CA2341360A1 - Nucleic acid and polypeptide p10 of a borna disease virus (bdv) and their use for diagnostic and immunization purposes - Google Patents
Nucleic acid and polypeptide p10 of a borna disease virus (bdv) and their use for diagnostic and immunization purposes Download PDFInfo
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- CA2341360A1 CA2341360A1 CA002341360A CA2341360A CA2341360A1 CA 2341360 A1 CA2341360 A1 CA 2341360A1 CA 002341360 A CA002341360 A CA 002341360A CA 2341360 A CA2341360 A CA 2341360A CA 2341360 A1 CA2341360 A1 CA 2341360A1
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- Prior art keywords
- polypeptide
- nucleic acid
- test kit
- acid molecule
- specific binding
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- Peptides Or Proteins (AREA)
Abstract
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 o f the present invention is a specific binding member, such as an antibody, tha t 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 encode s a polypeptide having at least one bioactivity of a polypeptide p10 of a Born a Disease Virus. 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 presen t invention is a method of diagnosis that includes at least one of: a polypeptide of the present invention, a specific binding member of the prese nt invention or a nucleic acid molecule of the present invention. A seventh aspect of the present invention is a method of identifying a test compound o r bioactivity, preferably bioactivities that are useful in the present inventi on.
Description
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 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 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 WO 00/12548 PC'T/US99/19227 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 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 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.
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:
"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.
"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%).
WO 00/1254$ PCT/US99/19227 "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 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.
"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, 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 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 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.
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.
"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.
WO 00/12548 PC'T/US99/19227 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 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
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 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 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 WO 00/12548 PC'T/US99/19227 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 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 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.
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:
"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.
"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%).
WO 00/1254$ PCT/US99/19227 "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 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.
"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, 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 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 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.
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.
"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.
WO 00/12548 PC'T/US99/19227 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 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
Claims (78)
1. A polypeptide comprising at least one bioactivity of the polypeptide encoded by the nucleic acid sequence of SEQ ID NO:5.
2. The polypeptide of claim 1, wherein said polypeptide is encoded by a nucleic acid sequence that has substantial identity with at least a portion of the nucleic acid sequence of SEQ ID NO:5.
3. The polypeptide of claim 1, wherein said polypeptide is encoded by a nucleic acid sequence that selectively hybridizes with at least a portion of the nucleic acid sequence of SEQ ID NO:5.
4. The polypeptide of claim 1, wherein said polypeptide is encoded by a nucleic acid sequence that encodes conservative amino acid substitutions of at least a portion of the nucleic acid sequence of SEQ ID NO:5.
5. The polypeptide of claim 1, wherein said polypeptide has a molecular weight of about kilodaltons.
6. The polypeptide of claim 1, wherein said polypeptide is encoded by the nucleic acid sequence of SEQ ID NO:5.
7. The polypeptide of claim 1, wherein said polypeptide comprises at least a portion of the amino acid sequence of SEQ ID NO:6.
8. The polypeptide of claim 1, wherein said polypeptide has substantial identity with at least a portion of the amino acid sequence of SEQ ID NO:6.
9. The polypeptide of claim 1, wherein said polypeptide comprises an amino acid sequence having at least one conserved amino acid substitution of at least a portion of the amino acid sequence of SEQ ID NO:6.
10. The polypeptide of claim 1, wherein said protein binds with at least one antibody that binds with a polypeptide p10 of a Borna Disease Virus.
11. A fusion protein comprising the polypeptide of claim 1.
12. A specific binding member that binds with the polypeptide of claim 1.
13. The specific binding member of claim 11, wherein said specific binding member specifically binds with a polypeptide p10 of a Borna Disease Virus.
14. A nucleic acid molecule encoding a polypeptide comprising at least one bioactivity of the protein encoded by the amino acid sequence of SEQ ID NO:6.
15. The nucleic acid molecule of claim 14, wherein said nucleic acid molecule has substantial identity with at least a portion of the nucleic acid sequence of SEQ ID
NO:5.
NO:5.
16. The nucleic acid molecule of claim 14, wherein said nucleic acid molecule selectively hybridizes with at least a portion of the nucleic acid sequence of SEQ ID
NO:5.
NO:5.
17. The nucleic acid molecule of claim 14, wherein said nucleic acid molecule encodes conservative amino acid substitutions of at least a portion of the amino acid sequence of SEQ ID NO:6.
18. The nucleic acid molecule of claim 14, wherein said polypeptide has a molecular weight of about 10 kilodaltons.
19. The nucleic acid molecule of claim 14, wherein said protein is encoded by the nucleic acid sequence of SEQ ID NO:5.
20. The nucleic acid molecule of claim 14, wherein said protein binds with at least one specific binding member that binds with a polypeptide p10 of a Borna Disease Virus.
21. The nucleic acid molecule of claim 14 operably linked to at least one control sequence.
22. The nucleic acid molecule of claim 14 in a vector.
23. The nucleic acid molecule of claim 14 in a cell that does not normally express said nucleic acid molecule.
24. A test kit for detecting a Borna Disease Virus infection in a subject comprising the polypeptide of claim 1.
25. The test kit of claim 24, further comprising a solid support.
26. The test kit of claim 25, wherein said solid support comprises at least one polymer.
27. The test kit of claim 25, wherein said solid support comprises a plurality of beads.
28. The test kit of claim 25, wherein said solid support comprises a matrix material.
29. The test kit of claim 25, wherein said solid support comprises an assay platform.
30. The test kit of claim 25, wherein said solid support comprises a bibulous material.
31. The test kit of claim 25, wherein said solid support comprises a membrane.
32. The test kit of claim 25, wherein said protein of claim 1 is associated with at least one .
cell.
cell.
33. The test kit of claim 32, wherein said at least one cell is a eukaryotic cell.
34. The test kit of claim 33, wherein said eukaryotic cell is transfected with the nucleic acid molecule of claim 14.
35. The test kit of claim 24, further comprising at least one specific binding reagent.
36. The test kit of claim 35, wherein said at least one specific binding reagent is a detestably labeled specific binding reagent.
37. A test kit, comprising: the specific binding member of claim 12.
38. The test kit of claim 37, wherein said specific binding member is a detectably labeled specific binding member.
39. The test kit of claim 37, further comprising a solid support.
40. The test kit of claim 37, further comprising a second specific binding reagent.
41. The test kit of claim 40, wherein said second specific binding reagent is a detectably labeled specific binding reagent.
42. A test kit, comprising at least one nucleic acid molecule of claim 14.
43. The test kit of claim 42, wherein at least one of said at least one nucleic acid is detectably labeled.
44. The test kit of claim 42, wherein said at least one nucleic acid molecule comprises at least one DNA molecule.
45. The test kit of claim 42, wherein said at least one nucleic acid molecule comprises at least one RNA molecule.
46. A vaccine, comprising the polypeptide of claim 1.
47. A method of immunizing a subject, comprising: administering the vaccine of claim 46 to said subject.
48. An aminal having been administered the vaccine of claim 46.
49. An animal having an immunity to a Borna Disease Virus produced according to the method of claim 46.
50. A vaccine, comprising the nucleic acid molecule of claim 14.
51. A method of immunizing a subject, comprising: administering the vaccine of claim 50 to said subject.
52. An animal having been administered the vaccine of claim 50.
53. An animal having an immunity to a Borna Disease Virus produced according to the method of claim 50.
54. A vaccine, comprising the specific binding member of claim 12.
55. A method of immunizing a subject, comprising: administering the vaccine of claim 54 to said subject.
56. An animal having been administered the vaccine of claim 54.
57. An animal having an immunity to a Borna Disease Virus produced according to the method of claim 54.
58. A method of testing a subject for determining whether the subject has a been exposed to, infected with or vaccinated against a Borna Disease Virus, comprising the steps of:
a) providing a sample from a subject;
b) contacting said sample with the specific binding member of claim 12; and c) detecting the binding of said specific binding member with said sample.
a) providing a sample from a subject;
b) contacting said sample with the specific binding member of claim 12; and c) detecting the binding of said specific binding member with said sample.
59. The method of claim 58, further comprising the step of comparing said binding with a standard.
60. A method of testing a subject for determining whether the subject has a been exposed to, infected with or vaccinated against a Borna Disease Virus, comprising the steps of:
a) providing a sample from a subject;
b) contacting said sample with the polypeptide of claim 1; and c) detecting the binding of said polypeptide with said sample.
a) providing a sample from a subject;
b) contacting said sample with the polypeptide of claim 1; and c) detecting the binding of said polypeptide with said sample.
61. The method of claim 60, further comprising the step of comparing said binding with a standard.
62. A method of testing a subject for determining whether the subject has a been exposed to, infected with or vaccinated against a Borna Disease Virus, comprising the steps of:
1. providing a sample from a subject;
2. contacting said sample with the nucleic acid molecule of claim 14; and 3. detecting the binding of said nucleic acid molecule with said sample.
1. providing a sample from a subject;
2. contacting said sample with the nucleic acid molecule of claim 14; and 3. detecting the binding of said nucleic acid molecule with said sample.
63. The method of claim 62, further comprising the step of comparing said binding with a standard.
64. A method for identifying a test compound or bioactivity, comprising:
contacting a sample comprising at least one cell infected with a Borna Disease Virus with a test compound, and monitoring the course of infection with a Borna Disease Virus in said at least one cell.
contacting a sample comprising at least one cell infected with a Borna Disease Virus with a test compound, and monitoring the course of infection with a Borna Disease Virus in said at least one cell.
65. The method of claim 64, wherein said monitoring comprises measuring the expression of a polypeptide p10 in said at least one cell.
66. A compound identified by the method of claim 64.
67. A pharmaceutical composition identified by the method of claim 64.
68. A method for identifying a bioactivity, comprising: contacting a sample comprising at least one DBV with a test compound, and monitoring the infectivity of said at least one BDV.
69. The method of claim 68, wherein said monitoring comprises contacting said sample with a least one cell that can be infected with said at least one BDV.
70. A compound identified by the method of claim 68.
71. A pharmaceutical composition identified by the method of claim 68.
72. A system, comprising:
a)a storage structure;
b)a dispensation structure;
c)a detector structure;
d)a computing structure;
a)a storage structure;
b)a dispensation structure;
c)a detector structure;
d)a computing structure;
73. The system of claim 72, further comprising a polypeptide of claim 1.
74. The system of claim 72, further comprising at least one test compound.
75. The system of claim 72, further comprising a specific binding member of claim 12.
76. The system of claim 72, further comprising a nucleic acid of claim 14.
77. A composition identified using the system of claim 72.
78. A pharmaceutical composition identified using the method of claim 72.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US9790198P | 1998-08-26 | 1998-08-26 | |
| US60/097,901 | 1998-08-26 | ||
| PCT/US1999/019227 WO2000012548A1 (en) | 1998-08-26 | 1999-08-24 | Nucleic acid and polypeptide p10 of a borna disease virus (bdv) and their use for diagnostic and immunization purposes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2341360A1 true CA2341360A1 (en) | 2000-03-09 |
Family
ID=22265683
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002341360A Abandoned CA2341360A1 (en) | 1998-08-26 | 1999-08-24 | Nucleic acid and polypeptide p10 of a borna disease virus (bdv) and their use for diagnostic and immunization purposes |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP1105417A4 (en) |
| AU (1) | AU5493899A (en) |
| CA (1) | CA2341360A1 (en) |
| WO (1) | WO2000012548A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2003225495B2 (en) | 2002-04-05 | 2009-01-15 | Roche Innovation Center Copenhagen A/S | Oligomeric compounds for the modulation HIF-1alpha expression |
| JP2017524375A (en) * | 2014-06-10 | 2017-08-31 | アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル | Pharmaceutical composition for prevention or treatment of neurodegenerative diseases |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4851341A (en) * | 1986-12-19 | 1989-07-25 | Immunex Corporation | Immunoaffinity purification system |
| US5654401A (en) * | 1990-11-28 | 1997-08-05 | The Johns Hopkins University | Borna disease virus-specific protein and kits |
| EP0791654A1 (en) * | 1996-02-21 | 1997-08-27 | Jürgen A. Dr. Richt | Polypeptides corresponding to the amino acid sequences of proteins p57 or p9.5 of Borna disease virus, nucleic acid fragments coding therefore and their use for diagnostic and immunization purposes |
-
1999
- 1999-08-24 CA CA002341360A patent/CA2341360A1/en not_active Abandoned
- 1999-08-24 EP EP99941247A patent/EP1105417A4/en not_active Withdrawn
- 1999-08-24 AU AU54938/99A patent/AU5493899A/en not_active Abandoned
- 1999-08-24 WO PCT/US1999/019227 patent/WO2000012548A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| EP1105417A4 (en) | 2002-08-07 |
| AU5493899A (en) | 2000-03-21 |
| EP1105417A1 (en) | 2001-06-13 |
| WO2000012548A1 (en) | 2000-03-09 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |