Note: Descriptions are shown in the official language in which they were submitted.
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ENV-GLYCOPROTEIN VACCINE FOR PROTECTION
OF IiTLV-I AND -II INFECTION
This application is a continuation-in-part of copending
application Serial No. 08/681,054 filed July 22, 1996, which
is incorporated by reference herein in its entirety.
1. INTRODUCTION '
The present invention relates to novel protein antigens
for use in a vaccine to treat and prevent human T-cell
lymphotropic virus-I (HTLV-I) and HTLV-II infection, and
novel methods of efficiently producing such antigens. The
present invention further relates to the nucleotide sequences
encoding the novel antigen and vectors and expression
systems, both eucaryotic and procaryotic, to express the
novel antigen. More particularly, the present invention
relates to methods of producing recombinant HTLV envelope
(env) glycoproteins using insect and mammalian cell lines to
express useful amounts of the envelope glycoprotein.
2. BACKGROUND OF THE INVENTION
Human T-cell lymphotropic virus type I (HTLV-I) and type
II (HTLV-II) are genetically and serologically related
members of a group of retroviruses sharing a tropism for T
lymphocytes and an association with rare lymphotropic
diseases. (Hall et al., 1994, Seminars in Virology 5:165-
178) HTLV-I is endemic in a number of well established
geographic areas, where infection is associated with adult T
cell leukemia (ATL), a malignancy of mature T lymphocytes,
and a chronic encephalomyelopathy known both as HTLV-I
associated myelopathy and tropical spastic paraparesis
(HAM/TSP).
HTLV-I infection is endemic in southwestern Japan, the
Caribbean, South America and some regions of Africa. HTLV-II
infection has now been clearly shown to be endemic in a large
number of native American Indian populations, and high rates
of infection have also been demonstrated in intravenous drug
abusers (IVDAs) in North America and Europe. (Hall et al.,
1994, Seminars in Virology 5:165-178)
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The vast majority of infected individuals remain as
asymptomatic carriers, and serve as a source for further
transmission of the virus. Although the modes of
transmission of HTLV-II remain less well established than
those of HTLV-I, all evidence obtained to date suggests that
they are similar, if not identical. HTLV-I transmission
occurs by three major routes: vertically from mother to
child, which occurs primarily through breast-feeding;
heterosexual and homosexual transmission; and via
contaminated blood products, which may occur after blood
transfusion or by intravenous drug abuse (Hollsberg et al.,
1993, New England J. Medicine 328: 1173).
Over the past decade there has been accumulating
evidence that HTLV-II infection may be associated with a
spectrum of neurological, and possibly rare
lymphoproliferative disorders. At present it is unclear if
HTLV-II is less pathogenic than HTLV-I, or whether the
observed lack of clinical disorders may simply reflect the
comparatively small number of infected individuals so far
identified, and who have been clinically evaluated.
The size and the structural organization of HTLV-II
provirus has been shown to be very similar to that of HTLV-I.
(Shimotono et al., 1985 Proc. Natl. Acad) Sci. USA 82:3101-
3105). The similar identity of much of the primary amino
acid sequence would suggest antigenic cross-reactivity
between HTLV-I and HTLV-II. The genome is flanked by long
terminal repeats (LTRs) which contain the binding site for
the RNA polymerase, and sequences that regulate virus
transcription. Four major genes have been identified and
occupy the following positions in the genome LTR-gag-pol-env-
pX-LTR (Seiki et al., 1983 Proc. Natl. Acad. Sci. 80:3618-
3622 ) .
The gag gene encodes a polyprotein which is processed to
produce three internal virus structural proteins. The pot
gene encodes reverse transcriptase, RNase H, and integrase,
all of which are involved in the synthesis and integration of
provirus into the host genome. An open reading frame for a
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putative viral protease is located at the 3' end of the gag
gene. This extends into the pot region and is thought to be
expressed from mRNA via mechanisms involving ribosomal frame
shifting. (Shimotono et al., 1985 Proc. Natl. Acad. Sci. USA
82:3101-3105).
The env gene is located upstream of the 3~ end of the
pot gene and partially overlaps it. The env gene encodes a
precursor protein p63 which undergoes proteolytic cleavage,
and subsequent glycosylation to produce two glycoproteins,
to gp46 and gp2l. The gp46 protein constitutes the surface
projections observed by electron microscopy on native virus
particles, is believed to have receptor binding activity, and
contains domains responsible for the production of
neutralizing antibodies. The gp21 protein is the
transmembrane glycoprotein, and by analogy with HIV may be
involved in cell fusion activity. The env proteins interact
with as of yet unidentified cellular receptors to mediate
viral entry.
The env protein has been deduced by its nucleotide
2o sequence to have a hydrophobic signal sequence at its amino
terminus, five potential acceptor sites for N-glycosylation
linked carbohydrates in the central portion, and a second
cluster of hydrophobic amino acids in the putative
transmembrane domain (Seiki et al., 1983 Proc. Natl. Acad.
Sci. 80:3618-3622). Its sequence character suggests that it
has a typical structure of a cell membrane glycoprotein.
There has been much research focused on the development
of a vaccine against HTLV-I and HTLV-II. The env protein has
been a target for such a vaccine, however efforts to develop
a full length gp63 to use as an effective antigen have
failed. Previous attempts to express useful levels of the
HTLV env protein have been unsuccessful. Therefore a number
of groups have instead developed synthetic peptides derived
from the sequence of the env protein as antigens for HTLV-I
(U. S. 5,378,805, U.S. 5,066,579) and HTLV-II (U. S. 5,378,805,
U.S. 5,359,029). The primary uses of these peptides are for
diagnosis of disease and the development of vaccines.
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Synthetic peptides have been used increasingly to map
antigenic determinants on the surface of proteins and as
possible vaccines. These chemically synthesized peptides
have been utilized in highly sensitive assays to distinguish
between HTLV-I and -II infections and to develop vaccines
(U. S. 5,476,765).
Viral vectors capable of expressing the recombinant env
protein have been suggested as a vaccine for HTLV-I, such as
a live adenovirus recombinant virus expressing the HTLV-I
envelope protein (deThe et al., 1994, Ciba Foundation
Symposium 187:47-60). The HTLV-I env protein expressed in
vaccinia virus has also been formulated into a vaccine
preparation (Seiki et al., 1990, Virus Genes 3:235-249; Shida
et al., 1987, EMBO J. 6:3379-3384). Another group has
developed a vaccine consisting of a live recombinant poxvirus
expressing the full length envelope protein of HTLV-I
(Franchini et al., 1995, AIDS Research and Human Retroviruses
11:307-313). However, a combination of this vaccine with two
additional boosts of the gp63 protein subunit failed to
confer protection suggesting that the administration of the
gp63 protein subunit negated the protective efficacy of the
vaccine.
The purification of the full length glycosylated gp63
has been described for use in an assay to determine the
presence of anti-HTLV antibodies in a biological specimen.
In the same report it is suggested that the full length
glycosylated gp63 may be used in a vaccine formulation, but
the efficacy of such a vaccine is not described. (U. S.
4,743,678 and U.S. 5,045,448).
Therefore there remains a need for an effective full-
length HTLV env antigen to be used in vaccine formulations
and an efficient means of producing such an antigen.
3. SUMMARY OF THE INVENTION
The present invention relates to novel protein antigens
derived from the HTLV env protein, that are capable of being
used as a vaccine to aid in the prevention and treatment of
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HTLV-I and HTLV-II infections and novel methods for the
production of such an antigen. The present invention relates
to nucleotide sequences that encode the novel antigenic
protein, mutants and derivatives thereof. The present
invention further relates to methods of expressing the novel
antigen, including expression vectors and cell lines, both
eucaryotic and procaryotic. The invention still further
relates to methods of using this novel antigen as an
immunogen in vaccine preparations for the prevention and/or
treatment of HTLV-I and HTLV-II infections.
The present invention relates to an HTLV env protein
lacking all or a portion of its membrane spanning domain such
that the polypeptide, when expressed recombinantly, is not
anchored in the membrane of the host cell. In a preferred
embodiment of the invention, the soluble HTLV env protein is
lacking all or a portion of its amino terminus. The present
invention further relates to an amino truncated form of the
HTLV env protein which is soluble and accumulates in the
cytoplasm of the host cell, so that the HTLV env protein is
readily purified from lysed host cells.
More particularly, the present invention relates to
nucleotide sequences encoding an amino terminally truncated
form of the HTLV env protein, the expression of the
recombinant HTLV env protein in host cell lines and the use
of the resulting recombinant env protein in vaccine
preparations for the prevention of HTLV infection.
A present difficulty in mammalian recombinant gene
expression is that many proteins are resistant to expression
in many systems, therefore the likelihood of success is
difficult to predict. Previous attempts to express high
levels of the HTLV env protein have been unsuccessful. The
Applicant's invention has overcome this difficulty by
expressing a truncated form of the HTLV env.gene in a
baculovirus expression system. The transcription of a cDNA
corresponding to an amino terminally truncated form of the
HTLV env protein led to an unexpected abundance of
transcribed protein. It was found that an approximate
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50 fold increase (relative to expression of the full length
gene in mammalian cells) in the expression of an
immunologically useful HTLV-1 env protein could be achieved.
The invention is further based on the Applicant's
discovery that an antigenic protein having the amino acid
sequence of the HTLV-I or the HTLV-II env protein with the
amino terminal leader or signal sequence deleted, serves to
protect the recipient when challenged with an inoculation of
HTLV-I or HTLV-II. The immunogenicity of this protein is
unexpectedly strong.
In a preferred embodiment of the invention, the
nucleotide sequences encoding an amino terminally truncated
form of HTLV env protein, upon expression in an appropriate
host cell, produce a polypeptide that is antigenic or
immunogenic. Antigenic polypeptides are capable of being
immunospecifically bound by an antibody to the antigen.
Immunogenic polypeptides are capable of eliciting an immune
response to the antigen, e.a., when immunization with the
polypeptide elicits production of an antibody that
immunospecifically binds the antigen or elicits a cell-
mediated immune response directed against the antigen.
In another preferred embodiment of the invention, the
antigen protein of the present invention is expressed in a
baculovirus system to produce an unglycosylated antigen or
the antigen protein is expressed in a stably transfected T
cell line to produce a glycosylated antigen. In yet another
preferred embodiment of the invention the amino terminally
truncated HTLV env protein is expressed as a fusion protein
in order to facilitate purification of the protein.
In another aspect of the invention, methods of using
these novel antigenic proteins are described. These methods
include using these novel antigenic proteins in vaccine
preparations in a solely preventative way, and/or in a
therapeutic procedure after the recipient is already infected
with either HTLV-I or HTLV-II, or both. The novel antigenic
proteins of the invention also have utility in diagnostic
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immunoassays, passive immunotherapy, and generation of
antiidiotypic antibodies.
3.1. DEFINITIONS
As used herein, the following terms will have the
meanings indicated.
The term "gpb3" refers to the 63 kilodalton precursor
protein of the outer membrane protein or env protein of the
HTLV-I or -II virus. The term also refers to mutants,
variants or fragments of gp63.
The term "gp46" refers to 46 kilodalton outer membrane
protein or env protein of HTLV-I or -II virus. The term also
refers to mutants, variants or fragments of gp46.
The term "env protein" refers to polypeptides comprising
the native sequence of the HTLV-I and/or -II ~env protein,
full-length and truncated, as well as analog thereof.
Preferred analogs are those which are substantially
homologous to the corresponding native amino acid sequence,
and most preferably encode at least one native HTLV-I and -II
env epitope, such as a neutralizing epitope. A more
preferred class of HTLV-I and -II env polypeptides are those
lacking a sufficient portion of the C-terminal transmembrane
domain to promote efficient expression and/or secretion of
the HTLV-I and II env proteins at high levels from insect or
mammalian cell expression hosts of the present invention.
The term "effective amount" refers to an amount of
HTLV-I and -II env polypeptide sufficient to induce an immune
response in the subject to which it is administered. The
immune response may comprise, without limitation, induction
of cellular and/or humoral immunity.
The term "treating or preventing HTLV infection" means
to inhibit the replication of the HTLV virus, to inhibit HTLV
transmission, or to prevent HTLV from establishing itself in
its host, and to ameliorate or alleviate the symptoms of the
disease caused by HTLV infection. The treatment is
considered therapeutic if there is a reduction in viral load,
decrease in mortality and/or morbidity.
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The term "pharmaceutically acceptable carrier" refers to
a carrier medium that does not interfere with the
effectiveness of the biological activity of the active
ingredient, is chemically inert and is not toxic to the
patient to whom it is administered.
The term "therapeutic agent" refers to any molecule
compound or treatment, preferably an.antiviral, that assists
in the treatment of a viral infection or the diseases caused
thereby.
4. BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1. The nucleotide sequence and the amino acid sequence
of the HTLV-I env protein. The boxed portion of the sequence
corresponds to those sequences which are deleted in one
l5 embodiment of the amino terminally truncated form of the
HTLV-I env antigen of the present invention.
FIGURE 2. The nucleotide sequence and the amino acid
sequence of the HTLV-II env protein. The boxed portion of
the sequence corresponds to those sequences which are deleted
in one embodiment of the amino terminally truncated form of
the HTLV-II env antigen of the present invention.
FIGURE 3. Detection of the HTLV-II recombinant env
glycoprotein of 63 kDa (rgp63) expressed in the H5 cells by
western blotting using HTLV-II infected human serum as the
specific antibody (Lane 1). The protein was not detected in
non-infected insect cells (Lane 2). HTLV-II negative human
serum did not react with the HTLV-II env glycoprotein
expressed in insect cells (Lane 3).
FIGURE 4. Detection of antibody against env gp46 1 week
after immunization of R3 and R4 with rgp63 (lane 2 and 3,
respectively). The antigen used in the detection system was-
a GST HTLV-II gp46 fusion protein. The serum of the R3 prior
to immunization did not react with the antibody to the GST-
gp46 fusion protein (lane 1).
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FIGURE 5. FACS analysis of HTLV-II infected cell lines using
immunized rabbit, R3. Positive staining was detected in the
cell lines, Vines and Mo-T. In contrast CEM was negative.
Dotted line: control FITC labeled anti-rabbit antibody.
Solid line anti HTLV-II env protein rabbit serum used at a
1:10 dilution. F1 - fluorescence intensity.
FIGURE 6A. Antibody titers of the animals after inoculation
of HTLV-II-Vines examined by particle agglutination (PA)
to method. The left figure (A) shows the antibody titers of
non-immunized rabbit. The right figure (B) shows the
antibody response in rabbits preimmunized with the
recombinant gp63. Closed circle . R1, open circle: R2, open
square . R3, closed square . R4.
FIGURE 6B. Detection of antibody against GST-gp46 of R1
after inoculation of HTLV-IT Vines cells. Antibody was first
detected after 2 weeks and was present. The antigen used in
the detection system was a GST-gp46 fusion protein
transferred to a nylon membrane.
FIGURE 7. Detection of HTLV-II provirus DNA by southern
hybridization of nested PCR.
Number denotes the week after inoculation. Only alive
HTLV-II-Vines cells-injected Rabbits (R1, R2 and R5) showed
positivity after long time. In contrast, vaccinated animals
(R3, R4, R8 and R9) or injected with heat inactivated
HTLV-II-Vines cells (R6 and R7) showed a negative response.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a novel antigenic
protein derived from the HTLV env protein, that can be used
as an immunogen in a vaccine preparation to aid in the
prevention and treatment of HTLV-I and HTLV-II infections.
The invention also relates to methods for the production of
such an antigen.
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The present invention relates to an HTLV env polypeptide
lacking all or a portion of its membrane spanning domain,
such that the polypeptide when expressed recombinantly is not
anchored in the membrane of the host cell. In a preferred
embodiment of the invention, the soluble HTLV env protein is
lacking all or a portion of its amino terminus. The present
invention further relates to an amino terminally truncated
form of the HTLV env protein which is soluble and accumulates
in the cytoplasm of the host cell, so that the HTLV env
protein is readily purified from lysed host cells.
The invention is based, in part, on the Applicant's
discovery that an antigenic or immunogenic protein having the
amino acid sequence of the HTLV-I or the HTLV-II envelope
protein with the amino terminal leader or signal sequence
deleted, serves to protect the recipient when' challenged with
an inoculation with HTLV-I or HTLV-II. Furthermore, the
transcription of a corresponding cDNA transcript to this
novel antigenic protein, in either eucaryotic or procaryotic
expression systems, has led to an unexpected abundance of
transcribed protein.
The present invention relates to nucleotide sequences
that encode the novel antigenic protein, mutants and
derivatives thereof. The present invention further relates
to methods of expressing the novel antigen, including
expression vectors and cell lines, both eucaryotic and
procaryotic.
In a preferred embodiment of the invention, the
nucleotide sequences, upon expression in an appropriate host
cell, produce a polypeptide that is antigenic or immunogenic.
3o Antigenic polypeptides are capable of being
immunospecifically bound by an antibody to the antigen.
Immunogenic polypeptides are capable of eliciting an immune
response to the antigen, eTa., when immunization with the
polypeptide elicits production of an antibody that
immunospecifically binds the antigen or elicits a cell-
mediated immune response directed against the antigen.
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In another preferred embodiment of the invention, the
antigen protein of the present invention is expressed in a
baculovirus system to produce an unglycosylated antigen or
the antigen expressed in a stably transfected T cell line to
produce a glycosylated antigen. In yet another preferred
embodiment of the invention the amino terminally truncated
env protein is expressed as a fusion protein to facilitate
purification of the protein.
In another aspect of the invention, the method of using
to these novel antigenic proteins are described. These methods
include using these antigenic proteins in vaccine
preparations in a solely preventative way, and/or in a
therapeutic procedure after the recipient is already infected
with either HTLV-I or HTLV-II, or both. The novel antigenic
proteins of the invention also have utility in diagnostic
immunoassays, passive immunotherapy, and generation of
antiidiotypic antibodies.
5.1. NOVEL ENV-GLYCOPROTEIN ANTIGEN
The present invention is based, in part, on the
Applicant's surprising discovery that the difficulty in
expressing useful amounts of HTLV-env protein could be
overcome by expressing an amino truncated form of the HTLV
env protein in insect cells or mammalian T lymphocytes. The
transcription of a cDNA corresponding to an amino truncated
form of the HTLV env protein led to an unexpected abundance
of transcribed protein. It was found that an approximate
50 fold increase in expressing of immunologically useful HTLV
env protein could be achieved.
The invention is further based on the Applicant's
discovery that the antigenicity of this protein is
unexpectedly strong. An antigenic protein having the amino
acid sequence of the HTLV env protein with the amino terminal
leader sequence deleted, induced the production of anti-
HTLV-II antibodies in recipients and served to protect the
recipient when challenged with an inoculation of HTLV-I or
-II. Due to the high level of amino acid sequence identity
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between the amino acid sequences of the different regional
strains of HTLV-I and HTLV-II, the HTLV-I env antigen of the
present invention will serve to protect against the many
regional isolates of HTLV-I and the HTLV-II env antigen of
the present invention will serve to protect against the many
regional isolates of HTLV-II.
In a preferred embodiment of the invention, the antigen
protein of the present invention is expressed in a
baculovirus system to produce an unglycosylated antigen or
the antigen protein is expressed in a stably transfected T
cell line to produce a glycosylated antigen.
5.2. NUCLEOTIDE SE UENCES ENCODING THE ANTIGEN
The present invention encompasses nucleotide sequences
encoding the HTLV env protein, including fragments,
truncations and variants thereof. A preferred embodiment of
the invention encompasses the nucleotide sequences encoding
an amino truncated form of the HTLV env gene. The preferred
embodiment of the invention encompasses the nucleotide
sequences encoding a 33 amino acid truncation of the amino
terminal of the HTLV-I env protein. The invention further
encompasses nucleotide sequences encoding 1 to l0, l0 to 15,
15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45,
45 to 50, 50 to 55, 55 to 60, 60 to 65, or 65 to 70 amino
acid trunctations of the amino terminal of the HTLV-I env
protein. The present invention further encompasses a
nucleotide sequence encoding an amino terminally truncated
form of the HTLV I env protein such that the polypeptide when
expressed recombinantly is not anchored in the membrane of
the host cell, yet still retains antigenic activity similar
to the full length HTLV I env protein. Further the invention
encompasses internal deletions which comprise deleting a
sufficient portion of the signal sequence domain so that the
polypeptide when expressed recominantly is not anchored in
the membrane of the host cell, yet still retains antigenic
activity similar to the full length HTLV I env protein. The
HTLV-I env nucleotide sequences of the invention include the
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following DNA sequences: (1) any DNA sequence encoding a
HTLV-I env protein which is immunologically reactive with a
anti-HTLV-I env antibody; (2) any DNA sequence encoding a
HTLV-I env protein containing the amino acid as shown in FIG.
1; (3) any nucleotide sequence that hybridizes to the
complement of the DNA sequence as shown in FIG. 1 under
highly stringent conditions, eg., hybridization to filter-
bound DNA in 0.5M NaHP09, 7% sodium dodecyl sulfate (SDS), 1mM
EDTA at 65°C, and washing in 0.1 x SSC/0.1% SDS at 68°C
(Ausubel F.M. et al., eds., 1989, Current Protocols in
Molecular Biology , Vol.I, Green Publishing Associates, Inc.,
and John Wiley & Sons, Inc., New York, at p. 2~10~3) and
encodes a functionally equivalent gene product; (4) any
nucleotide sequence that hybridizes to the complement of the
DNA sequence as shown in FIG. 1 under less stringent
conditions, such as moderately stringent conditions, e.a.,
washing in 0.2 x SSC/0.1% SDS at 42°C (Ausubel et al., 1989,
su ra), yet which still encodes a functionally equivalent
HTLV env gene product; and (5) any nucleotide sequence that
hybridizes to the complement of the DNA sequence as shown in
FIG. 1 under less stringent conditions, such as low
stringency conditions, ea., washing in 0.2 x SSC/ 0.1% SDS
at 37°C, and encodes a functionally equivalent gene product.
A functionally equivalent gene product encompasses a gene
product that is produced at high levels and is
immunologically reactive with an anti-HTLV-I env antibody.
Another preferred embodiment of the invention
encompasses the nucleotide sequences encoding a 17 amino acid
truncation of the amino terminal of the HTLV-II env protein.
The invention further encompasses nucleotide sequences
encoding 1 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30
to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60
to 65, or 65 to 70 amino acid truncations of the amino
terminal of the HTLV-II env protein. The present invention
further encompasses a nucleotide sequence encoding an amino
terminally truncated form of the HTLV II env protein such
that the~polypeptide when expressed recombinantly is not
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anchored in the membrane of the host cell, yet still retains
antigenic activity similar to the full length HTLV II env
protein. Further the invention encompasses internal
deletions which comprise deleting a sufficient portion of the
signal sequence domain so that the polypeptide when expressed
recominantly is not anchored in the membrane of the host
cell, yet still retains antigenic activity similar to the
full length HTLV II env protein. Further the invention
encompasses internal deletions which delete a sufficient
l0 portion of the signal sequence domain so that the polypeptide
when expressed recombinantly is not anchored. The HTLV-II
env nucleotide sequences of the invention include the
following DNA sequences: (1) any DNA sequence encoding a
HTLV-II env protein which is immunologically reactive with a
HTLV-II env antibody; (2) any DNA sequence encoding a HTLV-II
env protein containing the amino acid as shown in FIG. 2;
(3) any nucleotide sequence that hybridizes to the complement
of the DNA sequence as shown in FIG. 2 under highly stringent
conditions, e-a-, hybridization to filter-bound DNA in 0.5M
NaHP09, 7% sodium dodecyl sulfate (SDS), 1mM EDTA at 65°C, and
washing in 0.1 x SSC/0.1% SDS at 68°C (Ausubel F.M. et al.,
eds., 1989, Current Protocols in Molecular Biology, Vol. I,
Green Publishing Associates, Inc. and John Wiley & Sons,
Inc., New York, at p. 2103) and encodes a functionally
equivalent gene product; (4) any nucleotide sequence that
hybridizes to the complement of the DNA sequence as shown in
FIG. 2 under less stringent conditions, such as moderately
stringent conditions, e.ct., washing in 0.2 x SSC/0.1% SDS at
42°C (Ausubel et al., 1989, supra), yet which still encodes a
functionally equivalent HTLV-II env gene product; and (5) any
nucleotide sequence that hybridizes to the complement of the
nucleotide sequence as shown in FIG. 2 under less stringent
conditions, such as low stringency conditions, e-cr., washing
in 0.2 x SSC/0.1% SDS at 37°C, and encodes a functionally
equivalent gene product. A functionally equivalent gene
product encompasses a gene product that is produced at high
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levels and is immunologically reactive with an anti-HTLV-II
env antibody.
The present invention also encompasses the expression of
nucleotide sequences encoding immunologically equivalent
fragments of the HTLV env protein. Such immunologically
equivalent fragments of HTLV env may be identified by making
analogs of the nucleotide sequence encoding the protein that
are truncated at the 5' and/or 3' ends of the sequence and/or
have one or more internal deletions, expressing the analog
nucleotide sequences, and determining whether the resulting
fragments immunologically interact with a HTLV antibody or
induce the production of such antibodies in vivo,
particularly neutralizing antibodies. For example, a
preferred embodiment of the invention encompasses the
expression of nucleotide sequences encoding a HTLV env
protein with deletions of the amino terminal signal sequence
domain and internal regions which may facilitate secretion of
the env protein.
The invention also encompasses the DNA expression
vectors that contain any of the foregoing coding sequences
operatively associated with a regulatory element that directs
expression of the coding sequences and genetically engineered
host cells that contain any of the foregoing coding sequences
operatively associated with a regulatory element that directs
the expression of the coding sequences in the host cell. As
used herein, regulatory elements include but are not limited
to, inducible and non-inducible promoters, enhancers,
operators and other elements known to those skilled in the
art that drive and regulate expression.
The env glycoprotein gene products or peptide fragments
thereof, may be produced by recombinant DNA technology using
techniques well known in the art. Thus, methods for
preparing the env glycoprotein gene polypeptides and peptides
of the invention by expressing nucleic acid containing env
glycoprotein gene sequences are described herein. Methods
which are well known to those skilled in the art can be used
to construct expression vectors containing env glycoprotein
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gene product coding sequences and appropriate transcriptional
and translational control signals. These methods include,
for example, in vitro recombinant DNA techniques, synthetic
techniques, and in vivo genetic recombination. See, for
example, the techniques described in Sambrook et al., 1989,
supra, and Ausubel et al., 1989, supra. Alternatively, RNA
capable of encoding env glycoprotein gene product sequences
may be chemically synthesized using, for example,
synthesizers. See, for example, the techniques described in
"Oligonucleotide Synthesis", 1984, Gait, M.J. ed., IRL Press,
Oxford, which is incorporated by reference herein in its
entirety.
The invention also encompasses nucleotide sequences that
encode peptide fragments of the HTLV env gene products. In a
preferred embodiment of the present invention relates to
polypeptides or peptides corresponding to the amino
terminally truncated form of the HTLV env protein which is
soluble and accumulates in the cytoplasm of the host cell, so
that the HTLV env protein is readily purified from lysed host
cells. For example, polypeptides or peptides corresponding
to the extracellular domain of HTLV env protein may be useful
as "soluble" protein which would facilitate secretion. The
HTLV env protein gene product or peptide fragments thereof,
can be linked to a heterologous epitope that is recognized by
a commercially available antibody is also included in the
invention. A durable fusion protein may also be engineered;
i.e., a fusion protein which has a cleavage site located
between the HTLV env sequence and the heterologous protein
sequence, so that the HTLV env can be cleaved away from the
heterologous moiety. For example, a collagenase cleavage
recognition consensus sequence may be engineered between the
HTLV env protein or peptide and the heterologous peptide or
protein. The HTLV env domain can be released from this
fusion protein by treatment with collagenase. In a preferred
embodiment of the invention, a fusion protein of glutathione
S-transferase and HTLV env 46 kd protein may be engineered.
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5.3. HTLV ENV ANTIGENIC PROTEINS AND POLYPEPTIDES
The present invention relates to a HTLV env polypeptide
lacking all or a portion of its signal sequence or membrane
spanning domain such that the polypeptide when expressed
recombinantly is not anchored in the membrane of the host
cell. In a preferred embodiment of the invention, the
soluble HTLV env protein is lacking all or a portion of its
amino terminus. The preferred embodiment of the invention
encompasses the HTLV-I env polypeptide lacking 33 amino acids
of the amino terminus and the HTLV-II env polypeptide lacking
17 amino acids of the amino terminus.
The HTLV env protein, polypeptides and peptides,
mutated, truncated or deleted forms of the HTLV env proteins
can be prepared for vaccine preparations and as
pharmaceutical reagents useful in the treatment and
prevention of HTLV-I and -II infection.
The env protein has been deduced by its nucleotide
sequence to have a hydrophobic signal sequence at its amino
terminus, five potential acceptor sites for N-glycosylation
linked carbohydrates in the central portion, and a second
cluster of hydrophobic amino acids in the putative
transmembrane domain (Seiki et al., 1983 Proc. Natl. Acad.
Sci. 80:3618-3622). Its sequence character suggests that it
has a typical structure of a cell membrane glycoprotein.
The env gene encodes a precursor protein gp63 which
undergoes proteolytic cleavage, and subsequent glycosylation
to produce two glycoproteins, gp46 and gp2l. The gp46
protein constitutes the surface projections observed by
electron microscopy on native virus particles, is believed to
have receptor binding activity, and contains domains
responsible for the production of neutralizing antibodies.
The gp21 protein is the transmembrane glycoprotein, and may
be involved in cell fusion activity.
In addition, the invention also encompasses proteins
that are functionally equivalent to the HTLV env proteins
encoded by the nucleotide sequences described in Section
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5.2., as judged by a number of criteria, including but not
limited to, the ability to be recognized by a HTLV env
antibody. Such equivalent HTLV env gene products may contain
deletions, additions, substitutions of amino acid residues
within the amino acid sequence encoded by the HTLV env gene
sequences described above, but which result in a silent
change, thus producing a functionally equivalent HTLV env
gene product. Amino acid substitutions may be made on the
basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or amphipathic nature of
the residues involved. For example, nonpolar (hydrophobic)
amino acids include alanine, leucine, isoleucine, valine,
proline, phenylalanine, tryptophan, and methionine; polar
neutral amino acids include glycine, serine, threonine,
cysteine, tyrosine, asparagine, and glutamine; positively
charged (basic) amino acids include arginine, lysine and
histidine; and negatively charged amino acids include
aspartic acid and glutamic acid. "Functionally equivalent",
as utilized herein, refers to a protein capable of being
recognized by a HTLV env antibody, that is a protein capable
of eliciting a substantially similar immunological response
as the endogenous HTLV env gene products described above.
While random mutations can be made to the HTLV env
nucleotide sequences (using random mutagenesis techniques
well known to those skilled in the art) and the resulting
HTLV env proteins tested for activity, site directed
mutations of the HTLV env coding sequence can be engineered
(using site-directed mutagenesis techniques well known to
those in the art) to generate mutant HTLV env proteins with
increased function, e.a., leading to enhanced expression or
antigenicity.
The HTLV env proteins of the present invention for use
in vaccine preparations are substantially pure or homogenous.
The protein is considered substantially pure or homogenous
when at least 60 to 75% of the sample exhibits a single
polypeptide sequence. A substantially pure protein will
preferably comprise 60 to 90% of a protein sample, more
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preferably about 95% and most preferably 990. Methods which
are well known to those skilled in the art can be used to
determine protein purity or homogeneity, such as
polyacrylamide gel electrophoresis of a sample, followed by
visualizing a single polypeptide band on a staining gel.
Higher resolution may be determined using HPLC or other
similar methods well known in the art.
The present invention encompasses polypeptides which are
typically purified from host cells expressing recombinant
nucleotide sequences encoding these proteins. Such protein
purification can be accomplished by a variety of methods well
known in the art. In a preferred embodiment, the HTLV env
protein of the present invention is expressed as a fusion
protein with glutathione-S-transferase. The resulting
l5 recombinant fusion proteins purified by affinity
chromatography and the HTLV env domain is cleaved away from
the heterologous moiety resulting in a substantially pure
HTLV env protein sample. Other methods may be used, see for
example, the techniques described in "Methods In Enzymology",
1990, Academic Press, Inc., San Diego, "Protein Purification:
Principles and practice", 1982, Springer-Verlag, New York.
5.4. EXPRESSION SYSTEMS
The present invention encompasses expression. systems,
both eucaryotic and procaryotic expression vectors, which may
be used to express both truncated and full-length forms of
the HTLV env protein.
In a preferred embodiment of the invention, the
nucleotide sequences of FIG. 1, deleted of the boxed region,
encoding the truncated HTLV-I env protein are expressed in
either eucaryotic or procaryotic expression vectors. In
another preferred embodiment of the invention, the nucleotide
sequences of FIG. 2, deleted of the boxed region, encoding
the truncated HTLV-II env protein are expressed in either
eucaryotic or procaryotic expression vectors.
A preferred embodiment of the invention encompasses the
expression of both full-length and truncated forms of the
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HTLV env gene products in a baculovirus system to produce an
unglycosylated antigen.
Another preferred embodiment of the invention
encompasses the expression of full-length and truncated forms
of the HTLV env gene products in a stably transfected T cell
line to produce a glycosylated antigen.
A variety of host-expression vector systems may be
utilized to express the env glycoprotein gene coding
sequences of the invention. Such host-expression systems
represent vehicles by which the coding sequences of interest
may be produced and subsequently purified, but also represent
cells which may, when transformed or transfected with the
appropriate nucleotide coding sequences, exhibit the env
glycoprotein gene product of the invention in situ. These
include but are not limited to microorganisms such as
bacteria (eq., E. coli, B. subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA
expression vectors containing env glycoprotein gene product
coding sequences; yeast (eq., Saccharomyces, Yichia)
transformed with recombinant yeast expression vectors
containing the env glycoprotein gene product coding
sequences; insect cell systems infected with recombinant
virus expression vectors (e.q., baculovirus) containing the
env glycoprotein gene product coding sequences; plant cell
systems infected with recombinant virus expression vectors
(e~ct., cauliflower mosaic virus, CaMV; tobacco mosaic virus,
TMV) or transformed with recombinant plasmid expression
vectors (e. q., Ti plasmid) containing env glycoprotein gene
product coding sequences; or mammalian cell systems (e-a.,
COS, CHO, BHK, 293, 3T3) harboring recombinant expression
constructs containing promoters derived from the genome of
mammalian cells (e-a., metallothionein promoter) or from
mammalian viruses (e-cL, the adenovirus late promoter; the
vaccinia virus 7.5K promoter).
In bacterial systems, a number of expression vectors may
be advantageously selected depending upon the use intended
for the env glycoprotein gene product being expressed. For
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example, when a large quantity of such a protein is to be
produced, for the generation of pharmaceutical compositions
of env glycoprotein protein or for raising antibodies to env
glycoprotein protein, for example, vectors which direct the
expression of high levels of fusion protein products that are
readily purified may be desirable. Such vectors include, but
are not limited, to the E. coli expression vector pUR278
(Ruther et al., 1983, EMBO J. 2:1791), in which the env
glycoprotein gene product coding sequence may be ligated
individually into the vector in frame with the lac Z coding
region so that a fusion protein is produced; pIN vectors
(Inouye & hnouye, 1985, Nucleic Acids Res. 13:3101-3109; Van
Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and
the like. pGEX vectors may also be used to express foreign
polypeptides as fusion proteins with glutathione S-
transferase (GST). In general, such fusion proteins are
soluble and can easily be purified from lysed cells by
adsorption to glutathione-agarose beads followed by elution
in the presence of free glutathione. The pGEX vectors are
designed to include thrombin or factor Xa protease cleavage
sites so that the cloned target gene product can be released
from the GST moiety.
In an insect system, Autographa californica nuclear
polyhedrosis virus {AcNPV) is used as a vector to express
foreign genes. The virus grows in Spodoptera frugiperda
cells. The env glycoprotein gene coding sequence may be
cloned individually into non-essential regions (for example
the polyhedrin gene) of the virus and placed under control of
an AcNPV promoter (for example the polyhedrin promoter).
Successful insertion of env glycoprotein gene coding sequence
will result in inactivation of the polyhedrin gene and
production of non-occluded recombinant virus (i.e., virus
lacking the proteinaceous coat coded for by the polyhedrin
gene). These recombinant viruses are then used to infect
Spodoptera frugiperda cells in which the inserted gene is
expressed. (E -a-, see Smith et al., 1983, J. Virol. 46: 584;
Smith, U.S. Patent No. 4,215,051).
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In mammalian host cells, a number of viral-based
expression systems may be utilized. In cases where an
adenovirus is used as an expression vector, the env
glycoprotein gene coding sequence of interest may be ligated
to an adenovirus transcription/translation control complex,
ea., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome
by in vitro or in vivo recombination. Insertion in a non-
essential region of the viral genome (e.a., region E1 or E3)
will result in a recombinant virus that is viable and capable
of expressing env glycoprotein gene product in infected
hosts. (E. a., See Logan & Shenk, 1984, Proc. Natl. Acad.
Sci. USA 81:3655-3659). Specific initiation signals may also
be required for efficient translation of inserted env
glycoprotein gene product coding sequences. 'These signals
include the ATG initiation codon and adjacent sequences. In
cases where an entire env glycoprotein gene, including its
own initiation codon and adjacent sequences, is inserted into
the appropriate expression vector, no additional
translational control signals may be needed. However, in
cases where only a portion of the env glycoprotein gene
coding sequence is inserted, exogenous translational control
signals, including, perhaps, the ATG initiation codon, must
be provided. Furthermore, the initiation codon must be in
phase with the reading frame of the desired coding sequence
to ensure translation of the entire insert. These exogenous
translational control signals and initiation codons can be of
a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., 1987, Methods in
Enzymol. 153:516-544).
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5.5. CELL LINES
The present invention encompasses the expression of HTLV
env glycoprotein in animal and insect cell lines. In a
preferred embodiment of the present invention, the env
glycoprotein is expressed in a baculovirus vector in an
insect cell line to produce an unglycosylated antigen. In
another preferred embodiment of the invention, the env
glycoprotein is expressed in a stably transfected T
lymphocyte cell line to produce a glycosylated antigen.
Host cell strain may be chosen which modulates the
expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired.
Such modifications (e. g., glycosylation) and processing (e. g.
cleavage) of protein products may be important for the
function of the protein. Different host cells have
characteristic and specific mechanisms for the post-
translational processing and modification of proteins and
gene products. Appropriate cell lines or host systems can be
chosen to ensure the correct modification of the foreign
protein expressed. To this end, eucaryotic host cells which
possess the cellular machinery for proper processing of the
primary transcript, glycosylation, and phosphorylation of the
gene product may be used. Such mammalian host cells include
but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293,
3T3 and WI38 cell lines.
For long term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell
lines which stably express the env glycoprotein gene product
may be engineered. Rather than using expression vectors
which contain viral origins of replication, host cells can be
transformed with DNA controlled by appropriate expression
control elements (e.g.,promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and
a selectable marker. Following the introduction of the
foreign DNA, engineered cells may be allowed to grow for 1-2
days in an enriched media, and then are switched to a
selective media. The selectable marker in the recombinant
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plasmid confers resistance to the selection and allows~cells
to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded
into cell lines. This method may advantageously be used to
engineer cell lines. This method may advantageously be used
to engineer cell lines which express the env glycoprotein
gene products. Such cell lines would be particularly useful
in screening and evaluation of compounds that affect the
endogenous activity of the env glycoprotein gene product.
A number of selection systems may be used, including but
not limited to the herpes simplex virus thymidine kinase
(Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817)
genes can be employed in tk~, hgprt- or aprt- cells,
respectively. Also, antimetabolite resistance can be used as
the basis of selection for the following genes: dhfr, which
confers resistance to methotrexate (Wigler, et al., 1980,
Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 2981, Proc.
Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance
to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl.
Acad. Sci. USA 78:2072); neo, which confers resistance to the
aminoglycoside G-418 (Colberre-Garapin, et al., 2981, J. Mol.
Biol. 150:1); and hygro, which confers resistance to
hygromycin (Santerre, et al., 1984, Gene 30:147).
Alternatively, any fusion protein may be readily
purified by utilizing an antibody specific for the fusion
protein being expressed. For example, a system described by
Janknecht et al. allows for the ready purification of non-
denatured fusion proteins expressed in human cell lines
(Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88:
8972-8976). In this system, the gene of interest is
subcloned into a vaccinia recombination plasmid such that the
gene's open reading frame is translationally fused to an
amino-terminal tag consisting of six histidine residues.
Extracts from cells infected with recombinant vaccinia virus
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are loaded onto Ni2'~nitriloacetic acid-agarose columns and
histidine-tagged proteins are selectively eluted with
imidazole-containing buffers.
5.6. VACCINE FORMULATIONS AND METHODS OF ADMINISTRATION
Since the HTLV env protein antigen of the present
invention can be produced in large amounts, the antigen thus
produced and purified has use in vaccine preparations. The
HTLV env protein also has utility in immunoassays, ea., to
to detect or measure in a sample of body fluid from a vaccinated
subject the presence of antibodies to the antigen, and thus
to diagnose infection and/or to monitor immune response of
the subject subsequent to vaccination.
The preparation of vaccines containing an immunogenic
polypeptide as the active ingredient is known to one skilled
in the art.
5.6.1. DETERMINATION OF VACCINE EFFICACY
The immunopotency of the HTLV env antigen can be
determined by monitoring the immune response in test animals
following immunization with the HTLV env antigen, or by use
of any immunoassay known in the art. Generation of a humoral
(antibody) response and/or cell-mediated immunity, may be
taken as an indication of an immune response. Test animals
may include mice, hamsters, dogs, cats, monkeys, rabbits,
chimpanzees, etc., and eventually human subjects.
Methods of introducing the vaccine may include oral,
intracerebral, intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal or any other standard
routes of immunization. The immune response of the test
subjects can be analyzed by various approaches such as: the
reactivity of the resultant immune serum to the HTLV env
antigen, as assayed by known techniques, e.g., immunosorbant
assay (ELISA), immunoblots, radioimmunoprecipitations, etc.,
or in the case where the HTLV env antigen displays
antigenicity or immunogenicity, by protection of the
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immunized host from infection by HTLV and/or attenuation of
symptoms due to infection by HTLV in the immunized host.
As one example of suitable animal testing of an HTLV env
vaccine, the vaccine of the invention may be tested in
rabbits for the ability to induce an antibody response to the
HTLV env antigen. Male specific-pathogen-free (SPF) young
adult New Zealand White rabbits may be used. The test group
each receives a fixed concentration of the vaccine. A
control group receives an injection of 1 mM Tris-HC1 pH 9.0
without the HTLV env antigen.
Blood samples may be drawn from the rabbits every one or
two weeks, and serum analyzed for antibodies to the HTLV env
protein. The presence of antibodies specific for the antigen
may be assayed, e.a., using an ELISA.
5.6.2. VACCINE FORMULATIONS
Suitable preparations of sucr~ vaccines include
injectables, either as liquid solutions or suspensions; solid
forms suitable for solution in, suspension in, liquid prior
to injection, may also be prepared. The preparation may also
be emulsified, or the polypeptides encapsulated in Iiposomes.
the active immunogenic ingredients are often mixed with
excipients which are pharmaceutically acceptable and
compatible with the active ingredient. Suitable excipients
are, for example, water saline, dextrose, glycerol, ethanol,
or the like and combinations thereof. In addition, if
desired, the vaccine preparation may also include minor
amounts of auxiliary substances such as wetting or
emulsifying agents, pH buffering agents, and/or adjuvants
which enhance the effectiveness of the vaccine.
Examples of adjuvants which may be effective, include,
but are not limited to: aluminum hydroxide, N-acetyl-muramyl-
L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-
alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-
isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-
hydroxyphosphoryloxy)-ethylamine.
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The effectiveness of an adjuvant may be determined by
measuring the induction of antibodies directed against an
immunogenic polypeptide containing a HTLV env polypeptide
epitope, the antibodies resulting from administration of this
polypeptide in vaccines which are also comprised of the
various adjuvants.
The polypeptides may be formulated into the vaccine as
neutral or salt forms. Pharmaceutically acceptable salts
include the acid addition salts (formed with free amino
l0 groups of the peptide) and which are formed with inorganic
acids, such as, for example, hydrochloric or phosphoric
acids, or organic acids such as acetic, oxalic, tartaric,
malefic, and the like. Salts formed with free carboxyl groups
may also be derived from inorganic bases, such as, for
example, sodium potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine and
the like.
The vaccines of the invention may be multivalent or
univalent. Multivalent vaccines are made from recombinant
viruses that direct the expression of more than one antigen.
Many methods may be used to introduce the vaccine
formulations of the invention; these include but are not
limited to oral, intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal routes, and via
scarification (scratching through the top layers of skin,
e.g., using a bifurcated needle).
The patient to which the vaccine is administered is
preferably a mammal, most preferably a human, but can also be
a non-human animal including but not limited to cows, horses,
sheep, pigs, fowl (e. g., chickens), goats, cats, dogs,
hamsters, mice and rats.
The vaccine formulations of the invention comprise an
effective immunizing amount of the HTLV env protein and a
pharmaceutically acceptable carrier or excipient. Vaccine
preparations comprise an effective immunizing amount of one
or more antigens and a pharmaceutically acceptable carrier or
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excipient. Pharmaceutically acceptable carriers are well
known in the art and include but are not limited to saline,
buffered saline, dextrose, water, glycerol, sterile isotonic
aqueous buffer, and combinations thereof. One example of
such an acceptable carrier is a physiologically balanced
culture medium containing one or more stabilizing agents such
as stabilized, hydrolyzed proteins, lactose, etc. The
carrier is preferably sterile. The formulation should suit
the mode of administration.
The composition, if desired, can also contain minor
amounts of wetting or emulsifying agents, or pH buffering
agents. The composition can be a liquid solution,
suspension, emulsion, tablet, pill, capsule, sustained
release formulation, or powder. Oral formulation can include
25 standard carriers such as pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc.
Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free
concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent.
Where the composition is administered by injection, an
ampoule of sterile diluent can be provided so that the
ingredients may be mixed prior to administration.
In a specific embodiment, a lyophilized HTLV env
polypeptide of the invention is provided in a first
container; a second container comprises diluent consisting of
an aqueous solution of 50% glycerin, 0.25% phenol, and an
antiseptic (e. g., 0.005% brilliant green).
The precise dose of vaccine preparation to be employed
in the formulation will also depend on the route of
administration, and the nature of the patient, and should be
decided according to the judgment of the practitioner and
each patient's circumstances according to standard clinical
techniques. An effective immunizing amount is that amount
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sufficient to produce an immune response to the antigen in
the host to which the vaccine preparation is administered.
Use of purified antigens as vaccine preparations can be
carried out by standard methods. For example, the purified
proteins) should be adjusted to an appropriate
concentration, formulated with any suitable vaccine adjuvant
and packaged for use. Suitable adjuvants may include, but
are not limited to: mineral gels, e.g., aluminum hydroxide;
surface active substances such as lysolecithin, pluronic
polyols; polyanions; peptides; oil emulsions; alum, and MDP.
The immunogen may also be incorporated into liposomes, or
conjugated to polysaccharides and/or other polymers for use
in a vaccine formulation. In instances where the recombinant
antigen is a hapten, i.e., a molecule that is antigenic in
that it can react selectively with cognate antibodies, but
not immunogenic in that it cannot elicit an immune response,
the hapten may be covalently bound to a carrier or
immunogenic molecule; for instance, a large protein such as
serum albumin will confer immunogenicity to the hapten
coupled to it. The hapten-carrier may be formulated for use
as a vaccine.
Effective doses (immunizing amounts) of the vaccines of
the invention may also be extrapolated from dose-response
curves derived from animal model test systems.
The invention also provides a pharmaceutical pack or kit
comprising one or more containers comprising one or more of
the ingredients of the vaccine formulations of the invention.
Associated with such containers) can be a notice in the form
prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological
products, which notice reflects approval by the agency of
manufacture, use or sale for human administration.
The present invention thus provides a method of
immunizing an animal, or treating or preventing various
diseases or disorders in an animal, comprising administering
to the animal an effective immunizing dose of a vaccine of
the present invention.
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5.6.3. USE OF ANTIBODIES GENERATED
BY THE VACCINES OF THE INVENTION
The antibodies generated against the antigen by
immunization with the HTLV env protein of the present
invention also have potential uses in diagnostic
immunoassays, passive immunotherapy, and generation of
antiidiotypic antibodies.
The generated antibodies may be isolated by standard
techniques known in the art (e. g., immunoaffinity
chromatography, centrifugation, precipitation, etc.) and used
in diagnostic immunoassays. The antibodies may also be used
to monitor treatment and/or disease progression. Any
immunoassay system known in the art, such as those listed
supra, may be used for this purpose including but not limited
to competitive and noncompetitive assay systems using
techniques such as radioimmunoassays, ELISA (enzyme-linked
immunosorbent assays), "sandwich" immunoassays, precipitin
reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-
fixation assays, immunoradiometric assays, fluorescent
immunoassays, protein A immunoassays and
immunoelectrophoresis assays, to name but a few.
The vaccine formulations of the present invention can
also be used to produce antibodies for use in passive
lmmunotherapy, in which short-term protection of a host is
achieved by the administration of pre-formed antibody
directed against a heterologous organism.
The antibodies generated by the vaccine formulations of
the present invention can also be used in the production of
antiidiotypic antibody. The antiidiotypic antibody can then
in turn be used for immunization, in order to produce a
subpopulation of antibodies that bind the initial antigen of
the pathogenic microorganism (Jerne, 1974, Ann. Immunol.
(Paris) 125c:373; Jerne, et al., 1982, EMBO J. 1:234).
In immunization procedures, the amount of immunogen to
be used and the immunization schedule will be determined by a
physician skilled in the art and will be administered by
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reference to the immune response and antibody titers of the
subject.
5.6.4. PACKAGING
The compositions may, if desired, be presented in a pack
or dispenser device which may contain one or more unit dosage
forms containing the active ingredient. The pack may for
example comprise metal or plastic foil, such as a blister
pack. The pack or dispenser device may be accompanied by
instructions for administration. Compositions comprising a
compound of the invention formulated in a compatible
pharmaceutical carrier may also be prepared, placed in an
appropriate container, and labelled for treatment of an
indicated condition.
6. EXAMPLE: EXPRESSION OF HTLV-II ENV PROTEINS
In the example presented herein, the expression of an
amino terminally truncated form of the HTLV env protein is
demonstrated. It is further demonstrated that the resulting
recombinant HTLV env protein is immunoreactive.
6.1. MATERIALS AND METHODS
Construction of baculovirus transfer vector and GST fusion
vector.
The nucleotide sequence of the HTLV-II env gene used in
these constructs is shown in FIG. 2. The nucleotide
sequences deleted from the HTLV-II env gene are indicated by
the boxed region in FIG. 2. The primers used to amplify the
truncated form of HTLV-II env gene are underlined in FIG. 2.
The nucleotide sequence of the HTLV-I env gene that may
be expressed in the baculotransfer vector and GST fusion
vector is shown in FIG. 1. The nucleotide sequences deleted
from the HTLV-I env gene are indicated by the boxed region in
FIG. 1. The primers used to amplify the truncated form of
the HTLV-I env gene are underlined in FIG. 1.
HTLV-II env gene was amplified by PCR from plasmid Mo
which contains the 3' half genome (Shimotono) using taq
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WO 98/03197 PCT/US97/12776
polymerase (fetus), 30 cycles of amplification (94°C, 30 sec-
56 °C, 30 sec,'72°C, 1 min) and the following oligonucleotide
primers: 5'-AAGGATCCATGGGTAATGTTTTCTTC-3'5180-5197 and 5'-
AAGGATCCTTATAGCATGGTTTCTGG-3'6643-6626 (BamHl site is
indicated as underlined and sequence numbers were derived
from the published sequence of HTLV-II-Mo (Shimotono). PCR-
amplified products was digested with BamHl and following
electrophoresis, purified in low-melting temperature agarose
gels; DNA bands were excised and ligated to baculovirus
transfer vector pVLl 392 at the BamH I site which is located
downstream of the polyhedron promotor. The baculovirus
vector used to make these constructs is pVLl 392 was obtained
from Invitrogen, San Diego, CA. This recombinant plasmid was
used to transfect insect cells.
Similarly fusion proteins of glutathione S-transferase
(GST) and HTLV-11 env cleaved protein, gp46 was prepared by
amplifying plasmid Mo-T with primers
5'-AAGGATCCATGGGTAATGTTTTCTTC3'(5180-5197) and
5'AAGAATTCACGGCGGCGTCTTGTCGCGCCAGG3'(6103-6086, BamHI
and EcoRI stickey ends were introduced with these primers and
used to ligate the fragments together. Using the expression
plasmid pGEX2T (Pharmacia, Upsala, Sweden) GST-gp46 protein
was expressed and purified according to the manufacturer's
instructions.
Production of recombinant baculovirus
To generate recombinant baculovirus, monolayers
consisting 106 insect cells (High Five(H5), Invitrogen, San
Diego, CA) were cotransfected with transfer vector DNA
containing env CDNA as described above, together with
linearized baculovirus DNA (Baculogold, PharMingen, San
Diego, CA), using calciumphosphate method. Single plaque
including recombinant virus was purified from supernatant and
amplified in H5 monolayers cells.
Western immunoblot analysis
- 32 -
T_. _.__._~s.-_ __..___.. .__.._ _~_._ _ _..._.. ______ ~ ...
CA 02262007 1999-O1-22
WO 98103197 PCT/US97/12776
Western blot was used to assess the reactivity of the
env protein expressed in baculovirus infected cells with
human antisera. Total cell extracts from H5 cells infected
with recombinant baculovirus were subject to electrophoresis
through 10% SDS polyacrylamide gels (SDS-PAGE) and
transferred to PVDF membrane (Immobilon, Bedford, MA).
Filters were probed with HTLV-11 infected patient's serum
(Hall) at 200 times dilutions. Bound antibody was detected
by inoculation of the filter with horseradish'peroxidase-
conjugated antibody, anti-human (DAKO A/S, Denmark), at a
1:5000 dilution, followed by development with
chemiluminescence (ECL, Amerciam, Buckinghamshire, England)
Cells and viruses
The T-cell lines CEM was used for T cell~control
negative for HTLV-II, and B-cell line, BJAB for fusion assay.
HTLV-II-Vines was isolated from a male intravenous drug
abuser who was not infected with human immunodeficiency virus
and was used to establish an HTLV-II carrying human lymphoid
cell line (Hall). HTLV-II-Mo-T is a HTLV-II-infected
lymphoblastoid T cell line from patient Mo with a T-cell
variant of hairy cell leukemia (Saladoon). All the cell
lines were maintained in RPMI 1640 medium supplemented with
10% fetal calf serum, 2%glutamine and 50 ug/ml of
gentamycine, and cultured at 37°C in 5% CO2.
The insect cell, High Five (H5, Invitrogen, San Diego,
CA) was maintained in TC100 medium (Gibco BRL, Gaithersburg,
MD) including 100 of calf fetal serum and 50ug/ml of
kanamycin.
6.2. RESULTS
The purified recombinant baculovirus containing HTLV-II
env gene was used to infect monolayers of H5 insect cells.
The infected insect cells were harvested 4 days after
infection and examined for expression of the HTLV-II envelope
polypeptides. Proteins from lysed cells were separated by
polyacrylamide gel and transferred to PVDF membrane and
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CA 02262007 1999-O1-22
WO 98/03197 PCT/LJS97/12776
probed with HTLV-II infected patient's sera(Hall) (FIG. 2).
A protein with an apparent molecular mass of 63 kDa was
immunoreactive.
The location of the recombinant protein gp63 (rgp63) in
the insect cells was examined by immunofluorescence.
Similarly infected insect cells were harvested 2 days after
infection and incubated HTLV-II infected patient's serum.
The majority of infected insect cells bound the antibody,
suggesting recombinant gp63 localizing to the surface of
infected insect cells (data not shown).
These results demonstrate that an immunoreactive HTLV
env antigen was successfully expressed in a baculovirus
infected insect cell line.
7. EXAMPLE: IMMUNIZATION WITH HTLV-II ENV PROTEINS
The following analysis was conducted to determine the
effects of inoculating rabbits with the recombinant gp63 env
protein. In this analysis, rabbits were immunized with gp63
expressing insect cells, and serum from the rabbits was
assayed for antibodies to the HTLV env protein. The presence
of anti-HTLV env antibodies was measured by: (1) the ability
to detect recombinant GST-gp46 fusion protein expressed in
bacteria, and (2) the ability to recognize HTLV-II infected
human cells in FACS analysis.
7.1. MATERIALS AND METHODS
Cells and viruses
The T-cell lines CEM was used for T cell control
negative for HTLV-II, and B-cell line, BJAB for fusion assay.
HTLV-II-Vines was isolated from a male intravenous drug
abuser who was not infected with human immunodeficiency virus
and was used to establish an HTLV-II carrying human lymphoid
cell line(Hall). HTLV-II-Mo-T is a HTLV-II-infected
lymphoblastoid T cell line from patient Mo with a T-cell
variant of hairy cell leukemia(Saladoon). All the cell lines
were maintained in RPMI 1640 medium supplemented with 10%
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_ _. ___ _
CA 02262007 1999-O1-22
WO 98/03197 PCT/US97/12776
fetal calf serum, 2% glutamine and 50 ug/ml of gentamycine,
and cultured at 37°C in 5% C02.
The insect cell, High Five (H5, Invitrogen, San Diego,
CA) was maintained in TC1 00 medium (Gibco BRL, Gaithersburg,
MD) including 100 of calf fetal serum and 50ug/ml of
kanamycin.
FACS analysis
HTLV-II-Vines, Mo-T, and CEM cell lines were stained with
immunized rabbits sera, following 3 times of washing with PBS
incubated with FITC conjugated goat anti-human sera (DAKO
A/S, Denmark) at 1-50, in the presence of 2% calf fetal
serum. Relative fluorescence intensity was detected by flow
cytometry.
Rabbits
2.5kg, specific pathogen free, female New Zealand White
rabbits were obtained from a commercial rabbitry (SCL,
Shizuoka, Japan). Groups of rabbits were inoculated
intravenously with 5x10' HTLV-II infected cells or heat
inactivated cells (70°C for 20 min.) as shown Table 1.
HTLV-II infected cells were 90% infected, as determined by
fluorescent antibody assay using an HTLV-II infected
patient's serum (FIG. 5).
Syncytium inhibition assay.
HTLV-II-Vines cells were suspended in RPMI medium at 106
cells per ml, aliquots (50u1 per well) were incubated with
heat-inactivated, 100u1 of diluted Rabbit serum in 96-well
plates at 37°C for 15 min, and then 50u1 of BJAB cell
suspension(106 cells per ml) was added to each well. After
incubation at 37°C for 16 hrs in a 5% C02 incubator, each well
was examined for syncytia (giant multinuclear cells) with an
inverted microscope. Neutralization titers of antibody
samples were expressed as the reciprocal of the sample
dilution at which the syncytium formation was completely
(100%) inhibited in the microcultures.
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Immunization of Rabbits.
Recombinant-baculovirus-infected H5 cells were pelleted,
washed once in PBS, and resuspended in PBS at a concentration
of lo' cells/ml. Samples (10' cells) which were emulsified in
complete (day 0) or incomplete (day 14, 28, and 42) Freund's
adjuvant, were injected intramuscularly into New Zealand
White female rabbits (2.5kg). Rabbits were purchased from
SLC, Shizuoka, Japan. Immune sera from rabbits were
collected on day 56.
7.2. RESULTS
Rabbits were immunized with recombinant gp63 expressing
insect cells, and the serum was assayed for the detection of
recombinant GST-fusion protein with a cleaved form of the env
protein, gp46, expressed in bacteria. Because antigenicity
of insect cells are different from bacteria, rabbit antisera
after immunization does not show cross reactivity to this
fusion protein. One week after immunization, serum from
Rabbit 3 and 4 showed reactivity against GST-gp46 (FIG. 4,
lane 2 and 3), however the serum of R3 prior to immunization
did not react (lane 1).
Rabbits immunized with recombinant gp63 expressing
insect cells, and the serum was assayed for the detection of
HTLV-II infected human cells by FACS analysis. The FACS
analysis showed positive staining in the cell lines, Vines
and Mo-T (FIG. 5). In contrast the uninfected T cell line,
CEM showed a negative staining pattern, suggesting that the
rabbit serum is including antibody against HTLV-II env
proteins.
35
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CA 02262007 1999-O1-22
WO 98/03197 PCT/US97/12776
TABLE 1
IMMUNIZATION CHALLENGE OF DETECTION OF PROVIRUS
HTLV-II-Vines (weeks post infection)
EXp 1 2 4 6 8 20 70
Rl - 5 x 10' - + + + + +
R2 - 5 x 10' + + + + + +
R3 + 5 x 10'
_ _ _
R4 + 5 x 10'
Exp 2
R5 - 5 x 10' + +
R6 - 5 x 10' (heat inactivat)- -
R7 - 5 x l0' (heat inactivat)- -
R8 - 5 x 10' - -
R9 + 5 x 10' - -
8. NEUTRALIZATION ACTIVITY OF SERA FROM ~,IACCINATED
ANIMALS
HTLV-II infected cells induce cell to cell fusion after
cocultivation with B-cell line, BJAB (Hall). Therefore,
serum from rabbits immunized with recombinant gp63 was
assayed for its ability to block cell to cell fusion.
8~1. MATERIALS AND METHODS
Rabbits
2.5kg, specific pathogen free, female New Zealand White
rabbits were obtained from a commercial rabbitry(SCL,
Shizuoka, Japan). Groups of rabbits were inoculated
Intravenously with 5x10' HTLV-II infected cells or heat
inactivated cells (70°C for 2o min.) as shown Table 1.
HTLV-II infected cells were 90% infected, as determined by
fluorescent antibody assay using an HTLV-II infected
patient's serum (FIG. 4).
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WO 98/03197 PCTlUS97/12776
Syncytium inhibition assay.
HTLV-II-Vines cells were suspended in RPMI medium at 106
cells per ml, aliquots (50u1 per well) were incubated with heat-
inactivated, 100u1 of diluted Rabbit serum in 96-well plates
at 37°C for 15 min, and then 50u1 of BJAB cell suspension (106
cells per ml) was added to each well. After incubation at
37°C for 16 hrs in a 5% COZ incubator, each well was examined
for syncytia (giant multinuclear cells) with an inverted
microscope. Neutralization titers of antibody samples were
expressed as the reciprocal of the sample dilution at which
the syncytium formation was completely (100%) inhibited in
the microcultures.
Immunization of Rabbits.
Recombinant-baculovirus-infected H5 cells were pelleted,
washed once in PBS, and resuspended in PBS at a concentration
of 10 7 cells/mi. Samples (10' cells) which were emulsified
in complete (day 0) or incomplete (day 14, 28, and 42)
Freund's adjuvant, were injected intramuscularly into New
Zealand White female rabbits (2.5kg). Rabbits were purchased
from SLC, Shizuoka, Japan. Immune sera from rabbits were
collected on day 56.
Particle Agglutination method
The titer of rabbit sera against HTLV-II antigen was
calculated by particle agglutination kit (Fujirebio inc.,
Tokyo, Japan). Beads attached with purified HTLV-I particles
were incubated with various dilutions of sera and maximum
dilution that leads to agglutination were described as
antibody titers.
8.2. RESULTS
The results of incubating HTLV-II infected cells with
various dilutions of antisera to gp62 demonstrated that at
most 1:50 dilution of sera from samples R3, R4, R8 and R9 was
enough to completely inhibit fusion at the first bleeding.
Fusion was blocked by incubation with antisera but not with
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_._.____ __._ _.. . _._...___ __
CA 02262007 1999-O1-22
WO 98/03197 PCT/LTS97/12776
preimmune sera. This suggests that the epitopes eliciting
the fusion activity are located within the gp63, env protein.
Previous studies showed that sera that can block cell fusion
always show neutralization of virus infection.
In addition, since a cell free infection is not possible in
HTLV-I and II infection, blockage of cell to cell fusion
induced by the rabbit gp63 immunized sera indicates blockage
of HTLV-II infection. Therefore these results indicate that
rabbit gp63 immunized sera has neutralizing activity against
HTLV-II.
9. PROTECTION OF RABBITS FROM HTLV-II INFECTION
In the Example presented herein, the ability of the HTLV
env antigen of the present invention was assayed for its
ability to protect rabbits against HTLV-II infection.
9.1. MATERIALS AND METHODS
HTLV-II challenge and detection of provirus by PCR.
HTLV-II-Vines cells were washed with PBS and injected
intravenously into rabbits(5x10' cells) after with heat-
inactivation or without inactivation. Every week
postinfection, PBL were isolated from heparinized blood
samples by density separation medium for rabbit lymphocytes,
lympholyte-Rabbit (Cedarlane Laboratories, Hornby, Ontario,
Canada).
DNA samples were prepared by DNAzol (Gibco BRL, Gaitherburg,
MD) from PBMC, and 1 ug DNA samples were subjected to PCR
analysis. The primers used for PCR to amplify tax region
were SK43 5'TGGATA CCC CGT CTA CGT GT3' (7248 to 7267) and
SK44, 5'GAG CTG ACA ACG CGT CCA TCG3' (7406 to 7386), and
those used for the second step of PCR were SK43', 5GCG ATT
GTG TAC AGG CCG ATT GGT3'(7271 to 7294) which locates just
downstream of SK43 and together with SK44 works as nested
primer.
(SK43 and 44 were derived from PCR protocols M.A. Innis,
David H. Gelfand, J.J. Sninsky and T.J. White. Academic
press.)
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CA 02262007 1999-O1-22
WO 98/03197 PCT/US97/12776
Southern hybridization
Ten microliters (from a 50-ul PCR samples) of nested
amplified DNA was separated on 1.5o agarose gels and blotted
to nylon membranes (Schlicher & Schuell). Membranes were
hybridized for 2 hrs at 68°C in HybriQuick solution
(Strategen, San Diego, CA). PCR fragment made with SK43 and
44 primers, were purified from agarose gel and was used for
probe after random-primed-labelled with (a-32P)dCTP.
9.2. RESULTS
Rabbits were inoculated intravenously with 5x10' HTLV-II-
Vines cells. In the first experiment, the nonimmunized
groups Rl and R2 were seroconverted for HTLV-II 2 weeks after
challenge, with the antibody titer rising to a maximum at the
following 8 weeks (FIG. 4). Western blot analysis of R1
demonstrated the presence of antibody against the recombinant
cleaved env-fusion-protein after challenge (FIG. 6). In
rabbits, R3 and R4, immunized with HTLV-II env expressing
insect cells, respectively, antibody titer continued to
plateau in the following 10 weeks after challenge (FIG. 5).
The presence of HTLV-II nucleotide sequences in
peripheral blood lymphocytes (PBL) isolated from the
immunized rabbits was assayed by PCR analysis. In the first
experiment, as summarized in table 1, HTLV-II provirus was
detected in DNA samples from PBLs of nonimmunized rabbits but
was not been detected in PBLs from immunized rabbits for 20
weeks. Also in the second challenge HTLV-II provirus was
detected only in immunized rabbits. To confirm HTLV-II
infection, heat inactivated HTLV-II-Vines was also
challenged, expectedly provirus was not detected after 20
weeks. To confirm the absence of provirus in immunized
rabbits and the presence of provirus in non-immunized
rabbits, HTLV tax gene PCR products were amplified after both
challenges subjected to southern hybridization (FIG. 6). The
results of the second PCR analysis demonstrated that after
challenge with HTLV-II, non-immunized rabbits contained HTLV
provirus, while in immunized rabbits no HTLV-II provirus
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CA 02262007 1999-O1-22
WO 98/03197 PCT/US97/12776
could be detected. These results indicate that the HTLV env
antigen of the present invention conferred protection in
immunized rabbits against challenge with the HTLV-II virus.
The present invention is not to be limited in scope by
the specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from
the foregoing description and accompanying figures. Such
modifications are intended to fall within the scope of the
appended claims.
Various publications are cited herein, the disclosures
of which are incorporated by reference in their entireties.
20
30
- 41 -
CA 02262007 1999-07-19
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: The Rockefeller University
(ii) TITLE OF THE INVENTION: ENV-GLYCOPROTEIN VACCINE FOR
PROTECTIOF7 OF HTLV-I AND HTLV-II INFECTION
(iii) NUMBER OF SEQUENCES: 20
(iv) CORRESPONDENCE; ADDRESS:
(A) ADDRESSEE: 0~>ler, Hoskin & Harcourt
(B) STREET: 50 O'Connor Street
(C) CITY: Ottawa
(D) STATE: Ontario
(E) COUNTRY: Canada
(F) ZIP:K1P 6L2
(v) COMPUTER READAE~LE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBNf Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,262,007
(B) FILING DATE: 22-JUL-1397
(C) CLASSIFICATION: Corresponding to PCT/US97/12776
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: David W. Aitken
(B) REFERENCE/DOCKET NUMBER: 13456
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613) 235-7234
(B) TELEFAX: (613) 235-2867
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1467 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 1...1464
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCR:LPTION: SEQ ID NO: l:
ATG GGT AAG TTT CTC GCC ACT TTC ATT TTA TTC TTC CAG TTC TGC CCC 48
Met Gly Lys Phe Leu Ala Thr Phe Ile Leu Phe Phe Gln Phe Cys Pro
1 5 10 15
CTG ATC TTC GGT GAT TAC A(~C CCC AGC TGC TGT ACT CTC ACA ATT GCA 96
Leu Ile Phe Gly Asp Tyr Ser Pro Ser Cys Cys Thr Leu Thr Ile Ala
20 25 30
-4i/i-
CA 02262007 1999-07-19
GTCTCCTCA TACCACTCT AAACCCTGC AATCCTGCCCAG CCAGTTTGT 144
ValSerSer TyrHisSer LysProCys AsnProAlaGln ProValCys
35 40 q5
TCGTGGACC CTCGACCTG C;TGGCCCTT TCAGCAGATCAG GCCCTACAG 192
SerTrpThr LeuAspLeu I~euAlaLeu SerAlaAspGln AlaLeuGln
50 55 60
CCCCCCTGC CCTAACCTA GTAAGTTAC TCCAGCTACCAT GCCAACTAT 290
ProProCys ProAsnLeu ValSerTyr SerSerTyrHis AlaAsnTyr
65 70 75 80
TCCCTATAT CTATTCCCT C;ATTGGACT AAGAAGCCAAAC CGAAATGGC 288
SerLeuTyr LeuPhePro HisTrpThr LysLysProAsn ArgAsnGly
85 90 95
GGAGGCTAT TATTCAGCC TCTTATTCA GACCCTTGTTCC TTAAAGTGC 336
GlyGlyTyr TyrSerAla ~;erTyrSer AspProCysSer LeuLysCys
100 105 210
CCATACCTG GGGTGCCAA TCATGGACC TGCCCCTATACA GGAGCCGTC 389
ProTyrLeu GlyCysGln w'~erTrpThr CysProTyrThr GlyAlaVal
115 120 225
TCCAGCCCC TACTGGAAG T'TTCAACAC GATGTCAATTTT ACTCAAGAA 932
SerSerPro TyrTrpLys F~heGlnHis AspValAsnPhe ThrGlnGlu
130 135 140
GTTTCACGA CTCAATATT AATCTCCAT TTTTCAAAATGC GGTTTTCCC 980
ValSerArg LeuAsnIle A.snLeuHis PheSerLysCys GlyPhePro
195 150 155 160
TTCTCCCTT CTAGTCGAC GCTCCAGGA TATGACCCCATC TGGTTCCTT 528
PheSerLeu LeuValAsp AlaProGly TyrAspProIle TrpPheLeu
165 170 175
AATACCGAA CCCAGCCAA CTGCCTCCC ACCGCCCCTCCT CTACTCCCC 576
AsnThrGlu ProSerGln LeuProPro ThrAlaProPro LeuLeuPro
180 185 190
CACTCTAAC CTAGACCAC ATCCTCGAC CCCTCTATACCA TGGAAATCA 624
HisSerAsn LeuAspHis IleLeuAsp ProSerIlePro TrpLysSer
195 200 205
AAACTCCTG ACCCTTGTC C.AGTTAACC CTACAAAGCACT AATTATACT 672
LysLeuLeu ThrLeuVal GlnLeuThr LeuGlnSerThr AsnTyrThr
210 215 220
TGCATTGTC TGTATCGAT C~~TGCCACC CTCTCCACTTGG CACGTCCTA 720
CysIleVal CysIleAsp ArgAlaThr LeuSerThrTrp HisValLeu
225 230 235 240
TACTCTCCC AACGTCTCT G'rTCCATCC TCTTCTTCTACC CCCCTCCTT 768
TyrSerPro AsnValSer Va ProSer SerSerSerThr ProLeuLeu
l
245 250 255
TACCCATCG TTAGCGCTT CCAGCCCCC CACCTGACGTTA CCATTTAAC 816
TyrProSer LeuAlaLeu P:roAlaPro HisLeuThrLeu ProPheAsn
260 265 270
TGGACCCAC TGCTTTGAC CCCCAGATT CAAGCTATAGTC TCCTCCCCC 864
TrpThrHis CysPheAsp ProGlnIle GlnAlaIleVal SerSerPro
275 280 285
TGTCATAAC TCCCTCATC CTGCCCCCC TTTTCCTTGTCA CCTGTTCCC 912
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CA 02262007 1999-07-19
CysHis AsnSerLeuIle :LeuProPro PheSerLeu SerProVal Pro
290 295 300
ACCCTA GGATGCCGCTCC CGCCGAGCG GTACCGGTG GCGGTCTGG CTT 960
ThrLeu GlyCysArgSer i~rgArgAla ValProVal AlaValTrp Leu
305 310 315 320
GTCTCC GCCCTGGCCATG GGAGCCGGA GTGGCTGGC GGGATTACC GGC 1008
ValSer AlaLeuAlaMet GlyAlaGly ValAlaGly GlyIleThr Gly
325 330 335
TCCATG TCCCTCGCCTCA GGAAAGAGC CTCCTACAT GAGGTGGAC AAA 1056
SerMet SerLeuAlaSer <31yLysSer LeuLeuHis GluValAsp Lys
340 345 350
GATATT TCCCAGTTAACT C:AAGCAATA GTCAAAAAC CACAAAAAT CTA 1104
AspIle SerGlnLeuThr GlnAlaIle ValLysAsn HisLysAsn Leu
355 360 365
CTCAAA ATTGCGCAGTAT GCTGCCCAG AACAGACGA GGCCTTGAT CTC 1152
LeuLys IleAlaGlnTyr AlaAlaGln AsnArgArg GlyLeuAsp Leu
370 ~t75 380
CTGTTC TGGGAGCAAGGA GGATTATGC AAAGCATTA CAAGAACAG TGC 1200
LeuPhe TrpGluGlnGly GlyLeuCys LysAlaLeu GlnGluGln Cys
385 390 395 404
CGTTTT CCGAATAATAAC F,ATTCCGAT GTCCCAATA CTACAAGAA AGA 1248
ArgPhe ProAsnAsnAsn F,snSerAsp ValProIle LeuGlnGlu Arg
405 410 415
CCCCCC CTTGAGAATCGA GTCCTGACT GGCTGGGGC CTTAACTGG GAC 1296
ProPro LeuGIuAsnArg V'alLeuThr GlyTrpGIy LeuAsnTrp Asp
420 425 430
CTTGGC CTCTCACAGTGG GCTCGAGAG GCCTTACAA ACTGGAATC ACC 1344
LeuGly LeuSerGlnTrp A.laArgGlu AlaLeuGIn ThrGlyIle Thr
435 440 445
CTTGTT GCGCTACTCCTT CTTGTTATC CTTGCAGGA CCATGCATC CTC 1392
LeuVal AlaLeuLeuLeu LeuValIle LeuAlaGly ProCysIle Leu
450 455 460
CGTCAG CTACGACACCTC CCCTCGCGC GTCAGATAC CCCCATTAC TCT 1440
ArgGln LeuArgHisLeu ProSerArg ValArgTyr ProHisTyr Ser
465 470 475 480
CTTATA AAACCTGAGTCA TCCCTGTAA 1467
LeuIle LysProGluSer SerLeu
985
(2)INFORMATION FORSEQID N0:2:
(i ) QUENCE CTERISTICS:
SE CHARA
(A)LENGTH:488 amino ids
ac
(B)TYPE: arid
amino
(C)STRANDEDNESS :
(D)TOPOLOGY: known
un
(i i) OLECULETYPE: otein
M pr
(x i) EQUENCEDESCR:EPT ION:SEQ ID N0:2:
S
MetGly LysPheLeuAla PheIle LeuPhePhe GlnPheCys Pro
Thr
1 5 10 15
-41/3-
CA 02262007 1999-07-19
Leu Ile Phe Gly Asp Tyr Ser Pro Ser Cys Cys Thr Leu Thr Ile Ala
20 25 30
Val Ser Ser Tyr His Ser :~ys Pro Cys Asn Pro Ala Gln Pro VaI Cys
35 40 45
Ser Trp Thr Leu Asp Leu Leu Ala Leu Ser Ala Asp Gln Ala Leu Gln
50 !i5 60
Pro Pro Cys Pro Asn Leu Val Ser Tyr Ser Ser Tyr His Ala Asn Tyr
65 70 75 80
Ser Leu Tyr Leu Phe Pro His Trp Thr Lys Lys Pro Asn Arg Asn Gly
85 90 95
Gly Gly Tyr Tyr Ser Ala Ser Tyr Ser Asp Pro Cys Ser Leu Lys Cys
100 105 110
Pro Tyr Leu Gly Cys Gln Ser Trp Thr Cys Pro Tyr Thr Gly Ala Val
115 120 225
Ser Ser Pro Tyr Trp Lys f?he Gln His Asp Val Asn Phe Thr Gln Glu
130 :.35 140
Val Ser Arg Leu Asn Ile Asn Leu His Phe Ser Lys Cys Gly Phe Pro
145 150 155 260
Phe Ser Leu Leu Val Asp Ala Pro Gly Tyr Asp Pro Ile Trp Phe Leu
165 170 175
Asn Thr Glu Pro Ser Gln Leu Pro Pro Thr AIa Pro Pro Leu Leu Pro
180 185 I90
His Ser Asn Leu Asp His l:Ie Leu Asp Pro Ser Ile Pro Trp Lys Ser
195 200 205
Lys Leu Leu Thr Leu Val Gln Leu Thr Leu Gln Ser Thr Asn Tyr Thr
210 215 220
Cys Ile Val Cys Ile Asp Arg Ala Thr Leu Ser Thr Trp His Val Leu
225 230 235 240
Tyr Ser Pro Asn Val Ser Val Pro Ser Ser Ser Ser Thr Pro Leu Leu
245 250 255
Tyr Pro Ser Leu Ala Leu Pro Ala Pro His Leu Thr Leu Pro Phe Asn
260 265 270
Trp Thr His Cys Phe Asp Pro Gln IIe Gln Ala Ile Val Ser Ser Pro
275 280 285
Cys His Asn Ser Leu Ile Leu Pro Pro Phe Ser Leu Ser Pro Val Pro
290 295 300
Thr Leu Gly Cys Arg Ser F,rg Arg Ala Val Pro VaI Ala Val Trp Leu
305 310 315 320
Val Ser Ala Leu Ala Met Gly Ala Gly Val Ala Gly Gly Ile Thr Gly
325 330 335
Ser Met Ser Leu Ala Ser G'~ly Lys Ser Leu Leu His Glu Val Asp Lys
340 345 350
Asp Ile Ser Gln Leu Thr Gln Ala Ile Val Lys Asn His Lys Asn Leu
355 360 365
Leu Lys Ile AIa Gln Tyr A,la Ala Gln Asn Arg Arg Gly Leu Asp Leu
370 375 380
Leu Phe Trp Glu Gln Gly Gly Leu Cys Lys Ala Leu Gln Glu GIn Cys
385 390 395 900
Arg Phe Pro Asn Asn Asn A.sn Ser Asp Val Pro Ile Leu Gln Glu Arg
405 410 425
Pro Pro Leu Glu Asn Arg Val Leu Thr Gly Trp Gly Leu Asn Trp Asp
420 425 430
Leu Gly Leu Ser Gln Trp Ala Arg Glu Ala Leu Gln Thr Gly Ile Thr
435 440 445
Leu Val Ala Leu Leu Leu Leu Val Ile Leu Ala GIy Pro Cys IIe Leu
450 455 460
Arg Gln Leu Arg His Leu Pro Ser Arg Val Arg Tyr Pro His Tyr Ser
465 470 475 480
Leu Ile Lys Pro Glu Ser Ser Leu
485
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1461 base pairs
(B) TYPE: nucleic acid
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 1....1458
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: N0:3:
SEQ
ID
ATGGGTAAT GTTTTCTTC C;TACTTTTA TTCAGTCTCACA CATTTTCCA 48
MetGlyAsn ValPhePhe LeuLeuLeu PheSerLeuThr HisPhePro
1 5 10 15
CTAGCCCAG CAGAGCCGA TGCACACTC ACGATTGGTATC TCCTCCTAC 96
LeuAlaGln GlnSerArg C;ysThrLeu ThrIleGlyIle SerSerTyr
20 25 30
CACTCCAGC CCCTGTAGC C;CAACCCAA CCCGTCTGCACG TGGAACCTC 144
HisSerSer ProCysSer F'roThrGln ProValCysThr TrpAsnLeu
35 40 45
GACCTTAAT TCCCTAACA F~CGGACCAA CGACTACACCCC CCCTGCCCT 192
AspLeuAsn SerLeuThr ThrAspGln ArgLeuHisPro ProCysPro
50 55 60
AACCTAATT ACTTACTCT GGCTTCCAT AAGACTTATTCC TTATACTTA 240
AsnLeuIle ThrTyrSer GlyPheHis LysThrTyrSer LenTyrLeu
65 70 75 80
TTCCCACAT TGGATAAAA AAGCCAAAC AGACAGGGCCTA GGGTACTAC 288
PheProHis TrpIleLys L,ysProAsn ArgGlnGlyLeu GlyTyrTyr
85 90 95
TCGCCTTCC TACAATGAC CCTTGCTCG CTACAATGCCCC TACTTGGGC 336
SerProSer TyrAsnAsp ProCysSer LeuGlnCysPro TyrLeuGly
100 105 110
TGCCAAGCA TGGACATCC CGATACACG GGCCCCCTCTCC AGTCCATCC 384
CysGlnAla TrpThrSer ArgTyrThr GlyProLeuSer SerProSer
115 120 125
TGGAAGTTT CATTCAGAT GTAAATTTC ACCCAGGAACTC AGCCAAGTG 432
TrpLysPhe HisSerAsp ValAsnPhe ThrGlnGluLeu SerGlnVal
130 135 240
TCCCTTCGA CTACACTTC TCTAAGTGC GGCTCCTCCATG ACCCTCCTA 480
SerLeuArg LeuHisPhe SerLysCys GlySerSerMet ThrLeuLeu
145 150 155 160
GTAGATGCC CCTGGATAT GATCCTTTA TGGTTCATCACC TCAGAACCC 528
ValAspAla ProGlyTyr AspProLeu TrpPheIleThr SerGluPro
165 170 175
ACTCAGCCT CCACCAACT TCTCCCCCA TTGGTCCATGAC TCCGACCTT 576
ThrGlnPro ProProThr SerProPro LeuValHisAsp SerAspLeu
180 185 190
GAACATGTC CTAAACCCC T~~CACGTCC TGGACGACCAAA ATACTCAAA 624
GluHisVal LeuAsnPro S~~rThrSer TrpThrThrLys IleLeuLys
195 200 205
TTTATCCAG CTGACCTTA C:~1GAGCACC AATTACTCCTGC ATGGTTTGC 672
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PheIleGln LeuThrLeu GlnSerThr AsnTyrSerCys MetValCys
210 :?15 220
GTGGATAGA TCCAGCCTC TCATCCTGG CATGTACTCTAC ACCCCCACC 720
ValAspArg SerSerLeu SerSerTrp HisValLeuTyr ThrProThr
225 230 235 240
ATCTCCATT CCCCAACAA ACCTCCTCC CGAACCATCCTC TTTCCTTCC 768
IleSerIle ProGlnGln 7.'hrSerSer ArgThrIleLeu PheProSer
295 250 255
CTTGCCCTG CCCGCTCCT C:CATCCCAA CCCTTCCCTTGG ACCCATTGC 816
LeuAlaLeu ProAlaPro F'roSerGln ProPheProTrp ThrHisCys
260 265 270
TACCAACCT CGCCTACAG GCGATAACA ACAGATAACTGC AACAACTCC 864
TyrGlnPro ArgLeuGln AlaIleThr ThrAspAsnCys AsnAsnSer
275 280 285
ATTATCCTC CCCCCTTTT TCCCTCGCT CCCGTACCTCCT CCGGCGACA 9I2
IleIleLeu ProProPhe ~;erLeuAla ProValProPro ProAIaThr
290 2:95 300
AGACGCCGC CGTGCCGTT C'CAATAGCA GTGTGGCTTGTC TCCGCCCTA 960
ArgArgArg ArgAlaVal F'roIleAla VaITrpLeuVal SerAlaLeu
305 310 315 320
GCGGCCGGA ACAGGTATC GCTGGTGGA GTAACAGGCTCC CTATCTCTG 1008
AlaAlaGly ThrGIyIle A.laGlyGly ValThrGlySer LeuSerLeu
325 330 335
GCTTCCAGT AAAAGCCTT CTCCTCGAG CTTGACAAAGAC ATCTCCCAC 1056
AlaSerSer LysSerLeu LeuLeuGlu LeuAspLysAsp IleSerHis
340 345 350
CTTACCCAG GCCATAGTC AAAAATCAT CAAAACATCCTC CGGGTTGCA 1109
LeuThrGln AlaIleVal LysAsnHis GInAsnIIeLeu ArgValAla
355 360 365
CAGTATCGA GCCCAAAAT AGACGAGGA TTAGACCTCCTA TTCTGGGAA 2152
GlnTyrArg AIaGlnAsn ArgArgGIy LeuAspLeuLeu PheTrpGlu
370 375 380
CAAGGGGGT TTGTGCAAG GCCATACAG GAGCAATGTTGC TTCCTCAAC 1200
GlnGlyGly LeuCysLys AlaIleGln GluGlnCysCys PheLeuAsn
385 390 395 900
ATCAGTAAC ACTCATGTA TCCGTCCTC CAGGAACGGCCC CCTCTTGAA 1248
IleSerAsn ThrHisVal SerValLeu GlnGluArgPro ProLeuGlu
405 410 415
AAACGTGTC ATCACCGGC TGGGGACTA AACTGGGATCTT GGACTGTCC 1296
LysArgVal IleThrGly TrpGlyLeu AsnTrpAspLeu GlyLeuSer
420 425 430
CAATGGGCA CGAGAAGCC C'FCCAGACA GCCATAACCATT CTCGCTCTA 1344
GlnTrpAla ArgGluAla LeuGlnThr AlaIleThrIle LeuAlaLeu
435 440 445
CTCCTCCTC GTCATATTG T'FTGGCCCC TGTATCCTCCGC CAAATCCAG 2392
LeuLeuLeu ValIleLeu P;zeGlyPro CysIleLeuArg GlnIleGln
450 4.55 460
GCCCTTCCA CAGCGGTAT CAAAACCGA CATAACCAGTAT TCCCTTATC 1440
AlaLeuPro GlnArgTyr G.LnAsnArg HisAsnGlnTyr SerLeuIle
465 470 475 480
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AAC CCA GAA ACC ATG CTA ~.'AA 14 61
Asn Pro Glu Thr Met Leu
485
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 486 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: unl~:nown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCF;IPTION: SEQ ID N0:4:
Met Gly Asn Val Phe Phe Leu Leu Leu Phe Ser Leu Thr His Phe Pro
1 5 10 15
Leu Al.a Gln Gln Ser Arg C',ys Thr Leu Thr Ile Gly Ile Ser Ser Tyr
20 25 30
His Ser Ser Pro Cys Ser Fro Thr GIn Pro Val Cys Thr Trp Asn Leu
35 90 45
Asp Leu Asn Ser Leu Thr Thr Asp Gln Arg Leu His Pro Pro Cys Pro
50 55 60
Asn Leu Ile Thr Tyr Ser Caly Phe His Lys Thr Tyr Ser Leu Tyr Leu
65 70 75 80
Phe Pro His Trp Ile Lys L~ys Pro Asn Arg Gln Gly Leu Gly Tyr Tyr
85 90 95
Ser Pro Ser Tyr Asn Asp Pro Cys Ser Leu Gln Cys Pro Tyr Leu Gly
100 105 110
Cys Gln Ala Trp Thr Ser A.rg Tyr Thr Gly Pro Leu Ser Ser Pro Ser
115 120 125
Trp Lys Phe His Ser Asp Val Asn Phe Thr Gln Glu Leu Ser Gln Val
130 135 140
Ser Leu Arg Leu His Phe Ser Lys Cys Gly Ser Ser Met Thr Leu Leu
145 150 155 160
Val Asp Ala Pro Gly Tyr Asp Pro Leu Trp Phe Ile Thr Ser Glu Pro
165 170 175
Thr Gln Pro Pro Pro Thr Ser Pro Pro Leu Val His Asp Ser Asp Leu
180 185 190
Glu His Val Leu Asn Pro Ser Thr Ser Trp Thr Thr Lys Ile Leu Lys
195 200 205
Phe Ile Gln Leu Thr Leu Gln Ser Thr Asn Tyr Ser Cys Met Val Cys
210 215 220
Val Asp Arg Ser Ser Leu Ser Ser Trp His Val Leu Tyr Thr Pro Thr
225 230 235 240
Ile Ser Ile Pro Gln Gln Thr Ser Ser Arg Thr Ile Leu Phe Pro Ser
245 250 255
Leu Ala Leu Pro Ala Pro Pro Ser Gln Pro Phe Pro Trp Thr His Cys
260 265 270
Tyr Gln Pro Arg Leu Gln Ala Ile Thr Thr Asp Asn Cys Asn Asn Ser
275 280 285
Ile Ile Leu Pro Pro Phe Ser Leu Ala Pro Val Pro Pro Pro Ala Thr
290 295 300
Arg Arg Arg Arg Ala Val Pro Ile Ala Val Trp Leu Val Ser Ala Leu
305 310 325 320
Ala Ala Gly Thr Gly Ile Ala Gly Gly VaI Thr Gly Ser Leu Ser Leu
325 330 335
Ala Ser Ser Lys Ser Leu Leu Leu Glu Leu Asp Lys Asp Ile Ser His
340 345 350
Leu Thr Gln Ala Ile Val Lys Asn His Gln Asn Ile Leu Arg Val Ala
355 360 365
Gln Tyr Arg Ala Gln Asn Arg Arg Gly Leu Asp Leu Leu Phe Trp Glu
370 375 380
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Gln Gly Gly Leu Cys Lys Ala Ile Gln Glu Gln Cys Cys Phe Leu Asn
385 390 395 400
Ile Ser Asn Thr His Val :>er Val Leu Gln Glu Arg Pro Pro Leu Glu
405 410 415
Lys Arg Val Ile Thr Gly Trp Gly Leu Asn Trp Asp Leu Gly Leu Ser
420 425 430
Gln Trp Ala Arg Glu Ala Leu Gln Thr Ala Ile Thr Ile Leu Ala Leu
435 440 445
Leu Leu Leu Val Ile Leu E'he Gly Pro Cys Ile Leu Arg Gln Ile Gln
450 955 460
Ala Leu Pro Gln Arg Tyr Gln Asn Arg His Asn Gln Tyr Ser Leu Ile
465 470 475 480
Asn Pro Glu Thr Met Leu
485
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic' acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
AAGGATCCAT GGGTAATGTT TTCTTC 26
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
AAGGATCCTT ATAGCATGGT TTCTGG 26
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
AAGAATTCAC GGCGGCGTCT TGTCGCGCCA GG 32
(2) INFORMATION :EOR SEQ ID N0:8:
(i) SEQUENCE CHARAC'PERISTICS:
(A) LENGTH: 20 baae pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
TGGATACCCC GTCTACGTGT 20
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic: acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCF;IPTION: SEQ ID N0:9:
GAGCTGACAA CGCGTCCATC G 21
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GTGTACAGGC CGATTGGT lg
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