Note: Descriptions are shown in the official language in which they were submitted.
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NUCLEIC ACID AND POLYPEPTIDE P10 OF A BORNA
DISEASE VIRUS (BDV) AND THEIR USE FOR DIAGNOSTIC
AND IMMUNIZATION PURPOSES
RELATED APPLICATIONS
S This application claims the benefit of United States provisional patent
application No.
60/097,901 filed August 26, 1998, which is incorporated herein by reference in
its entirety.
GOVERNMENT SUPPORT
This invention was made partially with government support awarded by the
National
Institute of Mental Health, the National Institutes of Health, the Public
Health Service (Grant
No. MH57740). The United States Government may have certain rights in the
invention.
TECHNICAL FILED
The present invention is directed to the 10 kilodalton polypeptide p 10 of a
Borna Disease
Virus (BDV) and its coding nucleic acid sequence. Both can be used in the
detection of, and the
vaccination against, a BDV and related infections and diseases.
IS BACKGROUND
The Borna Disease virus (BDV) is an enveloped, negative sense, nonsegmented,
single-
stranded RNA virus which causes Borna Disease (BD), a transmissible
polioencephalomyelitis,
in susceptible animals. The Borna Disease was originally described in horses
and sheep, but
cattle, rabbits, goats, deer, llamas, alpacas, cats and ostriches can also be
naturally infected.
Recent reports indicate that the BDV also can infect humans. The virus can be
isolated from the
naturally infected hosts. 'The isolates from different species exhibit high
degrees of homology,
but it is not clear whether they are the same virus originally described as
the causative agent of
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BD in horses or they are closely related viruses. However, viral proteins from
one isolate can
react with BDV-specific antibodies in the serum of another species, and vice
versa.
There is general agreement that the virus is transmitted through saliva and
nasal
secretions. Animals become infected by direct contact with secretions or by
exposure to
contaminated food or water. It is likely that the nose is the main site of
viral entry into the body.
Contact experiments in horses have shown that persistently infected animals,
not presenting overt
disease, such as virus carriers, may represent a source of infection. This
observation is of
eminent importance for the introduction of BDV and BDV-related infection into
stables, herds
or breeding colonies without a previous history of BD. There is a great need
to develop a
laboratory based diagnostic test for the detection of BDV and BDV-related
infection as well as
carriers. There also is a great need to develop a vaccine against these
infections.
The BDV is strictly neurotropic and is disseminated by infra-axonal transport
from the
site of infection, for example through the olfactory nerve, or other cerebral
nerve endings
terminating in the mucous membrane of the oropharyngeal region. The virus
localizes
preferentially in certain parts of the brain such as the grey matter, nucleus
niger, hippocampus
or olfactory bulb, and may spread centrifugally to the peripheral nerves
whereby the virus can
reach the ganglia of some organs. Involvement of certain regions of the brain
may give certain
focal symptoms, for example involvement of the nucleus niger may explain the
appearance of
motor disorder. The clinical expression of BDV and BDV-related infection is
variable and is
dependent on the virus strain and the species infected. Hence, diagnosis of
BDV infection based
on clinical signs is often difficult, unless the infected animal or pet
presents the classical
symptoms of BD. There is a need io develop a laboratory test to detect
infection by this virus to
aid in the diagnosis of BDV and BDV-related infection and associated diseases.
Traditionally, horses, sheep and cattle are economically important to
agriculture. They
are also susceptible to BDV and BDV-related infection. More recently,
agricultural husbandry
has diversified to include llamas and alpacas for their wool, deer for
venison, and ostriches for
their meat, feathers and skin. Some of these animals are not indigenous and
have to be imported.
For example, llamas, alpacas and ostriches imported from South America to the
United States,
and ostriches imported from Africa to Israel. These animals also are
susceptible to BDV and
BDV-related infection. Importation of virus carriers into domestic herds or
breeding colonies
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may decimate a young and potentially blooming agriculture business. There is a
great need in
developing a laboratory test to detect the infected animals at the port of
entry. More important,
recent evidence that BDV and BDV-related infection in cats may cause a
neurologic disease and
that BDV may infect humans are disturbing, because it raises the concern that
BDV-infected cats
may be a viral reservoir of human infections. It is necessary to develop a
laboratory test to aid
the diagnosis of BDV-associated neurological diseases and other BDV diseases
in cats and in
other mammals, including humans. It also is important to develop an
efficacious vaccine against
BDV and BDV-related infection in these animals.
Antibodies or immunoglobulins are complex proteins made by lymphocytes of a
host in
response to foreign substance, proteins or pathogens called antigens.
Antibodies can bind
antigens. All antibodies have the same overall shape, but each antibody has
unique regions that
make it fit to one antigen but not to another. As a result of this
specificity, an antibody specific
for BDV will not bind to wart virus or influenza virus. A specific antibody is
made only after the
lymphocyte has encountered the antigen. The specific antibody is released into
the blood stream,
lymph, colostrum, saliva, cerebral spinal fluid and into the lumens of the
gastrointestinal,
respiratory and urinary tracts. Hence, detection of specific antibodies to
BDV, or to any one of
the viral proteins, in any one of these body fluid suggests that the host has
been exposed to or
infected infected with a BDV.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts the identification of the recombinant GST-BDV p10 fusion
protein by
serum from a rabbit infected with BDV from horse. Western blot analyses of GST
protein (lane
1 ) and GST-BDV pl 0 fusion protein (lane 2) by use of a serum from a rabbit
infected with BDV.
FIG. 2 depicts the nucleotide sequence of OItFxI-FLAG DNA fragment (SEQ ID NO:
5) in the pOIZFxI-FLAG eukaryotic expression construct (GeneBank Accession
Number:
030353). 'The underlined nucleotides represents the FLAG moiety, not
underlined section is the
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sequence of OltFx 1. The not underlined section is provided as SEQ ID N0:7 and
is part of the
present invention.
F1G. 3 depicts the amino acid sequence of the ORFxI-FLAG (SEQ ID N0:6). The
amino acid sequence was derived by computer analysis of the OltFx 1-FLAG DNA
sequence
S shown in FIG. 2 by use of the PCgene software (Intellegenetic Suite, CA).
One letter symbols
for the codons are given. The FLAG amino acids are underlined. Amino acid
sequence of
ORFxl encoding a polypeptide pl 0 is not underlined. The not underlined
section is provided as
SEQ ID N0:8 and is part of the present invention.
FIG. 4 depicts the specificity ofthe anti-BDV polypeptide pl 0 rabbit
antiserum to a BDV
polypeptide p10. Total protein cell-free lysate from the rat glial cell C6
(ATCC accession
number CCL-107) (lanes l and 2) and from the BDV-infected C6BV cells (lanes 3
and 4) were
tested by western blot against sera collected from a rabbit before
immunization (lanes 1 and 3)
and after immunization (lanes 2 and 4) with the affinity column-purified GST-
BDV polypeptide
p10 fusion protein. A protein band with an apparent molecular weight of
approximately 10
kilodalton was identified by the immune serum in the protein sample from the
infected cells.
FIG. SA, FIG. SB, F1G. SC and FIG. SD depict the specificity of the anti-BDV
polypeptide p10 rabbit antiserum to BDV. The Madin-Darby canine kidney cells
MDCK
(ATCC accession number CCL-34) infected (panels A and C) and not infected
(panels B and D)
with BDV were stained in immunofluorescence assays (IFA) by the anti-BDV
polypeptide p10
rabbit serum (panels A and B) prepared as described in Example 3, and
previously tested as
shown in FIG. 4. Only the infected cells were stained (panel A). The staining
was observed in
the nucleus and in the cytoplasm of the infected cells. The preimmune serum
did not stain the
infected (panel C) or the non-infected (panel D) cells.
FIG. 6A, FIG. 6B, FIG. 6C and FIG. 6D depict the specificity of the anti-BDV
polypeptide p10 rabbit antiserum to cells expressing BDV polypeptide p10. The
primate cells
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COS-7 (ATCC accession number CRL-1651) were transfected with the eukaryotic
expression
vector pORFx 1-FLAG created as described in Example 2. The transfected cells
expressing BDV
polypeptide p10 (F1G. 6A and FIG. 6C) and the non-transfected cells (FIG. 6B
and FIG. 6D)
were stained in immunofluorescence assays (IFA) by the anti-BDV polypeptide p
10 rabbit serum
5 (panels A and B) prepared as described in Example 3, and previously tested
as shown in FIG.
4. Only the transfected cells expressing BDV polypeptide p10 were stained
(FIG. 6A). The
preimmune serum did not stain the transfected (FI G. 6C) or the non-
transfected (FIG. 6D) cells.
FIG. 7 depicts the detection of anti-BDV polypeptide p10 antibodies in serum
from a
BDV-infected rabbit. Affinity column-purified GST protein (lanes 1 and 2) and
GST-BDV
polypeptide p10 fusion protein (lanes 3 and 4) were used as antigen substrates
in western blot to
test for antigen-specific antibodies in serum from a rabbit experimentally
infected (lanes 2 and
4) and not infected (lanes 1 and 3) with BDV.
FIG. 8 depicts the detection ofanti-BDV antibodies in serum from a BDV-
infected horse.
Affinity column-purified GST protein (lanes 3 and 6), GST-BDV polypeptide p10
fusion protein
(lanes 1 and 4) and GST-BDV p24 fusion protein (lanes 2 and 5) were used as
antigen substrates
in western blot to test for antigen-specific antibodies in sera from horses
naturally infected (lanes
1, 2 and 3) and not infected (lanes 4, 5 and 6) with BDV. The GST-BDV
polypeptide p10 fusion
protein detected anti-BDV polypeptide p10 specific antibodies in serum of the
infected horse.
Construction of the plasmid expressing the GST-BDV p24 BDV protein has
previously been
described, and detection of antibodies specific to this protein suggested BDV
infection of the
horse (Kishi et al. FEBS Lett. 364:293-297 (1995).
FIG. 9 depicts the subcellular localization of polypeptide p10 in BDV-infected
cells.
Antiserum specific for polypeptide p10 was used to stain C6BV (FIG. 9A) and C6
(FIG. 9B)
cells via IFA. C6BV (FIG. 9C) and C6 (F1G. 9D) cells were also stained with
prebleed serum
as a control. The FITC-conjugated protein A was used as a second antibody. The
stained cells
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were examined using an epifluroescence microscope at 160X magnification. The
staining was
observed in the nucleus and in the cytoplasm of the infected cells.
SUMMARY
The BDV has not been fully characterized, but overlapping nucleic acid
fragments of
its genome have been cloned from cells infected by cell-adapted BDV strains.
These cell-
adapted BDV nucleic acid fragments of its genome had been sequenced to give
the complete
nucleic acid sequence of the genome (Briese, Proc. Natl. Acad. Sci. USA
91:4362 (1994);
Cubitt, J. Virol. 68:1382 (1994)). It is possible to translate the BDV genomic
nucleotide
sequence into amino acids. From the amino acid sequence, one may predict the
number of
open reading frames (OR.Fs) encoding hypothetical proteins. In the case with
BDV, the
prediction was at least S to 6 ORFs, and one of these ORFs, ORFxl, would give
a protein of
approximately 10 kilodalton (p10). Cloning of BDV mRNAs as cDNAs and
expression
studies identified a 18 kilodalton, a 24 kilodalton and a 38/40 kilodalton
protein as BDV-
specific. However, cloning of BDV cDNAs, including one containing the ORFxI,
did not
provide BDV polypeptide p10 (United State Patent No. 5,654,401 to Clements et
al., issued
August 5, 1997, and United States Patent No. 5,854,417 to Clements et al.,
issued December
29, 1998 ). Hence, from the prior art it was uncertain whether the BDV
polypeptide p10
protein actually exists or whether it is an hypothetical protein not produced
naturally. In the
course of this invention, it was found that the BDV polypeptide p10 is indeed
naturally
produced.
One aspect of the present invention is a polypeptide having at least one
bioactivity of a
polypeptide p10 of a Borna Disease Virus.
A second aspect of the present invention is a specific binding member, such as
an
antibody or polypeptide p40 or polypeptide p24, that binds with at least a
portion of a
polypeptide p10 of a Borna Disease Virus.
A third aspect of the present invention is a nucleic acid molecule that
encodes a
polypeptide having at least one bioactivity of a polypeptide of a Borna
Disease Virus.
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A fourth aspect of the present invention is a test kit that includes at least
one of a
polypeptide of the present invention, a specific binding member of the present
invention or a
nucleic acid molecule of the present invention.
A fifth aspect of the present invention is a vaccine and method of
immunization that
includes at least one of a polypeptide of the present invention, a specific
binding member of
the present invention or a nucleic acid molecule of the present invention.
A sixth aspect of the present invention is a method of diagnosis that includes
at least
one of a polypeptide of the present invention, a specific binding member of
the present
invention or a nucleic acid molecule of the present invention.
A seventh aspect of the present invention is a method of identifying test
compounds or
bioactivities, preferably test compounds or bioactivities that are useful in
the present
invention.
DETAILED DESCRIPTION
DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Generally, the nomenclature used herein and the laboratory procedures
in cell culture,
chemistry, microbiology, molecular biology, cell science and cell culture
described below are
well known and commonly employed in the art. Conventional methods are used for
these
procedures, such as those provided in the art and various general references
(Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press,
Cold Spring
Harbor, N.Y. (1989)). Where a term is provided in the singular, the inventors
also contemplate
the plural of that term. The nomenclature used herein and the laboratory
procedures described
below are those well known and commonly employed in the art. As employed
throughout the
disclosure, the following terms, unless otherwise indicated, shall be
understood to have the
following meanings:
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"Isolated polynucleotide" refers to a polynucleotide of genomic, cDNA, or
synthetic
origin, or some combination thereof, which by virtue of its origin, the
isolated polynucleotide (1)
is not associated with the cell in which the isolated polynucleotide is found
in nature, or (2) is
operably linked to a polynucleotide that it is not linked to in nature. The
isolated polynucleotide
can optionally be linked to promoters, enhancers, or other regulatory
sequences.
"Isolated protein" or "isolated polypeptide" refers to a protein or
polypeptide of DNA,
cDNA, RNA, recombinant RNA, recombinant DNA or synthetic origin, or some
combination
thereof, which by virtue of its origin the isolated protein or isolated
polypeptide (I) is not
associated with proteins normally found within nature, or (2) is isolated from
the cell in which
it normally occurs, or (3) is isolated free of other proteins from the same
cellular source, for
example, free of cellular proteins), or (4) is expressed by a cell from a
different species, or (5)
does not occur in nature.
"Polypeptide" is used herein as a generic term to refer to a molecule
comprising at least
one peptide bond, such as, for example, a protein or a fragment, analogue or
active fragment
thereof.
"Active fragment" refers to a fragment of a parent molecule, such as an
organic molecule,
nucleic acid molecule, or polypeptide, or combinations thereof, that retains
at least one activity
of the parent molecule.
"Naturally occurring" refers to the fact that an object can be found in
nature. For
example, a polypeptide or polynucleotide sequence that is present in an
organism, including
viruses, that can be isolated from a source in nature and which has not been
intentionally
modified by man in the laboratory is naturally occurring.
"Molecular weight" refers to an apparent size estimation under the
circumstances and
methods used described in the individual examples. The true molecular mass can
only be
determined after sequencing the full length protein.
"Operably linked" refers to a juxtaposition wherein the components so
described are in
a relationship permitting them to function in their intended manner. A control
sequence operably
linked to a coding sequence is ligated in such a way that expression of the
coding sequence is
achieved under conditions compatible with the control sequences.
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"Control sequences" refer to polynucleotide sequences that effect the
expression of
coding and non-coding sequences to which they are ligated. The nature of such
control
sequences differs depending upon the host organism; in prokaryotes, such
control sequences
generally include promoter, ribosomal biding site, and transcription
termination sequences; in
eukaryotes, generally, such control sequences include promoters and
transcription termination
sequences. The tenor control sequences is intended to include components whose
presence can
influence expression, and can also include additional components whose
presence is
advantageous, for example, leader sequences and fusion partner sequences.
"Polynucleotide" refers to a polymeric form of nucleotides of a least ten
bases in length,
either ribonucleotides or deoxynucleotides or a modified from of either type
of nucleotide. The
term includes single and double stranded forms of DNA or RNA.
"Directly" in the context of a biological process or processes, refers to
direct causation
of a process that does not require intermediate steps, usually caused by one
molecule contacting
or binding to another molecule (the same type or different type of molecule).
For example,
molecule A contacts molecule B, which causes molecule B to exert effect X that
is part of a
biological process.
"Indirectly" in the context of a biological process or precesses, refers to
indirect causation
that requires intermediate steps, usually caused by two or more direct steps.
For example,
molecule A contacts molecule B to exert effect X which in turn causes effect
Y.
"Sequence homology" refers to the proportion of base matches between two
nucleic acid
sequences or the proportion of amino acid matches between two amino acid
sequences. When
sequence homology is expressed as a percentage, for example 50%, the
percentage denotes the
proportion of matches of the length of sequences from a desired sequence that
is compared to
some other sequence. Gaps (in either of the two sequences) are permitted to
maximize matching;
gap lengths of 15 bases or less are usually used, 6 bases or less are
preferred with 2 bases or less
more preferred. When using oligonucleotides as probes or treatments, the
sequence homology
between the target nucleic acid and the oligonucleotide sequence is generally
not less than 17
target base matches out of 20 possible oligonucleotide base pair matches
(85%); preferably not
less than 9 matches out of 10 possible base pair matches (90%), and most
preferably not less than
19 matches out of 20 possible base pair matches (95%).
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"Selectively hybridize" refers to detectably and specifically bind.
PolynucIeotides,
oligonucleotides and fragments thereof selectively hybridize to target nucleic
acid strands, under
hybridization and wash conditions that minimize appreciable amounts of
detectable binding to
nonspecific nucleic acids. High stringency conditions can be used to achieve
selective
S hybridization conditions as known in the art. Generally, the nucleic acid
sequence homology
between the polynucleotides, oligonucleotides, and fragments thereof and a
nucleic acid sequence
of interest will be at least 30%, and more typically and preferably of at
least 40%, 50%, 60%,
70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
Hybridization and washing conditions are typically performed at high
stringency
10 according to conventional hybridization procedures. Positive clones are
isolated and sequenced.
For example, a full length polynucleotide sequence can be labeled and used as
a hybridization
probe to isolate genomic clones from an appropriate target library as they are
known in the art.
Typical hybridization conditions and methods for screening plaque lifts and
other purposes are
known in the art (Benton and Davis, Science 196:180 (1978); Sambrook et al.,
supra, (1989)).
Two amino acid sequences are homologous if there is a partial or complete
identity
between their sequences. For example, 85% homology means that 85% of the amino
acids are
identical when the two sequences are aligned far maximum matching. Gaps (in
either of the two
sequences being matched) are allowed in maximizing matching; gap lengths of 5
or less are
preferred with 2 or less being more preferred. Alternatively and preferably,
two protein
sequences (or polypeptide sequences derived from them of at least 30 amino
acids in length) are
homologous, as this term is used herein, if they have an alignment score of at
least 5 (in standard
deviation units) using the program ALIGN with the mutation data matrix and a
gap penalty of
6 or greater (Dayhoff, in Atlas of Protein Sequence and Structure, National
Biomedical Research
Foundation, volume 5, pp. 101-110 {1972) and Supplement 2, pp. 1-10). The two
sequences or
parts thereof are more preferably homologous if their amino acids are greater
than or equal to
30% identical when optimally aligned using the ALIGN program.
"Corresponds to" refers to a polynucleotide sequence is homologous (for
example is
identical, not strictly evolutionarily related) to all or a portion of a
reference polynucleotide
sequence, or that a polypeptide sequence is identical to all or a portion of a
reference polypeptide
sequence. In contradistinction, the term "complementary to" is used herein to
mean that the
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complementary sequence will base pair with all or a portion of a reference
polynucleotide
sequence. For illustration, the nucleotide sequence TATAC corresponds to a
reference sequence
TATAC and is complementary to a reference sequence GTATA.
The following terms are used to describe the sequence relationships between
iwo or more
polynucleotides: "reference sequence," "comparison window;" "sequence
identity," "percentage
of sequence identity," and "substantial identity." A reference sequence is a
defined sequence
used as a basis for a sequence comparison; a reference sequence can be a
subset of a larger
sequence, for example, as a segment of a full length cDNA or gene sequence
given in a sequence
listing, or may comprise a complete cDNA or gene sequence. Generally, a
reference sequence
is at least 20 nucleotides in length, frequently at least 25 nucleotides in
length, and often at least
50 nucleotides in length. Since two polynucleotides can each (1) comprise a
sequence (for
example a portion of the complete polynucleotide sequence) that is similar
between the two
polynucleotides, and (2) may further comprise a sequence that is divergent
between the two
polynucleotides, sequence comparisons between two (or more) polynucleotides
are typically
1 S performed by comparing sequences of the two polynucleotides over a
"comparison window" to
identify and compare local regions of sequence similarity. A comparison widow,
as used herein,
refers to a conceptual segment of at least 20 contiguous nucleotide positions
wherein a
polynucleotide sequence may be compared to a reference sequence of at least 20
contiguous
nucleotides and wherein the portion of the polynucleotide sequence in the
comparison window
can comprise additions and deletions (for example, gaps) of 20 percent or less
as compared to
the reference sequence (which would not comprise additions or deletions) for
optimal alignment
of the two sequences. Optimal alignment of sequences for aligning a comparison
window can
be conducted by the local homology algorithm (Smith and Waterman, Adv. Appl.
Math., 2:482
(1981)), by the homology alignment algorithm (Needleman and Wunsch, J. Moi.
Bio., 48:443
(1970)), by the search for similarity method (Pearson and Lipman, Proc. Natl.
Acid. Sci. U.S.A.
85:2444 (1988)), by the computerized implementations of these algorithms such
as GAP,
BESTFIT, FASTA and TFASTA (Wisconsin Genetics Software Page Release 7.0,
Genetics
Computer Group, Madison, WI), or by inspection. Preferably, the best alignment
(for example,
the result having the highest percentage of homology over the comparison
window) generated
by the various methods is selected.
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"Sequence identity" means that two polynucleotide sequences are identical (for
example,
on a nucleotide-by-nucleotide basis) over the window of comparison.
"Percentage of sequence identity" is calculated by comparing two optimally
aligned
sequences over the window of comparison, determining the number of positions
at which the
identical nucleic acid base occurs in both sequences to yield the number of
matched positions,
dividing the number of matched positions by the total number of positions in
the window of
comparison (for example, the window size), and multiplying the result by 100
to yield the
percentage of sequence identity.
"Substantial identity" as used herein denotes a characteristic of a
polynucleotide
sequence, wherein the polynucleotide comprises a sequence that has at least
about 30 percent to
about 70 percent sequence identity; preferably at least about 60 % to about 90
% sequence
identity; more usually at Ieast about 91 %, at least about 92 %, at least 93
%, at least about 94 %,
at least about 95 %, at least about 96 %, at least about 97 %, at least about
98 % or at least about
99 % sequence identity as compared to a reference sequence over a comparison
window of at
least 20 nucleotide positions, frequently over a window of at least 25 to 50
nucleotides, wherein
the percentage of sequence identity is calculated by comparing the reference
sequence to the
polynucleotide sequence that rnay include deletions or addition which total 20
percent or less of
the reference sequence over the window of comparison.
"Substantial identity" as applied to polypeptides herein means that two
peptide sequences,
when optimally aligned, such as by the programs GAP or BESTFIT using default
gap weights,
share at least about 30 percent to about 70 percent sequence identity;
preferably at least about 60
to about 90 % sequence identity; more usually at least about 91 %, at least
about 92 %, at least
93 %, at least about 94 %, at least about 95 %, at least about 96 %, at least
about 97 %, at least
about 98 % or at least about 99 % sequence identity. Preferably, residue
positions, which are not
identical, differ by conservative amino acid substitutions.
"Conservative amino acid substitutions" refer to the interchangeability of
residues having
similar side chains. For example, a group of amino acids having aliphatic side
chains is glycine,
alanine, valine, leucine, and isoleucine; a group of amino acids having
aliphatic-hydroxyl side
chains is serine and threonine; a group of amino acids having amide-containing
side chains is
asparagine and glutamine; a group of amino acids having aromatic side chains
is phenylalanine,
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tyrosine and tryptophan; a group of amino acids having basic side chains is
lysine, arginine and
histidine; a group of amino acids having acidic side chains is aspartic acid
and glutamic acid; and
a group of amino acids having sulfur-containing side char is cystein and
methionine. Preferred
conservative amino acid substitution groups are: valine-leucine-isoleucine;
phenylalanine-
tyrosine; lysine-arginine; alanine-valine; glutamate-aspartate; and asparagine-
glutamine.
"Modulation" refers to the capacity to either enhance or inhibit a functional
property of
a biological activity or process, for example, enzyme activity or receptor
binding. Such
enhancement or inhibition may be contingent on the occurrence of a specific
event, such as
activation of a signal transduction pathway and/or may be manifest only in
particular cell types.
"Modulator" refers to a chemical (naturally occurring or non-naturally
occurring), such
as a biological macromolecule (for example, nucleic acid, protein, non-peptide
or organic
molecule) or an extract made from biological materials, such as prokaryotes,
bacteria, eukaryotes,
plants, fungi, multicellular organisms or animals, invertebrates, vertebrates,
mammals and
humans, including, where appropriate, extracts of: whole organisms or portions
of organisms,
cells, organs, tissues, fluids, whole cultures or portions of cultures, or
environmental samples or
portions thereof. Modulators are typically evaluated for potential activity as
inhibitors or
activators (directly or indirectly) of a biological process or processes (for
example, agonist,
partial antagonist, partial agonist, antagonist, antinevplastic, cytotoxic,
inhibitors of neoplastic
transformation or cell proliferation, cell proliferation promoting agents,
antiviral agents,
antimicrobial agents, antibacterial agents, antibiotics, and the like) by
inclusion in assays
described herein. The activity of a modulator may be known, unknown or
partially known.
"Test chemical" or "test compound" refers to a chemical or extract to be
tested by at least
one method of the present invention to be a putative modulator. A test
chemical is usually not
known to bind to the target of interest. "Control test chemical" or "control
test compound" refers
to a chemical known to bind to the target (for example, a known agonist,
antagonist, partial
agonist or inverse agonist). Test chemical does not typically include a
chemical added to a
mixture as a control condition that alters the function of the target to
determine signal specificity
in an assay. Such control chemicals or conditions include chemicals that (1)
non-specifically or
substantially disrupt protein structure (for example denaturing agents such as
urea or guandium,
sulfliydryl reagents such as dithiotritol and beta-mercaptoethanol), (2)
generally inhibit cell
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metabolism (for example mitochondria) uncouples) and (3) non-specifically
disrupt electrostatic
or hydrophobic interactions of a protein (for example, high salt
concentrations or detergents at
concentrations sufficient to non-specifically disrupt hydrophobic or
electrostatic interactions).
The term test chemical also does not typically include chemicals known to be
unsuitable for a
therapeutic use for a particular indication due to toxicity of the subject.
Usually, various
predetermined concentrations of test chemicals are used for determining their
activity. If the
molecular weight of a test chemical is known, the following ranges of
concentrations can be
used: between about 0.001 micromolar and about 10 millimolar, preferably
between about 0.01
micromolar and about 1 millimolar, more preferably between about 0.1
micromolar and about
100 micromolar. When extracts are uses a test chemicals, the concentration of
test chemical used
can be expressed on a weight to volume basis. Under these circumstances, the
following ranges
of concentrations can be used: between about 0.001 micrograrns/ml and about 1
milligram/ml,
preferably between about 0.01 micrograms/ml and about 100 micrograms/ml, and
more
preferably between about 0.1 micrograms/ml and about 10 micrograms/ml. A test
chemical or
test compound can have at least one bioactivity.
"Target" refers to a biochemical entity involved in a biological process.
Targets are
typically proteins that play a useful role in the physiology or biology of an
organism. A
therapeutic chemical typically binds to a target to alter or modulate its
function. As used herein,
targets can include, but not be limited to, cell surface receptors, G-
proteins, G-protein coupled
receptors, kinases, phosphatases, ion channels, lipases, phosholipases,
nuclear receptors,
intracellular structures, tubules, tubulin, and the like.
"Label" or "labeled" refers to incorporation of a detectable marker, for
example by
incorporation of a radiolabled compound or attachment to a polypeptide of
moieties such as
biotin that can be detected by the binding of a section moiety, such as marked
avidin. Various
methods of labeling polypeptide, nucleic acids, carbohydrates, and other
biological or organic
molecules are known in the art. Such labels can have a variety of readouts,
such as radioactivity,
fluorescence, color, chemiluminescence or other readouts known in the art or
later developed.
The readouts can be based on enzymatic activity, such as beta-galactosidase,
beta-lactamase,
horseradish peroxidase, alkaline phosphatase, luciferase; radioisotopes such
as 3H, '4C, 3sS, 'ZSI
or'3'I); fluorescent proteins, such as green fluorescent proteins; or other
fluorescent labels, such
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as FITC, rhodamine, and lanthanides. Where appropriate, these labels can be
the product of the
expression of reporter genes, as that term is understood in the art. Examples
of reporter genes
are beta-lactamase (U.S. Patent No. 5,741,657 to Tsien et al., issued April
21, 1998) and green
fluorescent protein (U.S. Patent No. 5,777,079 to Tsien et al., issued July 7,
1998; U.S. Patent
5 No. 5,804,387 to Cormack et al., issued September 8, 1998).
"Substantially pure" refers to an object species or activity that is the
predominant species
or activity present (for example on a molar basis it is more abundant than any
other individual
species or activities in the composition) and preferably a substantially
purified fraction is a
composition wherein the object species or activity comprises at least about 50
percent (on a
10 molar, weight or activity basis) of all macromolecules or activities
present. Generally , as
substantially pure composition will comprise more than about 80 percent of all
macromolecular
species or activities present in a composition, more preferably more than
about 85%, 90%, 95%
and 99%. Most preferably, the object species or activity is purified to
essential homogeneity,
wherein contaminant species or activities cannot be detected by conventional
detection methods)
15 wherein the composition consists essentially of a single macromolecular
species or activity. The
inventors recognize that an activity may be caused, directly or indirectly, by
a single species or
a plurality of species within a composition, particularly with extracts.
"Pharmaceutical agent or drug" refers to a chemical, composition or activity
capable of
inducing a desired therapeutic effect when properly administered by an
appropriate dose, regime,
route of administration, time and delivery modality.
A "bioactive compound" is a compound or composition that exhibits at least one
of
following bioactivities: antiviral activity, binding with p40 nucleoprotein N
of a BDV, binding
with the 24 kd viral phosphoprotein of a BDV, nuclear localization in a
eukaryotic cell, at least
one epitope, immunogenic activity, T-cell activating activity and antigenic
activity. Preferably,
a bioactive compound is specific for a BDV, preferably a polypeptide p 10 of a
BDV, such as, for
example, for use as a therapeutic, diagnostic, vaccine or other aspect of the
invention for a BDV,
but that need not be the case. A bioactive compound can be of any chemical
composition such
as, for example, a chemical, a small molecule such as a drug, a polypeptide, a
nucleic acid, a
lipid, a carbohydrate, a nucleic acid molecule such as DNA or RNA or both, or
other compound
or composition.
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A "bioactivity" is a composition or compound that exhibits at least one
activity of a
bioactive compound.
A "bioactive derivative" is a modification of a bioactive compound or
bioactivity that
retains at least one characteristic bioactivity of the parent compound.
A "bioactive precursor" is a precursor of a bioactive compound or bioactivity
that exhibits
at least one characteristic activity of the resulting bioactive compound or
bioactivity.
An "antiviral activity" is an activity that reduces the infectivity of at
least one virus
particle in a sample, such as in a sample including at least one virus,
including a subject. An
antiviral activity can also prevent or decrease the severity of a viral
disease state, such as
infection with a BDV and/or the resulting disease state. An antiviral activity
can act in any
appropriate manner, such as interfering with the attachment, penetration or
replication of a virus
in any manner; altering the virus particle to render the virus particle less
infective or non-
infective; or by mounting an immune response, cellular or humoral or both,
against a virus or a
viral infection, including a virus infected cell.
A "patient"or "subj ect" is a whole organism in need of treatment, such as a
farm animal,
companion animal or human. An animal is any animal, but does not include
humans.
A "specific binding member" refers to molecules that have specific binding
activity
towards a polypeptide of the present invention. Such specific binding members
can take part in
receptor-ligand type reactions and are generally characterized as binding with
their binding mate
by non-covalent reactions, such as hydrogen bonds, van der Walls interactions,
hydrophobic
interactions, and the like. A specific binding member can be at least a
portion of a molecule,
such as a protein, such as p40 nucleoprotein N of BDV or the 24 kd viral
phosphoprotein P of
BDV, that binds with a polypeptide of the present invention. A specific
binding member can also
be an immunoglobulin of any class, a polyclonal antibody, a monoclonal
antibody, or an active
fragment thereof.
"Binds with" in the context of specific binding members, refers to the binding
of one
specific binding member with its target, such as an antibody binding with a
polypeptide of the
present invention, and does not infer that the specific binding member will
not bind with moieties
other than a polypeptide of the present invention.
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"Specifically binds with" refers to a specific binding member that delectably
binds with
a polypeptide of the present invention preferentially or with greater affinity
than a moiety other
than a polypeptide of the present invention. A specific binding member that
specifically binds
with one polypeptide of the present invention can also specifically bind with
a second
polypeptide of the present invention.
"Immobilized" in the context of a test kit refers to a moiety attached to a
surface such that
the moiety remains substantially immobilized in an aqueous phase as opposed to
being
substantially mobile in an aqueous phase, such as, for example, in an
immunochromatographic
device and/or method.
"Vaccine" refers to a composition or compound, that when administered to a
subject in
an appropriate dose by an appropriate route of administration and an
appropriate regime, can
prevent the likelihood of the occurrence of the infection or the severity of
an infection with a
BDV in a non-infected subject through a physiological response, such as an
immune response.
A vaccine also refers to a composition or compound that, when administered to
a subject that has
been exposed to a BDV in an appropriate dose by an appropriate route of
administration and an
appropriate regime, can reduce the likelihood of infection or reduce the
severity of infection. A
vaccine also refers to a compound or composition that, when administered to a
subject that has
become infected with a BDV in an appropriate dose by an appropriate route of
administration and
an appropriate regime, can reduce the severity of infection.
"Borna Disease Virus" or "BDV" refers to a virus that is the etiological agent
for "Borna
Disease" or "BD" in horses. BDV from horses can infect other animals, such as
rats, to cause
a "BDV-infection." Viruses that have substantial nucleic acid homology with
BDV ("BDV-
associated viruses") have been isolated from a variety of species and are
considered BDVs.
BDV-associated viruses cause "BDV-related infection" which can progress to a
"BDV-associated
disease" in an appropriate host animal or host cell. "A BDV" includes BDV and
BDV-associated
viruses. Thus, a BDV can cause a BDV-infection that can progress to BD or a
BDV-associated
disease. A BDV, a BDV-infection, BD or a BDV-associated disease can be
detected using the
methods of the present invention. Symptoms of the disease can also be
monitored to follow the
course of BD or a BDV-associated disease.
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Other technical terms used herein have their ordinary meaning in the art that
they are
used, as exemplified by a variety of technical dictionaries, such as the
McGraw-Hill Dictionary
of Chemical Terms and the Stedman's Medical Dictionary.
Introduction
The present invention recognizes that a polypeptide of a Borna Disease Virus
is expressed
as part of the natural course of infectivity of that virus. The present
invention relates to
polypeptides, specific binding members, nucleic acid molecules, test kits,
vaccines, methods of
vaccination, vaccinated patients and methods ofdiagnosis as they relate to a
Borna Disease Virus
in general, and polypeptide p10 of a Borna Disease Virus in particular.
As a non-limiting introduction to the breath of the present invention, the
present invention
includes several general and useful aspects, including:
1) a polypeptide having at least one bioactivity of a polypeptide pI0 of a
Borna
Disease Virus;
2) a specific binding member, such as an antibody, that binds with at least a
portion
I S of a polypeptide of a Borna Disease Virus;
3) a nucleic acid molecule that encodes a polypeptide having at least one
bioactivity
of a polypeptide p10 of a Borna Disease Virus;
4) a test kit that includes at least one of a polypeptide of 1), a specific
binding
member of 2) or a nucleic acid molecule of 3);
5) a vaccine and method of immunization that includes at least one of a
polypeptide
of 1 ), a specific binding member of 2) or a nucleic acid molecule of 3);
6) a method of diagnosis including at least one of a polypeptide of 1), a
specific
binding member of 2) or a nucleic acid molecule of 3); and
7) a method of identifying a test compound or bioactivity, preferably test
compounds
or bioactivities that are useful in the present invention.
These aspects of the invention, as well as others described herein, can be
achieved by
using the methods, articles of manufacture and compositions of matter
described herein. To gain
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a full appreciation of the scope of the present invention, it will be further
recognized that various
aspects of the present invention can be combined to make desirable embodiments
of the
invention.
S I. A POLYPEPTIDE HAVING AT LEAST ONE BIOACTIVITY OF A
