease Virus hemagglutinin (NDV MN), or a Newcastle Disease Virus fusion (NDV F) protein. 12. The recombinant fowlpox virus of claim 8, wherein the A polypeptide is encoded by a gene contained in a virus selected 22/11 '00 WED 13:49 [TX/RX NO 6922] 22-11-00;12:45 ;DAVIES COLLISON CAVE Pat.&Trad ;61 3368 2262 18/ -118- from the group consisting of: human herpesvirus, herpes simplex virus-1, herpes simplex virus-2, human cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, human herpesvirus-6, human herpesvirus-7, human influenza, human immunodeficiency virus, rabies virus, measles virus, hepatitis f virus and hepatitis C virus. 13. The recombinant fowlpox virus of claim 8, wherein the polypeptide is Hepatitis B Virus core protein or Hepatitis B Virus surface protein. 14. The recombinant fowlpox virus of claim 8, wherein the polypeptide is Equine Influenza virus neuraminidase or Equine Influenza Virus hemagglutinin. 15. The recombinant fowlpox virus of claim 8, wherein the polypeptide is selected from the group consisting of: Equine Influenza Virus type A/Alaska 91 neuraminidase, Equine Influenza Virus type A/Kentucky 92 neuraminidase, Equine Influenza Virus type A/Prague 56 neuraminidase, Equine Influenza Virus type A/Miami 63 neuraminidase, Equine Influenza Virus type A/Kentucky 81 neuraminidase, Equine Herpesvirus type 1 glycoprotein B, and Equine Herpesvirus type 1 glycoprotein D. 16. The recombinant fowlpox virus of claim 8, wherein the polypeptide is selected from the group consisting of: Hog Cholera Virus glycoprotein El, Hog Cholera Virus glycoprotein E2, Swine Influenza Virus hemagglutinin, Swine Influenza Virus 22/11 '00 WED 13:49 [TX/RX NO 6922] ZZ-11-UU;12:4b ;L)AVIES COLLISON CAVL Pat. &Trad ;61 7 3368 2262 19/ -119- neuraminidase, Swine Influenza Virus matrix, Swine Influenza Virus nucleoprotein, Pseudorabies Virus glycoprotein B, Pseudorabies Virus glycoprotein C, Pseudorabies virus glycoprotein D, and PRRS Virus ORF7. 17. The recombinant fowlpox virus of claim 8, wherein the polypeptide is selected from the group consisting of: Infectious Bovine Rhinotracheitis Virus glycoprotein E, Bovine Respiratory Syncytial Virus attachment protein (BRSV G) Bovine Respiratory Syncytial Virus fusion protein (BRSV F), Bovine Respiratory Syncytial Virus nucleocapsid protein (BRSV Bovine Parainfluenza Virus type 3 fusion protein, Bovine parainfluenza Virus type 3 hemagglutinin Bovine Parainfluenza Virus type 3_neuraminidase. 18. The recombinant fowlpox virus of claim 8, wherein the polypeptide is Bovine viral Diarrhea Virus glycoprotein 48 or Bovine Viral Diarrhea Virus glycoprotein
  2. 53. 19. The recombinant fowlpox virus of claim 8, wherein the S: polypeptide is selected from the group consisting of: Feline Immunodeficiency Virus gag, Feline immunodeficiency Virus env, Infectious Laryngotracheitis Virus glycoprotein B, Infectious Laryngotracheitis Virus glycoprotein 1, Infectious Laryngotracheitis Virus glycoprotein D, Infectious Bovine Rhinotracheitis Virus glycoprotein G, Infectious Bovine Rhinotracheitis Virus glycoprotein E, Pseudorabies Virus glycoprotein 50, Pseudorabies Virus II glycoprotein B, Pseudorabies virus III glycoprotein C, Pseudorabies Virus 22/11 '00 WED 13:49 [TX/RX NO 6922] Z -11-UO;12:45 ;DAVIES COLLISON CAVE Pat.&Trad ;61 7 3368 2262 20/ -120- glycoprotein E, Pseudorabies Virus glycoprotein H, Marek's Disease virus glycoprotein A, Marek's Disease Virus glycoprotein B, Marek's Disease Virus glycoprotein D, Newcastle Disease Virus hemagglutinin, Newcastle Disease virus neuraminidase, Newcastle Disease Virus fusion, Infectious Bursal Disease Virus VP2, Infectious Bursal Disease Virus VP3, Infectious Bursal Disease Virus VP4, Infectious Bursal Disease Virus polyprotein, Infectious Bronchitis Virus spike, and Infectious Bronchitis Virus matrix. 20. The recombinant fowlpox virus of any one of claims 1 to 19, wherein the cytokine is chicken myelomonocytic growth factor (cMGF) or a chicken interferon (cIFN). 21. The recombinant fowlpox virus of any one of claims 1 to 19, wherein the cytokine is selected from the group consisting of interleukin-2, interleukin-6, interleukin-12, an interferon, a granulocyte-macrophage colony stimulating factor, and an interleukin receptor. 22. The recombinant fowlpox virus of any one of claims 1 to 21, wherein the first or second foreign DNA sequence is under the control of an endogenous upstream poxvirus promoter. 23. The recombinant fowlpox virus of any one of claims 1 to 21, wherein the first or second foreign DNA sequence is under the control of a heterologous upstream promoter. 24. The recombinant fowlpox virus of claim 23, wherein the 22/11 '00 WED 13:49 [TX/RX NO 6922] ;01 1 3300 440e af 4/ b -121- promoter is a synthetic pox viral promoter. The recombinant fowlpox virus of claim 23, wherein the promoter is selected from the group consieting of synthetic pox viral promoter, pox synthetic late promoter i. pox synthetic late promoter 2 early promoter 2, pox OIL promoter, pox 14L promoter, pox 13L promoter, pox 12L promoter, pox IL promoter, and pox ElOR promoter. B, 26. A homology vector for producing a recombinant fowlpox virus by inserting foreign DNA encoding an avian 0 cytokine into a fowlpox virus genome which comprises a double-stranded DNA molecule consisting essentially of foreign DNA encoding an avian cytokine inserted into double-stranded fowlpox virus DNA within a region corresponding to a 2.8 kb, 3.5 kb or 4.2 kb EcoRI fragment of the fowlpox virus viral genome. 27. The homology vector of claim 26 wherein the avian cytokine is chicken myelomonocytic growth factor (cMGF) or a chicken 08/12 '00 FRI 10:51 [TX/RX NO 71791 -122- interferon (cIFN). 28. A vaccine for immunizing an animal which comprises an effective immunizing amount of the recombinant fowlpox virus of any one of claims I to 25 and a suitable carrier. 29. A method of immunizing an animal which comprises administering to the animal the vaccine of claim 28. 30. A method of enhancing an avian immune response which comprises administering to an avian an effective immunizing dose of the recombinant fowlpox virus of any one of claims 1 to 25 and a suitable carrier. 31. A recombinant fowlpox virus comprising a foreign gene encoding chicken interferon and a gene encoding E. coli lacZ inserted at the unique SnaBI restriction endonuclease site in the 2.8 kB EcoRI fowlpox virus genome fragment, wherein the chicken interferon gene is under the control of a synthetic late/early promoter LP2EP2, and the E. coli lacZ gene is under the control of a synthetic late promoter LP1. 32. A recombinant fowlpox virus comprising a foreign gene encoding chicken myelomonocytic growth factor and a foreign gene encoding E. coli lacZ inserted at the unique SnaBI restriction endonuclease site in the 2.8 kB EcoRI fowlpox virus genomic fragment, wherein the gene encoding chicken myelomonocytic growth factor is under the control of a synthetic late/early promoter LP2EP2, and the gene encoding E. coli lacZ is under 22/11 '00 WED 13:49 [TX/RX NO 6922] I. I C H 40 0 1 i NU -123- the control of a synthetic late promoter LP1. 33. A recombinant fowlpox virus which comprises a foreign gene encoding chicken interferon and a foreign gene encoding E. coli lacZ both of which are inserted at the unique SnaBI restriction endonuclease site in the 2.8 kB EcoRI fowlpox virus genomic fragment, wherein the gene encoding chicken interferon is under the control of a synthetic late/early promoter LP2EP2, and the gene encoding E. coli lacZ is under the control of a synthetic late promoter LP1, and a foreign gene encoding Newcastle Disease virus P protein and a foreign gene encoding IIN protein inserted at the unique SnaBI restriction endonuclease site in the 2.8 kB EcoRI fowlpox virus genomic fragment wherein the gene encoding F protein and the gene encoding RN protein are each under the control of a synthetic early/late promoter EP1LP2. e a 34. A recombinant fowlpox virus according to any one of claims 1 to 25 or 31 to 33 or a homology vector according to any one of claims 26 or 27 or a vaccine according to claim 28 or a method according to any one of claims 29 or substantially as hereinbefore defined with reference to the Figures and/or Examples. 22/11 '00 WED 13:49 [TX/RX NO 6922]
AU64819/96A 1995-06-07 1996-06-04 Recombinant fowlpox viruses and uses thereof Ceased AU729518B2 (en)

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Publication number Priority date Publication date Assignee Title
WO1998044093A1 (en) * 1997-03-28 1998-10-08 Nippon Zeon Co., Ltd. Novel dna vector and vaccines containing novel recombinant dna vectors as the active ingredient
EP0974660A1 (en) * 1998-06-19 2000-01-26 Stichting Instituut voor Dierhouderij en Diergezondheid (ID-DLO) Newcastle disease virus infectious clones, vaccines and diagnostic assays
MXPA02011545A (en) * 2000-05-24 2003-06-06 Merial Sas Porcine reproductive and respiratory syndrome virus (prrsv) recombinant avipoxvirus vaccine.
WO2002102404A1 (en) * 2001-06-18 2002-12-27 Institut National De La Recherche Agronomique Uses of cytokines
CN1330376C (en) * 2001-09-30 2007-08-08 扬州大学 Recombinant Borrel virus combined vaccine
DE10221411B4 (en) * 2002-05-14 2004-07-08 GSF-Forschungszentrum für Umwelt und Gesundheit GmbH Recombinant fowlpox virus
GB2402391A (en) * 2003-06-04 2004-12-08 Oxxon Pharmaccines Ltd Fowlpox recombinant genome
EP1773387B1 (en) * 2004-06-25 2013-06-12 Merial Ltd. Avipox recombinants expressing foot and mouth disease virus genes
AP2013007166A0 (en) 2011-04-06 2013-10-31 Univ Paris Descartes Inst De Rech Pour Le Dev Ird Pharmaceutical compositions for preventing and/or treating an HIV disease in humans
JP2014236748A (en) * 2014-08-26 2014-12-18 一般財団法人阪大微生物病研究会 Recombinant polyvalent vaccine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU684070B2 (en) * 1993-01-21 1997-12-04 Virogenetics Corporation Recombinant virus immunotherapy

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5505941A (en) * 1981-12-24 1996-04-09 Health Research, Inc. Recombinant avipox virus and method to induce an immune response
DE3751664T2 (en) * 1986-08-01 1996-06-05 Commw Scient Ind Res Org RECOMBINANT VACCINE
JP2531729B2 (en) * 1987-03-27 1996-09-04 日本ゼオン株式会社 Recombinant avipox virus
US5286639A (en) * 1987-09-16 1994-02-15 Nippon Zeon Co., Ltd. Recombinant avipoxvirus
FR2632863B2 (en) * 1987-10-29 1990-08-31 Transgene Sa RECOMBINANT FOWLPOX VIRUS AND VACCINES DERIVED FROM SUCH VIRUSES
CA1341251C (en) * 1988-02-12 2001-06-12 David Bernard Boyle Pox virus vectors
GB8917059D0 (en) * 1989-07-26 1989-09-13 Nat Res Dev Avipox virus promoters
US5369025A (en) * 1991-06-28 1994-11-29 The United States Of America As Represented By The Secretary Of Agriculture Recombinant fowlpox vaccine for protection against Marek's disease
US5403582A (en) * 1993-01-21 1995-04-04 Nippon Zeon Co., Ltd. Vaccine comprising fowlpox virus recombinants expressing the envelope glycoprotein of an avian reticuloendotheliosis retrovirus
WO1994019014A1 (en) * 1993-02-26 1994-09-01 Syntro Corporation Recombinant fowlpox virus s-fpv-043 and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU684070B2 (en) * 1993-01-21 1997-12-04 Virogenetics Corporation Recombinant virus immunotherapy

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. OF VIROLOGY, 68(12), DEC 1994, K. LEONG ET AL. PP 8125-30 *
VIRUS RES., VOL 10, 1988, W.M. SCHRUTZLEIN ET AL. PP 65-76 *

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