AU727278B2 - Recombinant fowlpox viruses and uses thereof II - Google Patents

Description

J,

AUSTRALIA

PATENTS ACT 1990

ORIGINAL

COMPLETE SPECIFICATION 0* 0 go 0e 0 0 S Name of Applicant: Address of Applicant: Actual Inventor(s): Syntro Corporation 9669 Lackman Road, Lenexa, Kansas 66219, United States of America Mark D. COCHRAN Address for Service: DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

Complete Specification for the invention entitled: "Recombinant Fowlpox viruses and uses thereof II" The following statement is a full description of this invention, including the best method of performing it known to us: -1- RECOMBINANT FOWLPOX VIRUSES AND USES THEREOF II 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.

.*15 Background of the Invention

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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 25 cloned DNA sequences by insertions, deletions and single Sor 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 the 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 *0 (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 Se involved in RNA synthesis, and enzymes associated with '.15 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 (W) 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 W, 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 upon 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 S 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 S" 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 S" chickens and turkeys. Typically, the vaccine viruses are c*I" attenuated by serial passage in cell culture selecting 2p for strains that have altered growth and/or virulence Sproperties. 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 vaccines. 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 *0 foreign gene of interest (GOI), its suitability for genetic engineering, its probability of being licensed by regulatory agencies, etc. are all factors in the S* 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 0 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 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 0 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.

0'.5 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 o 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 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 0vectoring in the present invention is Infectious Laryngotracheitis virus (ILT). ILT is a member of the 20 herpesviridiae family, and this pathogen causes an acute S 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 the -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, 0 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.

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a E0t 15 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 types. Such polyvalent vaccines while generally ore effective in disease control, complicate the vaccine regime.

P:\OPER\MRO\2126880.SPE -3/12/98 -8- Summary of the Invention The present invention relates generally to recombinant fowlpox viruses comprising foreign DNA inserted into one or more regions of fowlpox virus genomic DNA which are nonessential, in particular within an approximately 2-8 kB EcoRI fragment and/or an approximately 3.5 kB EcoRI fragment and/or an approximately 4.2 kB EcoRI fragment, wherein the foreign DNA sequence is capable of being expressed in a host cell infected with a fowlpox virus. More particularly, the foreign DNA of the presently described recombinant fowlpox virus is inserted into the SnaBI restriction site of the 10 approximately 2.8 kB EcoRI fragment and/or into the Hpal restriction site of the approximately 3.5 kB EcoRI fragment and/or into the Stul restriction site of the approximately 4.2kB EcoRI fragment of fowlpox virus genomic DNA.

The foreign DNA is preferably expressed to produce a polypeptide, preferably an antigenic polypeptide in the case of vaccine applications, such as foreign DNA which encodes an antigenic polypeptide selected from infectious laryngotracheitis virus glycoprotein B; infectious laryngotracheitis virus gD; marek's disease virus glycoprotein A, marek's disease virus glycoprotein B; marek's disease virus glycoprotein D; l newcastle disease virus hemagglutinin or neuraminadase; newcastle disease virus fusion polypeptide; infectious bronchitis virus spike polypeptide; infectious bronchitis virus matrix polypeptide, and infectious bronchitis virus nucleocapsid. Alternatively or in addition, the foreign DNA may be expressed to produce a detectable marker, such as but not limited to P-galactosidase or 1-glucuronidase.

Those skilled in the art will be aware that expression of the foreign DNA may be achieved by placing the foreign DNA operably under the control of a suitable promoter derived from a fowlpox virus or another source. Particularly preferred promoters include endogenous poxvirus promoters and synthetic poxvirus promoters such as but not limited to the pox synthetic late promoters 1 and 2, pox synthetic early promoter and pox synthetic late promoter 2 early promoter 2, amongst others.

P:\OPER\MRO\2126880.SPE 3/12/98 -9- In a particularly preferred embodiment, this aspect of the invention is described with reference to the recombinant fowlpox viruses designated S-FPV-072 and S-FPV-079, which are described herein (Example 5) for the purposes of providing a non-limiting exemplification of the invention.

The present invention further provides vaccines comprising an effective immunizing amount of the subject recombinant fowlpox viruses in combination with a suitable carrier. The invention further provides a method of immunizing an animal against an animal pathogen which comprises administering to the animal an effective immunizing 10 dose of the vaccine of the present invention.

S o A further aspect of the present invention provides a recombinant fowlpox virus designated S-FPV-097 (ATCC Accession No. VR 2446).

The present invention also provides a vaccine which comprises an effective immunizing amount of the recombinant fowlpox virus designated S-FPV-097 and a suitable carrier. The present invention further provides a method of immunizing an animal against disease caused by fowlpox virus, Newcastle disease virus, and infectious laryngotracheitis virus which comprises administering to 0. an animal an effective immunizing dose of the vaccine of the present invention.

.0.015 The present invention further provides recombinant fowlpox viruses designated S-FPV-095, S-FPV-074, S-FPV- 081 and S-FPV-085. The present invention further provides a vaccine which comprises an effective immunizing amount of the recombinant fowlpox virus S-FPV- 095, S-FPV-074, S-FPV-081 or S-FPV-085 and a suitable carrier. The present invention further provides a method of immunizing an animal against disease caused by fowlpox virus, Newcastle disease virus, infectious laryngotracheitis virus or Marek's disease virus which comprises administering to an animal an effective immunizing dose of the vaccines of the present invention.

-11- Brief Description of the Figures Fig. 1A, Fig. 1B and Fig. 1C 00 0 0 15 0 a o 00 0 0 0 00 0 15 00 0 0 Detailed description of the SfiI 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.

0 -12- Detailed Descript-ion of the Invention The present invention also provides a recombinant fowlpox virus designated S-FPV-097. The S-FPV-097 was deposited on February, 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 vacc-ine 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 invention also provides a method of immunizing an animal, particularly poultry, against disease caused by fowlpox -13virus, 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.

*000 15 00 0 o 0 g '2 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 invention also provides a method of immunizing an animal, particularly poultry, against disease caused by fowlpox -14virus 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.

10 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 25 intramuscular, intraperitoneal, intravenous or intradermal injection. Alternatively, the vaccine may be administered intranasally, orally, or ocularly.

Suitable carriers for use with the recombinant fowlpox virus vaccines of the present invention are those well known 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 10-10 9 PFU/dose. Preferably, the effective immunizing amount is from about 1 0 1 0 5 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.

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00 0 0 -16- Materials 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 S 10 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 -700C, thawed and dispensed into 1.0 ml vials and refrozen at -70 0 C. Virus titers typically range between 10 8 and 10 7 PFU/ml.

PREPARATION OF FPV DNA. For fowlpox virus DNA isolation, S 25 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 0

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 0 C. FPV -17virions, present in the supernatant, were pelleted by centrifugation at 15,000 rpm for 20 minutes at 4 0 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 0 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 10 20% to 50% continuous sucrose gradient and centrifuged at ""16,000 rpm for 60 minutes in a SW41 rotor at 4 0 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 15 in a SW41 rotor at 4 0 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 0 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, ImM 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 -18- Sambrook et al (1989).

with minor variation.

Except as noted, these were used DNA SEQUENCING. Sequencing was performed using the BRL Sequenase Kit and 35 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 S 10 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 0 compared using Dnastar software. Manipulation and comparison of sequences obtained was performed with 15 Superclone and Supersee programs from Coral Software.

STRATEGY FOR THE CONSTRUCTION OF SYNTHETIC POX VIRAL S. PROMOTERS. For recombinant fowlpox vectors synthetic pox promoters offer several advantages including the ability 19 0 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:1l) 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 mthionines 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.

-19cDNA 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 S* 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 *.15 removed and the cells were lysed in 10 mis 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 mis of CsC1 solution (5.7 M CsC1, 25 mM sodium citrate pH 7.0) in a Beckman SW41 centrifuge tube. The samples were centrifuged for 18 hrs at 20°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 mis of guanidine solution (7.5 M guanidine-HC1, 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 40C at 10000 rpm in an SS34 rotor. The pellet was dissolved in 1.0 ml distilled water, recentrifuged at 13000 rpm, and the supernatant saved. 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 -20 0

C

for 18 hrs. The precipitated RNA was collected by centrifugation in the SS34 rotor at 4 0 C for 10 min at 10000 rpm. The pellet was dissolved in 1 ml distilled water and the concentration taken by adsorption at 10 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 Al distilled water.

Ten jg poly-A+ RNA was denatured in 20 mM methyl mercury hydroxide for 6 min at 22 0 C. f-mercaptoethanol was added to 75 mM and the sample was incubated for 5 min at 22 0

C.

The reaction mixture for first strand cDNA synthesis in 0.25 ml contained 1 pg oligo-dT primer (P-L Biochemicals) or 1 Mg 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 420C -21for 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 p1 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 NaC1). The leading edge of the eluted DNA **10 fractions were pooled, and DNA was concentrated by S lyophilization until the volume was about 100 Cl, then the DNA was precipitated with ammonium acetate plus i: ethanol as above.

0* S The entire first strand sample was used for second strand reaction which followed the Gubler and Hoffman (1983) method except that 50 pg/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 pg 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 p1 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 pmoles dCTP, and 25 units terminal -22deoxynucleotidyl transferase (Molecular Genetic Resources #S1001) in 50 pl. 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 pl of 0.01 M Tris pH 0.1 M NaC1, 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 Al 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.

*o 0 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-PoxT m 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 -23negative medium and incubated at 37 0 C in a humidified CO, incubator for five hours. The transfection procedure used is essentially that recommended for LipofectinTM Reagent (BRL). Briefly, for each 6 cm plate, micrograms of plasmid DNA were diluted up to 100 microliters with H,O. Separately, 50 micrograms of Lipofectin T M Reagent were diluted to 100 microliters with HO. The 100 microliters of diluted Lipofectin T M Reagent were added dropwise to the diluted plasmid DNA contained *0 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 S* washed once with CEF negative medium. Three mls of CEF :5 negative medium were added to the plasmid DNA/lipofectin mixture and the contents pipetted onto the cell S monolayer. Following overnight (about 16 hours) incubation at 37 0 C in a humidified 5% CO2 incubator, the medium was removed and replaced with 5 ml CEF complete o. 0 medium. The cells were incubated at 37'C in 5% CO, 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 -24dish 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 10 prehybridized with a solution containing 6X SSC, 3% skim milk, 0.5% SDS, salmon sperm DNA (50 Ag/ml) and incubated S 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- S* 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 70 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 wirh 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 horseradish 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 ig/ml 4-chloro-lnaphthol, 0.003% H202 in PBS). Plaques expressing the correct antigen stain black.

.0 06 SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. When the E. coli f-galactosidase (lacZ) or 0glucuronidase (uidA) marker gene was incorporated into a recombinant virus the plaques containing recombinants L5 were visualized by a simple assay. The enzymatic Ssubstrate was incorporated (300 Ag/ml) into the agarose overlay during the plaque assay. For the lacZ marker gene the substrates Bluogal T (halogenated indolyl-0-Dgalactosidase, Bethesda Research Labs) for blue plaques 20 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 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.

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 -26fragment 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 10 the NDV HN gene and was derived from the full length HN S cDNA clone. Note that the promoter and HN gene are fused so as to express a hybrid protein consisting of 4 amino

S

acids derived from the synthetic promoter followed by amino acids 2 to 577 of the HN gene. Both genes are in :'15 the opposite transcriptional orientation relative to the

S

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 o 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.

-27- 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 SfiI 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 SfiI is shown in figure 1.

The inserted SfiI fragment may be constructed utilizing S..l0 standard recombinant DNA techniques (Maniatis et al. and Sambrook et al., 1989), by joining restriction fragments from the following sources with the synthetic DNA Ssequences indicated in figure 1. Fragment 1 is e approximately 1811 base pair Avail to NaeI restriction -5 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 (B1 strain). Fragment 3 is an approximately 235 base pair PstI and Scal restriction fragment of the plasmid pBR322.

0S 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 -28fragment 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.

0 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 i-glucuronidase uidA marker gene preceeded by the ILT gB and gD genes was inserted as a cassette into the homology vector 451-08.22 at the unique *4 Sfil site. The cassette may be constructed utilizing go 0 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 -29the 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 S, 10 transcriptional orientation relative to ORF1 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 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), 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.

k* SHOMOLOGY VECTOR 538-51.27. The plasmid 538-51.27 was ,.15 constructed for the purpose of inserting the genes for Infectious Bronchitis virus (IBV) Massachusetts Spike protein (Mass Spike) and Massachusetts Matrix protein (Mass Matrix) into FPV. A lacZ marker gene and the genes for IBV Mass Spike and Mass Matrix were inserted as a '20 cassette into the homology vector 443-88.14 at the unique SfiI site. The inserted SfiI 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).

-31- 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 SfiI site. The inserted SfiI 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 5 fragment is the synthetic late promoter LP1 (SEQ ID NO: The fourth fragment contains the coding region of E.

coli uidA and is derived from plasmid pRAJ260 (Clonetech).

20 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 1B) at the unique Sfil site. The NDV HN and F 25 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 figure 1. 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 figure 1. Fragment 1 is an approximately 1811 base pair Avail to NaeI restriction fragment of the full length NDV HN cDNA clone .(Bl strain). Fragment 2 is an approximately 1812 base pair BamHI to PstI restriction fragment of the full length NDV F cDNA (B1 strain). Fragment 3 is an -32approximately 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).

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 :0 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 ;5 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 toQ 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.

-33- Examples 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 :15 synthetic DNA linkers (oligo 66.04; GGCGGCCGCGGCCCTCGAGGCCA-3' SEQ ID NO: 1 and oligo 66.05; TGGCCTCGAGGGCCGCGGCCGCC 3' SEQ ID NO: A 8galactosidase (lacZ) marker gene was inserted in each of the potential sites. A plasmid containing such a foreign 20 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.

34- Example 1A Homoloyv 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 Sail C and PstI F (Coupar et al., 1990). The NotI/Sfil 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

S*

.15 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.

20 This ORF shows no amino acid sequence homology to any known pox virus genes.

Example 1B .25 Homology 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 incomplete 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 1C Homolory 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 *5 of foreign DNA into FPV. The NotI/SfiI linker described above was inserted into a unique StuI site in this fragment. A unique MuI site is located approximately 500 base pairs away from the StuI insertion site. This site is designated the 540 insertion site.

f ftoftof ftOo t0 -36- 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 0-galactosidase (lacZ gene) and the gene for Newcastle Disease virus hemagglutinin-neuraminidase (HN) protein were inserted 20 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 *25 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 SPV. 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 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.

-37- 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-IG-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.

S" 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 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 5 RECOMBINANT SPV. The-transfection stock was screened by the PLAQUE HYBRIDIZATION PROCEDURE FOR PURIFYING RECOMBINANT FPV. The f inal result of plaque hybridization purification was the recombinant virus designated S-SPV-035.

S-FPV-035 was assayed f or 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 plaquesand not with S-FPV- 001 negative control plaques. All S-FPV-035 observed plaques reacted with the monoclonal antibody indicating -38that 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 B-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 Materials and Methods) and virus S-FPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING P* 02 RECOMBINANT SPV. 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 0-galactosidase expression, purity, 2'3 *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 -39that 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 0galactosidase (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.

15 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.

S-FPV-042 was derived from S-FPV-035. This was 0 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 SPV. The transfection stock was screened by the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC 0 25 MARKER GENES. The final result of red plaque purification was the recombinant virus designated S-FPV-042. 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-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 0@ 0 0 0.000 *0 0 0* 00 00 000 Ols 0000 0 0 0@ 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.

00 0 *0 0.09 0 a *0 a a Si 0I 00000g a 00 00 a 0 S-FPV-043 was derived from S-FPV-001. This was accomplished utilizing the homology vector 502-26.22 (see Materials and Methods) and virus S-FPV-001 in the :20 HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV. 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 25 designated S-SPV-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 -41

C.

Od*C

S

*4 a 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 15 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 with birds immunized with S-FPV-041. These results 0 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 125 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.

a.

C

0* *5~t

C

a.

'S

S

S~

B

S.

C

5 The duration of iununity 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 -42duration 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 4 egg production in laying hens.

a. .'15 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- FPV-043 and three weeks after vaccination, they were

*O.

challenged with either virulent NDV or virulent FPV.

Clinical responses were compared with non vaccinated 9* 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.

-43 Table 1. lImunity conferred by Fowipox recombinant vaccines vectoring different genes from Newcastle disease virus.

Chall~enge'

FPV

VIRUS~

FPV/h

FPV/

FPV/

RO Conti

MV-HN

DOSE b 8 x101 95 NT' IDV- F 2 X104 85 NT

TDV-HN+F

2 x10, 100 100 ols none 0 0 Percent protection following challenge 3 weeks postvac cinat ion PFU/O.l ml dose Not tested

NDV

e.g.

S.

0* C 25 .000 *0 a *e00@* .0 0 9* 9* 0 6

S

-44 Table 2. Ability of recombinant vaccine FPV/NDV-HN+F FPV-043) to vaccinate chicks with mraternal antibody.

Chall1engea History Flock vaccination Hen Ant ibody' NDV FPV NDV-HIc NDV ELISA FPV-AGPd Vacc. Con. Vacc. Con.

4'.

S

5h 54

C

C. p *4 *5 '4~t~

C

44* 6 .4.4

S

4* 5 25 C I ~C *4

C

*4

C.

.4 4.

C

0 4* 4 0 4 IlNDV +FPV 2 NDV FPV 3 NDV only 1:36 1:64 1:92 1: 1738 1: 2852 1:4324 Neg Neg Neg Neg 75 30 90 0 85 55 100 0 80 60 95 0 4 None Neg Neg 0 0 weeks post a Percent protection following challenge 3 vaccination.

b Every flock antibody.

C HI -Hemnagglutination Inhibition Assay d AGP -Agar Gel Precipitation Assay 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.

4; 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 15 RECOMBINANT FPV. The transfection stock was screened by the PLAQUE HYBRIDIZATION PROCEDURE FOR PURIFYING RECOMBINANT FPV. The final result of plaque S 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 1 25 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.

-46- 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, *0 and Newcastle disease virus.

o S-FPV-085 further expresses proteins from infectious ,e laryngotracheitis virus (ILTV), making them useful as 15 vaccines against ILTV.

Example 3A S-FPV-081 SS-FPV-081 is a recombinant fowlpox virus that expresses three foreign genes. The gene for E.coli 0-galactosidase (lacZ gene) and the genes for Marek's Disease virus (MDV) c. 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 -47virus 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-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 9 with the chicken antiserum indicating that the virus was *15 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 :'20 S-FPV-081 expresses foreign antigens from MDV. This virus is useful as a multi-valent vaccine against Marek's Disease and Fowlpox.

*SgS 00998: 0 :%,eeExample 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. coil 0-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 -48synthetic 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 SPV. 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 a-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.

SS-FPV-082 was assayed for expression of MDV specific 20 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 25 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 -49virus 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 &-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 0-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 *"15 synthetic early/late promoter EP1LP2.

0 a 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 :20 RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV.

SThe 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- 25 085. This virus is 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 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 Disease, Marek's Disease, Infectious Laryngotracheitis and Fowipox.

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-51- 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.

C

CC C

.S

S

Example 4A S-FPV-095 S-FPV-095 is a recombinant fowlpox virus that expresses three foreign genes. The gene for E.coli #-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.

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o* 25 eg 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 0glucuronidase expression, purity, and insert stability by multiple passages monitored by the blue plaque assay as described in the materials and methods. After the -52initial 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 S *e

S

S

S S

S.

15

S..

a a :20 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

o• 0e* 0

S

oooo 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 25 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-glucuronidase (uidA gene) and the genes for Infectious Laryngotracheitis virus (ILT) gD and gB were inserted into the 54a 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 accomplished utilizing the homology vector 586-36.6 (see Materials and Methods) and virus S-FPV-043 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING -53- RECOMBINANT SPV. 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 3-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.

9 *O 0* 3 33 3*5 .L 0Se 3 :20 3. 3 3 25 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 control of a synthetic early/late promoter EP1LP2. The gene for E.coli 0-glucuronidase (uidA gene) and the genes for Infectious Laryngotracheitis virus (ILTV) -54- 0 0e

O

0

U

0 9 0* 0 9e 0 0* 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- 15 097. This virus was 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 were blue 20 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 S' 25 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 ws- 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 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 55 stably expressing the NDV foreign genes.

S-FPV-097 expresses foreign antigens from NDV and ILTV.

This virus is useful as a muJ.ti-valent vaccine against Newcastle Disease, Infectious Laryngotracheitis and Fowipox.

9.

3 3 3 33 S 3S

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33 a a 3 0 9 9. a a* a S @3 3S@~ 3 3 a. 3. 3 03

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3 3 3. 9* .3 0 3 -56- 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 .'15 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 20 gene for E.coli 0-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 trom 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 -57passages 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 S* 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 20 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 S-galactosidase (lacZ gene) and the genes for Infectious Bronchitis virus (IBV) Massachusetts Spike -58protein (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 3glucuronidase (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 15 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 20 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 25 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 -59- 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.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer S. or step or group of integers or steps.

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. Mci. Biol. 210, 749- 769.

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. 3. Esposito, et al., Virology 165, 313.

7. F.A. Ferrari, et al., Journal of Bacteriology 161, 556-562, 1985.

0* U. Gubler and B.J. Hoffman, Gene 25, 263-269.

9. D. Hanahan, Molecular Biology 166, 557-580, 1983.

M.A. Innis, et 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).

-61 14. J. Saznbrook, et al., Molecular Cloning A Laboratory Manual Second Edition, Cold Spring Harbor Press, 1989.

15. J. Taylor, et al., Vaccine 9, 190-193, 1991.

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0e S 0 00 0 0*0.0~ .0 0 ~0 ~O 0 0- -62- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Syntro Corporation, et al.

(ii) TITLE OF INVENTION: Recombinant Fowlpox Viruses and Uses Thereof (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: John P. White STREET: 30 Rockefeller Plaza CITY: New York ge STATE: New York COUNTRY: USA 20 ZIP: 10112 Co*ege

C

COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk S. COMPUTER: IBM PC compatible 25 OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 0ooo 0 0 C*(vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: Not Yet Known FILING DATE: 28-FEB-1994

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: White. Esq, John P 35 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (212)977-9550 oo TELEFAX: (212)664-0525 TELEX: 422523 .40 oo INFORMATION FOR SEQ ID NO:l:

C

S(i) 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: 63 SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRAINDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (geriomi c) (iii) HYPOTHETICAL: NO (iv) AN'TI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: C.ATAATGGCC TCGAGGGCCG CGGCCGCC 28 INFORM4ATION FOR SEQ ID NO:3: Wi SEQUENCE CHARACTERISTICS: CA) LENGTH: 1507 base pairs TYPE: nucleic acid STRAZNDEDNESS: double :25 TOPOLOGY: linear see 0 :(ii)MOLECULE TYPE: DNA (genomic) ii)HYPOTHETICAL: No ATI- SENSE: NO (ix) FEATURE: NAM/KEY: CDS LOCATION: 260. .1411 Cxi) SEQUENCE DESCRI PTION: SEQ ID NO:3: 0:140 CTACTTCATA AAAAGTIrTAA ACCTICCGAA AGA7T~TTGG ATAAAAGTAG AGAACTCGCA TTGCGATrAT GCTCTAGGAC AATCCTGTAA AGTGTCTCGA TCTrAGCATA TAGATAAATG 120 *7TGAACTAA TAT CCTAAAQ CCTGTATGTA ACAGTTGGTG CCTATrGAAA GATACTGATr 180 ATCAAGGAGA AGAATAATAT AAALTCGTAAA AATAATACTr ATTATATAAT ATAATGTATA 240 ATAATATACA AAAACAGCC ATO ATA CGT ATT ATA ATA TTA TCG TTA TTA Trr 292 Met Ile Arg le Ile Ile Leu Ser Leu Leu Phe 1 5.10 ATT AAC GTA ACA ACA GAT ADT CAA GAA TCT TCA AAA AAT ATA CAA AAT 340 Ile Asn Val Thr Tkxr Asp Ser Gin Glu Ser Ser LYS An le Gin Asn i5 20 GTA TTG CAC OTT ACA GAA TAT ACT AGA ACT GGT GTA ACA GCT TOC 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 GOT TTA GAT CAA CCA AAA ACA Trr 436 Leu His Cys Phe Asp*Arg Ser Lys Gly Leu Asp Gin Pro Lys Thr Phe -64-

ATC

Ile 60 CTG CCT GOT AAA Leu Pro Gly Lys TAT AGC Tyr Ser 65 AAT AAC AGT Asn Asn Ser

ATA

Ile 70 AAA CTA GAA GTA Lys Lou Giu Vai

GCT

Ala ATT GAT ACA TAT Ile Asp Thr Tyr

AAA

Lys AAA GAT AGC GAC Lys Asp Ser Asp

TTC

Phe 85 AGT TAT TCT CAC Ser Tyr Ser His CCA TGT Pro Cys CAA ATA TTC Gin Ile Phe

CAG

Gin TTC TGT GTG TCT Phe Cys Vai Ser

GGT

Gly 100 AAT TTr AGT GGT Asn Phe Ser Gly AAA CGG TTC Lye Arg Phe 105 GAT ATT GCT Asp Ile Aia GAT CAT TAT Asp His Tyr 110 CTA TAT GGG TAT Leu Tyr Gly Tyr

ACA

Thr 115 ATT TCC GGA TTr Ile Ser Gly Phe

ATA

Ile 120 0 OS S

S

@0

S.

0 0 000 0 0@ 0 00 0 0 0000 0 .0 00 5 0 00@ CCA AAA Pro Lys 125 TAT TAT AGC GGT Tyr Tyr Ser Gly

ATG

Met 130 TCT ATA AGT ACT Ser Ile Ser Thr

ATT

Ile 135 ACT GT ATO CCA Thr Val Met Pro

TTA

Leu 140 CAA GAA OGA TCA Gin Giu Gly Ser

TTA

Leu 145 AAG CAT GAT GAT Lye His Asp Asp

GCC

Ala 150 GAT GAC TAT GAC Asp Asp Tyr Asp

TAC

Tyr 155 GAT GAT GAT TGT Asp Asp Asp Cys

GTT

Val 160 CCT TAT AAA GAA Pro Tyr Lys Glu

ACC

Thr i65 CAG CCT CGA CAT Gin Pro Arg His ATG CCA Met Pro 170 GAA TCG GTA Giu Ser Val

ATA

Ile 175 AAA GAA GGA TOT Lye Giu Gly ys

AAA

Lye 180 CCC ATT CCA Pro Ile Pro

CTA

Leu GAT OAA AAT GAC GAT CCT ACT TOT ATT ATO TAT TOG OAT Asp Giu Asn Asp Asp Pro Thr Cys Ile Met Tyr Trp Asp 190 195 200 CCA AGO TAT Pro Arg Tyr 185 CAC TCG TGG His Ser Trp AOT OAT CAC Ser Asp His 532 580 628 676 724 772 820 868 916 964 1012 1060 1108 1156 GAT AAT TAC TOT AAT OTT Asp Asn Tyr Cys Asn Vai 205

GGA

Oly 210 TTT TT AAT TCT Phe Phe Asn Ser

CTA

Leu 215

CAG

Gin

AAT

Asn 220 CCT CTG OTT. Trr Pro Leu Val Phe

CCG

Pro 225 TTA ACK AGT TAT Leu Thr Ser Tyr

TCT

Ser 230 OAT ATA AAC AAT Asp Ile Asn Asn

GCA

Ala 235 TT CAT OCT T=r CAA Phe His Ala Phe Gin 240 TCA TCT TAT TOT Ser Ser Tyr.Cys

AGA

Arg 245 TCA CTA GOC TT Ser Leu Oly Phe AAC CAA Asn Gln 250 TCA TAC AGT Ser Tyr Ser

OTA

Val 255 TGC OTA TCT ATA Cys Val Ser Ile

GGT

Oly 260 OAT ACA CCA TTr Asp Thr Pro Phe GAG OTT ACG Glu Val Thr 265 CAA GAA ATT Gin Giu Ile TAT CAT AOT Tyr His Ser 270 AAA ACA CTA Lye Thr Leu 285 TAT GAA AOT OTT Tyr Glu Ser Val

ACT

Thr 275 OTT GAT CAG TTA Val Asp Gin Leu

TTA

Leu 280 TAT GGA GAA Tyr Oly Glu

GAT

Asp 290 OCT GTA TAT GGA Ala Val Tyr Gly

TTA

Leu 295 CCG T7r AGA AAT Pro Ph. Arg Asn ATA ACT ATA AGG GCG CGT ACA CGG ATT CAA AGT TTA CCT CTT ACT AAC lie Thr Ile Arg Ala Arg Thr Arg Ile Gin Ser Leu Pro Leu Thr Asn 300 305 310 315 AAT ACC TGT ATC CCT AAA CAA GAC GAT GCT GAT GAT GTT GAC GAT GCT Asn Thr Cys Ile Pro Lys Gin 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 Leu Tyr Val Glu Thr Thr Pro Arg Val Pro Thr Ala Arg Lys Lye Pro 350 355 360 GTr ACA GAA GAA TAT AAT GAT ATA TIT AGT AGT TTT GAT AAT TIT GAC Val Thr Glu Glu Tyr Asn Asp Ile Phe Ser Ser Phe Asp Asn Phe Asp 365 370 375 ATG AAA AAG AAA TAAGACATAT TTATTAAAT CAAAAAGTCT GTCGAACTT Met Lys Lys Lys 380 25 TAGTGTTAA CCTATATCGA TTATGAT7'TTCCATGATG ATCCAGGCTA TGACTGACT 0 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 383 amino acids TYPE: amino acid TOPOLOGY: linear *e 35 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Ile Arg Ile e I Ile Leu Ser Leu Leu Phe Ile Asn Val Thr Thr 40 1 5 10 Asp Ser Gin Glu Ser Ser Lye Asn Ile Gin 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 Lye Gly Leu Asp Gin Pro Lye Thr Phe Ile Leu Pro Gly Lys 55 Tyr Ser.Asn Asn Ser Ile Lye Leu Glu Val Ala Ile Asp Thr Tyr Lys 70 75 Lye Asp Ser Asp Phe' Ser Tyr Ser His Pro Cys Gin Ile Phe Gin Phe as 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 lie Asp Ile Ala Pro Lye Tyr Tyr Ser 115 120 125 1204 1252 1300 1348 1396 1448 1507 -66- 0 @0 0 0 So

S

0 550 0

SOS.

0 0 S. 0 00 0-.

0 00.* 00 OS

S

000 1 1 2 2 3 34 4C 4 Gly Met 130 Leu Lys 145 Pro Tyr 0 Glu Gly Pro Thr Val Gly 210 Pro Leu 0 225 Ser Ser 5 Val Ser Ser Val 0 Glu Asp 290 Arg Thr 5 305 Lys Gin 0 Ala Asp Thr Pro Asn Asp 370 His Asp Lys Glu Cys Lys 180 Cys lie 195 Phe Phe Thr Ser Tyr Cys Ile Gly 260 Thr Val 275 Ala Val Arg Ile Asp Asp Asp Ala 340 Arg Val 355 Ile Phe Asp Thr 165 Pro Met Asn Tyr Arg 245 Asp Asp Tyr Gin Ala 325 Asp Pro Ser Ala 150 Gin lie Tyr Ser Ser 230 Ser Thr Gin Gly Ser 310 Asp Asp Thr Ser Ser Ile Ser Thr Ile Thr Val Met Pro Leu Gin Glu Gly Ser 135 Asp Pro Pro Trp Leu 215 Asp Leu Pro Leu Leu 295 Leu Asp Asp Ala Phe 375 v Asp Arg Leu Asp 200 Gin lie Gly Phe Leu 280 Pro Pro Val Asp Arg 360 Asp Tyr Asp His Met 170 Pro Arg 185 His Ser Ser Asp Asn Asn Phe Asn 250 Glu Val 265 Gin Glu Phe Arg Leu Thr Asp Asp 330 Asp Tyr 345 Lys Lys Asn Phe 140 Tyr Asp 155 Pro Glu Tyr Asp Trp Asp His Asn 220 Ala Phe 235 Gin Ser Thr Tyr Ile Lys Asn Ile 300 Asn Asn 315 Ala Asp Glu Leu Pro Val Asp Met 380 Asp Asp Cys Ser Val Ile 175 Glu Asn Asp 190 Asn Tyr Cys 205 Pro Leu Val His Ala Phe Tyr Ser Val 255 His Ser Tyr 270 Thr Leu Tyr 285 Thr lie Arg Thr Cys Ile Asp Val Asp 335 Tyr Val Glu .350 Thr Glu Glu 365 Lys Lys Lys Val 160 Lys Asp Asn Phe Gin 240 Cys Glu Gly Ala Pro 320 Asp Thr Tyr 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 67 (ix) FEATURE: NAME/KEY: COS LOCATION: 300. .1568 (ix) FEATURE:

NAME/KEY:

LOCATION:

CDS

complement (1685. .2848) (xi) SEQUENCE DESCRIPTION: SEQ ID AAGCCAGTI GAATrCAATA TTCATCGCCG ATAG7TGGTA GAAATACTAT TCATGAAATr TACCTTCTTC CGTGGCTTAA AAAC~rATrG TATGTACCAT TCATTATAAG ATCTGATACT ATCGGCATCT TCTAT~rrCC G.AGTTrrA CATCTrGTA CTAGTATCCA TGTrCGTCTA ATAAGAGGGA AGGAATATAT CTATCTACAT AAACATCATA AGGTTCTTTG ATAGA'T1AT ATCGCTAATA AAATATAAAT AATAATrAAA GATT~rATGA TATATCGAOC =TGCAAAA 0 00..

00000 0 S.

S.

S.

S.

0 0 005 S 005 S 0

S

0*

S

0 00 S .5 0 0 S *5 005...

S

OS SO 5 5

S

ATG TCT GTT OAT Met Ser Val Asp 25 1

TGG

Trp 5 CGT ACA GAA ATC Arg Thr Glu Ile TAT TCG Tyr Ser 10 GOT OAT ATA Gly Asp Ile TCC CTA Ser Leu GTA GAA AAA Val Glu Lys GAG GAA ACA Glu Glu Thr 35 ATA AAG AAT AAA Ile Lys Asn Lys

GOT

Gly AAT TGC ATC Asn Cys Ile AAT. ATA TCT GTA Ann Ile Ser Val ACA ACT CCG TTA Thr-Thr Pro Lau

ATA

Ile GAC OCT ATA AGA Asp Ala Ile Arg GGA AAT GCC Gly Ann Ala AAA ATA Lys Ile 50 AAT ACT 40 Ann Thr OTA GAA CTA Tr Val Giu Leu Phe AAG CAC GGA Lys His Gly GCG CAA Ala. Gin GTI' AAT CAT GTA Val Ann His Val AAA ATT CCT Lys Ile Pro

ANT

An 70 CCC TTG TTA ACA, Pro Leu Leu Thr ATC AAA ATA GGA le Lys Ile Oly

TCA

Ser so CAC GAT ATA GTA His Asp Ile Vai

AAA

Lys CTG CTG TTG ATT Leu Leu Leu Ile

AAC

Asn 90 GGA OTT GAT ACT Oly Val Asp Thr TCT A7T Ser Ile TrG CCA GTC Leu Pro Val AZT GOT GTG Ser Oly Val 115

CCC

Pro 100 TGC ATA AAT AAA Cys Ile Ann Lys

GAA

Glu 105 ATG ATA AAA ACT Met Ile Lys Thr ATA TrA OAT Ile Leu Asp 110 ACT TTC TTG Thr Phe Leu AAA GTA AAC ACA Lys Val Ann Thr

AAA

Lys 120 AAT GCT &AA TCT Ann Ala Lys 5cr

AAA

Lys 125 635 683 731, 779 CAT TAC His Tyr .130 GAG TAT Glu Tyr 145 GCG A7T AAG AAT Ala le Lys An

AAT

An 135 OAC TTA GAG OTr Asp Leu Glu Val AAA ATG CIT TIT Lye Met Leu Phe GGA OCT OAT Gly Ala Asp AAT ATA AAA OAT Ann Ile Lye Asp OAT AAC ATA TOT TAT TCT Asp Ann Ile Cys Tyr Ser 155 160 -68- ATA CAC ATA GCT ACT AGG AGT Ile His Ile Ala Thr Arg Ser 165 AAT TCA TAT Asn Ser Tyr 170 GAA ATC ATA AAA Glu Ile Ile Lys TTA CTA Leu Leu 175 TT'A GAA AAA Leu Glu Lys

GGT

Gly 180 GCT TAT GCA AAC Ala Tyr Ala Asn

GTA

Val 185 AAA GAC AAT TAT Lys Asp Asn Tyr GGT AAT TCT Gly Asn Ser 190 ATT AAA TTA Ile Lys Leu 827 875 923 CCG TIA CAT Pro Leu His 195 AAC GCG GCT AAA Asn Ala Ala Lys

TAT

Tyr 200 GGC CAT TAT GCT Gly Asp Tyr Ala

TGT

Cys 205 GTT TTA Val Leu 210 GAC CAT ACT AAT Asp His Thr As

AAC

Asn 215 ATA AGC AAT AAG Ile Ser Asn Lys

TGC

Cys 220 AAC AAC GGT TT Asn Asn Gly Val

ACA

Thr 225 CCG TTA CAT AAC Pro Leu His Asn

GCT

Ala 230 ATA CTA TAT AAT Ile Leu Tyr Asn 0 4 40

S.

4 4 4 .4@ 44 4 4. 4

AGA

Arg 235

GAT

Asp TCT GCC GTA GAA Ser Ala Val Glu

TTA

Leu 240 CTG ATT AAC AAT Leu Ile Asn As

CGA

Arg 245 TCT ATT AAT CAT Ser Ile Asn Asp

ACG

Thr 250 GTA CAC GGA Val Asp Gly TAT ACT Tyr Thr 255 25 CCA CTA CAT Pro Leu His

TAT

Tyr 260 GCT TTC CAA CCT Ala Leu Gin Pro

CCG

Pro 265 TGT ACT ATA CAT Cys Ser Ile Asp ATT ATA GAT Ile Ile Asp 270 AAT AAC GGA As Asn Gly ATA CTA CTA TAT Ile Leu Leu Tyr 275 AAC.AAC GCC Asn Asn Ala

GAT

Asp 280 ATA TCT ATA AAA Ile Ser Ile Lye

GAT

Asp 285 CGC AAT Arg Asn 290 CCT ATC CAT ACG Pro Ile Asp Thr

GCG

Ala 295 TTT AAG TAT Phe Lys Tyr ATT AAC Ile Asn 300 AGA CAT AGC CTT Arg Asp Ser Val

ATA

Ile 305 AAA GAA CTr CC Lys Glu Leu Leu

CGA

Arg 310 AAC CCC CTC Asn Ala Val TTA ATT Leu Ile 315 AAC GAG CTC CGT Asn Glu Val Cly

AAA

Lys 320 971 1019 1067 1115 1163 1211 1259 1307 1355 1403 1451 1499 1547 TTA AAA GAT-ACT Leu .e-s Asp Thr

ACT

Thr 325 ATC TTA GAA CAC Ile Leu Giu His

AAA

Lye 330 GAA ATA AAA CAC Glu Ile Lys Asp AAT ACC Asn Thr 335 CTG Trr TCA Val Phe Ser AAG AAA ACT Lys Lys Thr 355 ATA ACG TAT Ile.Arg Tyr 370

AAC

Asn 340 TT TC TAC CAA Phe Val Tyr Glu

TGT

Cys 345 AAT CAA CAA ATT Asn Glu Clu Ile AAA AAA ATC Lye Lye Met 350 GTA TAC ATG Vai Tyr Met AAA TCT CTC CGT Lys Cys Val Gly

CAC

Asp 360 TAT AGT ATC TTT Tyr Ser Met Phe

CAC

Asp 365 AAA CAC AAA Lye His Lys

TAT

Tyr 375 GAC GGT AAT AAG Asp Giy Asr Lye

CAT

Asp 380 AGT ATT AAA CAC Ser Ile Lye Asp

TAT

Tyr 385 TTG CGT TOT rr Leu Arg Cys Leu

GAT

Asp 390 CAT AAT.ACT ACT Asp Asn Ser Thr

CCT

Arg 395 ATG TTA AAA ACT Met Leu Lye Thr

ATA

Ile 400 CAT ATT AAT GAA TTT.CCT ATA TAT TCT ATC TAT CTC CTA ACA TCC CTA Asp Ile Asn Glu Phe Pro Ile Tyr Ser Met Tyr Leu Val Arg Cye Leu 69 405 410 415 TAT GAT ATG GTA ATA TAT TAAAAGAAAT GGGCTCTTGC ATACATAATC Tyr Asp Met Val Ile Tyr 420 GGTATAAAAA ATAACGAAAT TATTAGCGGT TACATATCTT ACGGCGGCCG CGGCCCTCGA 0* 0 0

S

V. S S 0*

S.

~8 000 0

CS..

0 0 V. *5 C S 0* CV eq C S 8* *0 d

C

0@ 0 0 OS So 0 0

C

GGCCAGTAGC

TTAGACCAAC

TI'ATGTCTAT

TAGTAAGTAT

I I 171ACTAA

TCTTAGTT

20

ATTTCTCTCA

ATATGTGCTA

25 CTACTATCTC AATGCTATrA

TCTAATAACA

GTAGATAAGT

CTCATATACG

35 AACGCTTCTA

ACAGCGGCAT

AATTCTAACA

40

GGAGTGATAT'

ATGTTTTCTG

TTTTAGCGT

GGTGCTTr.TA

TCAGTATTTC

CGCTAGAATC

TACTGGCTAA

TAATrCCTr

TAACGAATAT

CTTCCTTACA

AGGGGTTTAC

TGATATATCT

TAAGT1'CAGC

ATAAAGGATA

717-1rATAAC ATTTGTI-I-x-

GATTTGTTCC

TATCGGCCCC

AATGAATAGG

ACGTTATAAC

ACAGACTATC

TATCGTATCC

TAACATCTAT

CCGTGTAATG

CTATAAACTC

TAATATTTCA

GGATATGGAA

TATTATAGGA

ATCTAAAGAG

ACTCAACCAA

TITCACTTCTG.

AAAAGAAAAC

ACCATAATGT

TTGATrCCTT 7TCTGGTI'rA AGAA7TAACA

ATATAAGGCA

GTAATCTAAA

CGTCTTGTCA

TGTATCTTTA

AGCGGCGTrA

ATTCTTAGCC

TCCTCTI'ECC

CAAAGGAGTA

TAATATTGAG

TCTAA7=rG

GTTTAAGAC

TTATTTACTA

TAAAACATAT

ATACTTrAA

ATATCGTGAC

ATATrACTGT

AATAATATAT

ATGTTAATAG

CAATCTT

TTAGCTCCTC

AAATGTAAAA

AGA.GTGTrrA

CAATAATCTC

TATCTATCTA

ACATCTGCXAC

ATGAGATACA

AATAACTTGG

'1-1 rATAAA

AGITTGATAT

ATCTACTrI-r

GATCTCCGTA

AGTGATGTAA

TAATACGAAT

ACGTATCATC

GTATAAAATC

TAAGGCTCTT

TTACTATACC

CATTTACATC

TACACGCATA

TATCTATGAG

CCGTTCCTAT

TGATAACTAC

TAGCATrAC

GAGTAGAGGC

CCCGCA7TAT

GAGGAGTTTC

GTACTAGTCT

CATCTATAGA

CCGGAGAAGT

TI'CTAATATT

AT'rATAGAAA

CAGGTTCATG

ITTAGATATA

GCTTTGAATA

TTGTATACAT

ATCGATGACC

ATGATATTCT

CGCTCCGTrA

ATGCAACGGA

CG'rrrrTACA

CTTCTGCGAT

ATrGTTTCI-r

GTTCGCTCCC

TTGATGTA AT

TAAAGTTCTA

TCC'TrTAATG ACTrAACGAA A7TC 1595 1655 1715 1775 1835 1895 1955 2015 2075 2135 2195 2255 2315 2375 2435 2495 2555 2615 2675 2735 2795 2849 INFORMATION FOR SEQ ID NO:6: 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 Trp Arg Thr Glu Ile Tyr Ser Gly Asp Ile Ser Leu 1 5 10 Val Glu Lys Leu Ile Lys Asn Lys Gly Asn Cys Ile Asn Ile Ser Val 20 25 Glu Glu Thr Thr Thr Pro Leu Ile Asp Ala Ile Arg Thr Gly Asn Ala 40 Lys Ile Val Glu Leu Phe Ile Lys His Gly Ala Gin Val Asn His Val 55 Asn Thr Lys Ile Pro Asn Pro Leu Leu Thr Ala Ile Lys Ile Gly Ser 70 75 His Asp Ile Val Lys Leu Leu Leu Ile Asn Gly Val Asp Thr Ser Ile 90 Leu Pro Val Pro Cys Ile Asn Lys Glu Met Ile Lys Thr Ile Leu Asp 20 100 105 110 Ser Gly Val Lys Val Asn Thr Lys Asn Ala Lys Ser Lys Thr Phe Leu 115 120 125 25 His Tyr Ala Ile Lys Asn Asn Asp Leu Glu Val Ile Lys Met Leu Phe 130 135 140 C Glu Tyr Gly Ala Asp Val Asn Ile Lys Asp Asp Asn Ile Cys Tyr Ser 145 150 155 160 Ile His Ile Ala Thr Arg Ser Asn Ser Tyr Glu Ile Ile Lys Leu Leu 165 170 175 Leu Glu Lys Gly Ala Tyr Ala Asn Val Lys Asp Asn Tyr Gly Asn Ser S 35 180 185 190 Pro Leu His Asn Ala Ala Lys Tyr Gly Asp Tyr Ala Cys Ile Lys Leu 195 200 205 40 Val Leu Asp His Thr Asn Asn Ile Ser Asn Lys Cys Asn Asn Gly Val 210 215 220 66 Thr Pro Leu His Asn Ala Ile Leu Tyr Asn Arg Ser Ala Val Glu Leu 225 230 235 240 Leu Ile Asn Asn Arg Ser Ile Asn Asp Thr Asp Val Asp Gly Tyr Thr 245 250 255 Pro Leu His Tyr Ala Leu Gin Pro Pro Cys Ser Ile Asp Ile Ile Asp 260 265 270 Ile Leu Leu Tyr Asn Asn Ala Asp Ile Sec Ile Lys Asp Asn Asn Gly 275 280 285 Arg Asn Pro Ile Asp Thr Ala Phe Lys Tyr Ile Asn Arg Asp Ser Val 290 295 300 Ile Lys Glu Leu Leu Arg Asn Ala Val Leu Ile Asn Glu Val Gly Lys 305 310 315 320 Leu Lys Asp Thr Thr Ile Leu Glu His Lys Glu ile Lys Asp Asn Thr 325 330 335 Val Lys Ile Tyr 385 Asp Tyr Phe Lys Arg 370 Leu Ile Asp Ser Thr 355 Tyr Arg Asn Met Asn 340 Lys Lys Cys Glu Val 420 Phe Cys His Leu Phe 405 Ile Val Val Lys Asp 390 Pro Tyr Tyr Glu Gly Asp 360 Tyr Asp 375 .Asp Asn Ile Tyr -71- Cys Asn Glu Glu Ile Lys Lys Met 345 350 Tyr Ser Met Phe Asp Val Tyr Met 365 Gly Asn Lys Asp Ser Ile Lys Asp 380 Ser Thr Arg Met Leu Lys Thr Ile 395 400 Ser Met Tyr Leu Val Arg Cys Leu 410 415 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 'g 0 '-0 0* 0 09 0 s0,.

35 Ala Asp 40 Ala Gly 65 Gin Glu Ile Leu Val 145 Pro Val Lys 50 Ala Ala Leu His Asp 130 Leu Ser Asn 35 Asn Asp Ser Gly Tyr 115 Tyr His Leu Ser Arg 20 Ala Lys Asn Gly Tyr Asp Val Asn Ala 70 Thr Leu Asp Ala Asn Val 100 Ala Ala Val Gly Ala Asp Phe Ala Leu 150 Leu Ile Thr 55 Ala Arg Asn Arg Ile 135 Tyr Val Lys 40 Glu Asp Tyr Ala Asn 120 Glu Gly Pro 25 Gly Asn Ser Lys Arg 105 Asn Ala Thr 10 Lys Leu Glu Thr Ile Arg Leu Tyr 75 Asp Thr 90 Asp Tyr Val Val Leu Ser Asn Pro 155 Leu Pro Thr Ile Val Cys Ile Gin 140 Tyr Tyr Glu Leu Leu Thr Ile Asp Ile 125 Lys Met Thr Arg Tyr Ile Pro Thr Lys 110 Asn Ile Ser Val Gin Gly Ile Leu Met Met Arg Leu His Leu Leu Thr Pro Thr Leu Gly Thr Val Lys 160 Thr Leu Ile Asp Arg Gly Ala Asn Val Asn Ser Lys Asn Lys Tyr Leu -72 165 .170 175 Ser Thr Pro Leu His Tyr Ala Cys Lys Lys Asn Cys Lys Pro Giu Val 180 185 190 Ile Lys Met Leu Leu Asp Asn Gly Ala Asp Val Asn Ala Ile Asn Ile 195 200 205 Arg Asn Gin Tyr Pro Leu Leu Ile Ala Leu Giu Tyr His Gly Ile Val 210 215 220 Asn Ile Leu Leu His Tyr Gly Ala Giu Leu Arg Asp Ser Arg Val Ile 225 230 235 240 Asp Lys Ser Leu Asn Ser Asn Met Phe Ser Phe Arg Tyr Ile Ile Ala 245 250 255 His Ile Cys Ile Gin Asp Phe Ile Arg His Asp Ile Arg Ser Giu Val 260 265 270 20 **Asn Pro Leu Arg Gu Ile Ile Gin Ser Asp Asp Thr Phe Lys Ser Ile .275 280 285 S* Trp Leu Ser Cys Lys Giu Glu Leu Lys Asp Ile Ser Lys Ile Arg Ile 25 290 295 300 lot, Asn Met Phe Tyr Ser Leu Asp Ile Phe Val Ile Ser Lys Asn Met Asn 305 310 315 320 Leu Leu His His Leu Val Asn Asn Pro Ile Ile Lys Giu Ile Asn Thr 325 330 335 Tyr Tyr Phe Tyr Asn Tyr Giy Asp Arg Leu Lys Thr Ser Ile Ser Leu 340 345 350 Ala Ser Asn Arg His Lys Ile Leu Giu Lys Ser Arg Ser Lys Leu Asp 355 360 365 0 Gu Ile Leu Asp Ser Ser Gly Trp Ser Lys Leu Leu Arg Ile Ser Asn 40 370 37S 380 Ser GinTyr V.6 1o 045 INFORMATION FOR SEQ ID NO:S:

SEQUENCE*CHARACTERISTICS:

LENGTH: 40 base pairs TYPE: nucleic acid.

STRANDEDNESS: double (D),.TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: -73- AAAATTGAA AAACTATTCT AATTTATrGC 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: AATTCATT TGTrrTIrC TATGCTATAA AT 32 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: 0:060 LENGTH: 37 base pairs TYPE: nucleic acid 0 STRANDEDNESS: double 0* 0(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 0 0 0* (xi) SEQUENCE DESCRIPTION: SEQ ID GTATCCTAAA ATTGAATTGT AATTATCGAT AATAAAT 37 INFORMATION FOR SEQ ID NO:11: 45 SEQUENCE CHARACTERISTICS: 0 LENGTH: 42 base pairs TYPE: nucleic acid *0 0(C) STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: I I77MM-7- 7777=Z= GGCATATAAA TGAATTCGGA TC .42 /11+ 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:

LOCATION:

(ix) FEATURE: NAME/KEy:

LOCATION:

CDS

115. .186.0 CDs 2095. .3756 0@ S 0 OS SO 0 Oe 0 S S @0 S0 5 0 0 S S 00 0 0 5 0@ 5500

S

5* 0 @0 S 0 @0

S

0 0@ OS 0 S 0 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CATACTGGCC TCGAGOOCCG CGGCCGCCTG CAGGTCO.ACT CTAGAAAAAA TrQAAAAACT' ATTCTAATr'r ATTGCACGGA GATCrrZT 44'44411 TITrrGGCAT ATAA ATO met AAT TCG GAT 35 Asn Ser Asp CCG GAC CGC GCC Pro Asp Arg Ala GCA AAA AAT ACA Ala Lys Asn Thr MIT AGC Val Ser 10 CAA GTT GCG TTA Gin Val Ala Leu GAG AAT GAT Glu Asn Asp ATT GCA ATC Ile Ala Ile GAA AGA GAG Giu Arg Olu .20 CGC TTG ATA Trc Arg Leu Ile Phe

CG

Arg TTA TTC Leu Phe 35 TTA.ACA GTA GTG Leu Thr Val Val

ACC

Thr 40 TTG GCT ATA TCT Leu Ala Ile Ser

GTA

Val GCC TCC CTr TTA Ala Ser Leu Leu 117 165 213 261 309 357 405 453 45 TAT Tyr AGC ATO 000 GCT Ser Met Gly Ala

AGC

Ser ACA CCT AGC GAT Thr Pro Ser Asp Crr Leu OTA 0CC ATA CCG Val Gly Ile Pro

ACT

Thr AGG AT!' TCC AGO Arg Ile Ser Arg

GCA

Ala 70 GAA GAA AAO, AT Glu Giu Lys Ile

ACA

Thr TCT ACA cTT GOT Ser Thr Leu Gly TCC AAT Ser Asn .CAA GAT GTA Gin Asp Val OTA O AT Vai Asp as AGO ATA TAT Arg Ile Tyr ACT GAG ACC Thr Glu Thr 105 AAO CAA-GTG 0CC CT!' GAG TCT CCA Lys Gin Val Ala Leu Giu Ser Pro 90. 95 TTG OCA TA AAT Leu Ala Leu Leu Asn 100 ACA A" 1 ATO AAC Thr le Met Asn OCA ATA ACA TC!' Ala Ile Thr Ser CTC TCT TAT CArO AT!' AAT GA OCT GCA AAC AAC AGC 000 TGG 000 GCA Leu Ser Tyr Gin Ile Asn.Gly Ala Ala Asn Asn Ser Gly Trp Gly Ala CCT ATT Pro Ile 130 CAT GAC CCA His Asp Pro GAT TAT Asp Tyr 135 125 ATA GGG GGG ATA GGC AAA GAA CTC Ile Gly Gly Ile Gly Lys Glu Leu 140

ATT

Ile 145 GTA GAT GAT GCT Val Asp Asp Ala

AGT

Ser 150 GAT GTC ACA TCA Asp Val Thr Ser 7rC Phe 155 TAT CCC TCT GCA Tyr Pro Ser Ala TTT CAA Phe Gln 160 GAA CAT CTG Glu His Leu

AAT

Asn 165 TTT ATC CCG GCG Phe Ile Pro Ala ACT ACA GGA TCA Thr Thr Gly Ser GGT TGC ACT Gly Cys Thr 175 TAC ACC CAT Tyr Thr His is CGA ATA CCC Arg Ile Pro 180 TCA TTT GAC ATG Ser Phe Asp Met

AGT

Ser 185 GCT ACC CAT TAC Ala Thr His Tyr

TGC

Cys 190 597 645 693 741 789 AAT GTA ATA TTG TCT Asn Val Ile Leu Ser 195 TTA GCA CTT GGT GTG Leu Ala Leu Gly Val 210 GGA TGC Gly Cys 200 AGA GAT CAC TCA Arg Asp His Ser

CAC

His 205 TCA CAT CAG TAT Ser His Gin Tyr

CTC

Leu 215 CGG ACA TCT GCA Arg.Thr Ser Ala

ACA

Thr 220 GGG AGG GTA TTC Gly Arg Val Phe

TTT

Phe 225 TCT ACT CTG CGT Ser'Thr Leu Arg

TCC

Ser 230 ATC AAC CTG GAC Ile Asn Leu Asp

GAC

Asp 235 ACC CAA AAT CGG Thr Gin Asn Arg AAG TCT Lys Ser 240 TGC AGT G"rG Cys Ser Val

AGT

Ser 245 GCA ACT CCC CTG Ala Thr Pro Leu

GGT

Gly 250 TGT GAT ATG CTG Cys Asp Met Leu TGC TCG AAA Cys Ser Lys 255 CCT ACG CGG Pro Thr Arg GCC ACG GAG Ala Thr Glu 260 ATG GTA CAT Met Val His 275 ACA GAG GAA GAA Thr Glu Glu Glu

;GAT

Asp 265

TTC

Phe AT AAC TCA GCT yr Asn Ser Ala

GTC

Val 270 GGG AGG TTA Gly Arg Leu

'GGG

Gly 280 GAC GGC CAA sp Gly Gin

TAT

Tyr 285 CAC GAA AAG GAC His Glu Lys Asp

CTA

Leu 290 GAT GTC ACA ACA Asp Val Thr Thr

TTA

Leu 205 TrC GGG GAC TGG Phe Gly Asp Trp GCC AACTAC CCA Ala Asn Tyr Pro

GGA

Gly 305 837 885 933 981 1029 1077 1125 1173 1221 GTA GGG GGT GGA Val Gly Gly Gly

TCT

Ser 310 TTT A7T GAC AGC Phe Ile Asp Ser CGC GTG Arg'Val 315 TGG TTC TCA Trp Phe Ser GTC TAC Val Tyr 320 GGA GGG 7TA Gly Gly Leu

AAA

Lys 325 CCC AAT ACA CCC Pro Asn Thr Pro

AGT

Ser 330 GAC ACT GTA CAG ApjT Thr Val Gin GAA GGG AAA Glu Gly Lys 335 GAG CAA GAC Glu Gin Asp TAT GTG ATA Tyr Val Ile 340- TAC CAG ATT Tyr Gin Ile 355 TAC AAG CGA TAC Tyr Lys Arg Tyr

AAT

Asn 345 GAC ACA TGC CCA Asp Thr Cys Pro

GAT

Asp 350 CGA MG GCC Arg Met Ala

AAG

Lys 360 TCT TCG TAT AAG Ser Ser Tyr Lys CCT GGA CGG GGT Pro Gly Arg Phe Gly 365 76-

GGG

Giy 370 AAA CGC ATA CAG Lys Arg Ile Gin GCT ATC TTA TCT Ala Ile Leu Ser

ATC

Ile 380 AAA GTG TCA ACA Lys Val Ser Thr

TCC

Ser 385 ?TA GGC GAA GAC Leu Giy Giu Asp GTA CTG ACT GTA Vai Leu Thr Val

CCG

Pro 395 CCC AAC ACA GTC Pro Asn Thr Val ACA CTC Thr Leu 400 ATG GGG GCC Met Gly Ala TAT CAG CGA Tyr Gin .Arg

GAA

Giu 405 GGC AGA ATT CTC Gly Arg Ile Leu

ACA

Thr 410 GTA GGG ACA TCC Val Gly Thr Ser CAT TT1C TrG His Phe Leu 415 TAT CCT ATG Tyr Pro met GGG TCA TCA TAC Gly Ser Ser Tyr

TTC

Phe 42S TCT CCC GCG TrA Ser Pro Ala Leu TrA Leu 430 ACA GTC Thr Vai 435 AGC AAC AA.A ACA Ser Asn Lys Thr.

CC

Aia 440 ACT CTT CAT AGT Thr Leu His Ser

CCT

Pro 445 TAT ACA TTC AAT Tyr Thr Phe Asn

GCC

Al a 450 TTC ACT CGG CCA Phe Thr Arg Pro

CDT

Gly 455 AGT ATC CCT TGC Ser Ile Pro Cys

CAG

Gin 460 CCT TCA GCA AGA Ala Ser Ala Arg

TGC

Cys @0 S S 0606 6 .4000 0e 0 0 6 *0

S

606 0 0600 0 0

SS

S 0 0* 6@S* 6 0 OS S 00 0 6 0 *0 0 6~060e 0 @0 @0 0 0 6 0 CCC AAC TCA TGT Pro Asn Ser Cys GTT ACT GGA GTC TAT ACA CAT CCA TAT CCC CTA ATC Val Thr Gly Val Tyr Thr Asp Pro Tyr Pro Leu Ile 470 475 480 TTC TAT AGA Phe Tyr Arg OCT GAA CAA Gly Giu Gin 500

AAC

Asn 485 CAC ACC TTG CGA His Thr Leu Arg

GGG

Gly 490 GTA TTC CCC ACA Vai Phe Gly Thr ATG CTT CAT Met Leu Asp 495 CAT AGC ACA Asp Ser Thr 1265 1317 1365 1413 1461 1509 i1557 1605 1653 1701 1749 1797 1845 1897 1957 2017 OCA AGA CTT AAC Ala Arg Leu Asn

CCT

Pro 505 GCG TCT -GCA GTA Ala Ser Ala Val

TTC

Phe 510 TCC CC Ser Arg 515 ACT CGC ATA ACT Ser Arg Ile Thr

CGA

Arg 520 C TC AGT TCA AGC Val Ser Ser Ser

AGC

Ser 525 ATC AAA GCA GCA Ile Lys Ala Ala

TAC

Tyr 530 ACA ACA TCA ACT Thr Thr Ser Thr

TGT

Cys 535 Tr'r AAA CTG GTC Phe Lys Vai Val

AAG

Lys 540 ACC AAT AAG ACC Thr Asn Lys Thr

TAT

Tyr 545 TGT CTC AGC ATT Cys Leu Ser Ile

GCT

Ala 550 GAA ATA TCT ANT Giu Ile Ser Asri k1

ACT

Thr 555 CTC TTC GGA GAA Leu Phe Gly Giu 7TC AGA Phe Arg 560 ATC GTC CCG Ile-Val Pro

TTA

Leu 565 CTA CTr GAG ATC Leu Val Giu Ile

CTC

Leu 570 AAA CAT CAC GGC Lys Asp Asp Gly GTT ADA GAA Val Arg Giu 575 GCC ADD TCT GCC Ala Arg Ser Giy 580 TAGTTGADTC AACTATGAAA GA37r7GAAA GATGCATTGr TATCACCTAT CTr CTGCGAC ATCAAQAATC AAACCGAATG CCCCGATCCA TAATrAA7TA ATTAATI== ATCCCTCGAC TCTAGAAAAA A7rCAAAAAC TATTCTAATT TATrGCACGG AGATCTrTTT a a .a.a TITTrOCA TATAAATGAA TTCGGATCGA TCCCGG7TG 2077 77 CGCCCTCCAG GTGCAGG ATG GGC TCC AGA CCT TCT ACC AAG AAC CCA GCA Met Gly Ser Arg Pro Ser Thr Lys Asn Pro Ala 1 5 00 00 0 0000 00000 0 00 0 0 0 00 00 0 0 000 0 0000 S 0 00 0 00 0 0 00 0.00 0 *0 00 0 00 0 0 0 0 00 0 000000 0 00 @0 00 0

S

CCT

Pro

CCG

Pro

GTT

Val

TCA

Ser

TGT

Cys 25 CTC Leu

ACA

30 Thr

GGT

Gly

GCT

Ala 140

GAG

Glu 45 TTA Leu

GAC

Asp

CAG

Gin

GTA

Val 220

CAG

Gin

ATG

Met

GCA

Ala

ACA

Thr 45

ATC

Ile

GCG

Ala

ACC

Thr

TCT

Ser

GTG

Val 125

CTG

Leu

AGC

Ser

TCG

Ser

CAA

Gin

CAA

Gin 205

TTC

Phe

GCA

Ala

ATG

Met

AAC

Asn

GGA

Gly

ATA

Ile

AAA

Lys

CCC

Pro

GGA

Gly 110

GCT

Ala

ATA

Ile

ATT

Ile

CAA

Gin T7r Phe 190

GTT

Val

GGA

Gly Crr Leu CTG ACT ATC Leu Thr Ile TCC ATT CAT Ser Ile Asp GAC AAA GCA Asp Lys Ala GTI' AAG CTC Val Lys Leu 65 GCC CCC 7TG Ala Pro Leu so CTT GGT GAC Leu Gly Asp 95 GGG GGG AGA Gly Gly Arg C7T GGG GT Leu Gly Val CAA GCC AAA Gin Ala Lys 145 GCC GCA.ACC Ala Ala Thr 160 CTA GCA GTG Leu Ala Val 175 AAT AAA ACA Ann Lys Thr GGT GTA GAG Gly Val Giu CCA CAA ATC Pro Gin Ile 225 TAC AAT CTA Tyr Ann Leu Arg

GGC

CGiy

GTC

Val 50

CTC

Leu

CAT

Asp

TCT

Ser

CAG

Gin

GCA

Ala 130

CAA

Gin

AAT

An

GCA

Ala

OCT

Ala

CTC

Leu 210

ACT

Thr

GCT

Ala Val

AG

Arg 35

AAC

An

CCG

Pro

GCA

Ala

ATC

Ile

CCC

Gly 115

ACT

Thr

AAT

An

GAG

Giu GTr Val

CAG

Gin 195

AAC

An

TCA

Ser

GGT

Gly Ala Leu 20 CCT CTT Pro Leu ATA TAC Ile Tyr AAT CTG Asn Leu TAC AAC Tyr An 95 CGT AGG Arg Arg 100 CGC CTT Arg Leu GCC GCA Ala Ala CCT GCC Ala Ala GCT GTG Ala Val 165 CCC AAG Gly Lys 180 GAA TTA Ciu Leu CTG TAC Leu Tyr CCT GCC Pro Ala CCC AAT Gly An Val

GCA

Ala

ACC

Thr

CCA

Pro 70

AGG

Arg

ATA

Ile

ATA

Ile

CAA

Gin

AAC

An 150

CAT

His

ATG

Met

GAC

Asp

CTA

Leu

TTA

Leu 230

ATG

Me t Leu

GCT

Ala

TCA

Ser

AAG

Lys

ACA

Thr

CAA

Gin

GC

Giy

ATA

Ile 135

ATC

Ile

GAG

.Giu

CAG

Gin

TGC

CyB

ACC

Thr 215*

AAC

An

CAT

ASP

Ser

GCA

Ala

TCC

Ser

GAT

Asp Leu

GAG

Giu

CC

Ala 120

ACA

Thr

CTC

Leu

GTC

Val

CAG

Gin

ATC

Ile 200

GAA

Giu

AAG

Lys

TAC

Cys

GGA

Cly

CAC

Gin

AAG

Lys

ACC

Thr

TCT

Ser 105

ATT

Ile

GCG

Ala

CGA

Arg

ACT

Thr

TC

Phe 185

AAA

Lys

TCG

Ser

CTG

Leu TrA Leu CGC GTC GCG CTC GTA CTG ACT TOC ATC TGT Ile Cys A1-r CTG Ile.Val ACA GGA Thr Cly GAG GCA Clu Ala ACT TTG Thr Leu GTG ACT Val Thr ATT GCC.

Ile Giy CCC CCA Ala Ala CTT AAA Leu Lys 155 CAC GCA Asp Gly 170 GTT AAT Val An ATT GCA Ile Ala ACT ACA Thr Thr ACT AT Thr Ile 235 TTG ACT Leu Thr 2127 2'175 2223 2.271 2319 2367 2415 2463 2511 2559 2607 2655 2703 2751 2799 2847 78 AAG TTA GGT Lys Leu Giy TI'A AT-- ACC Leu Ile Thr 270

ATA

Ile 255 GGG AAC AAT CAA Giy Asn Asn Gin AGC TCA TIA Ser Ser Leu ATC'GGT AGC GGC Ile Gly Ser Gly 265 GGT AAC CCT ATT Gly Asn Pro Ile

CTA

Leu 275 TAC GAC TCA CAG Tyr Asp Ser Gin

ACT

Thr 280 CAA CTC TTG Gin Leu Leu GGT ATA Gly Ile 285 CAG GTA ACT CTA Gin Val Thr Leu

CCT

Pro 290 TCA GTC.GGG AAC Ser Val Giy Asn

CTA

Leu 295 AAT AAT ATG CGT Asn Asn Met Arg

GCC

Ala 300 ACC TAC TTG GAA Thr Tyr Leu Giu TTA TCC GTA AGC Leu Ser Val Ser

ACA

Thr 310 ACC AGG GGA Trr Thr Arg Gly Phe

GCC

Al a 315 TCG GCA*CTI' GTC Ser Ala Leu Val

CCA

Pro 320 AAA GTG GTG ACA Lys Val Val Thr

CGG

Arg 325 GTC GGT TCT GTG Val Gly Ser Val ATA GAA Ile Giu 330 000 0 0& a GAA CTT GAC Giu Leu Asp ACA AGA ATA Thr Arg Ile 350

ACC

Thr 335 TCA TAC TGT ATA Ser Tyr Cys Ile

GAA

Giu 340 ACT GAC TTA GAT Thr Asp Leu Asp TTA TAT TGT Leu Tyr Cys 345 TCC TGC TTG Ser Cys Lau GTA ACG TTC CCT Val Thr Phe Pro

ATG

Met 355 TCC CCT GGT ATI' Ser Pro Gly Ile

TAC

Tyr 360 AGC GGC Ser Qiy 365 AAT ACA TCG GCC Asn Thr Ser Ala

TGT

Cys 370 ATG TAC TCA AAG Met TyrSer Lys

ACC

Thr 375 GAA GGC GCA CTT Giu.Gly Ala Leu 2895 2943 2991 3039 3087 3135 3183 3231 3279 3327 3375 3423 3471 3519 3567 ACT ACA Thr Thr 380 CCA TAT ATG Pro Tyr Met

ACT

Thr 385 ATC AAA GOC .,TCA Ile Lys Gly Ser

GTC

Val 390 ATC GCT AAC TGC le Ala Asn Cys S S

S.

50@S S S S. S *5 0 5

C.

0

CC

S S ATG ACA ACA TGT Met Thr Thr Cys

AGA

Arg 400 TGT OTA AAC CCC Cys Val Ann Pro

CCG

Pro 405 GGT ATC ATA TCG Gly Ile Ile Ser CAA AAC Gin An 410 TAT GGA GAA Tyr Gly Giu TCC TTA GGt Ser Leu Gly 430

GCC

Ala 415 GTG TCT CTA ATA Val Ser Leu Ile

GAT

Asp 420 AAA CAA TCA TGC Lys Gin Ser Cys GGG ATA ACT TTA Gly Ile Thr Leu

AGG

Arg 435 CTC AGT GGG GAA Leu Ser Gly Giu

TTC

Phe 440 AAT GTT TA Ann Vai Leu 425 GAT GTA ACT Asp Vai Thr ATA ACA GGC Ile Thr Gly TAT CAG Tyr Gin 445 AAG AAT ATC Lys Asn Ile TCA ATA Ser Ile 450 CAA GAT TCT CAA Gin Asp Ser rzin GTA ATA Val Ile 455

AAT

An 460 CTr GAT ATC TCA Leu Asp Ile Ser

ACT

Thr 465 GAG Crr GGG AAT Giu. Leu Giy An

GTC

Val 470 AAC AAC TCG ATC Ann Ann Ser Ile

AGT

Ser 475 AAT GCC TTG AAT Ann Ala Leu An

AAG

Lys 480 TTA GAG GAA AGC Leu Glu Giu. Ser

AAC

An 485 AGA AAA CTA GAC Arg Lys Leu Asp AAA GTC Lys Val 490 9S

S

0*eS

S

S. S S S *5 5* a a 0 0 OS S *5

S.

00

SO

*5 *S es a 0 0@ @5 S. S a -79- AAT GTC AAA CTG ACC AGC ACA TCT GCT CTC ATT ACC Asn Vai Lys Leu Thr Ser Thr Ser Ala Leu Ile Thr 495 500 S ACT AT C ATA TCT CTr GTT TTr GGT ATA CTT AGC CTG Thr Ile Ile Ser Leu Val Phe Giy Ile Leu Ser Leu 510 515 TAC CTA ATG TAC AAG CAA AAG GCG CAA CAA AAG ACC Tyr Leu Met Tyr Lys Gin Lys Ala Gin Gin Lys Thr 525 530 535 GGG AAT AAT ACC CTA GAT CAG ATG AGA GCC ACT ACA Gly Asn Asn Thr Leu Asp Gin Met Arg Ala Thr Thr 540 545 550 TGAGGAACGA AGGTTrCCCT AATAGTAATT TGTGTGAAAG TTCI GGAGAGTTAA GAAAAAAAACCCCCCC CCCCCCCCCC CCC( TGGTGTCACG CTCGTCGTr'r GGTATGGCTT CATTCAGCTC CGG GAGTrACATG ATCCCCCATG TTGTGCAAAA AAGCGGTTAG CTC( 25 TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT CACTCATGGT TAT( CTCTrACTGT C.ATGCCATCC GTAAGATGCT TTTCTGTGAC TGG ATTAATTAAT TTTTATCCCG GGTCGACCTG CAGGCGGCCG CGG( INFORMATION FOR SEQ ID NO:i3: Wi SEQUENCE CHARACTERISTICS: LENGTH: 581 amino- acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i3: Met' Asn Ser Asp Pr o Asp Arg Ala Vai Ser Gin Val 1 5 10 45 Asp Glu Arg Glu Ala Lys Asn Thr .Trp Arg Leu Ile -25 Ile Leu Phe Leu Thr Val Vai Thr Leu Ala Ile Ser 50 35 40 Leu Tyr Ser Met Giy Ala Ser Thr Pro Sur-Asp Leu 55 Th r Arg Ile Ser Arg Ala Giu Giu Lys Ile Thr Ser 70 75 Asn Gin Asp Vai Val Asp Arg Ile Tyr Lys Gin Vai 90 Pro Leu Ala Leu Leu Asn Thr Giu Thr Thr Ile Met 100 105 TAT ATC Tyr Ile 505 ATT CTA Ile Leu 520 TTA TTA Leu Leu AAA ATG Lys Met rGGTAGTC

CCCCCCTC

rTCCCA.A C CTTCGGT C

GCAGCA

rGAGTcA i

CCCTCGAC

GTT 1TG Val Leu GCA TGC Ala Cys TGG CTr Trp Leu

TGAACACAGA

:TGTCAGTTC

;CAGGCATCG

:GATCAAGGC

:CTCCGATCG

TGCATAATr

CCATAATTA

;GCC

3615 3663 3711 3763 3823 3883 3943 4003 4063 4123 4177 Ala Phe Val Val Thr Ala Asn Leu Arg Ala Gly Leu Leu Ala 110 Giu is le 5cr Ile Gly Giu Ile Asn Ala Leui Pro Ser so.

Thr .s 4 0 04 S

S

*5 0*

S

0 0* 4

OS

S

0* Ser Ala Ile 145 Gin Thr His Tyr Phe 225 25 Ser Lys Arg Asp 35 Gly 305 Tyr Lys Asp Gly Ser 385 Leu Leu Leu Pro 130 Val Glu Arg Asn Leu 210 Ser Cys Ala Met Leu 290 Val Gly Tyr Gly 370 Leu Met Tyr Ser 115 Ile Asp His lie Val 195 Ala Thr Ser Thr Val 275 Asp Gly Gly.

Val Gin 355 Lys Gly Gly Gin Tyr His Asp Leu Pro 180 Ile Leu Leu Val Glu 260 His Val Gly Leu Ile 340 lie Arg Glu Ala Arg 420 Gin Asp Ala Asn 165 Ser Leu Gly Arg Ser 245 Thr Gly Thr Gly Lys 325 Tyr Arg lle Asp Glu 405 Gly Ile Pro Ser 150 Phe Phe Ser Val Ser 230 Ala Glu Arg Thr Ser 310 Pro Lys Met Gin Pro 390 Gly Ser Asn Asp 135 Asp lie Asp Gly Leu 215 lie Thr Glu Leu Leu 295 Phe Asn Arg Ala Gin 375 Val Arg Ser Gly 120 TIyr Val Pro Met Cys .200 Arg Asn Pro Glu Gly 280 Phe lie Thr Tyr Lys 360 Ala Leu Ile Tyr Ala Ala Asn Asn Ser Ile Thr Ala Ser 185 Arg Thr Leu Leu Asp 265 Phe Gly Asp Pro Asn 345 Ser lie Thr Leu Phe 425 Gly Ser Pro 170 Ala Asp Ser Asp Gly 250 Tyr Asp Asp Ser Ser 330 Asp Ser Leu Val Thr 410 Ser Gly Phe 155 Thr Thr His Ala Asp 235 Cys Asn Gly Trp Arg 315 Asp Thr Tyr Ser Pro 395 Val Pro Ile 140 Tyr Thr His Ser Thr 220 Thr Asp Ser Gin Vai 300 Val Thr Cys Lys lie 380 Pro Gly Ala 125 Gly Pro Gly Tyr His 205 Gly Gin Met Ala Tyr 285 Ala Trp Val Pro Pro 365 Lys Asn Thr Leu Pro 445 Gly Lys Ser Ser Cys 190 Ser Arg Asn Leu Val 270 His Asn Phe Gin Asp 350 Gly Val Thr Ser Leu 430 Trp Glu Ala Gly 175 Tyr His Val Arg Cys 255 Pro Glu Ser Glu 335 Glu Arg Ser Val His 415 Tyr Gly Leu Phe 160 Cys Thr Gin Phe Lys 240 Ser Thr Lys Pro Vai 320 Gly Gin Phe Thr Thr 400 Phe Pro Met Thr Val Ser Asn Lys Thr Ala 435 440 Thr Leu His Ser Tyr Thr Phe 81 Asn Ala Phe Thr Arg Pro Gly Ser Ile Pro Cy s Gin Ala Ser Ala Arg 450 455 460 Cys Pro Asn Ser Cys Val Thr Gly Val Tyr Thr Asp Pro Tyr Pro Leu 465 470 475 480 Ile Phe Tyr Arg Asn His Thr Leu Ar-g Gly Val Phe Gly Thr Met Leu 485 490 495 Asp Gly Giu Gin Ala Arg Leu Asn Pro Ala Ser Ala Val Phe Asp Ser 500 505 510 Thr Ser Arg Ser Arg Ile Thr Arg Val Ser Ser Ser Ser Ile Lys Ala 515 520 525 Ala Tyr Thr Thr Ser Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr 530 535 540 Tyr Cvs Leu Ser Ile Ala Giu Ile Ser Asn Thr Leu Phe Gly Giu Phe 545 550 555 560 Arg Ile Val Pro Leu Leu Val Giu Ile Leu Lys Asp Asp Gly Val Arg 565 570 575 Giu Ala Arg Ser Gly 0 580 030 INFORMUTION FOR SEQ ID NO:i14: so:*CiW SEQUENCE CHARACTERISTICS: LENGTH: 553 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ±D NO:i4: 4 as 0 Met Gly Ser.Arg Pro Ser Thr Lys Asn Pro Ala Pro Met.Met Leu Thr 1 10 00 *09 0& ole Arg Val Ala Leu Val Leu Ser Cys Ile Cys Pro Ala Asn Ser Ile 45 20 25 Asp Giy Arg Pro Leu Ala Ala Ala Gly Ile Val Val Thr Gly Asp Lys 40 Ala Val Asn Ile Tyr Thr Ser Ser Gin Thr Gly Ser Ile Ile Val Lys 55 Leu Leu Pro Asn Leu Pro Lys Asp Lys Giu Ala Cys Ala Lys Ala Pro 70 75 s0 Leu Asp Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly 8590 Asp Ser Ile Arg Arg Ile Gin Giu Ser Val Thr Thr Ser Gly Gly Gly 100 105 110 Arg Gin Gly .Arg LeuIle Gly Ala 11e Ile Giy Gly Val Ala Leu Gly -82c r r Ir r 'e

CI

r C

Y

r r L r r *r t C Val Lys 145 Thr Val Thr Glu Ile 225 Leu 25 Asn 30 Pro Leu Thr 305 Lys 40 Tyr 45 Phe Ala Thr 385 Cys Ser Ala 13.0 Gln Asn Ala Ala Leu 210 Thr Ala Asn Ile Pro 290 Leu Val ys Pro Cys 370 Ile Val Leu 115 Thr Asn Glu Val Gln 195 Asn Ser Gly Gln Leu 275 Ser Ser Val Ile Met 355 met Lys Asn Ile Ala Ala Ala Gly

ISO

Glu Leu Pro Gly Leu 260 Tyr Val Thr Glu 340 Ser Gly Pro Asp 420 Ala Ala Val 165 Lys Leu Tyr Ala Asn 245 Ser Asp Gly Ser Arg 325 thr Pro Ser Ser Pro 405 Lys Gln Asn

ISO

His met Asp Leu Leu 230 Met Ser Ser Asn Thr 310 Val Asp Gly Lys Val 390 Gly Gln Ile 135 Ile Glu Gln Cys Thr 215 Asn Asp Leu Gln Leu 295 Thr Gly Leu Ile Thr 375 Ile Ile Ser 120 Thr Leu Val Gln Ile 200 Glu Lys Tyr Ile Thr 2.80 Asn Arg Ser Asp 360 Glu Ala Ile ys Ala Arg Thr Phe 185 Lys Ser Leu Leu Gly 265 Gln Asn Gly Val Leu 345 Ser Gly Asn Ser Asn Ala Leu Asp 170 Val Ile Thr Thr Leu 250 Ser Leu Met Phe Ile 330 Tyr Cys Ala

CYS

Gln 410 Val Ala Ala 140 Lys Glu Gly Leu Asn Asp Ala Gln Thr Val 220 Ile Gln 235 Thr Lys Gly Leu Leu Gly Arg Ala 300 Ala Ser 315 Glu Glu Cys Thr Leu Ser .Leu Thr 380 Lys Met 3'95 Asn Tyr Leu Ser Leu Ser Ser Gln Gln 205 Phe Ala Leu Ile Ile 285 Thr Ala Leu Arg Gly 365 Thr Thr Gly Leu Gln 445 Ile Ile Gln Phe 190 Val Gly Leu Gly Thr 270 Gln Leu Asp Ile 350 Asn Pro Thr Glu Gly 430 125 Gln Ala Leu 175 Asn Gly Pro Tyrt Ile 255 Gly Val Leu Val Thr 335 Val Thr Tyr ys Ala 415 Gly Ala Ala 160 Ala Lys Val Gln Asn 240 Gly Asn Thr Glu Pro 320 Ser Thr Ser Met Arg 400 Val Ile 425 Thr Leu Arg 435 Leu Ser Gly Glu .Phe Asp Val Thr Tyr Lys Asn Ile 440 83 Ser Ile 450 Thr Giu 465 Leu Giu Gin Asp Ser Gin Vai Ile Ile Tkir Gly Asn 455 460 Leu Giy Asn Vai Asn Asn Ser Ile Ser Asn 470 475 Leu Asp Ile Ser Ala Leu Asn Lys 480 Giu Ser Asn Arg Lys Leu Asp Lys 490 Val Vai Asn Vai Lys Leu Thr 495 Ser Leu Ser Thr Ser Ala 500 Leu Ile Thr Leu Ser Leu Tyr Ile 505 Ile Leu 520 Leu Thr Ile Vai Phe Giy Ile 515 Aia Cys Tyr Leu 525 Asn Met Tyr Lys Asn Thr Leu Gin Lys 530 Asp Gin 545 Ala. Gin Met Arg Gin Lys Thr Leu Leu 535 Ala Thr Thr Lys Met 550 Trp Leu Giy 540 b C' t 4i C

S

5-4 iS h S U

C.

St. 595

S

C.

4.

C.

i.e.

4- 0

C

S SC 505*0'

S

t.t 09

C

a U INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH: 182 base pairs TYPE: nucleic acid STRANqDEDNESS: doubie CD) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO Civ) ANTI-SENSE: NO (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:iS: GGCCTCGAGG GCCGCGGCCG CCTGCAGGTC GACTCTAGAA AAAAT'rGAAA AACTAT'rCTA ATTTATrGCA CGG.AQATCTTrrT 1- a.r GCATATAAAT GAATrCGGAT 45 CCGGACCGCG*CCGTrAGCCA AGTrGCGTrA GAGAATGATG AAAGAGAGGC AAAAAATACA

TG

120 iso 182 INFORMATION FOR SEQ ID NqO:16: Wi SEQUENCE CHARACTERISTICS: LENGTH: 178 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (i)MOLECULE TYPE: DNA (genomic) (iii), HYPOTHETICAL: NO (iv) AN~TI-SENSE: NO 84 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: ATCTrrCTGCG ACATCAAGAA TCAAACCGAA TGCCCGGATC CATAATTAAT TAATTAATI-r TTATCCCTCG ACTCTAGAAA AAATTGAAAA ACTATTCTAA ",TrATGCAC GGAGATCT 12 T!J IT~GG CATATAAATG AATTCGGATC GATCCCGGTT GGCGCCCT INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genoniic) (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:1S: SEQUENCE CHARACTERISTICS: LENGTH: 120 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear 0 MOLECULE TYPE: DNA (genomic) *(iii) H07EIA:NO (iv) ANTI-SENSE: NO 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:lB: 0OATAATTCTCT TACTGTCATG CCATCCGTAA GATGCTTTC TGTGACTGGT GAGTGATCCA so 5 TAATTAATTA ATrAATT1-r ATC!CCGGGTC GACCTGCAGG CGGCCGCGGC CCTCGAGGCC 120 INFORMATION FOR SEQ ID NO:19: Wi SEQUENCE CHARACTERISTICS: (A).LENGTH: 1305 base .pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomi c) 85 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:

NAME/KEY:

LOCATION:

CDS

1. .1305 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

ATG

Met 1 CAC CGT CCT His Arg Pro CTC AGA CGG CAC Leu Arg Arg His TCG CGT TAC Ser Arg Tyr 10 TAC GCG AAA OGA Tyr Ala Lys Gly GAG GTG CTT Glu Val Leu GGC GCA GCT Gly Ala Ala 35 AAA CAC ATG Lys His Met GAT TGC Asp Cys GOT GGA AAA CGO Gly Gly Lys Arg TOC TGC TCA Cys Cys Ser ATT ATG CG le Met Arg 0O 0 0 0000 0 0 S. 0 S ~0 0O

S.

0 5'00 *5@e S S 0O S @0 0 0 0@ 50*5 9* S. S @0 5 *0 0 0*

S

*05505 0

S.

S 0 0

S

GTA TTC ACT CTT Val Phe Thr Leu

TTC

Phe 40 TGG ACT TGT GTC Trp Thr Cys Val

AGG

Arg 25 GAG CAT Glu His so ATC TGC TrT GTA Ile' Cys Phe Val

CGC

Arg 55 AAC GCT ATG GAC Ann Ala Met Asp

CGC

Arg CAT TTA IT Tro His Leu Phe Leu

AGG

Arg AAT GCT Tr'r TG Asn Ala Phe Trp

ACT

.Thr 70 ATC OTA CTO CTT Ile Val Leu Leu

TCT

Ser 75 TCC TTC OCT AGC Ser Phe Ala Ser

CAG

Gin so AGC ACC GCC GCC Ser Thr Ala Ala

GTC

Val as ACO TAC GAC TAC Thr Tyr Asp Tyr

ATT

le 90 TTA.GGC COT CGC Leu Gly Arg Arg GCG CTC Ala Leu GAC OCO CTA Asp Ala Leu

ACC

Thr 100 ATA CCG GCG Ile Pro Ala Val

GOC

Oly 105 CCG TAT AAC AGA Pro Tyr Ann Arg TAC CTC ACT Tyr Leu Thr 110 ATW TCT AAC le Ser An 144 192 240 288 336 384 432 480 528 AGO OTA TCA AGA Arg Val Ser Arg

:_I

GOC TOC GAC Oly Cys Asp Val 120 OTC GAG CTC AAC Val Glu Leu An GTO. GAC Val.Asp 130 GAC. ATm ATA TCG Asp Met Ile Ser

GCO

Ala 135 0CC AAA GAA AAA Ala Lys Glu Lys GAr.

Giu 140 AAG 000 GOGC CCT Lys Oly Gly Pro

TTC

Phe 145 GAG 0CC TCC OTC Glu Ala Ser Val

OTC

Val 150 TGG TTC'TAC Trp Phe Tyr OTO A7T Val Ile iss AAG GGC GAC GAC Lys Gly Asp Asp

GGC

Gly 160 GAG GAC AAG TAC Olu Asp Lys Tyr

TO.T

Cys 165 CCA ATC TAT AGA Pro le Tyr Arg

AAA

Lys 170 GAG TAC AGG GAA Giu Tyr Arg Glu TOT GGC Cys Gly 175 GAC OTA CAA Asp Val Gin

CTG

Leu 180 Leu.Ser Giu Cys OG= CCT AGC ACC

GCC

Ala 185 OTT CAA TCT GCA Val Gin Ser Ala CAG ATG TOG Gin Met Trp 190 GCA OTG GAC TAT Ala Val Asp Tyr Val lPro Ser Thr CTT OTA TCG CGA AAT GGC GCG OGA Leu Val Ser Arg Ann Oly Ala Gly -86- 200 CTG ACT Leu Thr 210 ATA TTC TCC CCC le Phe Ser Pro

ACT

Thr 215 GCT GCG CTC TCT Ala Ala Leu Ser

GGC

Gly 220 CAA TAC TTG CTG Gin Tyr Leu Leu

ACC

Thr 225 CTC AAA ATC GGG Leu Lys Ile Gly

AGA

Arg 230 TTT GCG CAA ACA Phe Ala Gin Thr

GCT

Ala 235 CTC GTA ACT CTA Leu Val Thr Leu

GAA

Glu 240 GT AAC GAT CGC Val Asn Asp Arg

TGT

Cys 245 TTA AAG ATC GGG Leu Lys Ile Gly CAG CTT AAC ITr Gin Leu Asn Phe TTA CCG Leu Pro 255 TCG AAA TGC Ser Lys Cys CAC CTT TAT 20 His Leu Tyr 275 TAT CGG GGA Tyr Arg Gly 290

TGG

Trp 260 ACA ACA GAA CAG Thr Thr Glu Gin

TAT

Tyr 265 CAG ACT GGA TTT Gin Thr Gly Phe CAA GGC GAA Gin Gly Glu 270 GAC GAC GTA Asp Asp Val CCG ATC GCA GAC Pro Ile Ala Asp

ACC

Thr 280 AAT ACA CGA CAC Asn Thr Arg His

GCG

Ala 285 TAC GAA GAT Tyr Glu Asp

ATT

Ile 295 CTG CAG CGC TGG Leu Gin Arg Trp

AAT

Asn 300 AAT TTG CTG AGG Asn Leu Leu Arg

S*

S

S

5SO 0 S. S

S*

S

S.

S S S. 0 *5

S

AAA

Lys 305 AAG AAT CCT AGC Lys Asn Pro Ser

GCG

Ala 310 CCA GAC CCT CGT Pro Asp Pro Arg CCA GAT Pro Asp 315 AGC GTC CCG Ser Val Pro

CAA

Gin 320 GAA ATr CCC GCT Glu Ile Pro Ala GTA ACC Val Thr 325 AAG AAA GCG Lys Lys Ala

GAA

Glu 330 GGG CGC ACC CCG Gly Arg Thr Pro GAC GCA Asp Ala 335 35 GAA AGC AGC Glu Ser Ser CAG GCA GAG 40 Gin Ala Glu 355 GAA GTC CCC Glu Vai Pro 370

GAA

Glu 340 AAG AAG GCC CCT Lye Lye Ala Pro

CCA

Pro 345 GAA GAC TCG GAG Glu Asp Ser Glu GAC GAC ATO Asp Asp Met 350 GAA GAC GAC Glu Asp Asp GCT TCT GGA GAA Ala Ser Gly Glu

AAT

Asn 360 CCT GCC GCC CTC Pro Ala Ala Leu

CCC

Pro 365 GAG GAC ACC Glu Asp Thr

GAG

Glu 375 CAC GAT GAT CCA His Asp Asp Pro

AAC

Asn 380 TCG GAT CCT GAC Ser Asp Pro Asp 960 1008 1056 1104 1152 1200 1248 1296.

1305

TAT

Tyr 385 TAC AAT GAC ATG Tyr Asn Asp Met

CCC

Pro 390

TCC

Ser GCC GTG ATC CCG Ala Val Ile Pro

GTG

Val 395 GAG GAG ACT ACT Glu Glu Thr Thr

AAA

Lye 400 AGT TCT AAT GCC Ser Ser Asn Ala

GTC

Val 405 ATO CCC ATA Met Pro Ile

TTC

Phe 410 GCG GCG TTC GTA Ala Ala Phe Val GCC TGC Ala Cys 415 GCG GTC GCG Ala Val Ala CGT AGC TAA Arg Ser 435

CTC

Leu 420 GTG GGG CTA CTG Val Gly Leu Leu GTT TGG AGC ATC OTA AAA TGC GCG Val Trp Ser Ile Val Lys Cys. Ala 425 430 -87- INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 434 amino acids S TYPE: amino acid TOPOLOGY: linear.

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID t Met His Arg Pro His Leu Arg Arg His Ser 1 5 0 0 0 0 00O0 at 0 :0.*0

OS

00 0 0

S

0S 0005 o 0 5 05 5 0 0 0 Glu Gly Glu Arg 25 65 Ser 30 Asp.

Arg 35 Val Phe 40 145 Glu J Asp Ala 7 Leu 2 Thr L 225 10 Gly G Va Al His Asn Thr Ala Val Asp 130 31u !.sp lal ral 'hr 10 1 Leu i Ala 35 Ile Ala Ala Leu Ser 115 Asp Ala L3e Gin Asp 195 Ile I Asr, 2C Val Cys Phe Ala Thr 100 Arg Met Ser Tyr ELeu 180 ryr ?he 1 Lys His Met Asp Cys 25 Phe Phe Trp Val Ile Gly le Val Cys 165 Leu Val Ser Thr Val Thr 70 Thr Pro Cys Ser Val 150 Pro Ser Pro Pro Arg 230 Leu Phe 40 Arg Asn 55 Ile Val Tyr Asp Ala Val Asp Val 120 Ala Ala 135 Trp Ph.

Ile Tyr Glu Cys.

Ser Thr 200 Thr Ala 215 Phe Ala Trp Thr Cys Val

I

Ala Met Asp Let Tyr Gly 105 Val Lye Tyr Arg Ala 185 Leu kla ;in 0:20: LrgTyr ;ly Lys Leu Ser 75 Ile Leu 90 Pro Tyr Glu Leu Glu Lys Val Ile 155 Lye Glu 170 Val Gin Val Ser Leu Ser Thr Ala 235 Ser Gin 250 Arg Ser Gly Asn Asn Glu 140 Lye Tyr Ser Arg Gly 220 Leu Leu

C

J

3 3

C

U

Tyr Ala Lys Gly Arg Cys Cys Ser Arg Ile Met Arg 45 His Lou Phe Leu Phe Ala Ser Gin so Arg Arg Ala Leu Arg Tyr Lou Thr 110 Pro Ile Ser Asn Lys Giy Giy Pro ;ly Asp Asp Gly 160 krg Glu Cys Gly 175 Lia Gin Met Trp 190 wsn Gly Ala Gly 05 n Tyr Leu Leu 'al Thr Lou Glu 240 an Phe Lou Pro 255 leu Lye Ile Gly Val Asn Asp Arg Cys 245 Ser Lys Cys Trp Thr 260 Leu Lye lie Gly Thr Giu Gin Tyr 265 Gin Thr Gly Phe Gin Gly Glu 270 88 His Leu Tyr ProIle Ala Asp Thr Asn Thr Arg His Ala Asp Asp Val 275 280 285 Tyr.Arg Gly Tyr Giu Asp Ile Leu Gin Ar g Trp Asn Asn Leu Leu Arg 290 295 300 Lys Lys Asn Pro Ser Ala Pro Asp Pro Arg Pro Asp Ser Val Pro Gin 305 310 315 320 Giu Ile Pro Ala Val Thr Lys Lys Ala Giu Gly Arg Thr Pro Asp Ala 325 330 335 Giu Ser Ser Glu Lys Lys Ala Pro Pro Glu Asp Ser Giu Asp Asp Met 340 345 350 Gin Ala Giu.Ala Ser Gly Glu Asn Pro Ala Ala Leu Pro Giu Asp Asp 355 360 365 Giu Val Pro Giu Asp Thr Glu His Asp Asp Pro Asn Ser Asp Pro Asp 370 375 380 Tyr Tyr Asn Asp Met Pro Ala Val Ile Pro Val Glu Giu Thr Thr Lys 385 390 395 400 :25 Ser Ser Asn Ala Val Ser Met Pro Ile Phe Ala Ala Phe Val Ala Cys 405 410 415 Ala Val Ala Lou Val Gly Leu Lou Val Trp Ser Ile Val Lys Cys Ala 30420 425 430 Arg Ser 9 *35

Claims (19)

1. A recombinant fowlpox virus comprising a foreign DNA sequence inserted into the fowlpox virus genomic DNA, wherein the foreign DNA sequence is inserted within an approximately 2.8 kB EcoRI fragment of the fowlpox virus genomic DNA and is capable of being expressed in a fowlpox virus infected host cell.

2. The recombinant fowlpox virus of claim 1, wherein 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.

3. A recombinant fowlpox virus comprising a foreign DNA sequence inserted into the fowlpox virus genomic DNA, wherein the foreign DNA sequence is inserted within an approximately 3.5 kB EcoRI fragment of the fowlpox virus genomic DNA and is capable of being expressed in a fowlpox virus infected host cell.

4. The recombinant fowlpox virus of claim 3, wherein the foreign DNA sequence is inserted within a Hpal restriction endonuclease site within the approximately 3.5 kB EcoRI fragment of the fowlpox virus genomic DNA. 4 A recombinant fowlpox virus comprising a foreign DNA sequence inserted into the fowlpox virus genomic DNA, wherein the foreign DNA sequence is inserted within an approximately 4.2 kB EcoRI fragment of the fowlpox virus genomic DNA and is capable of being expressed in a fowlpox virus infected host cell.

6. The recombinant fowlpox virus of claim 5, wherein the foreign DNA sequence is inserted within an Stul restriction endonuclease site within the approximately 4.2 kB EcoRI fragment of the fowlpox virus genomic DNA.

7. The recombinant fowlpox virus according to any one of claims 1 to 6, wherein P:\OPER\MRO\2126880.SPE 3/12/98 the foreign DNA sequence encodes a polypeptide.

8. The recombinant fowlpox virus of claim 7, wherein the polypeptide is an antigenic polypeptide.

9. The recombinant fowlpox virus of claim 7, further comprising a foreign DNA sequence which 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 antigenic polypeptide is derived or is derivable from the group consisting of infectious laryngotracheitis virus glycoprotein B; infectious laryngotracheitis virus gD; 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 polypeptide; infectious bronchitis virus spike polypeptide; infectious bronchitis virus matrix polypeptide, and infectious bronchitis virus nucleocapsid.

12. The recombinant fowlpox virus according to any one of claims 1 to 11, wherein the foreign DNA sequence is placed operably under control of a promoter.

13. The recombinant fowlpox virus of claim 12, wherein the promoter is an endogenous upstream poxvirus promoter.

14. The recombinant fowlpox virus of claim 12, wherein the promoter is a heterologous upstream promoter. The recombinant fowlpox virus of claim 14, wherein the promoter is selected P.\OPER\MRO\95219-98 cinldoc-21A9NyM -91 from the group consisting of: synthetic pox viral promoter, pox synthetic early promoter 1 late promoter 2, pox synthetic late promoter 1, pox synthetic late promoter 2 early promoter 2, pox synthetic late promoter 2, and pox synthetic early promoter.

16. The recombinant fowlpox virus of claim 1, designated S-FPV-072.

17. The recombinant fowlpox virus of claim 1, designated S-FPV-079.

18. A vaccine for immunising an animal against fowlpox virus which comprises an effective immunising amount of the recombinant fowlpox virus according to any one of claims 1 to 17 and a suitable carrier.

19. A method of immunising an animal against an animal pathogen which comprises administering to the animal an effective immunising dose of the vaccine of claim 18.

20. The recombinant fowlpox virus according to any one of claims 1 to 17 substantially 15 as hereinbefore described with reference to the accompanying Figures and/or Examples.

21. The vaccine of claim 18 substantially as hereinbefore described with reference to the Figures and/or Examples. :22. The method of claim 19 substantially as hereinbefore described with reference to S. 20 the Figures and/or Examples. DATED this 21 st day of September, 2000. Syntro Corporation By DAVIES COLLISON CAVE Patent Attorneys for the Applicants