Note: Descriptions are shown in the official language in which they were submitted.
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RECOMBINANT FOWLPOX Vl~U~S AND USES l~REOF
Within this application several publications are
referenced by arabic numerals within parentheses. Full
citations for these references may be found at the end of
the specification immediately preceding the claims. The
disclosures of these publications in their entireties are
hereby incorporated by reference into this application in
order to more fully describe the state of the art to
which this invention pertains.
BACKGROUND OF ~1~ lNV~-llON
The present invention relates to recombinant fowlpox
virus useful in live vaccine to protect fowl against
Newcastle disease virus and fowlpox virus.
The ability to isolate DNA and clone this isolated DNA
into bacterial plasmids has greatly expanded the
approaches available to make viral vaccines. The method
used to make the present invention involve modifying
cloned DNA sequences by insertions, deletions and single
or multiple base changes. The modified DNA is then
inserted into a viral genome, and the resulting virus may
then be used in a vaccine to elicit an immune response in
a host animal and provide protection to the animal
against disease.
Fowlpox virus (FPV) is a member o the poxviridiae family
of viruses. There are two sub~amilies in this
classification, and they are differentiated based upon
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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) (3~)
and lipid (5~), but in FPV the lipid component is -1/3 of
the dry weight. Polyacrylamide gel electrophoresis
(PAGE) of solubilized virions indicates that there are
~100 different proteins associated with the viruses that
include: structural polypeptides, enzymes associated with
translation of messenger ribonucleic acid (mRNA), enzymes
involved in RNA synthesis, and enzymes associated with
DNA replication. The genome of poxviruses consists
double-stranded DNA that varies in base composition (32
G+C to 64~ G+C) and length (140 kilobasepairs [kb] to 280
kb for FPV) depending upon individual virus. The
complete nucleotide sequence of the vaccina virus (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
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upon the specific poxvirus and strain (from 1~ to 30~ of
different mature W 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.
c
Fowlpox is unique throughout the world, but because its
host-range is limited to birds it is not considered to be
a public health hazard. All chickens can be infected by
the virus with a resulting decline in the growth rate of
the bird and temporary decreases in egg production.
Usually, transmission of FPV occurs through physical
contact of injured skin, but there are reports that the
virus is also transmitted via arthropod vectors. After
an incubation period of four to ten days, the disease is
typically manifested in the following ways: skin lesions
in non-~eathered areas, lesions of the nasal passages,
and lesions of the mouth. A normal FPV infection usually
lasts three to four weeks, and afterward the bird is
conferred life-long immunity to the disease.
Currently, conventionally derived FPV vaccines are being
used in commercial settings to provide protection to
chickens and turkeys. Typically, the vaccine viruses are
attenuated by serial passage in cell culture selecting
for strains that have altered growth and/or virulence
properties. The modified live vaccine is prepared by
growth in vitro in chicken embryo fibroblast cells or by
growth on the chorioallantoic membrane of the chicken
embryo. The vaccine virus is given to birds
subcutaneously.
The present invention concerns the use of FPV as a vector
for the delivery of specific vaccine antigens to poultry.
The idea of using live viruses as delivery systems for
antigens (vectoring) has a long history that is
associated with introduction o~ the first live viral
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--4--
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
foreign gene of interest (GOI), its suitability ~or
genetic engineering, its probability o~ being licensed by
regulatory agencies, etc. are all ~actors in the
configuration. The prior art does not teach these
questions of utility.
The presently preferred method for creating recombinant
poxviruses uses a plasmid of bacterial origin that
contains at least one cassette consisting of a poxvirus
promoter followed by the gene of interest. The
cassette(s) is flanked by poxvirus genomic DNA sequences
that direct the gene o~ interest to the corresponding
homologous nonessential region o~ the viral genome by
homologous recombination. Cells are initially in~ected
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
o~ interest be regulated by a promoter o~ poxvirus
origin. There are three characteristic poxvirus
promoters that are di~ferentiated based upon their
temporal regulation of gene expression relative to the
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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 non-
essential regions of DNA in various parts of the genome,
and that modifications of these regions can either
attenuate the virus, leading to a non-pathogenic strain
from which a vaccine may be derived, or give rise to
genomic instabilities that yield mixed populations of
virus. The degree of attenuation of the virus is
important to the utility of the virus as a vaccine.
Insertions or deletions which cause too much attenuation
or genetic deletions which cause too much attenuation or
genetic=instability of the virus will result in a vaccine
that fails to elicit an adequate immune response.
Although several examples of deletions/insertions are
known for poxviruses, the appropriate configuration is
not readily apparent.
Thus far, gene expression from foreign genes of interest
have been inserted into the genome of poxviruses has been
obtained for ~ive dif~erent pox viruses: vaccinia, canary
pox, pigeon pox, raccoon pox and fowlpox. Vaccinia virus
is the classically studied poxvirus, and it has been used
extensively to vector foreign genes of interest; it is
the subject of U.S. Patents 4,603,112 and 4,722,848.
Raccoon pox (Esposito, et al., 1988) and Canary pox
(Taylor, et al., 1991) have bene used to express antigens
from the rabies virus. More recently, FPV has been used
to vector a number of different foreign gene of interest,
and is the subject o~ patent applications (EPA 0 284 416,
PCT WO 89/03429, PCT WO 89/12684, PCT WO 91/02072, PCT WO
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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 re~uirement for
control of the disease. Typically, the gene of interest
encodes imml7nogenic polypeptides of the pathogen, and may
represent surface proteins, secreted proteins and
structural proteins.
One relevant avian pathogen that is a target for FPV
vectoring in the present invention is Infectious
Laryngotracheitis virus (ILT). ILT is a member of the
herpesviridiae family, and this pathogen causes an acute
disease of chickens which is characterized by respiratory
depression, gasping and expectoration of bloody exudate.
Viral replication is limited to cells of the respiratory
tract, where in the trachea the infection gives rise to
tissue erosion and hemorrhage. In chickens, no drug has
been effective in reducing the degree of lesion formation
or in decreasing clinical signs. Vaccination of birds
with various modified forms of the ILT virus derived by
cell passage and/or tedious regimes of administration
have conferred acceptable protection in susceptible
chickens. Because of the degree of attenuation of
current ILT vaccines, care must be taken to assure that
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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,
vaccination has been used to prevent disease, but because
of maternal antibody interference, life-span of the bird
and route of administration, the producer needs to adapt
immunization protocols to fit specific needs.
Marek's disease of poultry is a lymphoproliferative tumor
producing disease of poultry that primarily af~ects 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 o~ 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 o~ the various vaccine
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~ types. Such polyvalent vaccines while generally ore
ef~ective in disease control, complicate the vaccine
regime.
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SU~ RY OF I~IE lNV~N'l'lON
This invention provides a recombinant ~owlpox virus
comprising a foreign DNA sequence inserted into the
~owlpox virus genomic DNA, wherein the ~oreign DNA
sequence is inserted within a non-essential region of the
fowlpox virus genomic DNA and is capable o~ being
expressed in a fowlpox virus in~ected host cell.
The invention ~urther provides homology vectors, vaccines
and methods o~ immunization.
-10-
BRIEF DESCRIPTION OF THE FIGURES
Figures 1A-1C:
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.
Figures 2A-2D:
Detailed description of fowlpox virus S-FPV-099 and
S-FPV-101 and the DNA insertion in Homology Vector
751-07.D1. Diagram showing the orientation of DNA
fragments assembled in plasmid 751-07.D1. The
origin of each fragment is indicated in the table.
The sequences located at each of the junctions
between fragments is also shown. Figures 2A-2D show
the sequences located at Junction A (SEQ ID NO: ),
(SEQ ID NO: ), C (SEQ ID NO: ), D (SEQ ID NO: ) and
E (SEQ ID NO: ) between fragments and the sequences
located at the junctions. The restriction sites
used to generate each fragment as well as synthetic
linker sequences which are used to joint the
fragments are described for each junction. The
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--11--
location of several gene coding regions and
regulatory elements is also given. The following
two conventions are used: numbers in parentheses,
(), refer to amino acids, and restrictions sites in
brackets, [], indicate the remnants of sites which
are destroyed during construction. The following
abbreviations are used: fowlpox virus (FPV), chicken
interferon (cIFN), Escherichia coli (E. coli), pox
synthetic late promoter 2 early promoter 2 (LP2EP2),
pox synthetic late promoter 1 (LP1), base pairs
(BP), polymerase chain reaction (PCR).
Fiqures 3A-3D:
Detailed description of fowlpox virus S-FPV-100 and
the DNA insertion in Homology Vec~or 751-56.C1.
Diagram showing the orientation of DNA fragments
assembled in plasmid 751-56.C1. The origin of each
fragment is indicated in the table. The sequences
located at each of the junctions between fragments
is also shown. Figures 3A-3D show the sequences
located at Junction A (SEQ ID NOS: ), (SEQ ID NO: ),
C (SEQ ID NO: ), D (SEQ ID NO: ) and E (SEQ ID NO:
) between fragments and the sequences located at the
junctions. The restriction sites used to generate
each fragment as well as synthetic linker sequences
which are used to join the fragments are described
for each junction. The location of several gene
coding regions and regulatory elements is also
given. The following two conventions are used:
numbers in parentheses, (), refer to amino acids,
and restrictions sites in brackets, [], indicate the
remnants of sites which are destroyed during
construction. The following abbreviations are used:
fowlpox virus (FPV), chicken myelomoncytic growth
factor (cMGF), Escherichia coli (E. coli), pox
synthetic late promoter 2 early promoter 2 (LP2EP2),
pox synthetic late promoter 1 (LP1), base pairs
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(BP), polymerase chain reaction (PCR).
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DETz~TT~n DESCRIPTION OF THE lNV~;N'l'lON
This invention provides a recombinant fowlpox virus
comprising a foreign DNA sequence inserted into the
fowlpox virus genomic DNA, wherein the foreign DNA
sequence is inserted within a 2.8 kB EcoRI fragment of
the fowlpox virus genomic DNA and is capable of being
expressed in a fowlpox virus infected host cell.
In one embodiment the foreign DNA sequence is inserted
within a SnaBI restriction endonuclease site within the
approximately 2.8 kB EcoRI ~ragment of the fowlpox virus
genomic DNA.
This invention provides a recombinant fowlpox virus
comprising a foreign DNA sequence inserted into the
fowlpox virus genomic DNA, wherein the foreign DNA
sequence is inserted within a 3. 5 kB EcoRI fragment of
the fowlpox virus genomic DNA and is capable of being
expressed in a fowlpox virus infected host cell.
In ~one embodiment the recombinant fowlpox virus the
foreign DNA se~uence is inserted within a HpaI
restriction endonuclease site within the approximately
3.5 kB EcoRI fragment of the fowlpox virus genomic DNA.
The present invention provides a recombinant fowlpox
virus comprising a foreign DNA se~uence inserted into the
fowlpox virus genomic DNA, wherein the foreign DNA
sequence is inserted within a 4.2 kB EcoRI fragment of
the fowlpox virus genomic DNA and is capable of being
expressed in a fowlpox virus infected host cell.
.
In one embodiment of the recombinant fowlpox virus
3 5 foreign DNA sequence is inserted within a MluI
restriction endonuclease site within the approximately
4.2 kB EcoRI fragment of the fowlpox virus genomic DNA.
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The invention provides a recombinant fowlpox virus
comprising a foreign DNA sequence inserted into the
fowlpox virus genomic DNA, wherein the ~oreign DNA
sequence is inserted within a non-essential region of the
fowlpox virus genomic DNA and is capable of being
expressed in a fowlpox virus infected host cell.
In one embodiment this invention provides a recombinant
fowlpox virus wherein the foreign DNA sequence is
inserted into an open reading frame within the non-
essential region the fowlpox virus genomic DNA.
For purposes of this invention, "a recombinant fowlpox
virus capable of replication" is a live fowlpox virus
which has been generated by the recombinant methods well
known to those of skill in the art, e.g., the methods set
forth in HOMOLOGOUS RECOMBINATION PROCEDURE FOR
GENERATING RECOMBINANT FPV in Materials and Methods and
has not had genetic material essential for the
replication of the recombinant fowlpox virus deleted.
The invention further provides a foreign DNA sequence or
foreign RNA which encodes a polypeptide. Pre~erably, the
polypeptide is antigenic in the ~n;m~l. Preferably, this
antigenic polypeptide is a linear polymer of more than 10
amino acids linked by peptide bonds which stimulates the
animal to produce antibodies.
The invention further provides a recombinant fowlpox
virus capable of replication which contains a foreign DNA
encoding a polypeptide which is a detectable marker.
Preferably the detectable marker is the polypeptide E.
coli ,~-galactosidase or E. coli beta-glucuronidase.
In one embodiment of the recombinant fowlpox virus the
foreign DNA sequence encodes a cytokine. In another
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embodiment the cytokine is chicken myelomonocytic growth
factor (cMGF) or chicken interferon (cIFN). Cytokines
include, but are not limited to: transforming growth
- factor beta, epidermal growth ~actor family, fibroblast
growth factors, hepatocyte growth factor, insulin-like
growth ~actor, vascular endothelial growth factor,
interleukin 1, IL-1 receptor antagonist, interleukin-2,
interleukin-3, interleukin-4, interleukin-5, interleukin-
6, IL-6 soluble receptor, interleukin-7, interleukin-8,
interleukin-9, interleukin-10, interleukin-11,
interleukin-12, interleukin-13, angiogenin, chemokines,
colony stimulating factors, granulocyte-macrophage colony
stimulating factors, erythropoietin, interferon,
interferon gamma, c-kit ligand, leukemia inhibitory
factor, oncostatin M, pleiotrophin, secretory leukocyte
protease inhibitor, stem cell ~actor, tumor necrosis
factors, and soluble TNF receptors. These cytokines are
from humans, bovine, equine, feline, canine, porcine or
avian.
This invention provides a recombinant fowlpox virus
further comprising a newcastle disease virus
hemagglutinin (NDV HN), or a newcastle disease virus
fusion (NDV F).
Antigenic polypeptide of a human pathogen which are
derived from human herpesvirus include, but are not
limited to: hepatitis B virus and hepatitis C virus
hepatitis B virus surface and core antigens, hepatitis C
virus, human immunodeficiency virus, herpes simplex
virus-1, herpes simplex virus-2, human cytomegalovirus,
Epstein-Barr virus, Varicella-Zoster virus, human
herpesvirus-6, human herpesvirus-7, human influenza,
measles virus, hantaan virus, pneumonia virus,
rhinovirus, poliovirus, human respiratory syncytial
virus, retrovirus, human T-cell leukemia virus, rabies
virus, mumps virus, malaria (Plasmodium falciparum),
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Bordetella pertussis, Diptheria, Rickettsia prowazekii,
Borrelia berfdorferi, Tetanus toxoid, malignant tumor
antigens.
The antigenic polypeptide of an equine pathogen can
derived from equine influenza virus, or equine
herpesvirus. In one embodiment the antigenic polypeptide
is equine influenza neuraminidase or hemagglutinin.
Examples of such antigenic polypeptide are equine
influenza virus type A/Alaska 91 neuraminidase, equine
influenza virus type A/Prague 56 neuraminidase, equine
influenza virus type A/Miami 63 neuraminidase, equine
influenza virus type A/Kentucky 81 neuraminidase, equine
influenza virus type A/Kentucky 92 neuraminidase equine
herpesvirus type 1 glycoprotein B, equine herpesvirus
type 1 glycoprotein D, Streptococcus e~ui, equine
infectious anemia virus, equine encephalitis virus,
equine rhinovirus and equine rotavirus.
The present invention further provides an antigenic
polypeptide which includes, but is not limited to: hog
cholera virus gEl, hog cholera virus gE2, swine influenza
virus hemagglutinin, neuromanidase, matrix and
nucleoprotein, pseudorabies virus gB, gC and gD, and PRRS
virus ORF7.
For example, the antigenic polypeptide of derived from
infectious bovine rhinotracheitis virus gE, bovine
respiratory syncytial virus equine pathogen can derived
from equine influenza virus is bovine respiratory
syncytial virus attachment protein (BRSV G), bovine
respiratory syncytial virus fusion protein (BRSV F),
bovine respiratory syncytial virus nucleocapsid protein
(BRSV N), bovine parainfluenza virus type 3 fusion
protein, and the bovine parainfluenza virus type 3
hemagglutinin neuraminidase
-
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-17-
The present invention provides a recombinant fowlpox
virus wherein the foreign DNA sequence encodes an
antigenic polypeptide which is derived or derivable from
a group consisting of: feline ;mmllnodeficiency virus gag,
feline immunodeficiency virus env, infectious
laryngotracheitis virus glycoprotein B, infectious
laryngotracheitis virus gI, infectious laryngotracheitis
virus gD, infectious bovine rhinotracheitis virus
glycoprotein G, infectious bovine rhinotracheitis virus
glycoprotein E, pseudorabies virus glycoprotein 50,
pseudorabies virus II glycoprotein B, pseudorabies virus
III glycoprotein C, pseudorabies virus glycoprotein E,
pseudorabies virus glycoprotein H, marek's disease virus
glycoprotein A, marek's disease virus glycoprotein B,
marek's disease virus glycoprotein D, newcastle disease
virus hemagglutinin or neuraminadase, newcastle disease
virus fusion, infectious bursal disease virus VP2,
infectious bursal disease virus VP3, infectious bursal
disease virus VP4, infectious bursal disease virus
polyprotein, infectious bronchitis virus spike,
infectious bronchitis virus matrix, and chick anemia
virus.
The present invention provides a recombinant ~owlpox
virus wherein the foreign DNA sequence is under control
of a promoter. In one embodiment the foreign DNA sequence
is under control of an endogenous upstream poxvirus
promoter. In another embodiment the i~oreign DN~ sequence
is under control of a heterologous upstream promoter. In
another embodiment the promoter is selected from a group
consisting of: synthetic pox viral promoter, pox
synthetic late promoter 1, pox synthetic late promoter 2
early promoter 2, pox OlL promoter, pox I4L promoter, pox
I3L promoter, pox I2L promoter, pox IlL promoter, pox
=ElOR promoter, HCMV immediate early, BHV-1.1 VP8, marek's
disease virus glycoprotein A, marek's disease virus
glycoprotein B, marek's disease virus glycoprotein D,
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laryngotracheitis virus glycoprotein I, infectious
laryngotracheitis virus glycoprotein B, and in~ectious
laryngotracheitis virus gD.
The present invention also provides a recombinant fowlpox
virus designated S- FPV-097. The S-FPV-097 has been
deposited on February 25, 1994 pursuant to the Budapest
Treaty on the International Deposit of Microorganisms for
the Purposes of Patent Procedure with the Patent Culture
Depository of tke American Type Culture Collection, 12301
Parklawn Drive, Rockville, Maryland 20852 U.S.A. under
ATCC Accession No. VR 2446.
The present invention also provides a vaccine which
comprises an e~fective ;mml~n;zing 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
ef~ective immunizing dose o~ the vaccine o~ 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 e~fective
;mmllnizing 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
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invention also provides a method o~ ;mmnn;zing an animal,
particularly poultry, against disease caused by fowlpox
virus, Newca~tle disease virus and infectious
laryngotracheitis virus. This method comprises
a~min;stering to the animal an ef~ective 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 ~owlpox
virus designated S-FPV-074. The present invention also
provides a vaccine which comprises an e~fective
;mml~n;zing 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 pre~erred. The present
invention also provides a method of ;~m1ln;zing an animal,
particularly poultry, against disease caused by fowlpox
vlrus 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 a~m; n; .~tered 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 ~owlpox
virus designated S-FPV-081. The present invention also
provides a vaccine which comprises an e~~ective
;mml~n;zing 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 pre~erred. The present
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invention also provides a method o~ imm~ln;zing an animal,
particularly poultry, against disease caused by ~owlpox
virus and Marek's disease virus. This method comprises
administering to the animal an e~ective immunizing dose
o~ the vaccine of the present invention. The vaccine may
be a~mi n; .~tered by any o~ the methods well known to those
skilled in the art, ~or 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 ~owlpox
virus designated S-FPV-085. The present invention also
provides a vaccine which comprises an e~ective
~mmtln; zing amount o~ the recombinant virus designated S-
FPV-085 and a suitable carrier. The vaccine may contain
either inactivated or live ~owlpox virus S-FPV-085,
although live virus is presently preferred. The present
invention also provides a method of ;mmlln;zing an animal,
particularly poultry, against disease caused by ~owlpox
virus, Newcastle disease virus, infectious
laryngotracheitis virus and Marek's disease virus. This
method comprises administering to the animal an e~ective
;mmlln;zing dose o~ the vaccine o~ the present invention.
The vaccine may be administered by any o~ the methods
well known to those skilled in the art, ~or 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 ~owlpox
virus designated S-FPV-082, S-FPV-083, S-FPV-099, S-FPV-
100, and S-FPV-101.
. .
Suitable carriers for use with the recombinant ~owlpox
virus vaccines o~ the present invention are those well
CA 02223~91 1997-12-04
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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 o~ 102-109 PFU/dose. Preferably, the e~fective
immunizing amount is from about 103-105 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.
CA 02223~91 1997-12-04
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MATERIAL AND ~l~OVS
PREPARATION OF FOWLPOX VIRUS STOCK SAMPLES. Fowlpox virus
samples were prepared by infecting chicken embryo
fibroblast (CEF) cells at a multiplicity of in~ection of
0.01 PFU/cell in a 1:1 mixture of HAM's F10 medium and
Medium 199 (F10/199) containing 2 mM glutamine and
antibiotics (referred to as CEF negative medium). Prior
to infection, the cell monolayers were washed once with
CEF negative medium to remove fetal bovine serum. The
FPV contained in the initial inoculum (0.5 ml for 10 cm
plate; 10 ml for T175 cm flask) was allowed to absorb
onto the celi 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~C in 5~ CO2 until cytopathic effect
was complete. The medium and cells were harvested,
frozen at -70~C, thawed and dispensed into 1.0 ml vials
and refrozen at -70~C. Virus titers typically range
between 108 and 10~ PFU/ml.
PREPARATION OF FPV DNA. For fowlpox virus DNA isolation,
a confluent monolayer of CEF cells in a T175 cm2 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~C to 37~C).
After the last thaw, the cells (on ice) were sonicated
two times for 30 seconds each with 45 seconds cooling
time in between. Cellular debris was removed by
centrifuging (Sorvall RC-5B Superspeed Centrifuge) at
3000 rpm for 5 minutes in an HB4 rotor at 4~C. FPV
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virions, present in the supernatant, were pelleted by
centri~ugation at 15,000 rpm ~or 20 minutes at 4~C in a
SS34 rotor (Sorvall) and resuspended in 10mM Tris (pH
7.5). This ~raction was then layered onto a 36~ sucrose
gradient (w/v in 10 mM Tris pH 7.5) and centrifuged
(Beckman L8-70M Ultracentri~uge) at 18,000 rpm ~or 60
minutes in a SW41 rotor at 4~C. The virion pellet was
resuspended in 1.0 ml o~ 10 mM Tris pH 7.5 and sonicated
on ice for 30 seconds. This ~raction was layered onto a
20~ to 50~ continuous sucrose gradient and centri~uged at
16,000 rpm for 60 minutes in a SW41 rotor at 4~C. The
FPV virion band located about three quarters down the
gradient was harvested, diluted with 20~ sucrose and
pelleted by centri~ugation at 18,000 rpm ~or 60 minutes
in a SW41 rotor at 4~C. The resultant pellet was then
washed once with 10 mM Tris pH 7.5 to remove traces o~
sucrose and ~inally resuspended in 10mM Tris pH 7.5. FPV
DNA was then extracted ~rom the puri~ied virions by lysis
(~our hours at 60~C) ~ollowing the addition o~ EDTA, SDS,
and proteinase K to final concentrations o~ 20 mM, 0.5~
and 0.5 m~/ml, respectively. A~ter digestion, three
phenol-chloroform (1:1) extractions were conducted and
the sample precipitated by the addition of two volumes o~
absolute ethanol and incubated at -20~C ~or 30 minutes.
The sample was then centri~uged in an Eppendor~ mini~uge
~or ~ive minutes at ~ull speed. The supernatant was
decanted, and the pellet air dried and rehydrated in 0.01
M Tris pH 7.5, lmM EDTA at ~~C.
MOLECULAR BIOLOGICAL TECHNIQUES. Techniques ~or the
manipulation of bacteria and DNA, including such
procedures as digestion with restriction endonucleases,
gel electrophoresis, extraction o~ DNA ~rom gels,
ligation, phosphorylation with kinase, treatment with
phosphatase, growth o~ bacterial cultures, trans~ormation
o~ bacteria with DNA, and other molecular biological
methods are described by Maniatis et al ( 1982) and
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- -24-
Sambrook et al (1989). Except as noted, these were used
with minor variation.
DNA SEQu~N~l~. Sequencing was per~ormed using the BRL
Sequenase Kit and 35S-dATP (NEN) Reactions using both
the dGTP mixes and the dITP mixes were performed to
clarify areas of compression. Alternatively, compressed
areas were resolved on formamide gels. Templates were
double-stranded plasmid subclones or single stranded M13
subclones, and primers were either made to the vector
just outside the insert to be sequenced, or to previously
obtained sequence. Sequence obtained was assembled and
compared using Dnastar software. Manipulation and
comparison of sequences obtained was performed with
Superclone and Supersee programs from Coral Software.
STRATEGY FOR THE ~N~llw~-llON OF ~YN-l~-llC POX VIRAL
PROMOTERS. For recombinant fowlpox vectors synthetic pox
promoters of~er several advantages including the ability
to control the strength and timing of foreign gene
expression. We chose to design four promoter cassettes
EP1 (SEQ ID NO:8, LP1 (SEQ ID NO:9), EP2 (SEQ ID NO:10),
and LP2 (SEQ ID NO:11) based on promoters that have been
de~ined in the vaccinia virus (Bertholet et al. 1986,
Davidson and Moss, 1989a, and Davidson and Moss, 1989b).
Each cassette was designed to contain the DNA.sequences
defined in vaccina flanked by restriction sites which
could be used to combine the cassettes in any order or
combination. Initiator methionines were also designed
into each cassette such that in~rame fusions could be
made at either EcoRI or BamHi sites. A set o~
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 t
according to standard techniques and cloned into the
appropriate homology vectors.
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-25-
cDNA CLONING PRO~uK~. 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 Clonlng Kit that is very similar to the
procedures u5ed by applicants, and contains a set of
reagents and protocols that may be used to duplicate our
results.
For cloning virus mRNA species, a host cell line
sensitive to infection by the virus was infected at 5-10
plaque forming units per cell. When cytopathic effect
was evident, but before total destruction, the medium was
removed and the cells were lysed in 10 mls lysis buffer
(4 M guanidine thiocyanate, 0.1~ antifoam A, 25 mM sodium
citrate pH 7.0, 0.5~ N-lauroyl sarcosine, 0.1 M beta-
mercaptoethanol). The cell lysate was poured into a
sterilized Dounce homogenizer and homogenized on ice 8-10
times until the solution was homogenous. For RNA
purification, 8mls of cell lysate were gently layered
over 3. 5 mls of CsCl solution (5.7 M CsCl, 25 mM sodium
citrate pH 7.0) in a Beckman SW41 centrifuge tube. The
samples were centrifuged for 18 hrs at 20~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 ~l glass distilled water,
and 2.~ mls of guanidine solution (7.5 M guanidine-HCl,
2 5 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~C for 18 hrs to precipitate RNA. The
precipitate was collected by centrifugation in a Sorvall
centrifuge for 10 min at 4~C at 10000 rpm in an SS34
rotor. The pellet was dissolved in 1.0 ml distilled
water, recentrifuged at 13000 rpm, and the supernatant
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saved. RNA was re-extracted ~rom 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~C
for 18 hrs. The precipitated RNA was collected by
centrifugation in the SS34 rotor at 4~C for 10 min at
10000 rpm. The pellet was dissolved in 1 ml distilled
water and the concentration taken by adsorption at
A260/280. The RNA was stored at -70~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+ R~NA 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 buf~er and
eluted again in elution buffer. The sample was
precipitated with 200 mM sodium acetate and 2 volumes
cold ethanol at -20~C for 18 hrs. The RNA was
resuspended in 50 ~l distilled water.
Ten ~g poly-A+ RNA was denatured in 20 mM methyl mercury
hydroxide for 6 min at 22~C. ~-mercaptoethanol was added
to 75 mM and the sample was incubated for 5 min at 22~C.
The reaction mixture for first strand cDNA synthesis in
0.25 ml contained i ~g oligo-dT primer (P-L Bio-
chemicals) or 1 ~g synthetic primer, 28 units placental
ribonuclease inhibitor (Bethesda Research Labs #5518SA),
100 mM Tris pH 8.3, 140 mM KCl, 10 mM MgCl2, 0.8 mM dATP,
dCTP, dGTP, and dTTP (Pharmacia), 100 microcuries 32P-
labeled dCTP (New England Nuclear #NEG-013H), and 180
units AMV reverse transcriptase (Molecular Genetics
Resources #MG 101). The reaction was incubated at 42~C
-
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-27-
for 90 min, and then was terminated with 20 mM EDTA pH
8Ø The sample was extracted with an equal volume of
phenol/chloroform (1:1) and precipitated with 2 M
- ammonium acetate and 2 volumes of cold ethanol -20~C for
3 hrs. After precipitation and centrifugation, the
pellet was dissolved in 100 ~l 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
8.0, 100 mM NaCl). The leading edge of the eluted DNA
fractions were pooled, and DNA was concentrated by
lyophilization until the volume was about 100 ~l, then
the DNA was precipitated with ammonium acetate plus
ethanol as above.
The entire first strand sample was used for second strand
reaction which followed the Gubler and Hoffman (1983)
method except that 50 ,ug/ml dNTP's, 5.4 units DNA
polymerase I (Boerhinger M~nnheim #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/chloro~orm
extracted and precipitated. The DNA was resuspended in
10 ~l distilled water, treated with 1 ~g RNase A for 10
min at 22~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 ,ul
water.
Oligo-dC tails were added to the DNA to ~acilitate
cloning. The reaction contained the DNA, 100 mM
potassium cacodylate pH 7. 2, 0.2 mM dithiothreitol, 2 mM
CaCl2, 80 ~umoles dCTP, and 25 units terminal
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WO 96/40880 PCT~US96/11187
- -28-
deoxynucleotidyl transferase (Molecular Genetic Resources
#S1001) in 50 ~l. After 30 min at 37~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 ~l of 0.01 M Tris pH
7.5, 0.1 M NaCl, 1 mM EDTA pH 8.0 at 65~C for 2 min and
then 57~C for 2 hrs. Fresh competent E. coli DX-1 cells
were prepared and trans~ormed as described by ~n~h~n
(1983) using half the annealed cDNA sample in twenty 200
~l aliquots of cells. Transformed cells were plated on
L-broth agar plates plus 10 ~g/ml tetracycline. Colonies
were screened for the presence of inserts into the
ampicillin gene using Ampscreen\ (Bethesda ~esearch Labs
#5537 UA), and the positive colonies were picked for
analysis.
HOMOLOGOUS RECOMBINATION PRO~u~ FOR ~.NF.R~TING
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 Blo-Pox~ from
Agri-Bio Corporation, Gainsville, Georgia) at a
multiplicity of infection of 0.01 PFU/cell to introduce
replicating FPV (i.e. DNA synthesis) into the cells. The
plasmid homology vector DNA is then transfected into
these cells according to the "Infection-Transfection
Procedure".
INFECTION-~R~N.~ECTION 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
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WO 96/40880 PCT~US96/11187
-29-
negative medium and incubated at 37~C in a humidified 5~
CO2 incubator for five hours. The transfection procedure
used is essentially that recommended for Lipofectin~
Reagent (BRL). Briefly, for each 6 cm plate, 15
micrograms of plasmid DNA were diluted up to 100
microliters with H2O. Separately, 50 micrograms of
Lipofectin~ Reagent were diluted to lO0 microliters with
H2O. The 100 microliters of diluted Lipofectin~ Reagent
were added dropwise to the diluted plasmid DNA contained
in a polystyrene, 5 ml, snap cap tube and mixed gently.
The mixture was then incubated for 15-20 minutes at room
temperature. During this time, the virus inoculum was
removed from the 6 cm plates and the cell monolayers
washed once with CEF negative medium. Three mls of CEF
negative medium were added to the plasmid DNA/lipofectin
mixture and the contents pipetted onto the cell
monolayer. Following overnight (about 16 hours)
incubation at 37~C in a humidified 5~ CO2 incubator, the
medium was removed and replaced with 5 ml CEF complete
medium. The cells were incubated at 37-C in 5~ C~2 for 3-
7 days until cytopathic effect from the virus was 80-
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".
PI~QUE FnrRRTnIz~TIoN PROCEDlnRE FOR PlnRl~-YlN~ RECO~DBIN~NT
FPV. CEF cell monolayers were infected with various
dilutions of the infection/transfection viral stocks,
overlaid with nutrient agarose media (equal volumes of
1.2~-1.4~ 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
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-30-
dish and stored at 4~C. The CEF monolayer was overlaid
with a pre-wetted (PBS) NC membrane and pressure applied
to transfer the monolayer to the NC membrane. Cells
contained on the NC membrane were then lysed by placing
the membranes in 1.5 ml of 1.5 M NaCl 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
80~C for one hour. After this period the membranes were
prehybridized with a solution containing 6X SSC, 3~ skim
milk, 0.5~ SDS, salmon sperm DNA (50 ,ug/ml) and incubated
at 65~C for one hour. Radio-labeled probe DNA (alpha32P-
dCTP) was added and incubated at 65~C overnight (12
hours). After hybridization the NC membranes were washed
two times (30 minutes each) with 2X SSC at 65~C, followed
by two additional washes at 65~C with 0. 5X SSC. The NC
membranes were dried and exposed to X-ray film (Kodak X-
OMAT, AR) at -70~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~C. Typically, 5-6 rounds of plaque purification were
required to ensure purity of the recombinant virus.
2 5 SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINAN~ FPV
USING BLACK PLAQUE ASSAYS. To analyze expression of
foreign antigens expressed by recombinant fowlpox
viruses, monolayers of CEF cells were infected with
recombinant FPV, overlaid with nutrient agarose media and
incubated for 6-7 days at 37~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
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-31-
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
ncubated 15-30 minutes at room temperature with freshly
prepared substrate solution (100 ~g/ml 4-chloro-1-
naphthol, 0.003~ H2O2 in PBS). Plaques expressing the
correct antigen stain black.
SCREEN FOR RECOMBINA~T FPV EXPRESSING ENZYM~TIC ~Z~RRRR
GENES. When the E. coli ~-galactosidase (lacZ) or ~-
glucuronidase (uidA) marker gene was incorporated into a
recombinant virus the plaques containing recombinants
were visualized by a simple assay. The enzymatic
substrate was incorporated (300 ~g/ml) into the agarose
overlay during the plaque assay. For the l acZ marker
gene the substrates Bluogal~ (halogenated indolyl-~-D-
galactosidase, Bethesda Research Labs) for blue plaques
or CPRG (chlorophenol Red Galactopyranoside, Boehringer
m;:lnnh~im) for red plaques were used. For the uidA
marker gene the substrate X-Glucuro Chx (5-bromo-4-
chloro-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.
RNA ISOL~TED FROM CONCANAVALIN A STIMULATED CHICKEN
SPLEEN OELLS. Chicken spleens were dissected from 3 week
old SPAFAS hatched chicks, washed, and disrupted through
a syringe/needle to release cells. After allowing stroma
and debri to settle out, the cells were pelleted and
washed twice with PBS. The cell pellet was treated with
a hypotonic lysis buffer to lyse red blood cells, and
splenocytes were recovered and washed twice with PBS.
Splenocytes were resuspended at 5 x 106 cells/ml in RPMI
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W O 96/40880 PCT~US96/lllX7
containing 5~ FBS and 5 ~g/ml Concanavalin A and
incubated at 39O for 48 hours. Total RNA was isolated
from the cells using guanidine isothionate lysis reagents
and protocols ~rom the Promega RNA isolation kit (Promega
Corporation, Madison WI). 4~g of total RNA was used in
each 1st strand reaction containing the appropriate
antisense primers and AMV reverse transcriptase (Promega
Corporation, Madison WI). cDNA synthesis was performed in
the same tube following the reverse transcriptase
reaction, using the appropriate sense primers and Vent~
DNA polymerase (Life Technologies, Inc. Bethesda, MD).
HOMOLOGY VECTOR 451-79.95. The plasmid 451-79.95 was
constructed for the purpose of inserting the NDV HN gene
into FPV. A lacZ marker gene followed by the NDV HN gene
was inserted as a cassette into the homology vector 443-
88.14 at the unique 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 promoter LP1 (SEQ ID
NO:9). The second fragment contains the coding region of
E. coli lacZ and is derived from plasmid pJF751 (Ferrari
et al., 1985). Note that the promoter and lacZ gene are
fused so as to express a hybrid protein consisting of 4
amino acids derived from the synthetic promoter followed
by amino acids 10 to 1024 of the lacZ gene. The third
fragment is another copy of the synthetic late promoter
LP1. the fourth fragment contains the coding region of
the NDV HN gene and was derived from the full length HN
cDNA clone. Note that the promoter and HN gene are fused
so as to express a hybrid protein consisting of 4 amino
acids derived from the synthetic promoter followed by
amino acids 2 to 577 of the HN gene. Both genes are in
the opposite transcriptional orientation relative to the
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W O 96/40880 PCTrUS96/11187
-33-
ORF1 gene in the parental homology vector.
HOMOLOGY VECTOR 489-21.1. The plasmid 489-21.1 was
constructed for the purpose o~ 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 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 ~ollowing sources with the synthetic DNA
sequences indicated. The first ~ragment is the synthetic
early/late promoter EPlLP2 (SEQ ID NO:8/SEQ ID NO:11).
The second fragment contains the coding region o~ the NDV
HN gene and was derived ~rom the full length HN cDNA
clone. Note that the promoter and HN gene are fused so
as to express a hybrid protein consisting o~ 4 amino
acids derived ~rom the synthetic promoter ~ollowed by
amino acids 2 to 577 of the HN gene. The HN gene is in
the opposite transcriptional orientation relative to the
ORF in the parental homology vector.
HOMOLOGY VECTORS 502-26.22. The plasmid 502-26.22 was
constructed ~or the purpose of inserting the NDV HN and
F genes into FPV. The NDV HN and F genes were inserted
as a SfiI ~ragment (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 o~ the SfiI is shown in Figures
lA-lC. The inserted Sfi I ~ragment may be constructed
utilizing standard recombinant DNA techniques (Maniatis
et al. and Sambrook et al., 1989), by joining restriction
fragments ~rom the ~ollowing sources with the synthetic
DNA sequences indicated in Figures lA-lC. Fragment 1 is
approximately 1811 base pair AvaII to NaeI restriction
~ragment o~ the ~ull length NDV HN cDNA clone (Bl
strain). Fragment 2 is an approximately 1812 base pair
CA 02223~91 1997-12-04
W O 96/40880 PCT~US96/11187
34
BamHI to PstI restriction fragment of the full length NDV
F cDNA (Bl strain). Fragment 3 is an approximately 235
base pair PstI and ScaI restriction ~ragment of the
plasmid pBR322.
HOMOLOGY VECTOR 502-27 5. The plasmid 502-27.5 was
constructed for the purpose of inserting the NDV F gene
into FPV. A LacZ marker gene ~ollowed 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 beconstructed utilizing st~n~rd recombinant DNA techni~ues
(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 ~ollowed
by amino acids 10 to 1024 of the LacZ gene. The third
fragment is the synthetic early/late promoter EPlLP2 (SEQ
ID NO:8/SEQ ID NO:ll). 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 ~ollowed by amino acid 1 to
544 of the F gene. Both genes are in the opposite
transcriptional orientation relative to the ORF in the
parental homology vector.
HOMOLOGY VECTOR 586-36.6. The plasmid 586-36.6 was
constructed for the purpose of inserting the in~ectious
laryngotracheitis virus (ILT) gB and gD genes into the
FPV. An E. coli ~-glucuronidase uidA marker gene
-
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preceeded by the ILT gB and gD genes was inserted as a
cassette into the homology vector 451-08.22 at the unique
Sf iI 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 EPlLP2 (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 EPlLP2 (SEQ ID NO:8/SEQ ID NO:11). The fourth
fragment contains the coding region of the ILT gD gene
(SEQ ID NO:19) and was derived from an approximately 2060
base pair EcoRI to BclI restriction sub-fragment of the
ILT KpnI genomic restriction fragment #8 (10.6 KB). Note
that the promoter and gD gene are fused so as to express
a hybrid protein consisting of 3 amino acids dervied from
the synthetic promoter followed by amino acids 3 to 434
of the gD gene. The fifth fragment is the synthetic late
promoter LP1 (SEQ ID NO:9) The last fragment contains
the coding region of E. col i uidA and is derived from
plasmid pRAJ260 (Clonetech). Note that the promoter and
uidA gene are fused so as to express a hybrid protein
consisting of 3 amino acids derived from the synthetic
promoter followed by amino acids 1 to 602 of the uidA
gene. All three genes are in the opposite
transcriptional orientation relative to 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
,
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-36-
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 Sal I 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 EPlLP2 (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 ~used 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. col i LacZ and is derived from plasmid pJF751
(Ferrari, et al ., 1985). Note that the promoter and LacZ
gene are fused so as to express a hybrid protein
consisting of 4 amino acids derived from the synthetic
promoter followed by amino acids 10 to 1024 of the LacZ
gene. All three genes are in the opposite
transcriptional orientation relative to ORF1 in the
parental homology vector.
HOMOLOGY VECTOR 538-51.27. The plasmid 538-51.27 was
constructed for the purpose of inserting the genes for
Infectious Bronchitis virus (IBV) Massachusetts Spike
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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
cassette into the homology vector 443-88.14 at the unique
5 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 ~ragment is the synthetic early/late promoter
EPlLP2 (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 EPlLP2 (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 ~rom
an approximately 1500 base pair XbaI to SpeI IBV cDNA
fragment. The fifth fragment is the synthetic late
promoter LP1 (SEQ ID NO: 9). The sixth fragment contains
the coding region o~ E. coli lacZ and is derived ~rom
plasmid pJF751 (Ferrari, et al. 1985).
EOMOLOGY VECTOR 622-49.1. The plasmid 622-49.1 was
con~st~ructed 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 ~ragment 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
EPlLP2 (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
~ragment is the synthetic late promoter LP1 (SEQ ID NO:
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9). The fourth fragment contains the coding region of E.
coli uidA and is derived from plasmid pRAJ260
(Clonetech).
HOMOLOGY VECTORS 584-36.12. The plasmid 584-36.12 was
constructed for the purpose of inserting the NDV HN and
F genes into FPV. The NDV HN and F genes were inserted as
a SfiI fragment into the homology vector 443-88.14 (see
example lB) 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 fragment is shown in
Figures lA-lC. 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 with the
synthetic DNA sequences indicated in Figures lA-lC.
Fragment 1 is an approximately 1811 base pair AvaII to
NaeI restriction fragment of the full length NDV HN cDNA
clone (B1 strain). Fragment 2 is an approximately 1812
base pair BamHI to PstI restriction fragment of the ~ull
length NDV F cDNA (B1 strain). Fragment 3 is an
approximately 235 base pair PstI to ScaI restriction
~ragment 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 ~. coli ~-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 SfiI 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 EPlLP2 (SEQ ID NO:8/SEQ ID NO:11).
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The second fragment contains the coding region of ILTV gB
and is derived from an approximately 3000 base pair ILT
virus genomic EcoRI fragment. Note that the promoter and
gB gene are fused so as to express the complete coding
region of the gB gene (animo acids 1-883). The third
fragment is the synthetic early/late promoter EPlLP2 (SEQ
ID NO:8/SEQ ID NO:11). The fourth fragment contains the
coding region of the ILTV gD gene and was derived from an
approximately 2060 base pair EcoRI to BclI restriction
sub-~ragment of the ILTV KpnI genomic restriction
fragment ~8 (10.6 KB). Note that the promoter and gD gene
are fused so as to express a hybrid protein consisting of
3 amino acids derived from the synthetic promoter
followed by amino acids 3 to 434 of the gD gene. The
fifth fragment is the synthetic late promoter LPl (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.
HOMOLO~Y VECTOR 749-75.82. The plasmid 749-75.82 was used
to insert foreign DNA into FPV. It incorporates an E.
coli ,B-galactosidase (lacZ) marker gene and the
infectious bursal disease virus (IBDV) polymerase gene
flanked by FPV DNA. When this plasmid was used according
to the HOMOLOGOUS RECOMBINATION PROCBDURE FOR GENERATING
RECOMBINANT FPV a virus containing DNA coding for the
foreign genes results. Note that the ,B-galactosidase
(lacZ) marker gene is under the control of a synthetic
late pox promoter (LP1) and the IBDV polymerase gene is
under the control of a synthetic late/early pox promoter
(LP2EP2). The homology vector was constructed utilizing
standard recombinant DNA techniques (11 and 14), by
joining restriction fragments from the following sources
with the appropriate synthetic DNA sequences. The plasmid
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vector was derived from an approximately 2999 base pair
EcoRI restriction ~ragment of pSP64 (Promega). Fragment
1 is an approximately 1184 base pair EcoRI to SnaBI
restriction sub-fragment o~ the 2 8 kb EcoRI FPV genomic
fragment (SEQ ID NO. 5). Fragment 2 is an approximately
2700 EcoRI to AscI restriction fragment synthesized by
cDNA cloning and polymerase chain reaction (PCR) from an
IBDV RNA template. cDNA and PCR primers (5'-
CACGAATTCTGACATTTTCAACAGTCCACAGGCGC-3'; 12/93.4) (SEQ ID
NO: ) and 5'-GCTGTTGGACATCACGGGCCAGG-3'; 9/93.28) (SEQ ID
NO: ) were used to synthesize an approximately 1100 base
pair EcoRI to BclI fragment at the 5' end of the IBDV
polymerase gene. cDNA and PCR primers (5'-
ACCCGGAACATATGGTCAGCTCCAT-3'; 12/93.2) (SEQ ID NO: ) and
5'-GGCGCGCCAGGCGAAGGCCGGGGATACGG-3'; 12/93.3) (SEQ ID NO:
) were used to synthesize an approximately 1700 base pair
BclI to AscI fragment at the 3' end o~ the IBDV
polymerase gene. The two fragments were ligated at the
BclI site to form the approximately 2800 base pair EcoRI
to BclI fragment. Fragment 3 is an approximately 3002
base pair BamHI to P w II restriction fragment of plasmid
pJF751 (7). Fragment 4 is an approximately 1625 base pair
SnaBI to EcoRI restriction sub-fragment of the 2.8 kb
EcoRI FPV genomic fragment (SEQ ID NO. 5).
HOMOLOGY VECTOR 751-07.D1. The plasmid 7S1-07.D1 was used
to insert foreign DNA into FPV. It incorporates an E.
coli ~-galactosidase (lacZ) marker gene and the chicken
interferon (cIFN) gene flanked by FPV DNA. When this
plasmid was used according to the HOMOLOGOUS
RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV a
virus containing DNA coding for the foreign genes
results. Note that the ~-galactosidase (lacZ) marker gene
is under the control of a synthetic la~e pox promoter
(LP1) and the cIFN gene is under the control of a
synthetic late/early pox promoter (LP2EP2). The homology
vector was constructed utilizing standard recombinant DNA
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techniques (17), by joining restriction fragmen
ts from the following sources with the appropriate
synthetic DNA sequences. The plasmid vector was derived
from an approximately 2999 base pair EcoRI restriction
fragment of pSP64 (Promega). Fragment 1 is an
- approximately 1626 base pair EcoRI to SnaBI restriction
sub-fragment of the 2.8 kb EcoRI FPV genomic fragment
(SEQ ID NO. 5). Fragment 2 is an approximately 577 base
pair EcoRI to BglII fragment coding for the cIFN gene
(17) derived by reverse transcription and polymerase
chain reaction (PCR) (Sambrook, et al., 1989) of RNA
ISOLATED FROM CONCANAVALIN A STIMULATED CHICKEN SPLEEN
CELLS. The antisense primer (6/94.13) used for reverse
t r a n s c r i p t i o n and PCR was 5'
CGACGGATCCGAGGTGCGTTTGGGGCTAAGTGC-3' (SEQ ID NO: ). The
sense primer (6/94.12) used for PCR was 5'
CCACGGATCCAGCACAACGCGAGTCCCACCATGGCT-3' (SEQ ID NO: ).
The BamHI fragment resulting from reverse transcription
and PCR was gel purified and used as a template for a
second PCR reaction to introduce a unique EcoRI site at
the 5' end and a unique BglII site at the 3' end. The
second PCR reaction used primer 6/94.22 (5'
CCACGAATTCGATGGCTGTGCCTGCAAGCCCACAG-3'; SEQ ID NO: ) at
the 5' end and primer 6/94.34 (5'-
CGAAGATCTGAGGTGCGTTTGGGGCTAAGTGC-3'; SEQ ID NO: ) at the
3' end to yield an approximately 577 base pair fragment.
The DNA fragment contains the coding sequence from amino
acid 1 to amino acid 193 of the chicken interferon
protein (17) which includes a 31 amino acid signal
sequence at the amino terminus and 162 amino acids of the
mature protein encoding chicken interferon. Fragment 3 is
an approximately 3002 base pair BamHI to PvuII
restriction fragment of plasmid pJF751 (7). Fragment 4 is
an approximately 1184 base pair SnaBI to EcoRI
restriction sub-fragment of the 2.8 kb EcoRI FPV genomic
fragment (SEQ ID NO. 5).
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HOMOLOGY VECTOR 751-56.C1. The plasmid 751-56.C1 was used
to insert foreign DNA into FPV. It incorporates an E.
coli ,~-galactosidase (lacZ) marker gene and the chicken ~.
myelomonocytic growth factor (cMGF) gene flanked by FPV
DNA. When this plasmid was used according to the
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT FPV a virus containing DNA coding for the
foreign genes results. Note that the ~B-galactosidase
(lacZ) marker gene is under the control of a synthetic
late pox promoter (LP1) and the cMGF gene is under the
control of a synthetic late/early pox promoter (LP2EP2).
The homology vector was constructed utilizing standard
recombinant DNA techniques (11 and 14), by joining
restriction fragments from the following sources with the
appropriate synthetic DNA sequences. The plasmid vector
was derived from an approximately 2999 base pair EcoRI
restriction fragment of pSP64 (Promega). Fragment 1 is
an approximately 1184 base pair EcoRI to SnaBI
restriction sub-~ragment of the 2.8 kb EcoRI FPV genomic
fragment (SEQ ID NO. 5). Fragment 2 is an approximately
640 base pair EcoRI to BamHI fragment coding Eor the cMGF
gene (16) derived by reverse transcription and polymerase
chain reaction (PCR) (Sambrook, et al., 1989) of RNA
ISOLATED FROM CONCANAVALIN A STIMULATED CHICKEN SPLEEN
CELLS. The antisense primer (6/94.20) used for reverse
transcription and PCR was 5 '
CGCAGGATCCGGGGCGTCAGAGGCGGGCGAGGTG-3' (SEQ ID NO: ). The
sense primer (5/94.5) used for PCR was 5'
GAGCGGATCCTGCAGGAGGAGACACAGAGCTG-3' (SEQ ID NO: ). The
BamHI fragment derived :Erom PCR was subcloned into a
plasmid and used as a template for a second PCR reaction
using primer 6/94.16 (5'-GCGCGAATTCCATGTGCTGCCTCACCCCTGTG
3'; SEQ ID NO: ) at the 5' end and primer 6/94.20 (5'
CGCAGGATCCGGGGCGTCAGAGGCGGGCGAGGTG-3'; SEQ ID NO: ) at
the 3' end to yield an approximately 640 base pair
fragment. The DNA fragment contains the coding sequence
from amino acid 1 to amino acid 201 of the cMGF protein
- -
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(16) which includes a 23 amino acid signal sequence at
the amino terminus and 178 amino acids of the mature
protein encoding cMGF. Fragment 3 is an approximately
3002 base pair BamHI to PvuII restriction fragment of
plasmid pJF751 (7). Fragment 4 is an approximately 1626
- base pair SnaBI to EcoRI restriction sub-fragment of the
2. 8 kb EcoRI FPV genomic fragment (SEQ ID NO. 5).
-
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Example 1
Sites for Insertion of Foreiqn 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 ~ragments were subjected to restriction
mapping analysis. Unique blunt cutting restriction
~n~onllclease sites were identified and mapped within the
cloned FPV DNA regions. The blunt restriction sites were
converted to Not I and Sf i I sites through the use of
synthetic DNA linkers (oligo 66.04; 5'-
GGCGGCCGCGGCCCTCGAGGCCA-3' SEQ ID NO: 1 and oligo 66.05;
5' TGGCCTCGAGGGCCGCGGCCGCC 3' SEQ ID NO: 2). A ,B-
galactosidase (lacZ) marker gene was inserted in each of
the potential sites. A plasmid cont~;n;ng such a foreign
DNA insert may be used according to the HOMOLOGOUS
RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV to
construct a FPV containing the foreign DNA. For this
procedure to be successful it is important that the
insertion site be in a region non-essential to the
replication of the FPV and that the site be ~lanked with
FPV DNA appropriate for mediating homologous
recombination between virus and plasmid DNAs. The
plasmids cont~;n;ng the lacZ marker gene were utilized in
the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT FPV. The generation of recombinant virus was
determined by ~he SCREEN FOR RECOMBINANT FPV EXPRESSING
ENZYMATIC MARKER GENES. Three sites were successfully
used to generate a recombinant viruses. In each case the
resulting virus was easily purified to 100~, clearly
defining an appropriate site for the insertion of foreign
DNA. The three homology vectors used to define these
sites are described below.
Example lA
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Homoloqy Vector 443-88.8
The homology vector 443-88.8 contains a 3.5 KB FPV
genomic EcoRI fragment and is useful for the insertion
of foreign DNA into FPV. This EcoRI fragment maps to the
approximately 5.5 KB overlap of FPV genomic fragments
SalI C and PstI F (Coupar et al., 1990). The NotI/SfiI
linker described above was inserted into a unique HpaI
site in this fragment. This site is designated the 680
insertion site.
The homology vector 443-88.8 was characterized by DNA
sequence analysis. Approximately 1495 base pairs of DNA
sequence flanking the HpaI site was determined (SEQ ID
NO: 3). This sequence indicates that the open reading
frame of 383 amino acids spans the HpaI insertion site.
The HpaI site interrupts this ORF at amino acid 226.
This ORF shows no amino acid sequence homology to any
known pox virus genes.
Example lB
Homoloqv 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: 5). This sequence
indicates that the SnaBI site is flanked on one side by
a complete ORF of 422 amino acids (ORFl) reading toward
the restriction site and on the other side by an
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incomplete ORF o~ 387 amino acids (ORF2) also reading
toward the restriction site. Both ORF1 and ORF2 share
homology with the vaccinia virus MlL gene ~re~). The MlL
gene shares homology with the vaccinia virus KlL gene
which has been shown to be involved in viral host-range
~unctions.
Example lC
Homoloqv 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
o~ ~oreign DNA lnto FPV. The NotI /SfiI linker described
above was inserted into a unique StuI site in this
fragment. A unique MluI site is located approximately 500
base pairs away from the StuI insertion site. This site
is designated the 540 insertion site.
Exam~le 2
Bivalent Vaccines Aqainst 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).
Exam~le 2A
S-FPV-013
S-FPV-013 is a recombinant ~owlpox virus that expresses
two foreign genes. The gene for E. col 7 ~-galactosidase
(lacZ gene) and the gene for Newcastle Disease virus
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,
hemagglutinin-neuraminidase (HN) protein were inserted
into the 681 insertion site. The lacZ gene is under the
control of a synthetic late promoter LP1 and the HN gene
is under the control of the synthetic late promoter LP2.
S-FPV-013 was derived from S-FPV-001. This was
accomplished utilizing the homology vector 451-79.95 (see
Materials and Methods) and virus S-FPV-001 in the
HOMO~OGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT FPV. The transfection stock was screened by
the SCREEN FOR RECOMBINANT FPV EXPRE,SSING ENZYMATIC
MARKER GENES. The final result of red plaque
purification was the recombinant virus designated S-FPV-
013. This virus was assayed for ~-galactosidase
expression, purity, and insert stability by multiple
passages monitored by the blue plaque assay as described
in the materials and methods. After the initial three
rounds of purification all plaques observed were blue
indicating that the virus was pure, stable and expressing
the marker gene.
S-FPV-013 was assayed for expression of NDV specific
antigens using the BLACR PLAQUE SCREEN FOR FOREIGN GENE
EXPRESSION IN RECOMBINANT FPV. An NDV HN specific
monoclonal antibody (3-lG-5) was shown to react
specifically with S-FPV-013 plaques and not with S-FPV-
001 negative control plaques. All S-FPV-013 observed
plaques reacted with the monoclonal antibody antiserum
indicating that the virus was stably expressing the NDV
foreign gene.
Example 2B
S-FPV-035
S-FPV-035 is a recombinant fowlpox virus that express a
foreign gene. The Newcastle Disease virus HN gene was
inserted at the 680 insertion site (see example lA). The
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HN gene is under the control of the synthetic early/late
promoter EPlLP2.
S-FPV-035 was derived from S-FPV-001. This was
accomplished utilizing the homology vector 489-21.1 (see
Materials and Methods) and virus S-FPV-001 in the
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT FPV. The transfection stock was screened by
the PLAQUE HYBRIDIZATION PROCEDURE FOR PURIFYING
RECOMBINANT FPV. The final result of plaque
hybridization purification was the recombinant virus
designated S-FPV-035.
S-FPV-035 was assayed for expression of NDV specific
antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE
EXPRESSION IN RECOMBINANT FPV. An NDV HN speci~ic
monoclonal antibody (3-lG-5) was shown to react
specifically with S-FPV-035 plaques and not with S-FPV-
001 negative control plaques. All S-FPV-035 observed
plaques reacted with the monoclonal antibody indicating
that the virus was stably expressing the NDV foreign
gene.
Example 2C
S-FPV-041
S-FPV-041 is a recombinant fowlpox virus that expresses
two foreign genes. The gene for E. coli ~-galactosidase
(lacZ gene) and the gene for Newcastle Disease virus
fusion (F) protein were inserted into the 681 insertion
site. The l acZ 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 EPlLP2.
S-FPV-041 was derived from S-FPV-001. This was
accomplished utilizing the homology vector 502-27.5 (see
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Materials and Methods) and virus S-FPV-001 in the
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
R~COMBINANT FPV. The transfection stock was screened by
the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC
MARKER GENES. The final result of red plaque purification
was the recombinant virus designated S-FPV-041. This
virus was assayed for ~-galactosidase expression, purity,
and insert stability by multiple passages monitored by
the blue plaque assay as described in the materials and
methods. After the initial three rounds of purification
all plaques observed were blue indicating that the virus
was pure, stable and expressing the marker gene.
S-FPV-041 was assayed for expression of NDV specific
antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE
EXPRESSION IN RECOMBINANT FPV. An NDV F specific
monoclonal antibody (5-3F-2) was shown to react
specifically with S-FPV-041 plaques and not with S-FPV-
001 negative control plaques. All S-FPV-041 observed
plaques reacted wlth the monoclonal antibody indicating
that the virus was stably expressing the NDV foreign
gene.
Example 2D
S-FPV-042
S-FPV-042 is a recombinant fowlpox virus that expresses
three foreign genes. The gene for E. col i ~-
galactosidase (lacZ gene) and the gene for NewcastleDisease virus fusion (F) 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 EPlLP2.
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 EPlLP2.
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S-FPV-042 was derived ~rom S-FPV-035. This was
accomplished utilizing the homology vector 502-27.5 (see
Materials and Methods) and virus S-FPV-035 in the
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT FPV. The trans~ection stock was screened by
the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC
MARKER GENES. The ~inal result of red plaque purification
was the recombinant virus designated S-FPV-042. This
virus was assayed for ~-galactosidase expression, purity,
and insert stability by multiple passages monitored by
the blue plaque assay as described in the materials and
methods. A~ter the initial three rounds of puri~ication
all plaques observed were blue indicating that the virus
was pure, stable and expressing the marker gene.
S-FPV-042 was assayed ~or expression of NDV speci~ic
antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE
EXPRESSION IN RECOMBINANT FPV. Monoclonal antibodies
speci~ic ~or both HN (3-lG-5) and F (5-3F-2) were shown
to react speci~ically with S-FPV-042 plaques and not with
S-FPV-001 negative control plaques. All S-FP~-042
observed plaques reacted with the monoclonal antibodies
indicating that the virus was stably expressing the NDV
foreign genes.
Exam~le 2E
S-FPV-043
S-FPV-043 is a recombinant ~owlpox virus that expresses
two ~oreign genes. The genes ~or 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 o~ a synthetic early/late promoter EPlLP2.
S-FPV-043 was derived ~rom S-FPV-001. This was
accomplished utilizing the homology vector 502-26.22 (see
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Materials and Methods) and virus S-FPV-001 in the
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT FPV. The transfection stock was screened by
the PLAQUE HYBRIDIZATION PROCEDURE FOR PURIFYING
RECOMBINANT FPV. The final result of plaque
hybridization purification was the recombinant virus
designated S-FPV-043: The S-FPV-043 has been deposited
pursuant to the Budapest Treaty on the International
Deposit of Microorganisms for the Purposes of Patent
Procedure with the Patent Culture Depository of the
American Type Culture Collection, 12301 Parklawn Drive,
Rockville, Maryland 20852 U.S.A. under ATCC Accession
No. VR 2395 on December 9, 1992.
S-FPV-043 was assayed for expression of NDV specific
antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE
EXPRESSION IN RECOM3INANT FPV. Monoclonal antibodies
specific for both HN (3_1G_5) and F (5-3F-2) were shown
to react specifically with S-FPV-043 plaques and not with
S-FPV-001 negative control plaques. All S-FPV-043
observed plaques reacted with the monoclonal antibodies
antiserum indicating that the virus was stably expressing
the NDV ~oreign genes.
TESTING OF RECO~3INANT FPV EXPRESSING NDV ANTIGENS
Groups o~ one day old SPF chicks (HyVac Inc.) were
lmml~nl~ed with recombinant ~owlpox 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 1). The challenged chicks were
observed daily for 14 days ~or clinical signs and death
due to NDV. Non vaccinated control birds showed 100
mortality. S-FPV-043 vaccinated birds showed 100~
protection against FPV challenge. Birds vaccinated with
S-FPV-035 showed 95~ protection compared with 85~ seen
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with birds immunized with S-FPV-041. These results
suggest that recombinants expressing HN or F alone
provide only partial protection. When both NDV proteins
are combined into the same virus S-FPV-043, an
enhancement of protection against lethal NDV challenge is
obtained, resulting in a lower protective dose. The
chicks that were challenged with FPV were scored for pox
lesions. Non vaccinated control birds showed no
protection against FPV lesions. Birds vaccinated with
S-FPV-043 were completely protected from FPV lesion~.
The duration of ;mml~n;ty conferred by vaccination with S-
FPV-043 was ~m; ned A group of SPF chicks was
immunized with S-FPV-043 at one day o~ age and then
challenged six weeks post-vaccination with either NDV or
FPV. Complete protection was observed against both NDV
and FPV challenge in S-FPV-043 vaccinated birds, whereas
non vaccinated controls were totally susceptible to both
challenge viruses. These results suggest that the
duration of immunity af~orded by vaccination with S-FPV-
043 would span the life of a broiler bird (~ 6 weeks).
The effect of vaccinating hens in lay with the
recombinant S-FPV-043 was evaluated by assessing egg
production post-vaccination. One group of 50 hens was
vaccinated and a second group of 50 hens, housed under
conditions identical to the vaccinated group, served as
non vaccinated controls. Daily egg production was
monitored for four weeks post-vaccination. No differences
were observed in egg production between the two groups of
hens, indicating this vaccine will not adversely affect
egg production in laying hens.
A study was conducted to determine whether S-FPV-043
could actively immunize chicks in the presence of
maternal antibodies to both NDV and FPV. Chicks obtained
from NDV and FPV imml~nized flocks were vaccinated with S-
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FPV-043 and three weeks after vaccination, they were
challenged with either virulent NDV or virulent FPV.
Clinical responses were compared with non vaccinated
chicks from the same flock and with non-vaccinated chicks
from an antibody negative flock (Table 2). Chicks derived
from antibody negative flocks showed 100~ mortality after
NDV challenge. Protection against NDV challenge, in non-
vaccinated 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
85~ (flock 2) clearly demonstrate the ability of S-FPV-
043 to partially overcome maternal antibody to both NDVand FPV. A decrease in FPV protection (go~) was observed
in ~lock 1, suggesting some inhibition of FPV
replication.
,
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Table 1. Immunity con~erred by Fowlpox recombinant
vaccines vectoring di~ferent genes ~rom
Newcastle disease virus.
~ Challengea
VIRUS DOSEb NDV FPV
FPV/NDV-HN
8 x 105 95 NTc
FPV/NDV-F
2 x 104 85 NT
FPV/NDV-HN+F
. 2 x 103 100 100
Controls
none ~ ~
a Percent protection ~ollowing challenge 3 weeks post-
vaccination
b PFU/O.1 ml dose
c N~t tested
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--55--
Tab1Q 2. Ability of recombinant vaccine FPV/NDV-HN+F (S-
FPV-043) to vaccinate chicks with maternal
antibody.
Challengea
History
Flock
Vaccination Hen Antibodyb NDV FPV
NDV-HIC NDV ELISA FPV-AGPd Vacc. Con. Vacc. Con.
1 NDV + FPV
- 1:36 1:1738 Neg 75 30 90 0
2 NDV + FPV
1:64 1:2852 Neg 85 55 100 0
3 NDV only
1:92 1:4324 Neg 80 60 95 0
4 None
Neg Neg Neg -- 0 -- 0
a Percent protection following challenge 3 weeks
post-vaccination.
b Every flock antibody.
c HI - Hemagglutination Inhibition Assay
d AGP - Agar Gel Precipitation Assay
SU~STITVTE SHEET ~RULE 26)
-
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Example 2F
S-FPV-074
S-FPV-074 is a recombinant fowlpox virus that expresses
two foreign genes. The genes for Newcastle Disease virus
F protein and HN protein were inserted at the 681
insertion site. The F and HN genes are each under the
control of a synthetic late/early promoter LP2EP2.
S-FPV-074 was derived ~rom S-FPV-001. This was
accomplished utilizing the homology vector 584-36.12 (see
Materials and Methods) and virus S-FPV-001 in the
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT FPV. The trans~ection stock was screened by
the PLAQUE HYBRIDIZATION PROCEDURE FOR PURIFYING
RECOMBINANT FPV. The final result of plaque
hybridization purification was the recombinant virus
designated S-FPV-074.
S-FPV-074 was assayed for expression of NDV speci~ic
antigens using the BLACR PLAQUE SCREEN FOR FOREIGN GENE
EXPRESSION IN RECOMBINANT FPV. Monoclonal antibodies
specific for NDV HN (3-lG-5) and F (5-3F-2) were shown to
react specifically with S-FPV-074 plaques and not with S-
FPV-001 negative control plaques. All S-FPV-074 observed
plaques reacted with the monoclonal antibodies indicating
that the virus was stably expressing the NDV foreign
genes.
S-FPV-074 expresses foreign antlgens ~rom NDV. This
virus is useful as a multi-valent vaccine against
Newcastle Diseases and Fowlpox.
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Example 3
Recombinant fowlpox viruses expressing proteins from
Marek's disease virus (MDV) make vaccines protecting
against both fowlpox virus and Marek's disease virus. We
have constructed several recombinant FPV expressing MDV
proteins: S-FPV-081, S-FPV-082 and S-FPV-085. Of these
S-FPV-082 and S-FPV-085 also express proteins from
Newcastle disease virus. These viruses are useful for
vaccinating against fowlpox virus, Marek's disease virus,
and Newcastle disease virus.
S-FPV-085 further expresses proteins from infectious
laryngotracheitis virus (ILTV), making them useful as
vaccines against ILTV.
Exam~le 3A
S-FPV-081
S-FPV-081 is a recombinant fowlpox virus that expresses
three foreign genes. The gene for E.coli ~-galactosidase
tlacZ gene) and the genes ~or Marek's Disease virus ~MDV)
glycoprotein D (gD) and glycoprotein B (gB) were inserted
into the 681 insertion site. The lac Z gene is under the
control of a synthetic late promoter LP1 and the MDV gD
and gB genes are under the control of the synthetic
early/late promoters LP2EP2 and EPlLP2 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 o~ red plaque puri~ication
was the recombinant virus designated S-FPV-081. This
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virus was assayed for ~-galactosidase expression, purity,
and insert stability by multiple passages monitored by
the blue plaque assay as described in the materials and
methods. A~ter the initial three rounds o~ puri~ication
all plagues observed were blue indicating that the virus
was pure, stable and expressing the marker gene.
S-FPV-081 was assayed ~or expression o~ MDV speci~ic
antigens using the BLACK PLAQUE SCREEN FOR FOREIGN ~ENE
EXPRESSION IN RECOMBINANT FPV. Convalescent sera ~rom
MDV in~ected chickens was shown to react speci~ically
with S-FPV-081 plaques and not with S-FPV-001 negative
control plaques. All S-FPV-081 observed plaques reacted
with the chicken antiserum indicating that the virus was
stably expressing the MDV foreign genes. Western blot
assays o~ infected cell lysates using convalescent sera
from MDV-in~ected chickens indicated that ~-FPV-081 was
expressing a MDV glycoprotein B and MDV glycoprotein D.
S-FPV-081 expresses foreign antigens ~rom MDV. This virus
is useful as a multi-valent vaccine against Marek's
Disease and Fowlpox.
Exam~le 3B
~-FPV-082
S-FPV-082 is a recombinant ~owlpox 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 o~ a synthetic early/late promoter EPlLP2. The
gene ~or E. coli ~-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
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synthetic early/late promoters LP2EP2 and EPlLP2
respectively.
S-FPV-082 was derived from S-FPV-043. This was
accomplished utilizing the homology vector 608-10.3 (see
Materials and Methods) and virus S-FPV-043 in the
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT FPV. The transfection stock was screened by
the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC
M~RKER GENES. The final result of red plaque purification
was the recombinant virus designated S-FPV-082. This
virus was assayed for ~-galactosidase expression, purity,
and insert stability by multiple passages monitored by
the blue plaque assay as described in the materials and
methods. After the initial three rounds of purification
all plaques observed were blue indicating that the virus
was pure, stable and expressing the marker gene.
S-FPV-082 was assayed for expression of MDV specific
antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE
EXPRESSION IN RECOMBINANT FPV. Convalescent sera from
MDV infected chickens was shown to react specifically
with S-FPV-082 plaques and not with S-FPV-001 negative
control plaques. All S-FPV-082 observed plaques reacted
with the chicken antiserum indicating that the virus was
stably expressing the MDV foreign genes.
S-FPV-082 expresses foreign antigens from NDV and MDV.
This virus will be valuable as a multi-valent vaccine
against Newcastle Disease, Marek's Disease and Fowlpox.
Example 3C
-
S-FPV-085
S-FPV-085 is a recombinant fowlpox virus that expresses
eight foreign genes. The genes for Newcastle Disease
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virus F protein and HN protein are inserted at the 680
insertion site. The F and HN genes are each under the
control of a synthetic early/late promoter EPlLP2. 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 EPlLP2 respectively. The
gene for E.coli ~-glucuronidase (uidA gene) and the genes
for Infectious Laryngotracheitis virus (ILTV) gD and gB
are inserted into the 540 insertion site. The uidA gene
is under the control of a synthetic late promoter LP1 and
the ILTV gD and gB genes are each under the control of a
synthetic early/late promoter EPlLP2.
S-FPV-085 is derived from S-FPV-082. This is accomplished
utilizing the homology vector 586-36.6 (see Materials and
Methods) and virus S-FPV-082 in the HOMOLOGOUS
RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT FPV.
The transfection stock is screened by the SCREEN FOR
RECOMBINANT FPV EXPRESSING ENZYMATIC MARKER GENES. The
final result of blue plaque (~-glucuronidase)
purification is the recombinant virus designated S-FPV-
085. This virus is assayed for ~-glucuronidase
expression, purity, and insert stability by multiple
passages monitored by the blue plaque assay as described
in the materials and methods. After the initial three
rounds of purification all plaques observed 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
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Disease, Marek's Disease, Infectious Laryngotracheitis
and Fowlpox.
Example 4
Recombinant fowlpox virus (FPV) expressing proteins from
infectious laryngotracheitis virus (ILTV) make vaccines
protecting against both FPV and ILTV. We have
constructed several recombinant FPV expressing ILTV
proteins: S-FPV-095, S-FPV-083, and S-FPV-097. Of these,
S-FPV-083 and S-FPV-097 also express proteins from
Newcastle disease virus (NDV), making them useful as
vaccines against NDV as well.
Exam~le 4
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 LPl and the ILTV gD and gB genes are each
under the control of a synthetic early/late promoter
EPlLP2.
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 (~-glucuronidase) was the recombinant virus
designated S-FPV-095. This vlrus was assayed for ~-
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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 puri~ication all plaques observed
were blue indicating that the virus was pure, stable and
expressing the marker gene.
S-FPV-095 was assayed for expression o~ 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 speci~ically 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
~oreign genes.
S-FPV-095 expresses foreign antigens from ILTV. This
virus is useful as a multi-valent vaccine against
In~ectious Laryngotracheitis and Fowlpox.
Example 4B
S-FPV-083
S-FPV-083 is a recombinant fowlpox virus that expresses
five foreign genes. The genes for Newcastle Disease virus
F protein and HN protein were inserted at the 680
insertion site. The F and HN genes are each under the
control of a synthetic early/late promoter EPlLP2. The
gene for E. coli ~-glucuronidase (uidA gene) and the
genes for Infectious Laryngotracheitis virus (ILT) gD and
gB were inserted into the 540 insertion site. The uidA
gene is under the control of a synthetic late promoter - ~
LP1 and the ILT gD and gB genes are each under the
control of a synthetic early/late promoter (EPlLP2).
S-FPV-083 was derived from S-FPV-043. This was
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accomplished utilizing the homology vector 586-36.6 (see
Materials and Methods) and virus S-FPV-043 in the
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT FPV. The transfection stock was screened by
the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC
" MARKER GENES. The final result of blue plaque
purification was the recombinant virus designated S-FPV-
083. This virus was assayed for ~-glucuronidase
expression, purity, and insert stability by multiple
passages monitored by the blue plaque assay as described
in the materials and methods. After the initial three
rounds of purification all plaques observed were blue
indicating that the virus was pure, stable and expressing
the marker gene.
S-FPV-083 was assayed for expression of ILTV specific
antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE
EXPRESSION IN RECOMBINANT FPV. Convalescent sera from
ILTV infected chickens was shown to react specifically
with S-FPV-083 plaques and not with S-FPV-001 negative
control plaques. All S-FPV-083 observed plaques reacted
with the chicken antiserum indicating that the virus was
stably expressing the ILTV foreign genes.
S-FPV-083 expresses foreign antigens from NDV and ILTV.
This virus will be valuable as a multi-valent vaccine
against Newcastle Disease, Infectious Laryngotracheitis
and Fowlpox.
Example 4C
S-FPV-097
S-FPV-097 is a recombinant fowlpox virus that expresses
five foreign genes. The genes for Newcastle Disease virus
F protein and HN protein were inserted at the 680
insertion site. The F and HN genes are each under the
,
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control o~ a synthetic early/late promoter EPlLP2. 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 EPlLP2.
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 o~ blue plaque
purification was the recombinant virus designated S-FPV-
097. This virus was assayed for ~-glucuronidase
expression, purity, and insert stability by multiple
passages monitored by the blue plaque assay as described
in the materials and methods. After the initial three
rounds of purification all plaques observed were blue
indicating that the virus was pure, stable and expressing
the marker gene.
S-FPV-097 was assayed for expression of ILTV specific
antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE
EXPRESSION IN RECOMBINANT FPV. Antibodies to ILTV gB and
gD was shown to react speciflcally with S-FPV-097 plaques
and not with S-FPV-001 negative control plaques. All S-
FPV-097 observed plaques reacted with the antiserum
indicating that the virus was stably expressing the ILTV
foreign genes. All S-FPV-097 observed plaques reacted
with the chicken antiserum to ILTV indicating that the
virus was stably expressing the ILTV foreign genes.
Monoclonal antibodies speci~ic ~or NDV HN (3-lG-5) and F
(5-3F-2) were shown to react specifically with S-FPV-097
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plaques and not with S-FPV-001 negative control plaques.
All S-FPV-097 observed plaques reacted with the
monoclonal antibodies indicating that the virus was
stably expressing the NDV foreign genes.
S-FPV-097 expresses foreign antigens from NDV and ILTV.
This virus is useful as a multi-valent vaccine against
Newcastle Disease, Infectious Laryngotracheitis and
Fowlpox.
-
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Example 5
Recombinant fowlpox virus (FPV) expressing proteins ~rom
in~ectious 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 o~ these viruses also express
proteins ~rom Newcastle disease virus (NDV), making them
use~ul as vaccines against NDV.
Example 5A
S-FPV-072
S-FPV-072 is a recombinant fowlpox virus that expresses
~ive ~oreign genes. The genes for Newcastle Disease virus
F protein and HN protein were inserted at the ~80
insertion site. The F and HN genes are each under the
control of a synthetic early/late promoter EPlLP2. The
gene ~or E.coli ~-galactosidase (lacZ gene) and the genes
for In~ectious 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 o~ 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 EPlLP2.
S-FPV-072 was derived ~rom 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 trans~ection stock was screened by
the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC
MARKER GENES. The final result o~ red plaque
puri~ication was the recombinant virus designated S-FPV-
072. This virus was assayed ~or B-galactosidase
expression, purity, and insert stability by multiple
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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-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 speci~ically with S-FPV-072 plaques and not with S-
FPV-001 negative control plaques. All S-FPV-072 observed
plaques reacted with the monoclonal antibodies indicating
that the virus was stably expressing the IBV foreign
gene. Western blot assays of infected cell lysates using
monoclonal antibody 15-88 to the IBV Mass Spike protein
indicated that S-FPV-072 was expressing a 90 kD IBV Mass
Spike protein. Monoclonal antibodies specific for both
HN (3-lG-5) and F (5-3F-2) were shown to react
specifically with S-FPV-072 plaques and not with S-FPV-
001 negative control plaques. All S-FPV-072 observed
plaques reacted with the monoclonal antibodies indicating
that the virus was stably expressing the NDV foreign
genes.
S-FPV-072 expresses foreign antigens from NDV and IBV.
This virus is useful as a multi-valent vaccine against
Newcastle Diseases, Infectious Bronchitis, and Fowlpox.
Example 5B
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 EPlLP2. The
gene for E. coli ~-galactosidase (lacZ gene) and the genes
for Infectious Bronchitis virus (IBV) Massachusetts Spike
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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 o~ the synthetic early/late
promoter EPlLP2. The gene for the E. coli ~-
glucuronidase (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 EPlLP2.
S-FPV-079 was derived from S-FPV-072. This was
accomplished utilizing the Homology Vector 611-49.1 (see
Materials and Methods) and virus S-FPV-072 in the
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT FPV. The transfection stock was screened by
the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC
MARKER GENES. The ~inal result of red plaque
purification was the recombinant virus designated S-FPV-
079. This virus was assayed for B-galactosidase
expression, purity, and insert stability by multiple
passages monitored by the blue plaque assay as described
in the materials and methods. After the initial three
rounds of puri~ication, all plaques observed were blue
indicating that the virus was pure, stable and expressing
the marker gene.
S-FPV-079 was assayed for expression o~ NDV and IBV
speci~ic 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-~72 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
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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
90 kD IBV Mass Spike protein. Monoclonal antibodies
specific for both HN (3-lG-5) and F (5-3F-2) were shown
to react speci~ically wlth 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
~oreign 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.
Exam~le 6
Recombinant fowlpox virus, S-FPV-099 or S-FPV-101,
expressing chicken interferon (cIFN) or S-FPV-100,
expressing chicken myelomonocytic growth factor (cMGF),
are useful to enhance the immune response when added to
vaccines against diseases of poultry. Chicken
myelomonocytic growth factor (cMGF) is homologous to
m~mm~lian interleukin-6 protein, and chicken inter~eron
(cIFN) is homologous to m~mm~lian interferon Type I. When
used alone or in combination with vaccines against
specific avian diseases, S-FPV-099, S-FPV-100 and S-FPV-
101 provide enhanced mucosal, humoral, or cell mediated
immunity against avian disease-causing viruses including,
but not limited to, Marek's disease virus, Newcastle
disease virus, infectious laryngotracheitis virus,
infectious bronchitis virus, infectious bursal disease
virus.
S-FPV-099
S-FPV-099 is a recombinant fowlpox virus that expresses
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two foreign genes. The genes for chicken interferon
(cIFN) and E. coli lacZ were inserted at the uniqe SnaBI
restriction endonuclease site in the 2.8 kB EcoRI FPV
genomic fragment (681 insertion site). The cIFN gene is
under the control o~ a synthetic late/early promoter
LP2EP2, and the E. coli lacZ gene is under the control of
a synthetic late promoter LP1.
S-FPV-099 was derived from S-FPV-001. This was
accomplished utilizing the homolo~y vector 751-07.Dl (see
Materials and Methods) and virus S-FPV-001 in the
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT FPV. The transfection stock was screened by
the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC
MARKER GENES. The final result of red plaque purification
was the recombinant virus designated S-FPV-099. This
virus was assayed for ~-galactosidase expression, purity,
and insert stability by multiple passages monitored by
the blue plaque assay as described in Materials and
Methods. After the initial three rounds of purification,
all plaques observed were blue indicating that the virus
S-FPV-099 was pure, stable, and expressing the foreign
gene.
Supernatants ~rom S-FPV-099 have interferon activity in
cell culture. Addition o~ S-FPV-099 conditioned media to
chicken embryo fibroblast (CEF) cell culture inhibits
infection of the CEF cells by vesicular stomatitis virus
or by herpesvirus of turkeys. S-FPV-099 is useful to
enhance the immune response alone or when added to
vaccines against diseases of poultry.
S-FPV-100
S-FPV-100 is a recombinant fowlpox virus that expresses
two foreign genes. The genes for chicken myelomonocytic
growth factor (cMGF) and E. coli lacZ were inserted at
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the uniqe SnaBI restriction endonuclease site in the 2.8
kB EcoRI FPV genomic fragment (681 insertion site). The
cMGF gene is under the control of a synthetic late/early
promoter LP2EP2, and the E. coli lacZ gene is under the
control of a synthetic late promoter LP1.
S-FPV-100 was derived from S-FPV-001. This was
accomplished utilizing the homology vector 751-56.C1 (see
Materials and Methods) and virus S-FPV-001 in the
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT FPV. The transfection stock was screened by
the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC
MARKER GENES. The final result of red plaque purification
was the recombinant virus designated S-FPV-100. This
virus was assayed for ~-galactosidase expression, purity,
and insert stability by multiple passages monitored by
the blue plaque assay as described in Materials and
Methods. After the initial three rounds of purification,
all plaques observed were blue indicating that the virus
S-FPV-100 was pure, stable, and expressing the foreign
gene.
S-FPV-100 is useful to enhance the immune response alone
or when added to vaccines against diseases of poultry.
S-FPV-101
S-FPV-101 is a recombinant fowlpox virus that expresses
four foreign genes. The genes for chicken interferon
(cIFN~ and E. coli lacZ were inserted at the uniqe SnaBI
restriction endonuclease site in the 2.8 kB EcoRI FPV
genomic fragment (681 insertion site). The cIFN gene is
under the control of a synthetic late/early promoter
LP2EP2, and the E. coli lacZ gene is under the control of
a synthetic late promoter LP1. The genes for Newcastle
Disease virus F protein and HN protein were inserted at
the 680 insertion site. The F and HN genes are each under
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the control of a synthetic early/late promoter EPlLP2.
S-FPV-101 was derived from S-FPV-043. This was
accomplished utilizing the homology vector 751-07.D1 (see
Materials and Methods) and virus S-FPV-043 in the
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT FPV. The transfection stock was screened by
the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC
MARKER GENES. The final result of red pla~ue purification
was the recombinant virus designated S-FPV-101. This
virus was assayed for ~-galactosidase expression, purity,
and insert stability by multiple passages monitored by
the blue plaque assay as described in Materials and
Methods. After the initial three rounds of purification,
all plaques observed were blue indicating that the virus
S-FPV-101 was pure, stable, and expressing the foreign
gene.
Supernatants from S-FPV-101 have interferon activity in
cell culture. Addition of S-FPV-101 conditioned media to
chicken embryo fibroblast (CEF) cell culture inhibits
infection of the CEF cells by vesicular stomatitis virus
or by herpesvirus of turkeys. S-FPV-101 is useful to
enhance the immune response alone or when added to
vaccines against diseases of poultry. S-FPV-101 is useful
as a multi-valent vaccine against Newcastle Diseases and
Fowlpox.
Example 7
Recombinant ~owlpox virus expressing Newcastle's disease
virus HN and F proteins lacking the membrane anchor
sequences is a superior vaccine against fowlpox and
Newcastle's disease.
Day old chicks from hens which have been exposed to or
vaccinated against Newcastle's disease virus carry
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antibodies to NDV which may neutralize a vaccine
containing a recombinant fowlpox virus expressing the NDV
HN and F proteins. In vitro virus neutralization (VN)
assays using VN monoclonal antibodies specific for either
NDV HN or F proteins have been shown to neutralize
recombinant fowlpox virus expressing the NDV HN and F
proteins. These results suggest that the NDV HN and F
glycoproteins are incorporated into the fowlpox virus
virion. To increase the efficacy of a vaccine in the
presence on maternal antibodies against Newcastle's
disease virus, a recombinant fowlpox virus is constructed
which expresses the NDV HN and F proteins lacking the
membrane anchor domains of each protein. The resulting
recombinant virus produces NDV HN and F proteins secreted
into the serum of the vaccinated animal producing a
strong humoral and cell mediated immune response to the
Newcastle's disease virus. The NDV HN and F proteins are
not presented on the surface of the FPV particle and thus
evade neutralization by maternal antibodies present in
the vaccinated day old chicks.
The hemagglutinin-Neuraminidase (HN) and Fusion (F) genes
from the B1 strain of Newcastie Disease Virus (ATCC VR-
108) were isolated as cDNA clones, using oligo dT primed
poly A selected mRNA.
The fusion (F) protein mediates penetration of NDV into
host cells by fusion of the viral envelope with the host
cell plasma membrane. A posttranslational cleavage of
inactive precursors Fo into two disulfide-bonded
polypeptides, F1 and F2, is necessary to produce fusion
active F protein and thereby yield infectious virions.
The new hydrophobic N-terminus of F1 generated after
cleavage of Fo is responsible for the fusion
characteristic of paramyxoviruses and thus determines
virulence. The required proteolytic cleavage signal
(paired basic residues) in the NDV B1 strain is altered,
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thereby preventing cleavage of Fo into F1 and F2,
resulting in an attenuated NDV strain.
The addition of the NDV F signal sequence (aal-25) to VP2
(vFP147), resulted in the secretion of VP2 in the TC
fluid, but abolished its protective response (Paoletti,
et. al WO 93/03145). Three hydrophobic domains exist
within the F glycoprotein which interact with the lipid
bilayer : 1). The signal sequence at the N-terminus of
the primary translation product Fo; 2). the N-terminus of
F1; and 3). the transmembrane anchor domain near the C-
terminus of F1. The F glycoprotein of the B1 strain of
NDV is 544 amino acids in length with the transmembrane
anchor domain spanning 27 amino acids ~rom position 500
to 526 (LITYIVLTIISLVFGILSLILACYLMY). Amino acids 1-499
of the NDV F protein are expressed under the control of
a synthetic promoter element which functions as both an
early and late promoter, such as EPlLP2 or LP2EP2,
directing expression throughout the reproduction cycle.
This results in the deletion of amino acids 527-544, the
cytoplasmic tail, thought to interact with the inner
membrane protein (M) before or during virus assembly. A
recombinant fowlpox virus is constructed which expresses
the NDV F protein lacking the C-terminal membrane anchor
domain ~rom a synthetic early/late promoter.
The hemagglutinin-neuraminidase (HN) glycoprotein
provides NDV with the ability to agglutinate and elute
erythrocytes. The process consists of two stages:
attachment of the virus to the receptor on the red blood
cell sur~ace (agglutination) and destruction of the
receptor by the neuraminidase enzyme activity (elution).
The major hydrophobic anchor domain is present near the
N-terminus of HN, supporting the view that the N-terminus
is anchored to the lipid bilayer. The HN glycoprotein of
the B1 strain of NDV is 577 amino acids in length with
the transmembrane anchor domain spanning 28 amino acids
-
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from position 27 to 54 (IAILFLTVVTLAISVASLLYSMGASTPS).
The extreme N-terminal amino acids (1 to 26) are
relatively hydrophilic. Amino acids 55 to 577 of the HN
protein are expressed under the control of a synthetic
promoter element which functions as both an early and
late promoter, such as EPlLP2 or LP2EP2, directing
expression throughout the reproduction cycle. THE NDV HN
polypeptide has a membrane transport signal sequence,
such as the PRV gX signal sequence, at its amino terminus
to direct the protein to be secreted into the serum of a
vaccinated animal. A recombinant fowlpox virus is
constructed which expresses the NDV HN protein lacking
the N-terminal membrane anchor domain and containing an
N-terminal PRV gX signal sequence from a synthetic
early/late promoter. Alternatively the NDV HN polypeptide
contains a deletion of the transmembrane anchor domain
spanning 28 amino acids from position 27 to 54 and
retains amino acids 1 to 26 and 55 to 577. A recombinant
fowlpox virus is constructed which expresses the NDV HN
protein lacking the membrane anchor domain (amino acids
27 to 54) from a synthetic early/late promoter.
A recombinant fowlpox virus is constructed which
expresses both the NDV HN and F proteins lacking the
membrane anchor domains of each protein from a synthetic
early/late promoter. The resulting recombinant virus
produces NDV HN and F proteins secreted into the serum of
the vaccinated ~nim~l producing a strong humoral and cell
mediated immune response to the Newcastle's disease
virus. The NDV HN and F proteins are not presented on
the sur~ace of the FPV particle and thus evade
neutralizatlon by maternal antibodies present in the
vaccinated day old chicks.
-
Exam~le 8
,.
Recombinant fowlpox virus expressing cell surface
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receptors on the surface of the FPV viral particle useful
for targeting gene products to specific tissues or
organs.
Serum from chickens carrying maternal antibodies to
Newcastle's disease virus inhibits productive infection
and plaque formation by S-FPV-043 on chicken embryo
fibroblasts in cell culture. One explanation for this
result is that the antigenic epitopes of the NDV HN and
F proteins expressed in S-FPV-043 are displayed on the
surface of the fowlpox viral particle. Display of
proteins on the surface of the FPV particle is useful to
target specific gene products to specific normal cell
types or tumor cell types. Proteins which are displayed
on the surface of the FPV particle include but are not
limited to integrins which would target the virus to
integrin receptors on the cell surface; erythropoetin
which would target the virus to erythropoetin receptors
on the surface of red blood cells; antibodies or other
proteins which would target to specific proteins or
receptors on the surface of normal or tumor cells. The
fowlpox virus also delivers cytokines, interleukins,
interferons, or colony stimulating factors which
stimulate a strong humoral or cell mediated immune
response against a tumor or disease causing organism. The
proteins displayed on the sur~ace of the ~owlpox virus
are expressed from the fowlpox genome as ~usion proteins
to the membrane anchor domains of the NDV HN or F
proteins, or to other proteins containing membrane anchor
domains. The cytokines, interleuklns, interferons, or
colony stimulating factors are expressed as ~usion
proteins to PRV gX, E. coli ~-galactosidase or another
protein in a soluble, not membrane bound, form The
fusion protein stabilizes the cytokine protein and allows
it to diffuse in the serum of the animal to reach its
cellular target
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Example 9
S-FPV-098
S-FPV-098 is a recombinant fowlpox virus that expresses
two foreign genes. The genes for infectious bursal
disease virus (IBDV) polymerase gene and E. coli lacZ
were inserted at the 681 insertion site. The IBDV
polymerase gene is under the control of a synthetic
late/early promoter LP2EP2, and the E. coli lacZ gene i5
under the control of a synthetic late promoter LPl.
S-FPV-098 was derived from S-FPV-001. This was
accomplished utilizing the homology vector 749-75.82 (see
Materials and Methods) and virus S-FPV-001 in the
HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING
RECOMBINANT FPV. The transfection stock was screened by
the SCREEN FOR RECOMBINANT FPV EXPRESSING ENZYMATIC
MARKER GENES. The final result of red plaque purification
was the recombinant virus designated S-FPV-098. This
virus was assayed for ~-galactosidase expression, purity,
and insert stability by multiple passages monitored by
the blue plaque assay as described in Materials and
Methods. After the initial three rounds of purification,
all plaques observed were blue indicating that the virus
S-FPV-098 was pure, stable, and expressing the foreign
gene.
S-FPV-098 is useful for expression o~ IBDV polymerase
protein. S-FPV-098 is useful in an in vitro approach to
a recombinant IBDV attenuated vaccine. RNA strands from
the attenuated IBDV strain are synthesized in a bacterial
expression system using T3 or T7 promoters (pBlueScript
plasmid; Stratagene, Inc.) to synthesize double stranded
short and long segments of the IBDV genome. The IBDV
double stranded RNA segments and S-FPV-098 are
transfected into Vero cells. The fowlpox virus expresses
the IBDV polymerase but does not replicate in Vero cells.
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The IBDV polymerase produced from S-FPV-098 synthesizes
in~ectious attenuated IBDV virus from the double stranded
RNA genomic templates. The resulting attenuated IBDV
virus is use~ul as a vaccine against infectious bursal
disease in chickens.
As an alternative to the construction of a IBD vaccine
using a viral vectored delivery system and/or subunit
approaches, IBD virus RNA is directly manipulated re-
constructing the virus using full length RNA derived fromcDNA clones representing both the large (segment A) and
small (segment B) double-stranded RNA subunits.
Generation of IBD virus is this manner offers several
advantages over the ~irst two approaches. First, if IBD
virus is re-generated using RNA templates, one is able to
manipulate the cloned cDNA copies o~ the viral genome
prior to transcription (generation of RNA). Using this
approach, it is possible to either attenuate a virulent
IBD strain or replace the VP2 variable region o~ the
attenuated vaccine backbone with that of virulent
strains. In doing so, the present invention provides
protection against the virulent IBDV strain while
providing the safety and ef~icacy of the vaccine strain.
Furthermore, using this approach, the present invention
constructs and tests temperature sensitive IBD viruses
generated using the RNA polymerase derived ~rom the
related birnavirus infectious pancreatic necrosis virus
(IPNV) and the polyprotein derived from IBDV. The IPNV
polymerase has optimum activity at a temperature lower
than that of IBDV. If the IPNV polymerase recognizes the
regulatory signals present on IBDV, the hybrid virus is
expected to be attenuated at the elevated temperature
present in chickens. Alternatively, it is possible to
construct and test IBD viruses generated using the RNA
polymerase derived from IBDV serotype 2 viruse and the
polyprotein derived from IBDVserotype 1 virus.
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cDNA clones representing the complete genome of IBDV
(double stranded RNA segments A and B) is constructed,
initially using the BursaVac vaccine strain (Sterwin
Labs). Once cDNA clones representing full length copies
o~ segment A and B are constructed, template RNA is
prepared. Since IBDV exists as a bisegmented double-
stranded RNA virus, both the sense and anti-sense RNA
strands of each segment are produced using the
pBlueScript plasmid; Stratagene, Inc.). These vectors
utilize the highly specific phage promoters SP6 or T7 to
produce substrate amounts of RNA in vitro. A unique
restriction endonuclease site is engineered into the 3'
PCR primer to linearize the DNA for the generation of
run-off transcripts during transcription.
The purified RNA transcripts (4 strands) are transfected
into Vero cells to determine whether the RNA is
in~ectious. If IBD virus is generated, as determined by
black plaque assays using IBDV specific Mabs, no further
manipulations are required and engineering of the vaccine
strain can commence. The advantage of this method is
that engineered IBD viruses generated in this manner will
be pure and require little/no purification, greatly
decreasing the time required to generate new vaccines.
If negative results are obtained using the purified
RNA's, functional viral RNA polymerase is required by
use of a helper virus. Birnaviruses replicate their
nucleic acid by a strand displacement (semi-conservative)
mechanism, with the RNA polymerase binding to the ends of
the double-stranded RNA molecules forming circularized
ring structures (Muller & Nitschke, Virology 159, 174-
177, 1987). RNA polymerase open reading frame of about
878 amino acids in fowlpox virus is expressed and this
recombinant virus (S-FPV-098) is used to provide
functional IBDV RNA polymerase in t~ans. Fowlpox virus
expressed immunologically recognizable foreign antigens
in non-avian cells (Vero cells), where there are no signs
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of productive replication of the viral vector (Paoletti
e t al ., Technological Advances in Vaccine Development,
321-334, 1988, Alan R. Liss, Inc.). In the present
invention the IBDV polymerase protein is expressed in the
same cells as the transfected RNA using the fowlpox virus
vector without contaminating the cells with FPV
replication.
With the demonstration that IBD virus is generated in
vitro using genomic RNA, an improved live attenuated
virus vaccines against infectious bursal disease is
developed. Using recombinant DNA technology along with
the newly defined system of generating IBD virus,
specific deletions within the viral genome, facilitating
the construction of attenuated viruses are made. Using
this technology, the region of IBDV responsible for
virulence and generate attenuated, immunogenic IBDV
vaccines are identified. The present invention provides
a virulent IBD strain or replacement of the VP2 variable
region of the attenuated vaccine backbone with that of a
virulent strain, thus protecting against the virulent
strain while providing the safety and efficacy of the
vaccine strair,.
Exam~le 10
The chicken interferon (cIFN) gene was cloned into wild
type (FPV) viruses by homologous recombinant techniques.
Briefly, the entire coding region of cIFN was isolated
from activated chlcken spleen cell RNA by RT/PCR using
primer sequences from the recently published cIFN
sequence (Sekellick, M., et al., 1994). Recombinant FPV
viruses containing cIFN, and FPV/cIFN (S-FPV-099), were
engineered to contain the entire cIFN ORF under the
control of a synthetic pox virus promoter (LP2EP2), which
functions as both an early and late promoter, directing
expression throughout the entire viral replication cycle.
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A third recombinant virus, FPV/cIFN+NDV, (S-FPV-101) was
made in a similar manner, except that a FPV virus
previously engineered to contain the Newcastle Disease
(NDV) antigens HN and F was used as the parent virus
during homologous recombination, thus yielding a
f recombinant fowlpox virus co-expressing the cIFN and NDV
genes. All recombinant viruses contain the lac Z gene
engineered in tandem with cIFN under the control of a
synthetic late (LPl) pox promoter. All promoter/gene
constructs were sequenced at the promoter/cIFN junction
to confirm the integrity of the proper DNA coding frame.
Co-expression of ~-galactosidase facilitated the
isolation and plaque purification of the recombinant
viruses. Independent viral insertion sites were used
for insertion of the cIFN gene and the NDV genes in the
~owlpox virus. The insertion sites were found to
interrupt nonessential virus genes in both SPV and FPV.
To confirm the presence of the cIFN gene, recombinant
viral DNAs were analyzed by PCR, using cIFN specific
primers flanking the coding region. All viral DNA's
yielded the expected 600 bp amplified cIFN DNA product.
In addition, southern blot analysis on the viral DNA was
performed using a non-radioactive labeled cIFN cDNA
probe. ~ Plasmid constructs containing the cIFN gene
cassettes were sequenced across the transcriptional and
translational initiation/termina~ion signals, to confirm
the integrity of the ORF.
Growth Properties of Recombinant Viruses in Cell Culture.
Recombinant FPV/cIFN and FPV/cIFN+NDV were found to be
attenuated with respect to their growth in chicken embryo
fibroblast (CEF) cells. Plaque size was decreased
significantly and viral titers were 0.9-1.4 logs less
when compared to wild type FPV. We suggest that fowlpox
virus has anti-IFN mechanisms, similar to anti-IFN
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mech~n;~ms ~p~LLed for other pox viruses, e.g. vaccinia,
cowpox. And that these -~hAni~ help the virus to
overcome the inhibitory effects of exogenously expressed
cIFN. Therefore, fowlpox virus is able to infect,
replicate and retain a productive infectious state.
Tn vivo Proper~;es of Recomhin~nt FPV/cTFN V;rll~ in
Chicks.
10-day old chicks were inoculated, subcutaneously, with
recombinant FPV/cIFN (S-FPV-O99) virus at increasing
dosages. At 10 days post inoculation, all chicks were
inoculated with a mixture of sheep red blood cells (SRBC)
and Brucel l a abortus (BA) . At 15 days post FPV/cIFN
virus inoc~ tion, sera was collected, total body weights
and antibody responses to SRBC's and BA were measured,
and chicks were sacrificed for ne~,op~y analysis. These
data show that there were no significant differences in
chick body weight, SRBC and BA antibody responses or
gross pathologyC associated with inoculation of
recombinant FPV/cIFN virus, as c _~ed to chicks
inoculated with PBS alone. Therefore, this virus appears
to be safe in 10-day old chicks.
Table 3. Determination of safety of recombinant FPV/cIFN
virus in 10-day old chicks.
FPV/cIFN Total body Antibody titers Dd
(pfu/chick) weight
(grams)Db
BA SRBC
0 (PBS) 438 4.66 2.16
600 460 4.00 2.00
6,000 461 4.25 2.00
SUBSTITUTE SHEET (RUI E 26)
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60,000 460 4.62 2.00
a. Measured 15 days post FPV/cIFN virus inoculation
~. Mean body weight (n=8).
c There were no detectable gross pathoiogical changes
in any of the groups.
d, Mean antibody titers were determined by
agglutination assay and expressed as log2 (n=8).
One-day old chicks were inoculated
intranasally/intraocularly with NDV Bl (106 ELDso/chick)
alone or in addition to subcutaneous inoculation with
FPV/cIFN (103 pfu/chick). Chick mortality was recorded
2 weeks post vaccination. Chicks vaccinated with NDV Bl
alone or with NDV B1 plus FPV wild-type virus showed 20-
30% mortality c~ _-~ed to chickens co-vaccinated with
NDV-Bl and FPV/cIFN, in which group, all chicks r -;n~
alive. Subsequently, all chicks were challenged at 4
weeks post vaccination with a pathogenic strain of NDV
(GB-TX). All chicks were protected, except for those in
the "no treatment " control group. These data show that
NDV Bl vaccine induced mortality was reduced without
affecting the vaccine's protective ability.
Table ~. Effect of recombinant FPV/cIFN virus on NVD
Bl vaccine induced chick mortality and NDV B1
induced protection from NDV challenge.
SUBS H I UTE SHEET (RULE 26~
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--84--
Treatment Vaccine ~h~ n~e Po~2t vaccin:~tinn anti--
~ n~n~e~ i nrl~7~e~ NDV :~n~ iho~ly responEIes.
mortality.' mortality.b~C
Dead/Total Dead/Total 2 weeksd 4 week~~
No tr~a~ - L 0/25 15/15 cl <1
NDVBl alone 7/30 0/12 1.87 (0.31) 2.15 (0.32)
NDVB1 + FPV 9/30 0~10 1.96 (0.54) 1.99 (0.35)
NDVBl + 0/30 0/19 2.00 (O.42) 2.15 (O.37)
FPV/cIFN
~- Mortality was measured 2 weeks post vaccination.
0 b- Chicks were challenged 4 weeks post vaccination,
intramuscularly, with 10,000 ELD50NDV GB-TX.
c Mortality was measured 2 weeks post challen~e
d. Antibody titers were detel i n~ by NDV virus
neutralization and expressed as group mean (log1O).
17--day--oldchicken embryos were in~clllated with 500
pfu/embryo with FPV/cIFN/NDV virus, FPV wild-type virus
or PBS diluent (0.2 ml). Chicks were allowed to hatch
2 0 and then placed in an isolation unit and observed for
mortality for one week. These data show that
inoculation of chicken embryos with FPV/cIFN~NDV or FPV
wild-type does not interfere with normal hatching.
Table 5. Effect o~ FPV/cIFN/NDV virus in ovo.
Treatment Number of Eggs Mortality
Hatched/Total (Dead/Total) a
Diluent (PBS) 15/17 1/15
FPV (wild--type) 15/17 3/15
FPV/cIFN/NDV 14/18 0/14
a 1 week post hatch
SUBST1TI~TE SHEE r (RULE 26)
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Three week old SPF chicks were vaccinated,
subcutaneously, with 500 pfu/chick of FPV/cIFN/NDV
recombinant virus. Sera were collected 9 days and 28
days post vaccination to measure neutralizing antibody
responses raised against NDV. All chickens were
challenged 28 days post vaccination with a pathogenic
strain of NDV and observed for NDV induced mortality for
days. These data show that vaccinated chicks
developed detectable anti-NDV antibody resronC~C as
little as 9 days post vaccination with FPV/NDV/cIFN
recombinant virus. These antibody levels were
maintAin~ for at least 28 days. In addition, chickens
vaccinated with FPV/cIFN/NDV recombinant virus were all
protected against challenge with a virulent strain of
NDV.
Table 6. Protective efficacy of FPV/cIFN/NDV vaccine in
3-week-old-chickens.
Vaccine Post Post Vaccination Antibody
Challenge Responses
Mortality~
Dead/Total 9 days 28 days
None 19/19 < lb <lc
FPV-IFN-NDV 0/20 1.36 (0.12) 1.33 (0.31)
a. Chicks were challenged intraml~~c~ ~ly, 28 days post
vaccination, with 10,000 ELDsoNDV GB-TX.
b- Antibody responses were determined by VN test and
expressed as geometric mean titer (loglO) of 5
chickens
c Antibody responses were determined by VN test and
~ expressed as geometric mean titer (loglO) of 10
chickens
-
SUBSTITUTE SHEET (RULE 26)
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One day old SPF chicks were vaccinated, subcutaneously,
with 500 pfu/chick of FPV/cIFN/NDV recombinant virus.
Chicks were challenged intr~n~ ly/intraocularly at 4 ,
7 and 15 days post vaccination with virulent NDV (GB-TX),
and observed for NDV induced mortality for 15 days in
each case. These data show that vaccinated chicks are
resistant to virulent NDV when challenged at 7 days post
vaccination, but not as early as 4 days post vaccination.
Thus, onset of immunity to NDV following vaccination with
FPV/cIFN/NDV recombinant virus o~ between 4 and 7
days post vaccination.
~5 Table 6. Protective efficacy of FPV/cIFN/NDV vaccine in
one day old chicks.
Mortality following challenge
at 4, 7, and 15 days post
vaccination.
Experiment Vaccine 4-days 7-days 15-days
No.
Dead/Total Dead/Total Dead/Total
1 None NDa 10/10 10/10
FPV-IFN-NDV N~ 0/10 0/10
2 None 10/10 10/10 10/10
FPV-IFN-NDV 10/10 1/10 0/10
NDV-B1 4/10 0/lo O/lo
Not Done
SUBSTITUTE S~E~T (RULE 26)
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Conclusions
1. Recombinant fowlpox viruses express biologically
active chicken interferon into the supernatants o~
in~ected cells, as measured by protection of CEF cells
from VSV in~ection.
2. Chicken interferon expressed in supernatants from
recombinant SPV/cIFN infected cells has been shown to
protect=CEF cells against infection with HVT in a dose
dependent m~nn~r.
3. Chicken interferon expressed from SPV/cIFN acted
synergistically with LPS to activate chicken macrophages
as detected by nitric oxide induction.
4. Recombinant FPV/cIFN virus was found to be safe in lO
day old chicks at a dosage of 6 x 104 pfu/chick.
5. Recombinant FPV/cIFN virus was shown to reduce NDV B1
vaccine induced mortality without affecting the vaccine's
ability to protect chicks against NDV infection.
6. Inoculation of recombinant FPV/cIFN/NDV virus in ovo
does not appear to inter~ere with normal hatching.
7. Recombinant FPV/cIFN/NDV virus was shown to induce
anti-NDV neutralizing antibody in 3-week-old chicks as
early as 9 days post vaccination with sustained immunity
thru 28 days post vaccination. Furthermore, three-week-
old chicks were ~ully protected against virulent NDV
challenge at 28 days post vaccination.
CA 02223591 1997-12-04
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8. Recombinant FPV/cIFN/NDV virus was shown to protect
one-day-old chicks from virulent NDV challenge as early
as 7 days post vaccination.
9. The foregoing data indicate that recombinant fowlpox
viruses expressing chicken IFN may have bene~icial
applications as immune modulating agents in vitro, in
vivo and in ovo .
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Re~erences
1. C. Bertholet, et al., EMBO Journal 5, 1951-1957,
1986.
2. B.H. Coupar, et al., Virology 179, 159-167, 1990.
3. A.J. Davidson and B. Moss, J. Mol. Biol. 210, 749-
769.
4. A.J. Davidson and B. Moss, J. Mol. Biol., 210, 771-
784.
5. P.L. Earl, et al., Journal of Virology 64, 2448-
245r, 1990.
6. J. Esposito, et al., Virology 165, 313.
7. F.A. Ferrari, et al., Journal of Bacteriology 161,
556-562, 1985.
8. U. Gubler and B.J. Hoi~~man, Gene 25, 263-269.
9. D. ~n~ n, Molecular Biology 166, 557-580, 1983.
10. M.A. Innis, et al., PCR Protocols A Guide to Methods
and Applications, 84-91, Academic Press, Inc., San
Diego 1990.
11. Maniatis, et al., Molecular Cloning, Cold Spring
Harbor Laboratory, New York 19 82.
12. L.J.N. Ross, et al., Journal of General Virology,
70, 1789-1804 (1989).
13. L. J.N. Ross, et al., Journal of General Virology,
72, 949-954 (1991).
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--90--
14. J. Sambrook, e t al ., Mol ecul ar Cl oning A Labora tory
Manual Secon~ Edi tion, Cold Spring Harbor Press,
1989.
15. J. Taylor, et al ., Vaccine 9, 190-193, 1991.
16. A. Leutz, et al., EMB0 Journal 8: 175-182 (1989).
17. M.J. Sekellick, et al., Journal of Inter~eron
Reserch 14: 71-79 (1994).
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANTS: Mark D. Cochran and David E. Junker
(ii) TITLE OF lNv~NllON: Recor~in~nt Fowlpox Viruses and Uses
- 10 Thereof
(iii) NUMBER OF SEQUENCES: 20
(iv) CORRESPONDENCE ADDRESS:
(A) Ann~ s~ John P. White
(B) STREET: 1185 Avenue of the Americas
(C) CITY: New York
(D) STATE: New York
(E) CO~N1'K~: USA
(F) ZIP: 10036
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NVMBER: Not Yet Known
(B) FILING DATE: 07-JVN-1995
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: White, John P
(B) REGISTRATION NO: 28,678
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212)278-0400
(B) TELEFAX: (212)391-0526
(C) TELEX: 422523
(2) INFORMATION FOR SEQ ID NO:l:
~i) SEQUENCE CEARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) sTR~Nn~nN~s: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
CATAAGGCGG CCGCGGCC~l CGAGGCCA 28
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CEARACTERISTICS:
(A) LENGTE: 28 base pairs
(B) TYPE: nucleic acid
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(C) sTRANn~nN~cs double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(Xi) ~Q~'N~'~' DESCRIPTION: SEQ ID NO:2:
CATAATGGCC TCGAGGGCCG CGGCCGCC 28
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1507 base pairs
(B) TYPE: nucleic acid
(C) STRAN~ double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 260..1411
(Xi) ~:QU~:N~ DESCRIPTION: SEQ ID NO:3:
CTACTTCATA A~AAGTTTAA AC~l~lC~AA AGATTTTTGG ATAAAAGTAG AGAACTCGCA 60
TTGCGATTAT GCTCTAGGAC AALC~ AA A~1~1~1C~A TCTTAGCATA TAGATAAATG 120
TTTGAACTAA TATCCTAAAG CCTGTATGTA ACAGTTGGTG CCTATTGAAA GATACTGATT 180
ATCAAGGAGA AGAATAATAT A~ATCGTA~A AATAATACTT ATTATATAAT ATAATGTATA 240
AT~T~T~ AAAACAGCC ATG ATA CGT ATT ATA ATA TTA TCG TTA TTA TTT 292
Met Ile Arg Ile Ile Ile Leu Ser Leu Leu Phe
1 5 10
ATT AAC GTA ACA ACA GAT AGT CAA GAA TCT TCA A~A AAT ATA CAA AAT 340
Ile Asn Val Thr Thr Asp Ser Gln Glu Ser Ser Lys Asn Ile Gln Asn
GTA TTG CAC GTT ACA GAA TAT AGT AGA ACT GGT GTA ACA GCT TGC TCG 388
Val Leu His Val Thr Glu Tyr Ser Arg Thr Gly Val Thr Ala Cys Ser
30 35 40
TTA CAT TGT TTT GAT CGT TCC A~A GGT TTA GAT CAA CCA AaA ACA TTT 436
Leu His Cys Phe Asp Arg Ser LYB Gly Leu Asp Gln Pro Lys Thr Phe
45 50 55
ATC CTG CCT GGT A~A TAT AGC AAT AAC AGT ATA A~A CTA GAA GTA GCT 484
Ile Leu Pro Gly Lys Tyr Ser Asn Asn Ser Ile LYS Leu Glu Val Ala
ATT GAT ACA TAT A~A A~A GAT AGC GAC TTC AGT TAT TCT CAC CCA TGT 532
Ile Asp Thr Tyr LYS LYB Asp Ser Asp Phe Ser Tyr Ser His Pro Cys
==: =
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CAA ATA TTC CAG TTC TGT GTG TCT GGT AAT TTT AGT GGT AAA CGG TTC 580
Gln Ile Phe Gln Phe Cys Val Ser Gly Asn Phe Ser Gly Lys Arg Phe
95 loo 105
GAT CAT TAT CTA TAT GGG TAT ACA ATT TCC GGA TTT ATA GAT ATT GCT 628
Asp His Tyr Leu Tyr Gly Tyr Thr Ile Ser Gly Phe Ile Asp Ile Ala
llo 115 120
CCA A~A TAT TAT AGC GGT ATG TCT ATA AGT ACT ATT ACT GTT ATG CCA 676
Pro Lys Tyr Tyr Ser Gly Met Ser Ile Ser Thr Ile Thr Val Met Pro
125 130 135
TTA CAA GAA GGA TCA TTA AAG CAT GAT GAT GCC GAT GAC TAT GAC TAC 724
Leu Gln Glu Gly Ser Leu Lys His Asp Asp Ala Asp Asp Tyr Asp Tyr
140 145 150 155
GAT GAT GAT TGT GTT CCT TAT A~A GAA ACC CAG CCT CGA CAT ATG CCA 772
2 0 Asp Asp Asp Cys Val Pro Tyr Lys Glu Thr Gln Pro Arg His Met Pro
160 165 170
GAA TCG GTA ATA A~A GAA GGA TGT A~A CCC ATT CCA CTA CCA AGG TAT 820
Glu Ser Val Ile Lys Glu Gly Cys Lys Pro Ile Pro Leu Pro Arg Tyr
175 l80 185
GAT GAA AAT GAC GAT CCT ACT TGT ATT ATG TAT TGG GAT CAC TCG TGG 868
Asp Glu Asn Asp Asp Pro Thr Cys Ile Met Tyr Trp Asp His Ser Trp
lgo 195 200
GAT AAT TAC TGT AAT GTT GGA TTT TTT AAT TCT CTA CAG AGT GAT CAC 916
Asp Asn Tyr Cys Asn Val Gly Phe Phe Asn Ser Leu Gln Ser Asp His
205 210 215
AAT CCT CTG GTT TTT CCG TTA ACA AGT TAT TCT GAT ATA AAC AAT GCA 964
Asn Pro Leu Val Phe Pro Leu Thr Ser Tyr Ser Asp Ile Asn Asn Ala
220 22s 230 235
TTT CAT GCT TTT CAA TCA TCT TAT TGT AGA TCA CTA GGC TTT AAC CAA Io12
4 0 Phe His Ala Phe Gln Ser Ser Tyr Cys Arg Ser Leu Gly Phe Asn Gln
240 24s 250
TCA TAC AGT GTA TGC GTA TCT ATA GGT GAT ACA CCA TTT GAG GTT ACG 10 60
Ser Tyr Ser Val Cys Val Ser Ile Gly Asp Thr Pro Phe Glu Val Thr
255 260 26s
TAT CAT AGT TAT GAA AGT GTT ACT GTT GAT CAG TTA TTA CAA GAA ATT 110 8
Tyr His Ser Tyr Glu Ser Val Thr Val Asp Gln Leu Leu Gln Glu Ile
270 275 280
A~A ACA CTA TAT GGA GAA GAT GCT GTA TAT GGA TTA CCG TTT AGA AAT 1156
Lys Thr Leu Tyr Gly Glu Asp Ala Val Tyr Gly Leu Pro Phe Arg Asn
285 290 295
ATA ACT ATA AGG GCG CGT ACA CGG ATT CAA AGT TTA CCT CTT ACT AAC 12 04
Ile Thr Ile Arg Ala Arg Thr Arg Ile Gln Ser Leu Pro Leu Thr Asn
300 305 310 315
AAT ACC TGT ATC CCT A~A CAA GAC GAT GCT GAT GAT GTT GAC GAT GCT 1252
6 0 Asn Thr Cys Ile Pro Lys Gln Asp Asp Ala Asp Asp Val Asp Asp Ala
320 325 330
GAT GAT GTT GAC GAT GCT GAT GAT GCT GAC GAT GAT GAT GAT TAC GAG I300
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 A~A AAA CCC 1348
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Leu Tyr Val Glu Thr Thr Pro Arg Val Pro Thr Ala Arg Lys Lys Pro
350 355 360
GTT ACA GAA GAA TAT AAT GAT ATA TTT AGT AGT TTT GAT AAT TTT GAC 1396
Val Thr Glu Glu Tyr Asn Asp Ile Phe Ser Ser Phe Asp Asn Phe Asp
365 370 375
ATG A~A AAG A~A TAAGACATAT TTTATTAAAT CAAAAAGTCT GTCGAACTTT 1448
Met Lys Lys Lys
380
TA~~ lAA CCTATATCGA TTTATGATTT TTCCATGATG ATCCAGGCTA TGACTGACT 1507
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 383 amino acids
B) TYPE: amino acid
~:D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Ile Arg Ile Ile Ile Leu Ser Leu Leu Phe Ile Asn Val Thr Thr
1 5 10 15
Asp Ser Gln Glu Ser Ser Lys Asn Ile Gln Asn Val Leu His Val Thr
20 25 30
Glu Tyr Ser Arg Thr Gly Val Thr Ala Cys Ser Leu His Cys Phe Asp
Arg Ser Lys Gly Leu Asp Gln Pro Lys Thr Phe Ile Leu Pro Gly Lys
50 55 60
Tyr Ser Asn Asn Ser Ile Lys Leu Glu Val Ala Ile Asp Thr Tyr Lys
65 70 75 80
Lys Asp Ser Asp Phe Ser Tyr Ser His Pro Cys Gln IlÇ Phe Gln Phe
85 90 95
Cys Val Ser Gly Asn Phe Ser Gly Lys Arg Phe Asp His Tyr Leu Tyr
100 105 110
Gly Tyr Thr Ile Ser Gly Phe Ile Asp Ile Ala Pro Lys Tyr Tyr Ser
115 120 125
~0 Gly Met Ser Ile Ser Thr Ile Thr Val Met Pro Leu Gln Glu Gly Ser
130 135 140
Leu Lys His Asp Asp Ala Asp Asp Tyr Asp Tyr Asp Asp Asp Cys Val
145 150 155 160
.
Pro Tyr Lys Glu Thr Gln Pro Arg His Met Pro Glu Ser Val Ile Lys
165 170 175
Glu Gly Cys Lys Pro Ile Pro Leu Pro Arg Tyr Asp Glu Asn Asp Asp
180 185 190
Pro Thr Cys Ile Met Tyr Trp Asp His Ser Trp Asp Asn Tyr Cys Asn
195 200 205
Val Gly Phe Phe Asn Ser Leu Gln Ser Asp His Asn Pro Leu Val Phe
210 215 220
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Pro Leu Thr Ser Tyr Ser Asp Ile Asn Asn Ala Phe His Ala Phe Gln
225 230 235 240
Ser Ser Tyr Cys Arg Ser Leu Gly Phe Asn Gln Ser Tyr Ser Val Cys
245 250 255
Val Ser Ile Gly Asp Thr Pro Phe Glu Val Thr Tyr His Ser Tyr Glu
260 265 270
Ser Val Thr Val Asp Gln Leu Leu Gln Glu Ile Lys Thr Leu Tyr Gly
275 280 285
Glu Asp Ala Val Tyr Gly Leu Pro Phe Arg Asn Ile Thr Ile Arg Ala
290 295 300
Arg Thr Arg Ile Gln Ser Leu Pro Leu Thr Asn Asn Thr Cys Ile Pro
305 310 315 320
Lys Gln Asp Asp Ala Asp Asp Val Asp Asp Ala Asp Asp Val Asp Asp
325 330 335
Ala Asp Asp Ala Asp Asp Asp Asp Asp Tyr Glu Leu Tyr Val Glu Thr
340 345 350
~5 Thr Pro Arg Val Pro Thr Ala Arg Lys Lys Pro Val Thr Glu Glu Tyr
355 360 365
Asn Asp Ile Phe Ser Ser Phe Asp Asn Phe Asp Met Lys Lys Lys
370 375 380
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2849 base pairs
(B) TYPE: nucleic acid
(C) STRANn~n~ss: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 300..1568
(ix) FEATURE:
(A) NAME/ ~ Y: CDS
(B) LOCATION: complement (1685..2848)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
AAGCCAGTTT GAATTCAATA TTCATCGCCG ATA~lr~lA GAAATACTAT TCATGAAATT 60
TAC~~ C CGTGGCTTAA A~ACTTATTG TATGTACCAT TCATTATA~G ATCTGATACT 120
ATCGGCATCT TCTATTTTCC GA~llllllA CA'l~lG~llA CTAGTATCCA T~'11C~1~1A 180
ATAAGAGGGA AGGAATATAT CTATCTACAT A~ACATCATA AG~l~l~lll~G ATAGATTTAT 240
ATCGCTAATA A~ATATAAAT AATAATTA~A GATTTTATGA TATATCGAGC TTTGCA~AA 299
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ATG TCT GTT GAT TGG CGT ACA GAA ATC TAT TCG GGT GAT ATA TCC CTA 347
Met Ser Val Asp Trp Arg Thr Glu Ile Tyr Ser Gly Asp Ile Ser Leu
1 5 10 15
GTA GAA A~A CTT ATA AAG AAT A~A GGT AAT TGC ATC AAT ATA TCT GTA 395Val Glu Lys Leu Ile Lys Asn Lys Gly Asn Cys Ile Asn Ile Ser Val
GAG GAA ACA ACA ACT CCG TTA ATA GAC GCT ATA AGA ACC GGA AAT GCC 443
Glu Glu Thr Thr Thr Pro Leu Ile Asp Ala Ile Arg Thr Gly Asn Ala
AAA ATA GTA GAA CTA TTT ATC AAG CAC GGA GCG CAA GTT AAT CAT GTA 491
Lys Ile Val Glu Leu Phe Ile Lys His Gly Ala Gln Val Asn His Val
50 55 60
AAT ACT AAA ATT CCT AAT CCC TTG l'TA ACA GCT ATC AAA ATA GGA TCA 539
Asn Thr Lys Ile Pro Asn Pro Leu Leu Thr Ala Ile Lys Ile Gly Ser
65 70 75 80
CAC GAT ATA GTA A~A CTG CTG TTG ATT AAC GGA GTT GAT ACT TCT ATT 587
His Asp Ile Val Lys Leu Leu Leu Ile Asn Gly Val Asp Thr Ser Ile
85 90 95
TTG CCA GTC CCC TGC ATA AAT A~A GAA ATG ATA A~A ACT ATA TTA GAT 635
Leu Pro Val Pro Cys Ile Asn Lys Glu Met Ile Lys Thr Ile Leu Asp
100 105 110
AGT GGT GTG A~A GTA AAC ACA AAA AAT GCT A~A TCT AAA ACT TTC TTG 683
Ser Gly Val Lys Val Asn Thr Lys Asn Ala Lys Ser Lys Thr Phe Leu
115 120 125
CAT TAC GCG ATT AAG AAT AAT GAC TTA GAG GTT ATC AAA ATG CTT TTT 731
His Tyr Ala Ile Lys Asn Asn Asp Leu Glu Val Ile Lys Met Leu Phe
130 135 140
GAG TAT GGA GCT GAT GTT AAT ATA A~A GAT GAT AAC ATA TGT TAT TCT 779
Glu Tyr Gly Ala Asp Val Asn Ile Lys Asp Asp Asn Ile Cys Tyr Ser
145 150 155 160
ATA CAC ATA GCT ACT AGG AGT AAT TCA TAT GAA ATC ATA AAA TTA CTA 827
Ile His Ile Ala Thr Arg Ser Asn Ser Tyr Glu Ile Ile Lys Leu Leu
165 170 175
TTA GAA A~A GGT GCT TAT GCA AAC GTA A~A GAC AAT TAT GGT AAT TCT 875
Leu Glu Lys Gly Ala Tyr Ala Asn Val Lys Asp Asn Tyr Gly Asn Ser
180 185 190
CCG TTA CAT AAC GCG GCT A~A TAT GGC GAT TAT GCT TGT ATT A~A TTA 923
Pro Leu His Asn Ala Ala Lys Tyr Gly Asp Tyr Ala Cys Ile Lys Leu
195 200 205
GTT TTA GAC CAT ACT AAT AAC ATA AGC AAT AAG TGC AAC AAC GGT GTT 971
Val Leu Asp His Thr Asn Asn Ile Ser Asn Lys Cys Asn Asn Gly Val
210 215 220
ACA CCG TTA CAT AAC GCT ATA CTA TAT AAT AGA TCT GCC GTA GAA TTA 1019
Thr Pro Leu His Asn Ala Ile Leu Tyr Asn Arg Ser Ala Val Glu Leu
225 230 235 240
CTG ATT AAC AAT CGA TCT ATT AAT GAT ACG GAT GTA GAC GGA TAT ACT 1067
Leu Ile Asn Asn Arg Ser Ile Asn Asp Thr Asp Val Asp Gly Tyr Thr
245 250 255
CCA CTA CAT TAT GCT TTG CAA CCT CCG TGT AGT ATA GAT ATT ATA GAT 1115
Pro Leu His Tyr Ala Leu Gln Pro Pro Cys Ser Ile Asp Ile Ile Asp
260 . 265 270
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ATA CTA CTA TAT AAC AAC GCC GAT ATA TCT ATA AAA GAT AAT AAC GGA 1163
Ile Leu Leu Tyr Asn Asn Ala Asp Ile Ser Ile Lys Asp Asn Asn Gly
275 280 285
CGC AAT CCT ATC GAT ACG GCG TTT AAG TAT ATT AAC AGA GAT AGC GTT 1211
_ Arg Asn Pro Ile Asp Thr Ala Phe Lys Tyr Ile Asn Arg Asp Ser Val
290 295 300
ATA AAA GAA CTT CTC CGA AAC GCC GTG TTA ATT AAC GAG GTC GGT AAA 1259
r 10 Ile Lys Glu Leu Leu Arg Asn Ala Val Leu Ile Asn Glu Val Gly Lys
305 310 315 320
TTA A~A GAT ACT ACT ATC TTA GAA CAC A~A GAA ATA A~A GAC AAT ACC 1307
Leu Lys Asp Thr Thr Ile Leu Glu His Lys Glu Ile Lys Asp Asn Thr
325 330 335
GTG TTT TCA AAC TTT GTG TAC GAA TGT AAT GAA GAA ATT AAA AbA ATG 1355
Val Phe Ser Asn Phe Val Tyr Glu Cys Asn Glu Glu Ile Lys Lys Met
340 345 350
AAG AbA ACT A~A TGT GTC GGT GAC TAT AGT ATG TTT GAC GTA TAC ATG 1403
Lys Lys Thr Lys Cys Val Gly Asp Tyr Ser Met Phe Asp Val Tyr Met
355 360 365
ATA AGG TAT A~A CAC A~A TAT GAC GGT AAT AAG GAT AGT ATT A~A GAC 1451
Ile Arg Tyr Lys His Lys Tyr Asp Gly Asn Lys Asp Ser Ile Lys Asp
370 375 380
TAT TTG CGT TGT CTT GAT GAT AAT AGT ACT CGT ATG TTA AAA ACT ATA 1499
Tyr Leu Arg Cys Leu Asp Asp Asn Ser Thr Arg Met Leu Lys Thr Ile
385 390 395 400
GAT ATT AAT GAA TTT CCT ATA TAT TCT ATG TAT CTC GTA AGA TGC CTA 1547
Asp Ile Asn Glu Phe Pro Ile Tyr Ser Met Tyr Leu Val Arg Cys Leu
405 410 415
TAT GAT ATG GTA ATA TAT TAAAAGAAAT GGGCTCTTGC ATACATAATC 1595
Tyr Asp Met Val Ile Tyr
420
GGTATAAAAA ATAACGAbAT TATTAGCGGT TACATATCTT ACGGCGGCCG CGGCCCTCGA 1655
GGCCAGTAGC TCAGTATTTC CTATAbACTC TAATATTGAG AGTTTGATAT CCGGAGAAGT 1715
TTAGACCAAC CGCTAGAATC TAATATTTCA TCTAATTTTG ATCTACTTTT TTCTAATATT 1775
TTATGTCTAT TACTGGCTAA GGATATGGAA GTTTTAAGAC GATCTCCGTA ATTATAGAAA 1835
TAGTAAGTAT TAAl-llC~ll TATTATAGGA TTATTTACTA AGTGATGTAA CAGGTTCATG 1895
TTTTTACTAA TAACGAATAT ATCTAbAGAG TAbAACATAT TAATACGAAT TTTAGATATA 1955
~ llAGTT ~llC~llACA ACTCAACCAA ATACTTTTAA ACGTATCATC GCTTTGAATA 2015
A'111'~1~1~A AG~G~lllAC TTCACTTCTG ATATCGTGAC GTATAAAATC TTGTATACAT 2075
ATATGTGCTA TGATATATCT AbAAGA~AAC ATATTACTGT TAAGGCTCTT ATCGATGACC 2135
CTACTATCTC TAAGTTCAGC ACCATAATGT AATAATATAT TTACTATACC ATGATATTCT 2195
AATGCTATTA ATAbAGGATA TTGATTCCTT ATGTTAATAG CATTTACATC CGCTCCGTTA 2255
TCTAATAACA TTTTTATAAC 'l"l'~"l'W'l''l"l'A CAAll~llll TACACGCATA ATGCAACGGA 2315
GTAGATAAGT Alll~lllll AGAATTAACA TTAG~lC'~l~ TATCTATGAG C~lllllACA 2375
CTCATATACG GAlll~llCC ATATAAGGCA AbATGTAbAA CCGTTCCTAT CTTCTGCGAT 2435
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AACG~l~l~lA TALC~CCCC GTAATCTA~A AGA~l~L-llA TGATAACTAC All~lll~ll 2495
ACAGCGGCAT AATGAATAGG C~1~1"1~1~'A CAATAATCTC TAGCATTTAC GTTCGCTCCC 2555
AATTCTAACA ACGTTATAAC TGTATCTTTA TATCTATCTA GAGTAGAGGC TTGATGTAAT 2615
GGAGTGATAT ACAGACTATC AG~GC~llA ACATCTGCAC CCCGCATTAT TA~AGTTCTA 2675
A~ ~lG TATCGTATCC ATTCTTAGCC ATGAGATACA GAGGAGTTTC l~C-lllAATG 2735
TTTTTAGCGT TAACATCTAT l~l~-lllCC AATAACTTGG GTACTAGTCT ACTTAACGAA 2795
GGTGCTTGTA ~C~l~lAATG CA~AGGAGTA TTCTTATA~A CATCTATAGA ATTC 2849
(2) INFORMATION FOR SEQ ID NO:6:
(i) ~U~N~'~ CHARACTERISTICS:
(A) LENGTH: 422 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Xi) ~:QU~N~'~ DESCRIPTION: SEQ ID NO:6:
Met Ser Val Asp Trp Arg Thr Glu Ile Tyr Ser Gly Asp Ile Ser Leu
1 5 10 15
Val Glu Lys Leu Ile Lys Asn Lys Gly Asn Cys Ile Asn Ile Ser Val
20 25 30
Glu Glu Thr Thr Thr Pro Leu Ile Asp Ala Ile Arg Thr Gly Asn Ala
35 40 45
Lys Ile Val Glu Leu Phe Ile Lys His Gly Ala Gln Val Asn His Val
50 55 60
Asn Thr Lys Ile Pro Asn Pro Leu Leu Thr Ala Ile Lys Ile Gly Ser
65 70 75 80
His Asp Ile Val Lys Leu Leu Leu Ile Asn Gly Val Asp Thr Ser Ile
85 90 95
Leu Pro Val Pro Cys Ile Asn Lys Glu Met Ile Lys Thr Ile Léu Asp
100 105 110
Ser Gly Val Lys Val Asn Thr Lys Asn Ala Lys Ser Lys Thr Phe Leu
115 120 125
His Tyr Ala Ile Lys Asn Asn Asp Leu Glu Val Ile Lys Met Leu Phe
130 135 . 140
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
180 185 190
Pro Leu His Asn Ala Ala Lys Tyr Gly Asp Tyr Ala Cys Ile Lys Leu
195 200 205
CA 02223~91 1997-12-04.
W 096/40880 PCTrUS96/11187
_99 _
Val Leu Asp His Thr Asn Asn Ile Ser Asn Lys Cys Asn Asn Gly Val
210 215 220
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
~0 Pro Leu His Tyr Ala Leu Gln Pro Pro Cys Ser Ile Asp Ile Ile Asp
260 265 270
Ile Leu Leu Tyr Asn Asn Ala Asp Ile Ser 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
20 305 310 315 320
Leu Lys Asp Thr Thr Ile Leu Glu His Lys Glu Ile Lys Asp Asn Thr
325 330 335
Val Phe Ser Asn Phe Val Tyr Glu Cys Asn Glu Glu Ile Lys Lys Met
340 345 350
Lys Lys Thr Lys Cys Val Gly Asp Tyr Ser Met Phe Asp Val Tyr Met
355 360 365
Ile Arg Tyr Lys His Lys Tyr Asp Gly Asn Lys Asp Ser Ile Lys Asp
370 375 380
Tyr Leu Arg Cys Leu Asp Asp Asn Ser Thr Arg Met Leu Lys Thr Ile
35 385 390 395 400
Asp Ile Asn Glu Phe Pro Ile Tyr Ser Met Tyr Leu Val Arg Cys Leu
405 410 415
40 Tyr Asp Met Val Ile Tyr
420
45 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 387 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:7
Asn Ser Ile Asp Val Tyr Lys Asn Thr Pro Leu His Tyr Thr Val Gln
1 5 10 15
Ala Pro Ser Leu Ser Arg Leu Val Pro Lys Leu Leu Glu Arg Gly Ile
~ 20 25 30
Asp Val Asn Ala Lys Asn Ile Lys Gly Glu Thr Pro Leu Tyr Leu Met
Ala Lys Asn Gly Tyr Asp Thr Glu Asn Ile Arg Thr Leu Ile Met Arg
CA 02223~91 1997-12-04
W O 96/40880 PCTAUS96/11187
-100--
Gly Ala Asp Val Asn Ala Ala Asp Ser Leu Tyr Ile Thr Pro Leu His
65 70 75 80
Gln Ala Ser Thr Leu Asp Arg Tyr Lys Asp Thr Val Ile Thr Leu Leu
85 90 95
Glu Leu Gly Ala Asn Val Asn Ala Arg Asp Tyr Cys Asp Lys Thr Pro
100 105 110
Ile Xis Tyr Ala Ala Val Arg Asn Asn Val Val Ile Ile Asn Thr Leu
115 120 125
Leu Asp Tyr Gly Ala Asp Ile Glu Ala Leu Ser Gln Lys Ile Gly Thr
130 135 140
Val Leu His Phe Ala Leu Tyr Gly Thr Asn Pro Tyr Met Ser Val Lys
145 150 155 160
Thr Leu Ile Asp Arg Gly Ala Asn Val Asn Ser Lys Asn Lys Tyr Leu
2 0 165 170 175
Ser Thr Pro Leu His Tyr Ala Cys Lys Lys Asn Cys Lys Pro Glu Val
180 185 190
2 5 Ile Lys Met Leu Leu Asp Asn Gly Ala Asp Val Asn Ala Ile Asn Ile
195 200 205
Arg Asn Gln Tyr Pro Leu Leu Ile Ala Leu Glu Tyr His Gly Ile Val
210 215 220
Asn Ile Leu Leu His Tyr Gly Ala Glu 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
3 5 245 250 255
His Ile Cys Ile Gln Asp Phe Ile Arg His Asp Ile Arg Ser Glu Val
260 265 270
4 0 Asn Pro Leu Arg Glu Ile Ile Gln Ser Asp Asp Thr Phe Lys Ser Ile
275 280 285
Trp Leu Ser Cys Lys Glu Glu Leu Lys Asp Ile Ser Lys Ile Arg Ile
290 295 300
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 Glu Ile Asn Thr
325 330 335
Tyr Tyr Phe Tyr Asn Tyr Gly Asp Arg Leu Lys Thr Ser Ile Ser Leu
340 345 350
~5 Ala Ser Asn Arg His Lys Ile Leu Glu Lys Ser Arg Ser Lys Leu Asp
355 360 365
Glu Ile Leu Asp Ser Ser Gly Trp Ser Lys Leu Leu Arg Ile Ser Asn
370 375 380
Ser Gln Tyr *
385
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
CA 02223~91 1997-12-04
WO 96140880 PCT~US96/11187
- --101--
(B) TYPE: nucleic acid
(C) STRANn~nN~S: double
tD) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
l5 AAAAATTGAA A~ACTATTCT AATTTATTGC ACGGAGATCT 40
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D] TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(Xi) ~QU~N~'~ DESCRIPTION: SEQ ID NO:9:
35 AATTTCATTT '~ l"llC TATGCTATAA AT 32
(2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STR~Nn~nNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTIO~: SEQ ID NO:l0:
55 GTATCCTAAA ATTGAATTGT AA~TATCGAT AATAAAT 37
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
CA 02223~9l l997-l2-04
W 096/40880 PCT~US96/11187
- -102-
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
'l"l"l"L'l~l"l"l"l''l' '1"1"1"1"1"1"1''1"1"1' GGCATATA~A TGAATTCGGA TC 42
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4177 base pairs
(B) TYPE: nucleic acid
(C) sTRANn~nN~S: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 115... 1860
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2095.. .3756
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CATACTGGCC TCGAGGGCCG CGGCCGCCTG CAGGTCGACT CTAGA~AAAA TTGA~AAACT 60
ATTCTAATTT ATTGCACGGA GAl~lllLll llllll-llll TTTTTGGCAT ATAA ATG 117
Melt
AAT TCG GAT CCG GAC CGC GCC GTT AGC CAA GTT GCG TTA GAG AAT GAT 165
Asn Ser Asp Pro Asp Arg Ala Val Ser Gln Val Ala Leu Glu Asn Asp
5 10 15
GAA AGA GAG GCA A~A AAT ACA TGG CGC TTG ATA TTC CGG ATT GCA ATC 213
Glu Arg Glu Ala Lys Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala Ile
20 25 30
TTA TTC TTA ACA GTA GTG ACC TTG GCT ATA TCT GTA GCC TCC CTT TTA 261
Leu Phe Leu Thr Val Val Thr Leu Ala Ile Ser Val Ala Ser Leu Leu
35 40 45
TAT AGC ATG GGG GCT AGC ACA CCT AGC GAT CTT GTA GGC ATA CCG ACT 309
Tyr Ser Met Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Ile Pro Thr
50 55 60 65
AGG ATT TCC AGG GCA G~A GAA AAG ATT ACA TCT ACA CTT GGT TCC AAT 357
Arg Ile Ser Arg Ala Glu Glu Lys Ile Thr Ser Thr Leu Gly Ser Asn
CAA GAT GTA GTA GAT AGG ATA TAT AAG C~A GTG GCC CTT GAG TCT CCA 405
Gln Asp Val Val Asp Arg Ile Tyr Lys Gln Val Ala Leu Glu Ser Pro
TTG GCA TTG TTA AAT ACT GAG ACC ACA ATT A~G AAC GCA ATA ACA TCT 453
Leu Ala Leu Leu Asn Thr Glu Thr Thr Ile Met Asn Ala Ile Thr Ser
100 105 110
CA 02223~9l l997-l2-04
W O 96/40880 PCTrUS96/11187
-103-
CTC TCT TAT CAG ATT AAT GGA GCT GCA AAC AAC AGC GGG TGG GGG GCA 501
Leu Ser Tyr Gln Ile Asn Gly Ala Ala Asn Asn Ser Gly Trp Gly Ala
115 120 125
CCT ATT CAT GAC CCA GAT TAT ATA GGG GGG ATA GGC A~A GAA CTC ATT 549
- Pro Ile His Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Glu Leu Ile
130 135 140 145
GTA GAT GAT GCT AGT GAT GTC ACA TCA TTC TAT CCC TCT GCA TTT CAA 597
-10 Val Asp Asp Ala Ser Asp Val Thr Ser Phe Tyr Pro Ser Ala Phe Gln
150 155 160
GAA CAT CTG AAT TTT ATC CCG GCG CCT ACT ACA GGA TCA GGT TGC ACT 645
Glu His Leu Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr
165 170 175
CGA ATA CCC TCA TTT GAC ATG AGT GCT ACC CAT TAC TGC TAC ACC CAT 693
Arg Ile Pro Ser Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr His
180 185 190
AAT GTA ATA TTG TCT GGA TGC AGA GAT CAC TCA CAC TCA CAT CAG TAT 741
Asn Val Ile Leu Ser Gly Cys Arg Asp His Ser His Ser Xis Gln Tyr
195 200 205
2 5 TTA GCA CTT GGT GTG CTC CGG ACA TCT GCA ACA GGG AGG GTA TTC TTT 789
Leu Ala Leu Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Val Phe Phe
210 215 220 225
TCT ACT CTG CGT TCC ATC AAC CTG GAC GAC ACC CAA AAT CGG AAG TCT 837
3 0 Ser Thr Leu Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn Arg Lys Ser
230 235 240
TGC AGT GTG AGT GCA ACT CCC CTG GGT TGT GAT ATG CTG TGC TCG AAA 885
Cys Ser Val Ser Ala Thr Pro Leu Gly Cys Asp Met Leu Cys Ser Lys
245 250 255
GCC ACG GAG ACA GAG GAA GAA GAT TAT AAC TCA GCT GTC CCT ACG CGG 933
Ala Thr Glu Thr Glu Glu Glu Asp Tyr Asn Ser Ala Val Pro Thr Arg
260 265 270
ATG GTA CAT GGG AGG TTA GGG TTC GAC GGC CAA TAT CAC GAA AAG GAC 981
Met Val His Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Glu Lys Asp
275 280 285
4 5 CTA GAT GTC ACA ACA TTA TTC GGG GAC TGG GTG GCC AAC TAC CCA GGA 1029
Leu Asp Val Thr Thr Leu Phe Gly Asp Trp Val Ala Asn Tyr Pro Gly
290 295 300 305
GTA GGG GGT GGA TCT TTT ATT GAC AGC CGC GTG TGG TTC TCA GTC TAC 1077
Val Gly Gly Gly Ser Phe Ile Asp Ser Arg Val Trp Phe Ser Val Tyr
310 315 320
GGA GGG TTA A~A CCC AAT ACA CCC AGT GAC ACT GTA CAG GAA GGG A~A 1125
Gly Gly Leu Lys Pro Asn Thr Pro Ser Asp Thr Val Gln Glu Gly Lys
325 330 335
TAT GTG ATA TAC AAG CGA TAC AAT GAC ACA TGC CCA GAT GAG CAA GAC 1173
Tyr Val Ile Tyr I,ys Arg Tyr Asn Asp Thr Cys Pro Asp Glu Gln Asp
340 345 350
6Q
TAC CAG ATT CGA ATG GCC AAG TCT TCG TAT AAG CCT GGA CGG TTT GGT 1221
Tyr Gln Ile Arg Met Ala Lys Ser Ser Tyr IJYS Pro Gly Arg Phe Gly
355 . 360 365
GGG A~A CGC ATA CAG CAG GCT ATC TTA TCT ATC AAA GTG TCA ACA TCC 1269
Gly Lys Arg Ile Gln Gln Ala Ile Leu Ser Ile Lys Val Ser Thr Ser
370 375 - 380 385
CA 02223~91 1997-12-04
WO 96/40880 PCT~US96/11187
- -104-
TTA GGC GAA GAC CCG GTA CTG ACT GTA CCG CCC AAC ACA GTC ACA CTC 1317
Leu Gly Glu Asp Pro Val Leu Thr Val Pro Pro Asn Thr Val Thr Leu
390 395 400
ATG GGG GCC GAA GGC AGA ATT CTC ACA GTA GGG ACA TCC CAT TTC TTG 1365
Met Gly Ala Glu Gly Arg Ile Leu Thr Val Gly Thr Ser His Phe Leu
405 410 415
10 TAT CAG CGA GGG TCA TCA TAC TTC TCT CCC GCG TTA TTA TAT CCT ATG 1413
Tyr Gln Arg Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met
420 425 430
ACA GTC AGC AAC A~A ACA GCC ACT CTT CAT AGT CCT TAT ACA TTC AAT 1461
15 Thr Val Ser Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Thr Phe Asn
435 440 445
GCC TTC ACT CGG CCA GGT AGT ATC CCT TGC CAG GCT TCA GCA AGA TGC 1509
Ala Phe Thr Arg Pro Gly Ser Ile Pro Cys Gln Ala Ser Ala Arg Cys
20 450 455 460 465
CCC AAC TCA TGT GTT ACT GGA GTC TAT ACA GAT CCA TAT CCC CTA ATC 1557
Pro Asn Ser Cys Val Thr Gly Val Tyr Thr Asp Pro Tyr Pro Leu Ile
470 475 480
TTC TAT AGA AAC CAC ACC TTG CGA GGG GTA TTC GGG ACA ATG CTT GAT 1605
Phe Tyr Arg Asn His Thr heu Arg Gly Val Phe Gly Thr Met Leu Asp
485 490 495
3 0 GGT GAA CAA GCA AGA CTT AAC CCT GCG TCT GCA GTA TTC GAT AGC ACA 1653
Gly Glu Gln Ala Arg Leu Asn Pro Ala Ser Ala Val Phe Asp Ser Thr
500 505 510
TCC CGC AGT CGC ATA ACT CGA GTG AGT TCA AGC AGC ATC A~A GCA GCA 1701
3 5 Ser Arg Ser Arg Ile Thr Arg Val Ser Ser Ser Ser Ile Lys Ala Ala
515 520 525
TAC ACA ACA TCA ACT TGT TTT A~A GTG GTC AAG ACC AAT AAG ACC TAT 1749
Tyr Thr Thr Ser Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr Tyr
40 530 535 540 545
TGT CTC AGC ATT GCT GAA ATA TCT AAT ACT CTC TTC GGA GAA TTC AGA 1797
Cys Leu Ser Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Glu Phe Arg
550 555 560
ATC GTC CCG TTA CTA GTT GAG ATC CTC A~A GAT GAC GGG GTT AGA GAA 1845
Ile Val Pro Leu Leu Val Glu Ile Leu Lys Asp Asp Gly Val Arg Glu
565 570 575
50 GCC AGG TCT GGC TAGTTGAGTC AACTATGAAA GAGTTGGA~A GATGGCATTG 1897
Ala Arg Ser Gly
580
TATCACCTAT ~ GC~AC ATCAAGAATC A~ACCGAATG CCCGGATCCA TAATTAATTA 1957
ATTAATTTTT A'1'~:C~ ~AC TCTAGA~ A ATTGA~A~AC TATTCTAATT TATTGCACGG 2017
AGAl~ A TATAAATGAA TTCGGATCGA TCCCGGTTGG 2077
60 CGCC~:lCc~AG GTGCAGG ATG GGC TCC AGA CCT TCT ACC A~G AAC C.CA GCA 2127
Met Gly Ser Arg Pro Ser Thr Lys Asn Pro Ala
5 10
CCT ATG ATG CTG ACT ATC CGG GTC GCG CTG GTA CTG AGT TGC ATC TGT 2175
65 Pro Met Met Leu Thr Ile Arg Val Ala Leu Val Leu Ser Cys Ile Cys
CA 02223~91 1997-12-04
WO 96/40880 PCTnJS96/11187
- -105-
CCG GCA AAC TCC ATT GAT GGC AGG CCT CTT GCA GCT GCA GGA ATT GTG 2223
Pro Ala Asn Ser Ile Asp Gly Arg Pro Leu Ala Ala Ala Gly Ile Val
GTT ACA GGA GAC A~A GCA GTC AAC ATA TAC ACC TCA TCC CAG ACA GGA 2271
- Val Thr Gly Asp Lys Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gly
45 50 55
TCA ATC ATA GTT AAG CTC CTC CCG AAT CTG CCA AAG GAT AAG GAG GCA 2319
~10 Ser Ile Ile Val Lys Leu Leu Pro Asn Leu Pro Lys Asp Lys Glu Ala
60 65 70 75
TGT GCG AAA GCC CCC TTG GAT GCA TAC AAC AGG ACA TTG ACC ACT TTG 2367
Cys Ala Lys Ala Prp Leu Asp Ala Tyr Asn Arg Thr Leu Thr Thr Leu
80 85 90
CTC ACC CCC CTT GGT GAC TCT ATC CGT AGG ATA CAA GAG TCT GTG ACT 2415
Leu Thr Pro Leu Gly Asp Ser Ile Arg Arg Ile Gln Glu Ser Val Thr
100 105
ACA TCT GGA GGG GGG AGA CAG GGG CGC CTT ATA GGC GCC ATT ATT GGC 2463
Thr Ser Gly Gly Gly Arg Gln Gly Arg Leu Ile Gly Ala Ile Ile Gly
110 115 120
GGT GTG GCT CTT GGG GTT GCA ACT GCC GCA CAA ATA ACA GCG GCC GCA 2511
Gly Val Ala Leu Gly Val Ala Thr Ala Ala Gln Ile Thr Ala Ala Ala
125 130 135
GCT CTG ATA CAA GCC A~A CAA AAT GCT GCC AAC ATC CTC CGA CTT A~A 2559
Ala Leu Ile Gln Ala Lys Gln Asn Ala Ala Asn Ile Leu Arg Leu Lys
140 145 150 155
GAG AGC ATT GCC GCA ACC AAT GAG GCT GTG CAT GAG GTC ACT GAC GGA 2607
Glu Ser Ile Ala Ala Thr Asn Glu Ala Val His Glu Val Thr Asp Gly
160 165 170
TTA TCG CAA CTA GCA GTG GCA GTT GGG AAG ATG CAG CAG TTC GTT AAT 2655
Leu Ser Gln Leu Ala Val Ala Val Gly Lys Met Gln Gln Phe Val Asn
175 180 185
GAC CAA TTT AAT A~A ACA GCT CAG GAA TTA GAC TGC ATC A~A ATT GCA 2703
Asp Gln Phe Asn Lys Thr Ala Gln Glu Leu Asp Cys Ile Lys Ile Ala
190 195 200
CAG CAA GTT GGT GTA GAG CTC AAC CTG TAC CTA ACC GAA TCG ACT ACA 2751
Gln Gln Val Gly Val Glu Leu Asn Leu Tyr Leu Thr Glu Ser Thr Thr
205 210 215
GTA TTC GGA CCA CAA ATC ACT TCA CCT GCC TTA A~C AAG CTG ACT ATT 2799
Val Phe Gly Pro Gln Ile Thr Ser Pro Ala Leu Asn Lys Leu Thr Ile
220 225 230 235
CAG GCA CTT TAC AAT CTA GCT GGT GGG AAT ATG GAT TAC TTA TTG ACT 2847
Gln Ala Leu Tyr Asn Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Thr
240 245 250
AAG TTA GGT ATA GGG AAC AAT CAA CTC AGC TCA TTA ATC GGT AGC GGC 2895
Lys Leu Gly Ile Gly Asn Asn Gln Leu Ser Ser Leu Ile Gly Ser Gly
255 260 265
TTA ATC ACC GGT AAC CCT ATT CTA TAC GAC TCA CAG ACT CAA CTC TTG 2943
Leu Ile Thr Gly Asn Pro Ile Leu Tyr Asp Ser Gln Thr Gln Leu Leu
270 275 280
GGT ATA CAG GTA ACT CTA CCT TCA GTC GGG AAC CTA AAT AAT ATG CGT 2991
Gly Ile Gln Val Thr Leu Pro Ser Val Gly Asn Leu Asn Asn Met Arg
285 290 295
-
CA 02223~9l l997-l2-04
W O 96/40880 PCTAUS96/11187
- -106-
GCC ACC TAC TTG GAA ACC TTA TCC GTA AGC ACA ACC AGG GGA TTT GCC 3039
Ala Thr Tyr Leu Glu Thr Leu Ser Val Ser Thr Thr Arg Gly Phe Ala
300 305 310 315
TCG GCA CTT GTC CCA AAA GTG GTG ACA CGG GTC GGT TCT GTG ATA GAA 3087Ser Ala Leu Val Pro Lys Val Val Thr Arg Val Gly Ser Val Ile Glu
320 325 330
GAA CTT GAC ACC TCA TAC TGT ATA GAA ACT GAC TTA GAT TTA TAT TGT 3135
Glu Leu Asp Thr Ser Tyr Cys Ile Glu Thr Asp Leu Asp Leu Tyr Cys
335 340 345
ACA AGA ATA GTA ACG TTC CCT ATG TCC CCT GGT ATT TAC TCC TGC TTG 3183
Thr Arg Ile Val Thr Phe Pro Met Ser Pro Gly Ile Tyr Ser Cy5 Leu
350 355 360
AGC GGC AAT ACA TCG GCC TGT ATG TAC TCA AAG ACC GAA GGC GCA CTT 3231
Ser Gly Asn Thr Ser Ala Cys Met Tyr Ser Lys Thr Glu Gly Ala Leu
365 370 375
ACT ACA CCA TAT ATG ACT ATC AAA GGC TCA GTC ATC GCT AAC TGC AAG 3279
Thr Thr Pro Tyr Met Thr Ile Lys Gly Ser Val Ile Ala Asn Cys Lys
380 385 390 395
ATG ACA ACA TGT AGA TGT GTA AAC CCC CCG GGT ATC ATA TCG CAA AAC 3327
Met Thr Thr Cys Arg Cys Val Asn Pro Pro Gly Ile Ile Ser Gln Asn
400 405 410
TAT GGA GAA GCC GTG TCT CTA ATA GAT A~A CAA TCA TGC AAT GTT TTA 3375
Tyr Gly Glu Ala Val Ser Leu Ile Asp Lys Gln Ser Cys Asn Val Leu
415 420 .425
TCC TTA GGC GGG ATA ACT TTA AGG CTC AGT GGG GAA TTC GAT GTA ACT 3423
Ser Leu Gly Gly Ile Thr Leu Arg Leu Ser Gly Glu Phe Asp Val Thr
430 435 440
TAT CAG AAG AAT ATC TCA ATA CAA GAT TCT CAA GTA ATA ATA ACA GGC 3471
Tyr Gln Lys Asn Ile Ser Ile Gln Asp Ser Gln Val Ile Ile Thr Gly
445 450 455
AAT CTT GAT ATC TCA ACT GAG CTT GGG AAT GTC AAC AAC TCG ATC AGT 3519
Asn Leu Asp Ile Ser Thr Glu Leu Gly Asn Val Asn Asn Ser Ile Ser
460 465 470 475
AAT GCC TTG AAT AAG TTA GAG GAA AGC AAC AGA A~A CTA GAC A~A GTC 3567Asn Ala Leu Asn Lys Leu Glu Glu Ser Asn Arg Lys Leu Asp Lys Val
480 485 490
AAT GTC A~A CTG ACC AGC ACA TCT GCT CTC ATT ACC TAT ATC GTT TTG 3615
Asn Val Lys Leu Thr Ser Thr Ser Ala Leu Ile Thr Tyr Ile Val Leu
495 500 505
ACT ATC ATA TCT CTT GTT TTT GGT ATA CTT AGC CTG ATT CTA GCA TGC 3663
Thr Ile Ile Ser Leu Val Phe Gly Ile Leu Ser Leu Ile Leu Ala Cys
510 515 520
TAC CTA ATG TAC AAG C~A AAG GCG CAA CAA AAG ACC TTA TTA TGG CTT 3711
Tyr Leu Met Tyr Lys Gln Lys Ala Gln Gln Lys Thr Leu Leu Trp Leu
525 530 535
GGG AAT AAT ACC CTA GAT CAG ATG AGA GCC ACT ACA A~A ATG TGAACACAGA 3763
Gly Asn Asn Thr Leu Asp Gln Met Arg Ala Thr Thr Lys Met
540 545 550
TGAGGAACGA A~~ ~C~l AATAGTAATT l~l~l~AAAG TTCTGGTAGT CTGTCAGTTC 3823
GGAGAGTTA~ G~;AAAAAAa A~ACCCCCCC C(~CC(:'CCCCC CCCCCCCCCT GCAGGCATCG 3883
CA 02223~9l l997-l2-04
W O ~G/4~~0 PCTrUS96/11187
- -107-
TGGTGTCACG ~lC~lC~lll GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC 3943
GAGTTACATG ATCCCCCATG TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG 4003
TTGTCAGAAG TAA~ll~GCC G QGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT 4063
CTCTTACTGT CATGCCATCC GTAAGATGCT lll'~l~l~AC TGGTGAGTGA TCCATAATTA 4123
ATTAATTAAT TTTTATCCCG GGTCGACCTG CAGGCGGCCG CGGCC~lC~A GGCC 4177
1 0
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 581 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
txi) ~: U~N~ DESCRIPTION: SEQ ID NO:13:
Met Asn Ser Asp Pro Asp Arg Ala Val Ser Gln Val Ala Leu Glu Asn
1 5 10 15
Asp Glu Arg Glu Ala Lys Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala
20 25 30
Ile Leu Phe Leu Thr Val Val Thr Leu Ala Ile Ser Val Ala Ser Leu
35 40 45
Leu Tyr Ser Met Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Ile Pro
50 55 60
Thr Arg Ile Ser Arg Ala Glu Glu Lys Ile Thr Ser Thr Leu Gly Ser
65 70 75 80
Asn Gln Asp Val Val Asp Arg Ile Tyr Lys Gln Val Ala Leu Glu Ser
85 90 95
Pro Leu Ala Leu Leu Asn Thr Glu Thr Thr Ile Met Asn Ala Ile Thr
100 105 110
Ser Leu Ser Tyr Gln Ile Asn Gly Ala Ala Asn Asn Ser Gly Trp Gly
115 120 125
Ala Pro Ile His Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Glu Leu
130 135 140
Ile Val Asp Asp Ala Ser Asp Val Thr Ser Phe Tyr Pro Ser Ala Phe
145 150 155 160
Gln Glu Xi8 Leu Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys
165 . 170 175
Thr Arg Ile Pro Ser Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr
180 185 190
His Asn Val Ile Leu Ser Gly Cys Arg Asp His Ser His Ser His Gln
195 200 205
Tyr Leu Ala Leu Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Val Phe
210 215 220
Phe Ser Thr Leu Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn Arg Lys
225 230 235 240
CA 02223~91 1997-12-04
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--108--
Ser Cys Ser Val Ser Ala Thr Pro Leu Gly Cys Asp Met Leu Cys Ser
245 250 255
Lys Ala Thr Glu Thr Glu Glu Glu Asp Tyr Asn Ser Ala Val Pro Thr
260 265 270
Arg Met Val His Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Glu Lys
275 280 285
10 Asp Leu Asp Val Thr Thr Leu Phe Gly Asp Trp Val Ala Asn Tyr Pro
290 295 300
Gly Val Gly Gly Gly Ser Phe Ile Asp Ser Arg Val Trp Phe Ser Val
305 310 315 320
Tyr Gly Gly Leu Lys Pro Asn Thr Pro Ser Asp Thr Val Gln Glu Gly
325 330 335
Lys Tyr Val Ile Tyr ~ys Arg Tyr Asn Asp Thr Cys Pro Asp Glu Gln
340 345 350
Asp Tyr Gln Ile Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Arg Phe
355 360 365
2 5 Gly Gly Lys Arg Ile Gln Gln Ala Ile Leu Ser Ile Lys Val Ser Thr
370 375 380
Ser Leu Gly Glu Asp Pro Val Leu Thr Val Pro Pro Asn Thr Val Thr
385 390 395 400
Leu Met Gly Ala Glu Gly Arg Ile Leu Thr Val Gly Thr Ser HiS Phe
405 410 415
Leu Tyr Gln Arg Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro
420 425 430
Met Thr Val Ser Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Thr Phe
435 440 445
4 0 Asn Ala Phe Thr Arg Pro Gly Ser Ile Pro Cys Gln 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 Arg Gly Val Phe Gly Thr Met Leu
485 490 495
Asp Gly Glu Gln 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
55 Ala Tyr Thr Thr Ser Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr
530 535 540
Tyr Cys Leu Ser Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Glu Phè
545 550 555 560 A
Arg Ile Val Pro Leu Leu Val Glu Ile Leu Lys Asp Asp Gly Val Arg
565 570 575
Glu Ala Arg Ser Gly
580
CA 02223~9l l997-l2-04
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(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS-
(A) LENGTH: 553 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Met Gly Ser Arg Pro Ser Thr Lys Asn Pro Ala Pro Met Met Leu Thr
1 5 10 15
Ile Arg Val Ala Leu Val Leu Ser Cys Ile Cys Pro Ala Asn Ser Ile
20 25 30
Asp Gly Arg Pro Leu Ala Ala Ala Gly Ile Val Val Thr Gly Asp Lys
35 40 45
Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gly Ser Ile Ile Val Lys
50 55 60
Leu Leu Pro Asn Leu Pro Lys Asp Lys Glu Ala Cys Ala Lys Ala Pro
65 70 75 80
Leu Asp Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly
Asp Ser Ile Arg Arg Ile Gln Glu Ser Val Thr Thr Ser Gly Gly Gly
100 105 110
Arg Gln Gly Arg Leu Ile Gly Ala Ile Ile Gly Gly Val Ala Leu Gly
115 120 125
Val Ala Thr Ala Ala Gln Ile Thr Ala Ala Ala Ala Leu Ile Gln Ala
130 135 140
Lys Gln Asn Ala Ala Asn Ile Leu Arg Leu Lys Glu Ser Ile Ala Ala
145 150 155 160
Thr Asn Glu Ala Val His Glu Val Thr Asp Gly Leu Ser Gln Leu Ala
165 170 175
Val Ala Val Gly Lys Met Gln Gln Phe Val Asn Asp Gln Phe Asn Lys
180 185 190
Thr Ala Gln Glu Leu Asp Cys Ile Lys Ile Ala Gln Gln Val Gly Val
195 200 205
Glu Leu Asn Leu Tyr Leu Thr Glu Ser Thr Thr Val Phe Gly Pro Gln
210 215 220
Ile Thr Ser Pro Ala Leu Asn Lys Leu Thr Ile Gln Ala Leu Tyr Asn
225 230 235 240
Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Thr Lys Leu Gly Ile Gly
245 250 255
~0 Asn Asn Gln Leu Ser Ser Leu Ile Gly Ser Gly Leu Ile Thr Gly Asn
260 265 270
Pro Ile Leu Tyr Asp Ser Gln Thr Gln Leu Leu Gly Ile Gln Val Thr
275 280 285
Leu Pro Ser Val Gly Asn Leu Asn Asn Met Arg Ala Thr Tyr Leu Glu
290 295 300
CA 02223~91 1997-12-04
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- --110--
Thr Leu Ser Val Ser Thr Thr Arg Gly Phe Ala Ser Ala Leu Val Pro
305 310 315 320
Lys Val Val Thr Arg Val Gly Ser Val Ile Glu Glu Leu Asp Thr Ser
325 330 335
Tyr Cys Ile Glu Thr Asp Leu Asp Leu Tyr Cys Thr Arg Ile Val Thr
340 345 350
Phe Pro Met Ser Pro Gly Ile Tyr Ser Cys Leu Ser Gly Asn Thr Ser
355 360 365
Ala Cys Met Tyr Ser Lys Thr Glu Gly Ala Leu Thr Thr Pro Tyr Met
370 375 380
Thr Ile Lys Gly Ser Val Ile Ala Asn Cys Lys Met Thr Thr Cys Arg
385 390 395 400
Cys Val Asn Pro Pro Gly Ile Ile Ser Gln Asn Tyr Gly Glu Ala Val
2 0 405 410 415
Ser Leu Ile Asp Lys Gln Ser Cys Asn Val Leu Ser Leu Gly Gly Ile
420 425 430
2 5 Thr Leu Arg Leu Ser Gly Glu Phe Asp Val Thr Tyr Gln Lys Asn Ile
435 440 445
Ser Ile Gln Asp Ser Gln Val Ile Ile Thr Gly Asn Leu Asp Ile Ser
450 455 460
Thr Glu Leu Gly Asn Val Asn Asn Ser Ile Ser Asn Ala Leu Asn Lys
465 470 475 480
Leu Glu Glu Ser Asn Arg Lys Leu Asp Lys Val Asn Val Lys Leu Thr
485 490 495
Ser Thr Ser Ala Leu Ile Thr Tyr Ile Val Leu Thr Ile Tl e Ser Leu
500 505 510
~ 0 Val Phe Gly Ile Leu Ser Leu Ile Leu Ala Cys Tyr Leu Met Tyr Lys
515 520 525
Gln Lys Ala Gln Gln Lys Thr Leu Leu Trp Leu Gly Asn Asn Thr Leu
530 535 540
Asp Gln Met Arg Ala Thr Thr Lys Met
545 550
5 0 (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 182 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLBCULE TYPE: DNA (genomic)
(iii) HYPOTXETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CA 02223~91 1997-12-04
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- 111 -
GGCCTCGAGG GCCGCGGCCG CCTGCAGGTC GACTCTAGAA A~AATTGA~A AACTATTCTA 60
ATTTATTGCA CGGAGATCTT llll l"l"l''L'l"l' 'l"l"l"l"l"l"l''l''l'G GCATATA~AT GAATTCGGAT 120
5 CCGGACCGCG CCGTTAGCCA AGTTGCGTTA GAGAATGATG A~AGAGAGGC AAAAAATACA 180
TG 182
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 178 base pairs
(B) TYPE: nucleic acid
(C) sTRANn~nN~qs: double =.
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Al~llclGCG ACATCAAGAA TCAAACCGAA TGCC~Gr-~TC CATAATTAAT TAATTAATTT 60
TTAl~CC~l~G ACTCTAGA~A A~ATTGA~AA ACTATTCTAA TTTATTGCAC GGAGATCTTT 120
L~ lllf'~llLlllllGG CATATA~ATG AATTCGGATC GATCCCGGTT GGCGCCCT 178
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) sTR~Nn~n~ss: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
A~AAACCCCC CCCCCCCCCC CCCCCCCC~C CTGCAGGCAT CGTGGTGTCA CGCTCGTCGT 60
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 120 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
CA 02223~91 1997-12-04
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- -112-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
ATAATTCTCT TACTGTCATG CCATCCGTAA GATGCTTTTC TGTGACTGGT GAGTGATCCA 60
TAATTAATTA ATTAATTTTT A~LC~CGG~LC GACCTGCAGG CGGCCGCGGC CCTCGAGGCC 120
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1305 base pairs
(B) TYPE: nucleic acid
(C) STR~NnRnNR-~: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1305
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
ATG CAC CGT CCT CAT CTC AGA CGG CAC TCG CGT TAC TAC GCG AAA GGA 48
Met His Arg Pro His Leu Arg Arg His Ser Arg Tyr Tyr Ala Lys Gly
1 5 10 15
GAG GTG CTT AAC A~A CAC ATG GAT TGC GGT GGA A~A CGG TGC TGC TCA 96
Glu Val Leu Asn Lys His Met Asp Cys Gly Gly Lys Arg Cys Cys Ser
20 25 30
GGC GCA GCT GTA TTC ACT CTT TTC TGG ACT TGT GTC AGG ATT ATG CGG 144
Gly Ala Ala Val Phe Thr Leu Phe Trp Thr Cys Val Arg Ile Met Arg
35 40 45
GAG CAT ATC TGC TTT GTA CGC AAC GCT ATG GAC CGC CAT TTA TTT TTG 192
Glu His Ile Cys Phe Val Arg Asn Ala Met Asp Arg His Leu Phe Leu
~5 50 55 60
AGG AAT GCT TTT TGG ACT ATC GTA CTG CTT TCT TCC TTC GCT AGC CAG 240
Arg Asn Ala Phe Trp Thr Ile Val Leu Leu Ser Ser Phe Ala Ser Gln
AGC ACC GCC GCC GTC ACG TAC GAC TAC ATT TTA GGC CGT CGC GCG CTC 288
Ser Thr Ala Ala Val Thr Tyr Asp Tyr Ile Leu Gly Arg Arg Ala Leu
GAC GCG CTA ACC ATA CCG GCG GTT GGC CCG TAT AAC AGA TAC CTC ACT 336
Asp Ala Leu Thr Ile Pro Ala Val Gly Pro Tyr Asn Arg Tyr Leu Thr
100 105 110
AGG GTA TCA AGA GGC TGC GAC GTT GTC GAG CTC AAC CCG ATT TCT AAC 384
Arg Val Ser Arg Gly Cys Asp Val Val Glu Leu Asn Pro Ile Ser Asn
115 120 125
GTG GAC GAC ATG ATA TCG GCG GCC A~A GAA A~A GAG AAG GGG GGC CCT 432
Val Asp Asp Met Ile Ser Ala Ala Lys Glu Lys Glu Lys Gly Gly Pro
130 135 140
TTC GAG GCC TCC GTC GTC TGG TTC TAC GTG ATT AAG GGC GAC GAC GGC 480
CA 02223~91 1997-12-04
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Phe Glu Ala Ser Val Val Trp Phe Tyr Val Ile Lys Gly Asp Asp Gly
145 150 155 160
GAG GAC AAG TAC TGT CCA ATC TAT AGA A~A GAG TAC AGG GAA TGT GGC 528
Glu Asp Lys Tyr Cys Pro Ile Tyr Arg Lys Glu Tyr Arg Glu Cys Gly
165 170 175
GAC GTA CAA CTG CTA TCT GAA TGC GCC GTT CAA TCT GCA CAG ATG TGG 576
Asp Val Gln Leu Leu Ser Glu Cy5 Ala Val Gln Ser Ala Gln Met Trp
r10 180 185 190
GCA GTG GAC TAT GTT CCT AGC ACC CTT GTA TCG CGA AAT GGC GCG GGA 624
Ala Val Asp Tyr Val Pro Ser Thr Leu Val Ser Arg Asn Gly Ala Gly
195 200 205
CTG ACT ATA TTC TCC CCC ACT GCT GCG CTC TCT GGC CAA TAC TTG CTG 672
Leu Thr Ile Phe Ser Pro Thr Ala Ala Leu Ser Gly Gln Tyr Leu Leu
210 215 220
ACC CTG A~A ATC GGG AGA TTT GCG CAA ACA GCT CTC GTA ACT CTA GAA 720
Thr Leu Lys Ile Gly Arg Phe Ala Gln Thr Ala Leu Val Thr Leu Glu
225 230 235 240
GTT AAC GAT CGC TGT TTA AAG ATC GGG TCG CAG CTT AAC TTT TTA CCG 768
Val A~n Asp Arg Cys Leu Lys Ile Gly Ser Gln Leu Asn Phe Leu Pro
245 250 255
TCG A~A TGC TGG ACA ACA GAA CAG TAT CAG ACT GGA TTT CAA GGC GAA 816
Ser Lys Cys Trp Thr Thr Glu Gln Tyr Gln Thr Gly Phe Gln Gly Glu
260 265 270
CAC CTT TAT CCG ATC GCA GAC ACC AAT ACA CGA CAC GCG GAC GAC GTA 864
His Leu Tyr Pro Ile Ala Asp Thr Asn Thr Arg His Ala Asp Asp Val
275 280 285
TAT CGG GGA TAC GAA GAT ATT CTG CAG CGC TGG AAT AAT TTG CTG AGG 912
Tyr Arg Gly Tyr Glu Asp Ile Leu Gln Arg Trp Asn Asn Leu Leu Arg
290 295 300
A~A AAG AAT CCT AGC GCG CCA GAC CCT CGT CCA GAT AGC GTC CCG CAA 960
Lys Lys Asn Pro Ser Ala Pro Asp Pro Arg Pro Asp Ser Val Pro Gln
305 310 315 320
GAA ATT CCC GCT GTA ACC AAG A~A GCG GAA GGG CGC ACC CCG GAC GCA 1008
Glu Ile Pro Ala Val Thr Lys Lys Ala Glu Gly Arg Thr Pro Asp Ala
325 330 335
GAA AGC AGC GAA AAG AAG GCC CCT CCA GAA GAC TCG GAG GAC GAC ATG 1056
Glu Ser Ser Glu Lys Lys Ala Pro Pro Glu Asp Ser Glu Asp Asp Met
340 345 350
CAG GCA GAG GCT TCT GGA GAA AAT CCT GCC GCC CTC CCC GAA GAC GAC 1104
Gln Ala Glu Ala Ser Gly Glu Asn Pro Ala Ala Leu Pro Glu Asp Asp
355 360 365
GAA GTC CCC GAG GAC ACC GAG CAC GAT GAT CCA AAC TCG GAT CCT GAC 1152
Glu Val Pro Glu Asp Thr Glu His Asp Asp Pro Asn Ser Asp Pro Asp
370 375 380
TAT TAC AAT GAC ATG CCC GCC GTG ATC CCG GTG GAG GAG ACT ACT AAA 1200
Tyr Tyr Asn Asp Met Pro Ala Val Ile Pro Val Glu Glu Thr Thr Lys
385 390 395 400
AGT TCT AAT GCC GTC TCC ATG CCC ATA TTC GCG GCG TTC GTA GCC TGC 1248
Ser Ser Asn Ala Val Ser Met Pro Ile Phe Ala Ala Phe Val Ala Cys
405 410 415
CA 02223~9l l997-l2-04
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GCG GTC GCG CTC GTG GGG CTA CTG GTT TGG AGC ATC GTA AAA TGC GCG 1296
Ala Val Ala Leu Val Gly Leu Leu Val Trp Ser Ile Val Lys Cys Ala
420 425 430
CGT AGC TAA 1305
Arg Ser
435
10 (2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 434 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Met His Arg Pro His Leu Arg Arg His Ser Arg Tyr Tyr Ala Lys Gly
1 5 10 15
Glu Val Leu Asn Lys His Met Asp Cys Gly Gly Lys Arg Cys Cys Ser
20 25 30
Gly Ala Ala Val Phe Thr Leu Phe Trp Thr Cys Val Arg Ile Met Arg
35 40 45
Glu ~is Ile Cys Phe Val Arg Asn Ala Met Asp Arg His Leu Phe Leu
50 55 60
Arg Asn Ala Phe Trp Thr Ile Val Leu Leu Ser Ser Phe Ala Ser Gln
65 70 75 80
Ser Thr Ala Ala Val Thr Tyr Asp Tyr Ile Leu Gly Arg Arg Ala Leu
85 90 95
Asp Ala Leu Thr Ile Pro Ala Val Gly Pro Tyr Asn Arg Tyr Leu Thr
100 105 ilO
Arg Val Ser Arg Gly Cys Asp Val Val Glu Leu Asn Pro Ile Ser Asn
115 120 125
Val Asp Asp Met Ile Ser Ala Ala Lys Glu Lys Glu Lys Gly Gly Pro
130 135 140
Phe Glu Ala Ser Val Val Trp Phe Tyr Val Ile Lys Gly Asp Asp Gly
145 150 155 160
Glu Asp Lys Tyr Cys Pro Ile Tyr Arg Lys Glu Tyr Arg Glu Cys Gly
165 170 175
Asp Val Gln Leu Leu Ser Glu Cys Ala Val Gln Ser Ala Gln Met Trp
180 185 190
Ala Val Asp Tyr Val Pro Ser Thr Leu Val Ser Arg Asn Gly Ala Gly
195 200 205
~0 Leu Thr Ile Phe Ser Pro Thr Ala Ala Leu Ser Gly Gln Tyr Leu Leu
210 215 220
Thr Leu Lys Ile Gly Arg Phe Ala Gln Thr Ala Leu Val Thr Leu Glu
225 230 235 240
Val Asn Asp Arg Cys Leu Lys Ile Gly Ser Gln Leu Asn Phe Leu Pro
245 250 255
CA 02223~9l l997-l2-04
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- -115-
Ser Lys Cys Trp Thr Thr Glu Gln Tyr Gln Thr Gly Phe Gln Gly Glu
260 265 270
His Leu Tyr Pro Ile Ala Asp Thr Asn Thr Arg His Ala Asp Asp Val
275 280 285
Tyr Arg Gly Tyr Glu Asp Ile Leu Gln Arg Trp Asn Asn Leu Leu Arg
290 295 300
0 Lys Lys Asn~ Pro Ser Ala Pro Asp Pro Arg Pro Asp Ser Val Pro Gln
305 310 315 320
Glu Ile Pro Ala Val Thr Lys Lys Ala Glu Gly Arg Thr Pro Asp Ala
325 330 335
Glu Ser Ser Glu Lys Lys Ala Pro Pro Glu Asp Ser Glu Asp Asp Met
340 345 350
Gln Ala Glu Ala Ser Gly Glu Asn Pro Ala Ala Leu Pro Glu Asp Asp
355 360 365
Glu Val Pro Glu Asp Thr Glu His Asp Asp Pro Asn Ser Asp Pro Asp
370 375 380
2 5 Tyr Tyr Asn Asp Met Pro Ala Val Ile Pro Val Glu Glu Thr Thr Lys
385 390 395 400
Ser Ser Asn Ala Val Ser Met Pro Ile Phe Ala Ala Phe Val Ala Cys
405 410 415
Ala Val Ala Leu Val Gly Leu Leu Val Trp Ser Ile Val . Lys Cys Ala
420 425 430
Arg Ser
3 5
