WO1995029240A1 - Isolation and characterization of a novel primate t-cell lymphotropic virus and the use of this virus or components thereof in diagnostics assays and vaccines - Google Patents

Isolation and characterization of a novel primate t-cell lymphotropic virus and the use of this virus or components thereof in diagnostics assays and vaccines Download PDF

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WO1995029240A1
WO1995029240A1 PCT/US1995/004910 US9504910W WO9529240A1 WO 1995029240 A1 WO1995029240 A1 WO 1995029240A1 US 9504910 W US9504910 W US 9504910W WO 9529240 A1 WO9529240 A1 WO 9529240A1
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leu
pro
protein
stlv
pan
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PCT/US1995/004910
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French (fr)
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Genoveffa Franchini
Robert C. Gallo
Phillip Markham
Adriana Giri
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The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
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Priority to AU23927/95A priority Critical patent/AU2392795A/en
Publication of WO1995029240A1 publication Critical patent/WO1995029240A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
    • C12N2740/14021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
    • C12N2740/14022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to the field of retrovirology and more specifically, to the isolation and characterization of a novel primate T-cell lymphotropic virus and the use of this virus or its viral components in diagnostic assays and vaccines.
  • HTLV Human T-cell lymphotropic viruses
  • BLV bovine lymphotropic virus
  • STLV simian T-cell lymphotropic virus
  • HIV retrovirus
  • AIDS viruses are classified as members of the lentivirus subfamily of
  • Retroviridae The human T-cell lymphotropic viruses are divided into two types, HTLV-I and HTLV-II.
  • HTLV-I was first identified by Gallo and co-workers (Poiesz, B.J. et al. (1980) Proc. Natl. Acad. Sci. U.S.A., 77:7415-7419) in a T-lympho-blastoid cell line that had been established from a patient diagnosed with cutaneous T-cell lymphoma.
  • HTLV-II was first identified in a T-cell line established from a patient diagnosed with hairy-cell leukemia (Saxon, A. et al. (1978) Ann. Intern. Med., 88:323-326).
  • HTLV-I HTLV-I-associated myelopathy
  • BLV bovine lymphotropic virus
  • STLV-I lymphotropic virus type I
  • Lancet, ii:658 represents a widely used animal model for HTLV infection.
  • Virus-transformed lymphocyte cell lines from infected primates have been derived (Miyoshi, I. et al. (1983) Int. J. Cancer 32:333-336), and the nucleotide sequence of an infectious molecular clone of STLV-I has been determined and shown to share 90-95% nucleotide sequence homology with HTLV depending on precisely which isolates are compared (Watanabe, T. et al. (1986)
  • Virology 148:383-388.
  • the virus is capable of immortalizing both human and simian lymphocytes in vitro (Miyoshi, I. et al. (1983) Gann, 74:223-226).
  • the organization of the STLV-I is very similar to that of HTLV-I in that cells infected with these viruses contain the same classes of proteins - for
  • group antigen gag
  • reverse transcriptase pol
  • envelope env
  • env envelope
  • env envelope
  • env env
  • STLV pan-p lymphotropic virus
  • the invention also relates to primate cell cultures infected with STLV pan-p .
  • the invention further relates to an isolated and substantially pure preparation of the proviral DNA of the simian T-cell lymphotropic virus STLV pan-p .
  • the invention further relates to nucleic acid sequences derived from the genomic RNA, mRNA or proviral DNA of STLV pan-p . It is therefore an object of this
  • nucleic acid sequences capable of directing production of recombinant STLV pan-p proteins, as well as equivalent natural nucleic acids sequences.
  • nucleic acid sequences may be isolated from a cDNA library prepared from RNA or DNA isolated from STLV pan-p or from STLV pan-p infected cells, from proviral DNA segments by restriction digestion, by PCR amplification of RNA isolated from STLV pan-p infected cells or by PCR
  • nucleic acid sequence refers to RNA, DNA, cDNA or any synthetic variant thereof which encodes for protein.
  • nucleic acid sequences derived from the STLV pan-p genome are useful as probes to isolate naturally occurring variants of the virus in other primates.
  • the nucleic acid sequences of the present invention are also useful as probes to detect the presence of T-cell
  • lymphotropic viruses in biological samples.
  • the invention further relates to a method for detection of STLV pan-p viruses in biological samples based on selective amplification of STLV pan-p gene fragments utilizing primers derived from the STLV pan-p genome.
  • the invention also relates to the use of single stranded antisense poly- or oligonucleotides derived from the STLV pan-p genome which are useful to inhibit the
  • the invention also relates to isolated and substantially purified STLV pan-p proteins and analogs thereof.
  • the invention also relates to the use of the recombinant STLV pan-p proteins as diagnostic agents and as vaccines.
  • STLV pan-p viral particles may be used as diagnostic agents and as immunogens in the
  • the present invention further encompasses methods of detecting antibodies specific for STLV pan-p viruses in biological samples. Such methods are useful for diagnosis of infection and disease caused by STLV pan-p viruses and for monitoring the progression of such disease. Such methods are also useful for monitoring the efficacy of therapeutic agents during the course of treatment of STLV pan-p virus infection in a primate.
  • the present invention also relates to antibodies to STLV pan-p or parts thereof and to the use of these antibodies in immunoassays to detect STLV pan-p viruses in biological samples or as antiviral agents.
  • the invention also relates to pharmaceutical compositions useful in the prevention and treatment of STLV pan-p virus infection in mammals.
  • the invention further relates to cell culture systems and animal model systems infected with STLV pan-p and the use of these systems to screen antiviral agents.
  • Figure 1 shows a Western blot analysis of sera from 12 chimpanzees.
  • Figure 2 shows a genealogical tree and serological profile of the Pigmy chimpanzee colony from which STLV pan-p was isolated.
  • Figures 3A-3C show electron micrographs of cells from primate cocultures.
  • FIG. 4 shows polyacrylamide gel
  • Figures 5A and 5B show Southern blot analyses of cellular proviral DNA isolated from STLV pan-p -infected cells.
  • Figure 1 shows the results of Western blot analysis of sera from 12 chimpanzees in the Pan Paniscus colony. Sera from each animal and from two positive controls, strong reactive HTLV-I and -II, were tested at a 1:100 dilution using Western blot strips from Cellular Products (Buffalo, NY). The antigens present on the
  • Figure 2 shows a genealogical tree
  • the seropositive animals are represented with their gender symbol blackened.
  • the asterisk indicates animal sera that have not been tested because of unavailability.
  • the numbers included in the animal designation represent the estimated birth date for the wild born animals (LI-1954, BO-1971, MA-1970, KI-1974 and KI-1950) and the actual birth dates of the remaining animals.
  • Figures 3A-3C represent electron micrographs of cells from primate cell cultures positive for STLV pan-p .
  • Figure 3A represents an electron micrograph (60,000 magnification) of typical type C retroviral particles in a cell from coculture L93-79B obtained from animal LA-1967's peripheral blood mononuclear cells (PBMC).
  • Figure 3B shows an electron micrograph (90,000 magnification) of an immature virion present in a cell from coculture L93-79C originated from animal JI-1985.
  • Figure 3C shows an electron micrograph (90,000 magnification) of typical budding from type C retroviruses in a cell obtained from a transient coculture of L93-79C with the human B cell line B-JAB.
  • Figure 4 shows a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of 35 S- labelled proteins immunoprecipitated from the cell lines L93-79C, MT-2 (HTLV-I-infected) and MO-T (HTLV-II-infected) using the following sera: 1: serum from animal LI-1954, 2: serum from animal ZA-1984, 3: serum from one seronegative (on Western blot analysis) orangutan, 4 and 5: sera from HTLV-I- and HTLV-II-infected individuals, respectively.
  • p24 gag protein is indicated by an arrow.
  • Figures 5A and 5B shows the results of Southern blot analysis of DNA isolated from STLV pan-p -infected cell lines. Twenty micrograms of DNA from the cell lines L93-79A, B and C, the HTLV-I- and -II-infected cell lines C91/PL and MO-T, respectively and the uninfected T-cell line H9, were cleaved with the Pst-1 endonuclease, transferred to nitrocellulose and duplicate filters hybridized with probes specific to HTLV-I ( Figure 5B) or HTLV-II ( Figure 5A). The size of the internal Pst-1 fragments for HTLV-I and -II are indicated by arrows on the right-hand side of Figures 5A and 5B.
  • STLV pan-p lymphotropic virus designated STLV pan-p .
  • STLV pan-p means a primate T-cell lymphotropic virus characterized by having a genomic nucleic acid sequence, wherein the genomic nucleic acid sequence includes a nucleic acid sequence
  • substantially homologous to the nucleic acid sequence shown in SEQ ID NO: 7.
  • substantially homologous is meant at least 80% homologous, more preferably, greater than 90% homologous, and most preferably, greater than 95% homologous than SEQ ID NO: 7.
  • STLV pan-p was isolated from infected Pigmy chimpanzees.
  • peripheral blood mononuclear cells PBMC
  • PBMC peripheral blood mononuclear cells
  • Alternative methods of detecting virus expression include, but are not limited to, radioimmune or immunoblot analysis to detect other T-cell lymphotropic viral antigens such as gag, pol and env; electron microscopy; various nucleic acid hybridization techniques such as Southern blot analysis or PCR amplification, and assays for reverse transcriptase activity (Poiesz, B.J. et al. (1980) Proc. Natl. Acad. Sci. U.S.A.. 77:7415-7419).
  • STLV pan-p viral particles may be isolated from cell cultures infected with STLV pan-p or from sera or lymphoid tissues obtained from STLV pan-p - infected individuals by any of the methods known in the art,
  • STLVpan-p viral particles are isolated from cell cultures infected with STLV pan-p by
  • the STLV pan-p viral particles are found at a density of about 1.170 g/ml.
  • the presence of STLV pan-p may be detected by hybridization analysis of the extracted genome using probes derived from the STLV pan-p sequence described herein or by immunoassay utilizing as probes antibodies directed against antigens encoded by the STLV pan-p genome.
  • fractions obtained in the purification are treated under conditions which would cause the disruption of viral particles, for example, with detergents in the presence of chaotropic agents, and the presence of viral nucleic acid is determined by hybridization techniques.
  • the viral particles from the purified preparations may be utilized as antigens in diagnostic immunoassays or as immunogens in the production of antibodies.
  • STLV pan-p virus may be further characterized via the isolation of RNA from purified STLV pan-p viral particles.
  • STLV pan-p proviral DNA may be isolated from cell cultures infected with STLV pan-p or from sera or tissues obtained from STLV pan-p infected
  • RNA or DNA from such samples are known to those skilled in the art and include, but are not limited to, use of phenol chloroform
  • proviral DNA may be isolated from STLV pan-p infected cells as described in present Example 2. Once obtained, the STLV pan-p RNA or proviral DNA may be characterized as unique from the RNA or proviral DNA of previously isolated T-cell lymphotropic viruses by a variety of approaches.
  • the STLV pan-p proviral DNA can be subjected to Southern blot analysis wherein DNA is cleaved by a restriction enzyme or enzymes to yield a restriction pattern that is unique for STLV pan-p .
  • Restriction enzymes capable of generating such a unique restriction fragment pattern include, but are not limited to, Pst-1.
  • An example of Southern blot analysis following Pst-1 cleavage of proviral DNA is described in Example 4 of the present specification.
  • the STLV pan-p DNA of the present invention can be characterized by polymerase chain reaction analysis using specific primer sequences which are capable of amplifying STLV-I as well as all three of the known HTLV-I clades but not STLV pan-p .
  • specific primer sequences which are capable of amplifying STLV-I as well as all three of the known HTLV-I clades but not STLV pan-p .
  • primers include, but are not limited to, sequences shown below as SEQ ID NO:1
  • the DNA of the present invention can be characterized by PCR analysis using oligonucleotide primers which are incapable of amplifying STLV pan-p sequence but can amplify either HTLV-I or HTLV-II sequence.
  • primer pairs include, but are not limited to, sequences shown below as
  • TCATGAACCC CAGTGGTAA which amplify HTLV-II sequences. It would be understood by those skilled in the art that such PCR analysis could be carried out on viral RNA isolated from purified viral particles, from cell cultures infected by STLV pan-p or from the sera or tissues obtained from STLV pan-p -infected
  • extraction methods such as extraction by differential precipitation, extraction by organic solvents and
  • the STLV pan-p proviral DNA can be sequenced and its sequence compared with the nucleic acid sequence of other known T-cell lymphotropic viruses to demonstrate that STLV pan-p viral sequence is distinct from the sequences of previously identified
  • STLV pan-p A partial nucleic acid sequence of STLV pan-p is set forth below as SEQ ID NO: 7.
  • nucleotides are those standardly used in the art.
  • sequence information shown in SEQ ID NO:7 is useful for the design of probes for the isolation of additional sequences derived from other regions of the STLV pan-p genome.
  • probes containing a sequence of approximately eight or more nucleotides, preferably twenty or more nucleotides, which were derived from regions close to the 5'-termini or 3'-termini of the sequence shown in SEQ ID NO: 7 may be used to isolate overlapping DNA sequences of the STLV pan-p genome. Probes can then be derived from the 5 ' or 3' termini of these overlapping DNA sequences in order to isolate additional sequence from the STLV pan-p genome. This approach known as "genome walking" is known to those skilled in the art.
  • the T-cell lymphotropic viruses have regions of conserved nucleic acid sequences and nucleic acid probes containing these conserved
  • sequences may be used to design primers for use in systems which amplify the genome sequences of STLV pan-p .
  • the information provided herein makes it possible to clone and sequence the STLV pan-p genome.
  • LTR (U3, R, U5), the entire gag, protease and remaining portion of the polymerase genes is shown below as SEQ ID NO: 8
  • AACCCTGTCT TTCCGGTCAA AAAACCAAAC GGCAAGTGGA 2680 GATTTATTCA TGACCTGAGG GCCACCAATG CCATCACGAC 2720
  • STL tax LTR sequence from the 3' region (STL tax LTR) encompassing the tax/rex gene and 3' LTR is shown below as SEQ ID NO: 9
  • the information provided in the present application also enables the isolation of viral homologues of STLV pan-p from other primates such as humans.
  • a radioimmunoprecipitation assay may be used to immunoprecipitate virus or viral proteins from sera obtained from human patients exhibiting HTLV-I/HTLV-II indeterminant seropositivity.
  • HTLV-I/HTLV-II HTLV-I/HTLV-II
  • indeterminate seropositivity can be determined by Western blotting to detect antigens specific to HTLV-I and/or HTLV-II.
  • the patient's sera can be screened using immunoassays containing STLV pan-p or parts thereof as antigens.
  • nucleic acid sequences of viral homologues of STLV pan-p may be isolated from tissues or serum obtained from infected individuals via
  • tissues from which the virus may be isolated include, but are not limited to, peripheral blood leukocytes, lymph nodes and spleen.
  • Primers which may be used in PCR amplification of STLV pan-p variants in other primates may be derived from the proviral DNA of STLV pan-p .
  • the primers may be derived from regions of conserved nucleic acid sequences found within the family of T-cell lymphotropic viruses.
  • viral homologues of STLV pan-p may be isolated from infected individuals using antibodies raised to purified STLV pan-p viral particles, to proteins purified from these particles, to recombinant proteins produced from STLV pan-p nucleic acid sequences, or to chemically synthesized STLV pan-p proteins.
  • the nucleic acid sequence information derived from STLV pan-p and its viral homologues can be aligned with sequences from known T-cell lymphotropic viruses to determine areas of homology and heterogeneity within the viral genome (s) which could indicate the presence of different strains of the genome.
  • computer alignment of the STLV pan-p sequences with sequences of known T-cell lymphotropic viruses such as HTLV-I, HTLV-II and STLV-I can be used to identify regions of inter-strain homology and non-homology which would be useful in the design of strain-common and strain-specific
  • oligonucleotide and peptide probes useful in detecting STLV pan-p antigens or nucleic acids in biological samples.
  • An example of a computer program useful for carrying out such sequence alignments is GENALIGN (Intelligenetics, Inc., Mountainview, CA).
  • nucleic acid sequences of the present invention may be inserted into a suitable nucleic acid sequences of the present invention.
  • suitable expression vector by methods known to those skilled in the art.
  • suitable expression vector is meant a vector that is capable of carrying and expressing a nucleic acid sequence coding for protein.
  • the nucleic acid sequence encodes at least a single open-reading frame of a known T-cell lymphotropic gene.
  • genes include, but are not limited to, gag, pol, env, tax and rex.
  • Suitable expression vectors for the present invention include any vectors into which a nucleic acid sequence as described above can be inserted, along with any preferred or required operational elements, and which vector can then be subsequently transferred into a host organism and replicated in such organism.
  • Preferred vectors are those whose restriction sites have been well documented and which contain the operational elements preferred or required for transcription of the nucleic acid sequence.
  • the "operational elements" as discussed herein include at least one promoter, at least one operator, at least one leader sequence, at least one determinant, at least one terminator codon, and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the inserted nucleic acid sequence.
  • such vectors will contain at least one origin of replication recognized by the host organism along with at least one selectable marker and at least one promoter sequence capable of initiating transcription of the nucleic acid sequence.
  • cloning vector of the present invention it should additionally be noted that multiple copies of the nucleic acid sequence encoding STLV pan-p protein(s) and its attendant operational elements may be inserted into each vector. In such an embodiment, the host organism would produce greater amounts per vector of the desired protein(s). In a similar fashion, multiple different proteins may be expressed from a single vector by inserting into the vector a copy (or copies) of nucleic acid sequence encoding each protein and its attendant operational elements. In yet another embodiment, a polycistronic vector in which multiple proteins (either identical in sequence or different) may be expressed from a single vector is created by placing expression of each protein under the control of an internal ribosomal entry site (IRES) (Molla A. et al.
  • IRES internal ribosomal entry site
  • the number of multiple copies of the nucleic acid sequence encoding protein which can be inserted into the vector is limited only by the ability of the resultant vector, due to its size, to be transferred into, and replicated and transcribed in, an appropriate host organism.
  • Expression vectors suitable for the present method include those vectors capable of producing high efficiency gene transfer in vivo. Such vectors include but are not limited to retroviral, adenoviral and vaccinia viral vectors. Operational elements of such expression vectors are disclosed previously in the present
  • Preferred vectors are attenuated vaccinia or pox virus vectors.
  • An expression vector containing nucleic acid sequence capable of directing host cell synthesis of STLV pan-p protein can be administered in a pure or
  • a preferred substance having affinity for nucleic acid is a polycation such as polylysine.
  • Internalizing factors include, but are not limited to, antibodies to T-cell markers.
  • the present invention also relates to methods for detecting the presence of STLV pan-p viruses in primates.
  • the method for detecting the presence of STLV pan-p viruses comprises analyzing the DNA of a primate subject for the presence of a STLV pan-p nucleic acid sequence. For analysis of the DNA, a biological specimen is obtained from the subjects.
  • Preferred biological specimens are peripheral blood leukocytes and lymphoid tissues.
  • nucleic acid can be extracted from contaminating cell debris and other protein substances by extraction of the sample with phenol.
  • phenol extraction the aqueous sample is mixed with an approximately equal volume of redistilled phenol and centrifuged to separate the two phases.
  • the aqueous containing nucleic acid is removed and precipitated with ethanol to yield nucleic acid free of phenol.
  • Alternative methods of purifying DNA from biological samples are disclosed in Sambrook et al. ((1989) in "Molecular
  • Methods for analyzing the DNA for the presence of STLV pan-p viral nucleic acid sequences include, but are not limited to, Southern blotting after digestion with the appropriate restriction enzymes (restriction fragment length polymorphism, RFLP) (Botstein, D., Amer. J. Hum. Genet., (1980) 69:201-205), oligonucleotide hybridization (Conner, R. et al., EMBO J., (1984) 3:13321-1326), RNase A digestion of a duplex between a probe RNA and the target DNA (Winter, E. et al., Proc. Natl. Acad. Sci. U.S.A.,
  • PCR polymerase chain reaction
  • Patents 4,683,195 and 4,683,202 and ligase chain reaction (LCR) (European Patent Application Nos. 0,320,308 and 0,439,182).
  • DNA is analyzed by Southern blotting following digestion with one or more restriction enzymes.
  • restriction enzymes to be used in the present invention are those enzymes for whom the presence or absence of their recognition site is linked to STLV pan-p virus expression.
  • Preferred restriction enzyme include, but are not limited to, Pst-1.
  • the nucleic acid sequence used as a probe in Southern analysis can be labelled in single-stranded or double-stranded form. Labelling of the nucleic acid sequence can be carried out by techniques known to one skilled in the art. Such labelling techniques can include radiolabels and enzymes (Sambrook, J. et al. (1989) in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, Plainview, New York). In addition, there are known non-radioactive techniques for signal
  • the size of the probe can range from about 50 nucleotides to about several kilobases.
  • the nucleic acid sequences used as a probe in Southern analysis are derived from STLV pan-p genomic nucleic acid sequence. Once the separated DNA fragments are hybridized to the labelled nucleic acid probes, the restriction digest pattern can be visualized by
  • Amounts of DNA which can be analyzed by Southern blot range from about 5 micrograms to about 50 micrograms. Southern blot analysis of 20 ⁇ g of DNA prepared from STLV pan-p -infected cells is described in
  • the DNA is analyzed for the presence of STLV pan-p nucleic acid
  • each of the pairs of primers selected for use in PCR are designed to hybridize with sequences in the STLV pan-p genome which are an appropriate distance apart (at least about 50
  • Primer pairs which can specifically hybridize to such viral sequences can be derived from a T-cell lymphotropic virus genome containing a region of nucleic acid sequence substantially homologous to the DNA sequence shown in SEQ ID NO: 7.
  • Each primer of a pair is a single-stranded oligonucleotide of about 15 to about 50 bases in length which is complementary to a sequence at the 3' end of one of the strands of a double-stranded target sequence.
  • Each pair comprises two such primers, one of which is
  • the target sequence is generally about 100 to about 400 base pairs long but can be as large as 1,000 base pairs. Optimization of the amplification reaction to obtain sufficiently specific hybridization to STLV pan-p virus genomes is well within the skill in the art and is preferably achieved by adjusting the annealing
  • the present invention also provides purified and isolated pairs of primers for use in analysis of DNA for the presence of STLV pan-p viruses.
  • primers include, but are not limited to, the primers set forth in SEQ ID NOS: 10-12.
  • the primers of the present invention can be synthesized using any of the known methods of
  • oligonucleotide synthesis e.g., the phosphodiester method of Agarwal et al. (1972) Agnew. Chem. Int. Ed. Engl., 11:451, the phosphodiester method of Hsiung et al. (1979) Nucleic Acids Res., 6:1371, or the automated
  • oligonucleotides can be synthesized by automated
  • the primers can be derivatized to include a detectable label suitable for detecting and/or identifying the primer extension products (e.g., biotin, avidin, or radiolabeled dNTP's), or with a substance which aids in the isolation of the products of amplification (e.g., biotin, avidin, or radiolabeled dNTP's), or with a substance which aids in the isolation of the products of amplification (e.g.
  • the primers are synthetic oligonucleotides.
  • primer pairs can be selected to hybridize selectively to STLV pan-p nucleic acid sequences present in primates.
  • the selected primer pairs will hybridize sufficiently specifically to the STLV pan-p sequences such that non-specific hybridization to HTLV-I, HTLV-II and STLV-I sequences will not occur.
  • Primer pairs which hybridize selectively to STLV pan-p nucleic acid sequences can be used to amplify such sequences present in the DNA of a biological sample.
  • such primers are derived from STLV pan-p nucleic acid sequences encoding the env genes.
  • the amplification products of PCR can be detected either directly or indirectly.
  • direct detection of the amplification products is carried out via labelling of primer pairs. Labels suitable for labelling the primers of the present
  • the derived labels can be any suitable fluorescent molecules.
  • the derived labels can be any suitable fluorescent molecules.
  • the derived labels can be any suitable fluorescent molecules.
  • a preferred labelling procedure utilizes radiolabeled ATP and T4 polynucleotide kinase (Sambrook, J. et al. (1989) in "Molecular Cloning, A
  • the desired label can be incorporated into the primer extension products during the
  • the labelled amplified PCR products can be analyzed for the presence of STLV pan-p virus sequences via separating the PCR products by
  • unlabelled amplification products can be analyzed for the presence of virus nucleic acid sequences via hybridization with
  • STLV pan-p nucleic acid probes radioactively labelled or, labelled with biotin, in Southern blots or dot blots.
  • Nucleic acid probes useful in this embodiment are those described earlier for Southern analysis.
  • the present invention also encompasses methods for detecting the presence of STLV pan-p viruses comprising analyzing the RNA of a primate subject.
  • RNA is isolated from blood or tissue biopsies such as lymph nodes and spleen obtained from the subject.
  • the RNA to be analyzed can be isolated by methods known to those skilled in the art. Such methods include extraction of RNA by differential precipitation (Birnboim, H.C. (1988) Nucleic Acids Res., 16:1487-1497), extraction of RNA by organic solvents (Chomczynski, P. et al. (1987) Anal. Biochem.,
  • RNA 162:156-159
  • extraction of RNA with strong denaturants Chirgwin, J.M. et al. (1979) Biochemistry, 18:5294-5299).
  • a preferred method of isolating RNA utilizes guanidine thiocyanate.
  • RNA for STLV pan-p virus-specific mRNA expression include Northern blotting (Alwine, J.C. et al. (1977) Proc. Natl. Acad. Sci.,
  • RT-PCR reverse transcription-polymerase chain reaction
  • the invention also relates to isolated and substantially purified STLV pan-p proteins and analogs thereof where such proteins are derived from T-cell lymphotropic virus genomes which encode the amino acid sequences shown in SEQ ID NO: 10.
  • amino acids 18-57 of SEQ ID NO: 15 correspond to the amino acid sequence shown in SEQ ID NO: 10 (the single amino acid difference between SEQ ID NO: 10 and the amino acids 18-57 of SEQ ID NO: 15).
  • amino acids 40-78 of SEQ ID NO: 12 correspond to the amino acid sequence shown in SEQ ID NO: 11.
  • SEQ ID NO: 12 shows the amino acid sequence of rex where this sequence is encoded starting at nucleotide 5088 of SEQ ID NO: 8, continuing at nucleotide 1 of SEQ ID NO: 9, and ending at nucleotide 447 of SEQ ID NO: 9;
  • SEQ ID NO: 13 shows the amino acid sequence of gag where this sequence is encoded by nucleotides 748 to 2043 of SEQ ID NO: 8;
  • SEQ ID NO: 14 shows the amino acid sequence of pol where this sequence is encoded by nucleotides 2167 to 5151 of SEQ ID NO: 8 and SEQ ID NO: 15 shows the amino acid sequence of tax where this sequence is encoded by
  • nucleic acid sequences capable of directing production of proteins encoded by the STLV pan-p genome are intended to be
  • Such proteins include, but are not limited, to, gag (SEQ ID NO:13), env, pol (SEQ ID NO:14), tax (SEQ ID NO:15) and rex (SEQ ID NO: 12). Due to the degeneracy of the genetic code, it is to be understood that numerous choices of nucleotides may be made that will lead to a DNA sequence capable of directing production of the instant STLV pan-p proteins or their analogs.
  • analog refers to a protein having an amino acid sequence substantially identical to a sequence encoded by the STLV pan-p genome in which one or more
  • residues have been conservatively substituted with a functionally similar residue and which displays the functional aspects of the STLV pan-p proteins.
  • conservative substitutions include, for example, the substitution of non-polar (i.e. hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (i.e. hydrophilic) residue for another, such as a substitution between arginine and lysine, between glutamine and asparagine, or between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
  • conservative substitution may also include the use of a chemically derivatized residue in place of a non-derivatized residue provided that the resulting protein or polypeptide displays the requisite functional activity.
  • Chemical derivative refers to a protein or polypeptide having one or more residues chemically derivatized by reaction of a functional side group.
  • derivatized molecules include, but are not limited to, those molecules in which free amino groups have been derivatized to form, for example, amine
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.
  • the imidazole nitrogen of histidine may be derivatized to form N-imbenzylhistidine. Also included as chemical
  • derivatives are those proteins or peptides which contain one or more naturally-occurring amino acid derivatives of the twenty standard amino acids.
  • 4-hydroxyproline may be substituted for proline
  • 5-hydroxylysine may be substituted for lysine
  • 3-methylhistidine may be substituted for histidine;
  • a protein or polypeptide of the present invention also includes any protein or polypeptide having one or more additions and/or deletions of residues relative to the sequence of a protein or polypeptide whose sequence is encoded by the STLV pan-p genome so long as the requisite activity is maintained.
  • the overlapping DNA sequences encoding the STLV pan-p genome obtained by the methods
  • polypeptides derived from the STLV pan-p genome Such as
  • nucleic acid sequences may be natural or synthetic.
  • the recombinant protein can be composed of one open-reading frame (ORF) protein or a combination of the same or different ORF proteins.
  • ORF open-reading frame
  • the recombinant protein may be a fusion protein produced by ligating together STLV pan-p nucleic acid sequence encoding at least one ORF and a nucleic acid sequence encoding a protein capable of targeting the recombinant fusion protein to STLV pan-p infected cells. Proteins capable of targeting the recombinant fusion protein to virus-infected cells
  • recombinant fusion proteins include, but are not limited to antibodies to T-cell markers.
  • the production of such recombinant fusion proteins may be accomplished by techniques known to those skilled in the art.
  • STLV pan-p protein whether fused or unfused, may be cloned into a suitable expression vector capable of being
  • Suitable expression vectors can function in prokaryotic or eukaryotic cells.
  • Preferred expression vectors are those that function in eukaryotic cells. Examples of such vectors include but are not limited to retroviral vectors, vaccinia virus, adenovirus, and adeno-associated virus.
  • the expression vector may then be used for purposes of expressing the protein in a suitable eukaryotic cell system.
  • suitable eukaryotic cell systems include, but are not limited to cell lines such as SF9 insect cells, Chinese Hamster Ovary (CHO) cells, CEM, H7 and HeLa cells.
  • Preferred eukaryotic cell systems are SF9 insect cells.
  • the expressed protein may be detected by methods known in the art such as metabolic radiolabelling or Western blotting.
  • the protein expressed by the cells may be obtained as crude lysate or it may be purified by standard protein purification procedures known in the art which may include differential precipitation, molecular sieve chromatography, ion-exchange
  • the protein may be purified by passage through a column containing a resin which has bound thereto antibodies specific for the protein.
  • the STLV pan-p proteins of the present invention may be purified from STLV pan-p viral particles. Methods for purifying viral particles and for detecting them during the purification procedure have been previously described. Purified proteins may be obtained from the viral particles following disruption of the particles by treatment with, for example, detergents in the presence of chaotropic agents. The lysates produced by the disruption of the viral particles may then be subjected to the various protein purification
  • selected proteins present in the lysate such as the envelope proteins
  • the proteins present in the lysate produced from the disrupted viral particles may be separated via gel electrophoresis.
  • containing the protein of interest may then be excised from the gel and the protein electroeluted via techniques known to one skilled in the art.
  • the STLV pan-p proteins may be synthetic peptides derived from STLV pan-p nucleic acid sequences where those skilled in the art would be aware that the peptides of the present invention or analogs thereof can be synthesized by automated
  • analogs can further include branched or non-linear
  • the present invention therefore relates to the use of STLV pan-p proteins in immunoassays for diagnosing or prognosing STLV pan-p infection in a primate.
  • the immunoassay is useful in diagnosing
  • STLV pan-p proteins particularly the envelope proteins, provide a highly specific and sensitive method for
  • Immunoassays of the present invention may be a radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, immunohistochemical assay and the like. Standard
  • Such assays may be a direct, indirect, competitive, or
  • Bio samples appropriate for such detection assays include, but are not limited to, whole or heparinized blood, lymph nodes and spleen.
  • test serum is reacted with a solid phase reagent having surface-bound recombinant
  • STLV pan-p protein as an antigen, preferably an envelope protein or a combination of an envelope protein with different proteins.
  • the solid surface reagent can be prepared by known techniques for attaching protein to solid support material. These attachment methods include non-specific adsorption of the protein to the support or covalent attachment of the protein to a reactive group on the support. After reaction of the antigen with anti-STLV pan-p antibody, unbound serum components are removed by washing and the antigen-antibody complex is reacted with a
  • the label may be an enzyme which is detected by incubating the solid support in the presence of a suitable
  • fluorimetric or calorimetric reagent fluorimetric or calorimetric reagent.
  • Other detectable labels may also be used, such as radiolabels or colloidal gold, and the like.
  • the STLV proteins and analogs thereof may be prepared in the form of a kit, alone, or in combinations with other reagents such as secondary antibodies, for use in immunoassays.
  • STLV pan-p viral particles may be substantially isolated by previously described methods and used as an antigen in immunoassays of the present invention.
  • the STLV pan-p proteins and analogs thereof can also be used as a vaccine to protect mammals against challenge with STLV pan-p viruses.
  • the vaccine which acts as an immunogen, may be a cell lysate prepared from cells transfected with a recombinant expression vector or a cell culture supernatant containing the expressed protein.
  • the immunogen is a partially or
  • substantially purified recombinant protein a protein substantially purified from STLV pan-p viral particles or a synthetic peptide derived from STLV pan-p nucleic acid sequence.
  • the immunogen While it is possible for the immunogen to be administered in a pure or substantially pure form, it is preferable to present it as a pharmaceutical composition, formulation or preparation.
  • formulations of the present invention both for veterinary and for human use, comprise an immunogen as described above, together with one or more
  • the carrier (s) must be any suitable pharmaceutically acceptable carriers and optionally other therapeutic ingredients.
  • the carrier (s) must be any suitable pharmaceutically acceptable carriers and optionally other therapeutic ingredients.
  • the carrier (s) must be any suitable pharmaceutically acceptable carriers and optionally other therapeutic ingredients.
  • All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation.
  • Formulations suitable for intravenous intramuscular, subcutaneous, or intraperitoneal are Formulations suitable for intravenous intramuscular, subcutaneous, or intraperitoneal.
  • administration conveniently comprise sterile aqueous solutions of the active ingredient with solutions which are preferably isotonic with the blood of the recipient.
  • Such formulations may be conveniently prepared by
  • compositions of the present invention may incorporate a stabilizer.
  • stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids which may be used either on their own or as admixtures. These stabilizers are preferably incorporated in an amount of 0.11-10,000 parts by weight per part by weight of immunogen. If two or more stabilizers are to be used, their total amount is preferably within the range specified above. These stabilizers are used in aqueous solutions at the
  • anti-adsorption agent may be used.
  • Controlled release preparations may be achieved through the use of polymer to complex or absorb the proteins or their
  • the controlled delivery may be exercised by selecting appropriate macromolecules (for example
  • polyester polyamino acids
  • polyvinylpyrrolidone polyvinylpyrrolidone
  • carboxymethylcellulose, or protamine sulfate carboxymethylcellulose, or protamine sulfate
  • concentration of macromolecules as well as the methods of incorporation in order to control release.
  • Another possible method to control the duration of action by controlled-release preparations is to incorporate the proteins, protein analogs or their functional derivatives, into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers.
  • a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers.
  • poly(methylmethacylate) microcapsules respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • compositions may be combined with typical carriers, such as lactose, sucrose, starch, talc magnesium stearate,
  • crystalline cellulose methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate or gum arabic among others.
  • the STLV pan-p viral particles or viral proteins may be administered at a range from about 1 to about 1,000 ug of protein.
  • the immunogen may be an expression vector containing nucleic acid sequences capable of directing expression of STLV pan-p protein(s).
  • the immunogens of the present invention may be supplied in the form of a pharmaceutical composition as described above.
  • Vaccination can be conducted by conventional methods.
  • the immunogen can be used in a suitable diluent such as saline or water, or complete or incomplete adjuvants.
  • the immunogen may or may not be bound to a carrier to make it more immunogenic.
  • carrier molecules include but are not limited to bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid, and the like.
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • tetanus toxoid and the like.
  • the immunogen can be administered by any route appropriate for antibody production such as intravenous, intraperitoneal, intramuscular, subcutaneous, and the like.
  • the immunogen may be administered once or at periodic intervals until a significant titer of anti-STLV pan-p antibody is produced.
  • the antibody may be detected in the serum using any of the immunoassays described earlier.
  • the administration of the immunogen of the present invention may be for either a prophylactic or therapeutic purpose.
  • the immunogen is provided in advance of any exposure to
  • the immunogen serves to prevent or attenuate any subsequent STLV pan-p infection in a primate.
  • the immunogen is provided at (or shortly after) the onset of the infection or at the onset of any symptom of infection or disease caused by STLV pan-p viruses.
  • the therapeutic administration of the immunogen serves to attenuate the infection or disease.
  • the vaccine comprises an STLV pan-p envelope protein substantially purified from an isolated STLV pan-p viral particle.
  • the compositions can be used to prepare antibodies to STLV pan-p virus-like particles.
  • the immunogens of the present invention may include purified STLV pan-p viral particles, cells infected with STLV pan-p , or STLV pan-p proteins.
  • the antibodies produced in response to these immunogens can be used directly as antiviral agents.
  • a host animal is immunized using the virus particles or, as appropriate, non-particle antigens native to the virus particle are bound to a carrier as described above for vaccines.
  • the host serum or plasma is collected following an appropriate time interval to provide a composition comprising antibodies reactive with the virus particle.
  • the gamma globulin fraction or the IgG antibodies can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art.
  • the antibodies are substantially free of many of the adverse side effects which may be associated with other anti-viral agents such as drugs.
  • the antibody compositions can be made even more compatible with the host system by minimizing potential adverse immune system responses. This is accomplished by removing all or a portion of the Fc portion of a foreign species antibody or using an antibody of the same species as the host animal, for example, the use of antibodies from human/human hybridomas.
  • Humanized antibodies i.e., nonimmunogenic in a human
  • Such chimeric antibodies may contain the reactive or antigen binding portion of an antibody from one species and the Fc portion of an antibody
  • chimeric antibodies include but are not limited to, non-human mammal-human chimeras, rodent-human chimeras, murine-human and rat-human chimeras (Robinson et
  • Suitable "humanized” antibodies can be alternatively produced by CDR or CEA substitution (Jones et al., (1986) Nature, 321:552; Verhoeyan et al., 1988 Science 239:1534; Biedleret al. (1988) J. Immunol.,
  • the antibodies or antigen binding fragments may also be produced by genetic engineering.
  • the technology for expression of both heavy and light chain genes in E. coli is the subject the PCT patent applications;
  • the antibodies can also be used as a means of enhancing the immune response.
  • the antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody.
  • pooled gamma globulin is administered at 0.02-0.1 ml/lb body weight during the early incubation period of other viral diseases such as rabies, measles and hepatitis B to interfere with viral entry into cells.
  • antibodies reactive with the STLV pan-p virus particle can be passively administered alone or in conjunction with another anti-viral agent to a host infected with a STLV pan-p viruses to enhance the immune response and/or the effectiveness of an antiviral drug.
  • anti-STLV pan-p antibodies can be induced by administering anti-idiotype antibodies as immunogens.
  • antibody preparation prepared as described above is used to induce anti-idiotype antibody in a host animal.
  • the composition is administered to the host animal in a suitable diluent. Following administration, usually repeated administration, the host produces anti-idiotype antibody.
  • antibodies produced by the same species as the host animal can be used or the FC region of the
  • administered antibodies can be removed.
  • composition can be purified as described above for anti-STLV pan-p antibodies, or by affinity chromatography using anti-STLV pan-p antibodies bound to the affinity matrix.
  • anti-idiotype antibodies produced are similar in conformation to the authentic STLV pan-p -antigen and may be used to prepare an STLV pan-p vaccine rather than using an STLV pan-p particle antigen.
  • the manner of injecting the antibody is the same as for vaccination purposes, namely intramuscularly, intraperitoneally, subcutaneously or the like in an effective concentration in a physiologically suitable diluent with or without adjuvant.
  • One or more booster injections may be desirable.
  • Monoclonal anti-virus particle antibodies or anti-idiotype antibodies can be produced as follows. The spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art. (Goding, J.W. 1983. Monoclonal Antibodies: Principles and Practice, Pladermic Press, Inc., NY, NY, pp. 56-97). To produce a human-human hybridoma, a human lymphocyte donor is selected. A donor known to be infected with STLV pan-p (where infection has been shown for example by the presence of anti-virus antibodies in the blood or by virus culture) may serve as a suitable lymphocyte donor.
  • Lymphocytes can be isolated from a peripheral blood sample or spleen cells may be used if the donor is subject to splenectomy.
  • Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a human fusion partner can be used to produce human-human hybridomas.
  • Primary in vitro immunization with peptides can also be used in the generation of human monoclonal antibodies.
  • Antibodies secreted by the immortalized cells are screened to determine the clones that secrete
  • antibodies of the desired specificity For monoclonal anti-virus particle antibodies, the antibodies must bind to STLV virus particles. For monoclonal anti-idiotype antibodies, the antibodies must bind to anti-virus
  • Cells producing antibodies of the desired specificity are selected.
  • the above described antibodies and antigen binding fragments thereof may be supplied in kit form alone, or as a pharmaceutical composition for in vivo use.
  • the antibodies may be used for therapeutic uses,
  • the present invention also relates to the production of cell lines expressing STLV pan-p viruses.
  • T-cell lymphotropic viruses Methods for culturing T-cell lymphotropic viruses are known to those skilled in the art (Markham, P.D. et al.
  • suitable cells or cell lines for culturing T-cell lymphotropic viruses may include those known to support lymphotropic virus replication.
  • Such cell lines include, but are not limited to, B cell lines such as A-2 and B-JAB and T-cell lines such HUT-78 and SUP-T1. It is possible that
  • peripheral blood mononuclear cells can be cultured and then infected with STLV pan-p or alternatively, that
  • peripheral blood mononuclear cells can be derived from STLV pan-p infected individuals (e.g., human or
  • chimpanzees The latter case is an example of the cell which is infected in vivo being passaged in vitro.
  • Immortalization of such primary PBMCs may be achieved by cocultivating the virus-infected PBMCs with cells such as human blood cord leukocytes as described in Example 2. Also, primary cultures of infected PBMCs may be infected with transforming viruses or transfected with transforming genes in order to create permanent or semi-permanent cell lines.
  • infecting cells with T-cell lymphotropic viruses (Markham et al. (1983) and (1984)).
  • infection of cells with STLV pan-p viruses in vitro can be accomplished by cocultivation of the cells to be transformed with virus-infected cells.
  • cells to be transformed include, but are not limited to, peripheral blood or bone narrow leukocytes.
  • In vitro transformation of human peripheral blood cord blood lymphocytes typically results primarily in transformation of CD4 cells.
  • CD8 + cells can also be transformed by STLV pan-p viruses in vitro and immature CD4- CD8- cells from bone marrow can also be transformed.
  • primate B cell lines such as B- JAB and AA2 may be infected by STLV pan-p viruses. It is understood by one skilled in the art that cells
  • transformed in vitro may actively transcribe STLV pan-p RNA to produce virions and that they themselves may be used to transform other normal cells.
  • animal model systems may also be used to promote viral replication.
  • Animal systems in which T-cell lymphotropic viruses are known to replicate include chimpanzees or other non human primates, rats, rabbits and cattle.
  • cell culture and animal model systems for STLV pan-p produced by the present invention may be used to screen for antiviral agents which inhibit
  • the antiviral agents are tested at a variety of concentrations for their effect on preventing viral replication in cell culture systems and then for an inhibition of infectivity of viral pathogenicity and a low level of toxicity in an appropriate animal model system.
  • the methods provided herein for detecting STLV pan-p antigens and STLV pan-p nucleic acid sequences are useful for
  • the STLV pan-p nucleic acid probes described herein may be used to quantitate the amount of viral nucleic acid produced in a cell culture via a method such as PCR amplification.
  • the Pigmy Chimpanzees used in the following examples were from a colony housed at the Yerkes Regional Primate Center (Atlanta, GA). This Pigmy chimpanzee breeding colony was initiated in the mid-seventies with two founder females, LI-1954 and MA-1970, both wild born, with estimated birth dates of 1954 and 1970, which were obtained from the San Diego Zoo and Zaire, respectively. Another female, KI-1950, the oldest Pigmy chimpanzee
  • SC is a serum control introduced to each strip by Cellular Products which serves as a positive control to ensure that serum has been added to each strip.
  • the animal designations for the 12 chimpanzees and the designations for the two positive controls are shown at the top of Figure 1. The results of this Western blot analysis demonstrated that eight (numbers 1-8 at the bottom of Figure 1) of the 12 Pigmy chimpanzees tested for HTLV-I and -II antigens scored positive for the recombinant
  • Figure 2 show that the founder female (LI-1954) was seropositive (blackened symbol) for HTLV-I and -II
  • STLV pan-p was isolated by coculturing peripheral blood mononuclear cells (PBMCs) prepared from heparinized blood obtained from Pigmy Chimpanzees diagnosed as of HTLV- I/HTLV- II indeterminate seropositivity based on the Western blot analysis shown in Figure 1.
  • PBMCs peripheral blood mononuclear cells
  • ALB11 CD45RA
  • VCHL1 CD45RO
  • BW242/412 TCR ⁇
  • TS/1(TCR ⁇ ) T-cell Diagnostic, Cambridge, MA
  • FACScanTM Becton Dickinson
  • PCR is performed with both primer pairs to amplify fragments of DNA from the tax region of STLV pan-p related viruses.
  • 1 ⁇ g of DNA is mixed with PCR reaction mix containing 50 mM KCl, 10 mMTris-HCl pH 8.3, 1.6 mM MgCl 2 , 50 pmol of each primer, 200 ⁇ g dATP, dGTP, dCTP, dTTP, and 2.5 unite of taq DNA polymerase (Perkin Elmer Cetus), in 2 final volumes of 100 ⁇ l.
  • Enzymatic DNA amplification is performed in 2 DNA Thermal Cycler (Perkin Elmer) with the following thermal parameters: denaturation step (5 min at 95°C), 35 cycles of denaturation (1 min 94°C) and annealing (2 min 55°C) and at least synthesis step (72°C 7 min.).
  • T4 polynucleotide kinase labeled by T4 polynucleotide kinase.
  • 1.5 ⁇ 10 5 cpm of probe are mixed with 30 ⁇ l of amplified products in a final volume of 40 ⁇ l. The mixture is heated at 95°C for 5 min and then at 56°C for 15 min to allow hybridization to occur.
  • Six ⁇ l of 0.01% bromophenol blue and 30 ⁇ l chloroform are then added and loaded onto a 10%
  • 2X RIPA buffer 1% Triton, ) .1% SDS, 1% deoxycholate, 0.05M Tris pH 7.5, 0.25M NaCl
  • Samples are precleared with 100 ⁇ L of Protein A agarose beads (Boehringer Mannheim Co.) plus 10 ⁇ L of normal rabbit serum by incubation at 4 degrees for 5 hours.
  • Sera from STLV pan-p sero-positive Pigmy (positive control) chimpanzees and from humans are added at a 1:10 dilution (50 ⁇ L) and incubated overnight at 4 degrees shaking.
  • Samples are then immunoprecipitated with 30 ⁇ L of Protein A agarose beads rocking at 4 degrees for 45 minutes.
  • Cells from established human B-cell lines can be infected with STLV pan-p by co-cultivation with infected, primary, human cord leukocytes or irradiated (6000 rad) STLV pan-p transformed cord blood leukocyte cultures.
  • donor cells STLV pan-p -infected
  • target cells e.g. RPMI 1640 supplemented with 10% fetal calf serum, glutamine, pen./strep., at 37°C, 5% CO 2 .
  • Cultures are monitored for morphological changes and for release of virus as detected by antigen capture assays for antigens cross reacting with HTLV-I/II p24.
  • a unique characteristic of cultures treated as described is the frequent induction of multiple syncytia or giant cells similar, but not identical to those observed in some HIV-1-infected cells, but clearly distinguishable from the multi-lobulated nuclei-containing giant cells often observed in HTLV-I-infected cells.
  • the wells in polystyrene microtiter plates are coated with STLV pan-p prepared from culture supernatant fluids by density centrifugation or with purified viral structural proteins. This is done by incubating the appropriate concentrations of the antigens in the wells overnight at 4°C in a humid chamber. Following overnight incubation, the unbound protein is poured off, and the wells are coated with 1% BSA for two hours to block any open binding sites for proteins. The contents of the wells are then poured off, and the wells are washed with PBS-Tween (PBS containing 0.05% Tween-20).
  • PBS-Tween PBS containing 0.05% Tween-20
  • the coated wells are filled with the test sera expected to contain the antibodies, and the plates are incubated for 3 hours at room temperature in a humid chamber or overnight at 4°C. The unreacted material is washed off as before. Any antibody in the unknown sample remains attached to the antigen molecules on the well surface.
  • the conjugate consisting of enzyme-linked specific antibody reactive against the test antibody
  • the amount of the enzyme-linked specific antibody on the plate is determined as a measure of the amount of the test antibody in the immune complex. This is achieved by adding the appropriate substrate to the plate and following the rate of the enzyme reaction.
  • Viral load can also be assessed at the level of STLV pan-p viral RNA present in human serum, plasma or cells using one or more molecular amplification-detection systems such as, but not limited to, RT-PCR (Saksela, K. et al. (1994) Proc. Natl. Acad. Sci. U.S. A., 91:1104-1108 and NASBA (Kievits, T. et al. (1991) J. Virol. Methods. 35:273-286; Bruisten, S. et al. (1993) AIDS Res. Human Retroviruses, 9:259-265; van Gemen, B. et al. (1993) J. Virol. Methods, 43:177-188). Both procedures are
  • RT-PCR involves use of a preliminary reverse transcriptase reaction followed by amplification at high temperatures by conventional PCR procedures.
  • NASBA is a specific, isothermal amplification method which uses the coordinated activities of reverse transcriptase, RNase H, and T7 polymerase.
  • GTCTTCCCAA AAAGGCGGCA TACGTACTCT GGGACCAGAC 3920
  • CATCCTCCGC CATGACTCTA TCACATTGCC CCCCCATGGC 3960

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Abstract

The isolation and characterization of a novel primate T-cell lymphotropic virus designated STLVpan-p is disclosed. The invention relates to the proviral DNA of this virus and to the use of the nucleic acid sequence encoded by this proviral DNA to design nucleic acid probes and synthesize proteins which may be used in diagnostic and vaccines. Antibodies to these proteins or to the viral particle itself are also disclosed where such antibodies may be useful in immunoassays for detecting STLVpan-p viral antigens in individuals and where such antibodies may be used for treatment of STLVpan-p viral infections in individuals. The reagents provided in this invention also enable isolation of related T-cell lymphotropic viruses in other species. Finally, the present invention discloses the propagation of these viruses in tissue culture systems where such systems are useful for screening for anti-viral agents for STLVpan-p viruses.

Description

TITLE OF INVENTION
ISOLATION AND CHARACTERIZATION OF A NOVEL PRIMATE T-CELL
LYMPHOTROPIC VIRUS AND THE USE OF THIS VIRUS OR COMPONENTS
THEREOF IN DIAGNOSTICS ASSAYS AND VACCINES.
FIELD OF INVENTION
The present invention relates to the field of retrovirology and more specifically, to the isolation and characterization of a novel primate T-cell lymphotropic virus and the use of this virus or its viral components in diagnostic assays and vaccines.
BACKGROUND OF INVENTION
Human T-cell lymphotropic viruses (HTLV) are members of the class of retroviruses that include bovine lymphotropic virus (BLV) and simian T-cell lymphotropic virus (STLV). These viruses have been taxonomically grouped within the oncovirus subfamily of the Retroviridae and are distinct from the other pathogenic human
retrovirus, the AIDS viruses (HIV). AIDS viruses are classified as members of the lentivirus subfamily of
Retroviridae. The human T-cell lymphotropic viruses are divided into two types, HTLV-I and HTLV-II. HTLV-I was first identified by Gallo and co-workers (Poiesz, B.J. et al. (1980) Proc. Natl. Acad. Sci. U.S.A., 77:7415-7419) in a T-lympho-blastoid cell line that had been established from a patient diagnosed with cutaneous T-cell lymphoma. HTLV-II was first identified in a T-cell line established from a patient diagnosed with hairy-cell leukemia (Saxon, A. et al. (1978) Ann. Intern. Med., 88:323-326). Since the initial discovery of HTLV-I, the virus has been shown to be associated with human diseases such as adult T-cell leukemia (ATL) and the neurologic disorder known as tropical spastic paraparesis (TSP) or HTLV-I-associated myelopathy (HAM). By comparison, the limited number of individuals shown to harbor HTLV-II in association with a specific disease has precluded epidemiologic demonstration of a definitive etiologic role for HTLV-II in human malignancy.
As noted above, two naturally occurring animal retroviruses related to HTLV are known to exist; namely, bovine lymphotropic virus (BLV) and simian T-cell
lymphotropic virus type I (STLV-I). STLV-I, first
discovered in Japanese monkeys by virtue of its antigenic cross-reactivity to HTLV-I (Miyoshi, I. et al. (1982)
Lancet, ii:658), represents a widely used animal model for HTLV infection. Virus-transformed lymphocyte cell lines from infected primates have been derived (Miyoshi, I. et al. (1983) Int. J. Cancer 32:333-336), and the nucleotide sequence of an infectious molecular clone of STLV-I has been determined and shown to share 90-95% nucleotide sequence homology with HTLV depending on precisely which isolates are compared (Watanabe, T. et al. (1986)
Virology, 148:383-388). Like HTLV-I, the virus is capable of immortalizing both human and simian lymphocytes in vitro (Miyoshi, I. et al. (1983) Gann, 74:223-226).
Moreover, the organization of the STLV-I is very similar to that of HTLV-I in that cells infected with these viruses contain the same classes of proteins - for
example, group antigen (gag), reverse transcriptase (pol), envelope (env) and two additional nonstructural proteins, tax (trans-activator) and rex (regulator of expression).
Recent reports demonstrating an unusual serological profile against proteins of human T-cell lymphotropic virus types I and II (HTLV-I and -II) in several human Pigmy tribes in Zaire and Cameroon
(Kalyanaranan, V.S. et al. (1982) Science, 218:571-573; Goubau, P. et al. Aids Res. Hum. Retroviruses, 9:709-713 (1993); and Delaport, E. et al. Aids, 5:771-772 (1991)) suggest that an HTLV distinct from HTLV-I and HTLV-II may exist. More recently, an extensive phylogenetic study of HTLV- Is and STLV-Is indicated that multiple discreet interspecies transmission of ancestral viruses had occurred among primates (Koralnik, I.J., et al. (1994) Virology, in press). Based on the results of these studies, it was hypothesized that the HTLV producing the HTLV-I/II indeterminant seropositivity in the human Pigmy tribes might have originated in humans as a result of horizontal transmission of the virus from Pigmy
chimpanzees to human Pygmies. Human Pigmy tribes and the Pigmy chimpanzee (Pan Paniscus) have lived in a common habitat for thousands of years and the hunting practices of the human Pygmies can lead to accidental exposure to infected chimpanzee blood. This hypothesis, together with the observation that identification of simian
immunodeficiency virus had led to the discovery of human immunodeficiency virus Type-II (Kanki, P.J. et al. (1986) Science, 232:238-243 and Clavel, F. et al. (1986) Science 223:343-346), provided a basis for attempting to isolate the simian homologue of the above-noted human Pigmy HTLV from Pigmy chimpanzees.
SUMMARY OF INVENTION
The present invention relates to the isolation and characterization of a newly discovered T-cell
lymphotropic virus, designated STLVpan-p.
The invention also relates to primate cell cultures infected with STLVpan-p.
The invention further relates to an isolated and substantially pure preparation of the proviral DNA of the simian T-cell lymphotropic virus STLVpan-p.
The invention further relates to nucleic acid sequences derived from the genomic RNA, mRNA or proviral DNA of STLVpan-p. It is therefore an object of this
invention to provide synthetic nucleic acid sequences, especially nucleic acid sequences capable of directing production of recombinant STLVpan-p proteins, as well as equivalent natural nucleic acids sequences. Such nucleic acid sequences may be isolated from a cDNA library prepared from RNA or DNA isolated from STLVpan-p or from STLVpan-p infected cells, from proviral DNA segments by restriction digestion, by PCR amplification of RNA isolated from STLVpan-p infected cells or by PCR
amplification of cloned proviral DNA in a plasmid. For purposes of this application nucleic acid sequence refers to RNA, DNA, cDNA or any synthetic variant thereof which encodes for protein.
The nucleic acid sequences derived from the STLVpan-p genome are useful as probes to isolate naturally occurring variants of the virus in other primates. The nucleic acid sequences of the present invention are also useful as probes to detect the presence of T-cell
lymphotropic viruses in biological samples.
The invention further relates to a method for detection of STLVpan-p viruses in biological samples based on selective amplification of STLVpan-p gene fragments utilizing primers derived from the STLVpan-p genome.
The invention also relates to the use of single stranded antisense poly- or oligonucleotides derived from the STLVpan-p genome which are useful to inhibit the
expression of STLVpan-p genes.
The invention also relates to isolated and substantially purified STLVpan-p proteins and analogs thereof. The invention also relates to the use of the recombinant STLVpan-p proteins as diagnostic agents and as vaccines. Alternatively, STLVpan-p viral particles may be used as diagnostic agents and as immunogens in the
production of anti-STLVpan-p antibodies.
The present invention further encompasses methods of detecting antibodies specific for STLVpan-p viruses in biological samples. Such methods are useful for diagnosis of infection and disease caused by STLVpan-p viruses and for monitoring the progression of such disease. Such methods are also useful for monitoring the efficacy of therapeutic agents during the course of treatment of STLVpan-p virus infection in a primate.
The present invention also relates to antibodies to STLVpan-p or parts thereof and to the use of these antibodies in immunoassays to detect STLVpan-p viruses in biological samples or as antiviral agents.
The invention also relates to pharmaceutical compositions useful in the prevention and treatment of STLVpan-p virus infection in mammals.
The invention further relates to cell culture systems and animal model systems infected with STLVpan-p and the use of these systems to screen antiviral agents.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows a Western blot analysis of sera from 12 chimpanzees.
Figure 2 shows a genealogical tree and serological profile of the Pigmy chimpanzee colony from which STLVpan-p was isolated.
Figures 3A-3C show electron micrographs of cells from primate cocultures.
Figure 4 shows polyacrylamide gel
electrophoresis of 35S-labelled protein.
Figures 5A and 5B show Southern blot analyses of cellular proviral DNA isolated from STLVpan-p -infected cells.
DETAILED DESCRIPTION OF THE FIGURES
Figure 1 shows the results of Western blot analysis of sera from 12 chimpanzees in the Pan Paniscus colony. Sera from each animal and from two positive controls, strong reactive HTLV-I and -II, were tested at a 1:100 dilution using Western blot strips from Cellular Products (Buffalo, NY). The antigens present on the
Western blot strips are indicated on the righthand side of Figure 1 where MTA-1 and K55 are envelope proteins specific for HTLV-I and II strains, respectively; p24 and rgp21 are gag and envelope proteins common to both HTLV-I and HTLV-II strains and p19 is a major gag protein specific for HTLV-I. The animal designations for the 12 chimpanzees and the designations for the two positive controls are shown at the top of Figure 1.
Figure 2 shows a genealogical tree and
serological profile of the Pan Paniscus colony from which STLVpan-p was isolated. The seropositive animals are represented with their gender symbol blackened. The asterisk indicates animal sera that have not been tested because of unavailability. The numbers included in the animal designation represent the estimated birth date for the wild born animals (LI-1954, BO-1971, MA-1970, KI-1974 and KI-1950) and the actual birth dates of the remaining animals.
Figures 3A-3C represent electron micrographs of cells from primate cell cultures positive for STLVpan-p.
Figure 3A represents an electron micrograph (60,000 magnification) of typical type C retroviral particles in a cell from coculture L93-79B obtained from animal LA-1967's peripheral blood mononuclear cells (PBMC). Figure 3B shows an electron micrograph (90,000 magnification) of an immature virion present in a cell from coculture L93-79C originated from animal JI-1985. Figure 3C shows an electron micrograph (90,000 magnification) of typical budding from type C retroviruses in a cell obtained from a transient coculture of L93-79C with the human B cell line B-JAB.
Figure 4 shows a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of 35S- labelled proteins immunoprecipitated from the cell lines L93-79C, MT-2 (HTLV-I-infected) and MO-T (HTLV-II-infected) using the following sera: 1: serum from animal LI-1954, 2: serum from animal ZA-1984, 3: serum from one seronegative (on Western blot analysis) orangutan, 4 and 5: sera from HTLV-I- and HTLV-II-infected individuals, respectively. p24 gag protein is indicated by an arrow.
Figures 5A and 5B shows the results of Southern blot analysis of DNA isolated from STLVpan-p-infected cell lines. Twenty micrograms of DNA from the cell lines L93-79A, B and C, the HTLV-I- and -II-infected cell lines C91/PL and MO-T, respectively and the uninfected T-cell line H9, were cleaved with the Pst-1 endonuclease, transferred to nitrocellulose and duplicate filters hybridized with probes specific to HTLV-I (Figure 5B) or HTLV-II (Figure 5A). The size of the internal Pst-1 fragments for HTLV-I and -II are indicated by arrows on the right-hand side of Figures 5A and 5B.
DETAILED DESCRIPTION OF INVENTION
The present invention relates to the isolation and characterization of a novel primate T-cell
lymphotropic virus designated STLVpan-p. As used throughout the specification and claims, "STLVpan-p" means a primate T-cell lymphotropic virus characterized by having a genomic nucleic acid sequence, wherein the genomic nucleic acid sequence includes a nucleic acid sequence
"substantially homologous" to the nucleic acid sequence shown in SEQ ID NO: 7. By "substantially homologous" is meant at least 80% homologous, more preferably, greater than 90% homologous, and most preferably, greater than 95% homologous than SEQ ID NO: 7. STLVpan-p was isolated from infected Pigmy chimpanzees. In brief, peripheral blood mononuclear cells (PBMC) were prepared from heparinized blood obtained from the infected Pigmy chimpanzees and cocultured with human cord blood leukocytes. Five
independent cocultures were found positive for virus expression as determined by the detection via a p24 antigen capture assay of antigens cross-reacting with HTLV-I/HTLV-II p24 antigen in the supernatant of these cocultures. Three of the cocultures positive for virus expression were designated L93-79A, L93-79B and L93-79C, and L93-79C was deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 on April 13, 1994 and has ATCC accession number CRL 11615. Alternative methods of detecting virus expression include, but are not limited to, radioimmune or immunoblot analysis to detect other T-cell lymphotropic viral antigens such as gag, pol and env; electron microscopy; various nucleic acid hybridization techniques such as Southern blot analysis or PCR amplification, and assays for reverse transcriptase activity (Poiesz, B.J. et al. (1980) Proc. Natl. Acad. Sci. U.S.A.. 77:7415-7419).
STLVpan-p viral particles may be isolated from cell cultures infected with STLVpan-p or from sera or lymphoid tissues obtained from STLVpan-p- infected individuals by any of the methods known in the art,
including, for example, techniques based on size
discrimination, such as sedimentation or exclusion
methods, or techniques based on density, such as
ultracentrifugation in density gradients, or precipitation with agents such as polyethylene glycol, or chromatography on a variety of materials such as anionic or cationic exchange materials, and materials which bond due to
hydrophobicity, as well as affinity columns.
In one embodiment, STLVpan-p viral particles are isolated from cell cultures infected with STLVpan-p by
expanding the cultures and then subjecting the culture supernatants to ultracentrifugation in a sucrose density gradient. (Poiesz et al. (1980). The STLVpan-p viral particles are found at a density of about 1.170 g/ml.
During this isolation procedure, the presence of STLVpan-p may be detected by hybridization analysis of the extracted genome using probes derived from the STLVpan-p sequence described herein or by immunoassay utilizing as probes antibodies directed against antigens encoded by the STLVpan-p genome. For detection of STLVpan-p by nucleic acid hybridization, fractions obtained in the purification are treated under conditions which would cause the disruption of viral particles, for example, with detergents in the presence of chaotropic agents, and the presence of viral nucleic acid is determined by hybridization techniques. The viral particles from the purified preparations may be utilized as antigens in diagnostic immunoassays or as immunogens in the production of antibodies.
The STLVpan-p virus may be further characterized via the isolation of RNA from purified STLVpan-p viral particles. Alternatively, STLVpan-p proviral DNA may be isolated from cell cultures infected with STLVpan-p or from sera or tissues obtained from STLVpan-p infected
individuals. Methods for isolating RNA or DNA from such samples are known to those skilled in the art and include, but are not limited to, use of phenol chloroform
extraction. In one embodiment, proviral DNA may be isolated from STLVpan-p infected cells as described in present Example 2. Once obtained, the STLVpan-p RNA or proviral DNA may be characterized as unique from the RNA or proviral DNA of previously isolated T-cell lymphotropic viruses by a variety of approaches.
For example, the STLVpan-p proviral DNA can be subjected to Southern blot analysis wherein DNA is cleaved by a restriction enzyme or enzymes to yield a restriction pattern that is unique for STLVpan-p. Restriction enzymes capable of generating such a unique restriction fragment pattern include, but are not limited to, Pst-1. An example of Southern blot analysis following Pst-1 cleavage of proviral DNA is described in Example 4 of the present specification.
Alternatively, the STLVpan-p DNA of the present invention can be characterized by polymerase chain reaction analysis using specific primer sequences which are capable of amplifying STLV-I as well as all three of the known HTLV-I clades but not STLVpan-p. Examples of such primers include, but are not limited to, sequences shown below as SEQ ID NO:1
TTTGAGCGGC CGCTCAAGCT ATAGTCTCCT CCCCCTG
and SEQ ID NO:2
ACTTAGAATT CGGGAGGTGT CGTAGCTGAC GGAGG.
In yet another embodiment, the DNA of the present invention can be characterized by PCR analysis using oligonucleotide primers which are incapable of amplifying STLVpan-p sequence but can amplify either HTLV-I or HTLV-II sequence. Examples of such primer pairs include, but are not limited to, sequences shown below as
SEQ ID NO: 3
CCCTACAATC CCACCAGCTC AG
and SEQ ID NO: 4
GTACTTTACT GACAAAACCC GACCTAC
which amplify HTLV-I sequences and SEQ ID NO: 5
GTGGTGGATT TGCCATCGGG TTTT
and SEQ ID NO: 6
TCATGAACCC CAGTGGTAA which amplify HTLV-II sequences. It would be understood by those skilled in the art that such PCR analysis could be carried out on viral RNA isolated from purified viral particles, from cell cultures infected by STLVpan-p or from the sera or tissues obtained from STLVpan-p-infected
individuals. Methods of isolating viral RNA are known to those skilled in the art and include a variety of
extraction methods such as extraction by differential precipitation, extraction by organic solvents and
extraction with strong denaturants such as guanidine thiocyanate .
In another embodiment, the STLVpan-p proviral DNA can be sequenced and its sequence compared with the nucleic acid sequence of other known T-cell lymphotropic viruses to demonstrate that STLVpan-p viral sequence is distinct from the sequences of previously identified
T-cell lymphotropic viruses. A partial nucleic acid sequence of STLVpan-p is set forth below as SEQ ID NO: 7.
ATGGGGTCCC AGGTGAGCTG GTGCTCTGGG CAGGTGGCGA GAAGGGCGTT CCGGTGCAGG CGGGTGGAAC AAAGCCCACC TGAAACGGGA CACCAATCGG CTTGAACACA ATCGCTAAAC AC.
The abbreviations used for the nucleotides are those standardly used in the art.
The sequence information shown in SEQ ID NO:7 is useful for the design of probes for the isolation of additional sequences derived from other regions of the STLVpan-p genome. For example, probes containing a sequence of approximately eight or more nucleotides, preferably twenty or more nucleotides, which were derived from regions close to the 5'-termini or 3'-termini of the sequence shown in SEQ ID NO: 7 may be used to isolate overlapping DNA sequences of the STLVpan-p genome. Probes can then be derived from the 5 ' or 3' termini of these overlapping DNA sequences in order to isolate additional sequence from the STLVpan-p genome. This approach known as "genome walking" is known to those skilled in the art. Alternatively, it is known that the T-cell lymphotropic viruses have regions of conserved nucleic acid sequences and nucleic acid probes containing these conserved
sequences may be used to design primers for use in systems which amplify the genome sequences of STLVpan-p. Thus, the information provided herein makes it possible to clone and sequence the STLVpan-p genome. The sequence of the 5' region of STLVpan-p (STL gag/pol), spanning the complete 5'
LTR (U3, R, U5), the entire gag, protease and remaining portion of the polymerase genes is shown below as SEQ ID NO: 8
TGACAGTGAC TCTGACCCTG GGCCTTCTAG CCTCGGGGCC 40
CCCAGGGCGA GTCATCAGCT TAAAGGTCAC GCTGTCTCAC 80
ACAAACAATC CCGGGAACAG GCTCTGACGT TTCCCCCTGC 120
AGACCATTTG AGGAACCAGG AACCAGTCTC CAGAAAAATA 160
ACCTCACCCT TACCCACTTC CCCTTGCCTT GAAAAACAAA 200
GGCTCTGACG ACTACCCCCC CCACCCATAA AATTTGCCTA 240
CTCAATAAAG CCCAGGCCTA TAAAAGCGCA AGGACGGTTC 280
AGGAGGGGGT CACCTTCTTT CCACCTGCCC GCTGTGCCTA 320
CCTTGGAGAT CCATTCCGCC CGGGGCCTCG GTCGAGACGT 360
CTCGAGAAAA GCTCCGTCTC CCGTTCCAGA CACTCTGAAC 400
CGCGCCTTTC AGGGTAAGTC TCCCCCCGGT TGAGCTGGGC 440
TACGACTCCC GTAGTCGCTC CCGCAGTCGG TTGAGGCTCC 480
CTGACCCTCC GGCCGCGCAC GTTAGGCTCT GGTTTGTAAC 520
CCTACTTCCG CGTTCTTGTC TTATTCTGCG CCGAAACCGA 560
AAGCACAGCG CCTCTGGGTG CCACGCTGGC CCGGGGCCAG 600 CATCCTGTCC AGGGACGCGC GCCTACTGAA CCCGAAGGCG 640
CCTCAGGCCT CTCCGGGAGG GGCACTCGGC TCGGGCCCTT 680
GTTCTCTCCG GGAGAGACAA ACAAGTGGGG GCTCGTCCGG 720
GGATACCTAC CCCTGCCCTG TCGCATTATG GGACAAACCT 760
ACGGCCTCTC GCCTAGCCCA ATCCCCAAGG CCCCCAGAGG 800
TTTATCAACC CACCACTGGT TAAATTTCCT CCAAGCCTCC 840
TACCGGCTAC AACCTGGGCC CTCCGACTTT GATTTTCAGC 880
AACTACGTCG TTTCCTGAAA CTAGCTCTTA AAACACCTAT 920
TTGGTTAAAT CCAATCGATT ACTCCCTCCT GGCCAGCCTC 960
ATCCCCAAGG GTTACCCCGG GCGGACAAGC GAGATTATTA 1000
ATGTGTTAAT TAGAAATCAA GCGTCCCCCA CCCCGCCTCC 1040
TGCCCCGTCT CTACCCGAAC CGGCTAACCC ACCGCCCCTC 1080
CAGCAGCCCT CGGCTCCTCC GGAACCCCAT ACGCCCCCCC 1120
CCTATATAGA GCCTCCCGCT ACCCATTGCC TTCCCATACT 1160 ACATCCACAT GGGGCTCCCT CGGCTCACAG GCCATGGCAA 1200
ATGAAAGACC TGCAGGCCAT CAAACAGGAG GTCAATACCT 1240
CGGCCCCTGG GAGTCCCCAG TTCATGCAAA CAGTCCGGCT 1280
CGCAATTCAG CAATTCGACC CCACGGCCAA AGACTTACAA 1320
GATCTCTTGC AGTACCTCTG CTCCTCCCTA GTCGTCTCCC 1360
TTCACCATCA ACAACTCCAT ACCCTAATTA CCGAGGCTGA 1400
AACCAGGGGA ATGACAGGTT ATAATCCCAT GGCCGGACCC 1440
CTAAGGATGC AGGCCAACAA CCCCGCCCAG GAAGGACTCC 1480
GGAGGGAATA CCAAAACCTC TGGCTGGCAG CCTTTTCGGC 1520
TCTGCCAGGG AACACCCGCG ACCCATCTTG GGCGGCAATC 1560
TTGCAAGGGC TAGAAGAACC CTATTGCGCC TTGCTAGAGC 1600
GCCTTAATGT TGCTCTTGAC AACGGTCTCC CCGAGGGAAC 1640
CCCAAAGGAG CCCATCTTGC GCTCCCTGGC TTACTCCAAC 1680
GCCAACAAAG ACTGCCAAAA ACTGCTGCAG GCTCGGGGCC 1720
ATACTAATAG CCCCTTGGGA GACATGCTCC GAGCCTGTCA 1760
AGCATGGACA CCCAAGGACA AAGCCCGGGT CCTCGTCGTC 1800
CAATCACGAA AGCCCCCGCC CACGCAGCCC CTGTTCCGCT 1840
GTGGAAAGGC AGGGCATTGG AGCCGAGACT GCACCCTCCC 1880
ACGCCCCCCC CCTGGTCCAT GCCCCTTGTG CAAAGACCCT 1920
TCCCATTGGA AACGAGATTG TCCACAGCTC AAACCCCCCC 1960
CGACGGAGGA GGAACCCCTC CTGCTGGACC TGCCCTCAGA 2000
TGCCGTTGCC ACCGAGGAAA AAAACTCCCT AGGGGGGGAG 2040
ATCTAATCTC CCCCCAACAA ATCTCACTGC TCCCTCTCAT 2080
TCCTTTAGAG CAACAGCAGC AGCCCCTTCT AGACGTTCAG 2120
GTCTCCATTG CAGGCGCCCC CCCTCGACCT ACCCAGGCAC 2160
TCTTAGACAC AGGGGCTGAT CTCACCGTCC TGCCCCAGGC 2200
CCTTGCCCCC GAGTCAGAAG GTCTCTGACA CAACGGTTCT 2240
AGGCGCTGGC GGTCAGACCA GCTCCCAGTT CAAACTCCTC 2280
CGATCCCCCC TGTGTGTCTA CCTGCCCTTC CGGAAGGCCC 2320
CCGTTACTCT CCCGTCATGC CTTCTAGACA CCGATAATAA 2360
ATGGGCCATT ATTGGCCGTG ATATCCTCCA GCAATGCCAG 2400
AGTGTCCTGT ACCTCCCGGA GGACAATCTC TGCGAAGGTA 2440
CCCCCCGGCC CTCCGGCAGA ATGAATTCCC CCCGACTATT 2480
ACCCGTGGCC ACCCCCAGTG TCATCGGCCT TGAGCACTTC 2520
CCACCACCCC CACAGATAGA TCAGTTCCCC TTTAAACCTG 2560
AGCGCCTCCA GGCCTTGACT GACCTGGTCT CCAAGGCCCT 2600
GGAGGCTAGC TACATTGAAC CTTACTCTGG ACCAGGCAAC 2640
AACCCTGTCT TTCCGGTCAA AAAACCAAAC GGCAAGTGGA 2680 GATTTATTCA TGACCTGAGG GCCACCAATG CCATCACGAC 2720
CACCTTGGCC TCCCCGTCCC CCGGACCTCC CGATCTCACC 2760
AGCTTATCAA CAGCCCTCCC ATATGTGCAA ACTATAGACC 2800
TTGCTGACGC CTTCTTCCAA ATTCCCCTTC CAAAACAGTT 2840
CCAGCCATAC TTCGCCTTCA CTATCCCCCA GCCATGCAAT 2880
TATGGCCCCG GGGCTCGGTA TGCCTGGACT GTCCTTCCAC 2920
AAGGATTCAA AAATAGTCCC ACCCTGTTTG AGCAACAGCT 2960
GGCTGCCATC CTTTCCCCTA TCAGGAAAGC CTTCCCTACG 3000
TCCACTATCA TCCAATACAT GGATGATATA CTCTTGGCCA 3040
GTCCTGCGCA GGGGGAACTG CAACAACTCT CCAAGATGAC 3080
CCTCCAAGCG CTAGTCACCC ACGGCCTTCC AGTCTCCCAG 3120
GCCCCCCGGG AACAAACCCC TGGGCAAATA CGTTTCTTAG 3160
GTCAAGTTAT ATCTCCTGAC CATATTACCT ATGAAACCAC 3200
CCCCACCATT CCTATGAAAT CCCAGTGGAC TCTCGCTGAA 3240
CTACAGACTG TGCTAGGAGA GATCCAATGG GTCTCAAAAG 3280
GAACCCCCAT CCTCCGCAAA CACCTGCAGT GTTTATACTC 3320
CGCCCTTCGC GGATATCAGG ACCCCAGGGC ACACCTCCTC 3360
CTCCAGAAAC AACAGCTCCA TGCCCTCCAT GCCATCCAAC 3400
AGGCCTTACA GCACAATTGC CGCAGCCGTC TCAATCCTGC 3440
CTTGCCCATC CTGGGACTTA TCTCCTTGAG CTCGTCTGGT 3480
ACAACCTCCG TCCTCTTCCA AGCCCGGCAG CGATGGCCCC 3520
TGGTTTGGTT GCACACCCCC CATCCACCAA CCAGCCTTGG 3560
CCCCTGGGGT CACTTACTGG CCTGCACTAT CCTGACCTTA 3600
GACAAATACT CCCTTCAGCA CTATGGCCGG CTGTGCCAGT 3640
CCTTGGAGGA CAACATGTCC AACACAGCCC TCCACGATTT 3680
TGTGAAAAAC TCCCCCCACC CGAGGGTAGG CATCCTTATC 3720
CATCACATGA GCCGCTTTCA TAACCTCGGT AGTCAGCCAT 3760
CTGGCCCTTG GAAGGCTCTC TTACACCTTC CCGCTCTCCT 3800
CCAAGCGGCA CGGCTCCTCC GACCTCTCTT CACGCTATCT 3840
CCCGTGGTCT TAACAACGCA CCCTGTCTCT TCTCCGACGG 3880
GTCTTCCCAA AAAGGCGGCA TACGTACTCT GGGACCAGAC 3920
CATCCTCCGC CATGACTCTA TCACATTGCC CCCCCATGGC 3960
AGCAACTCAG CTCAAAGAGG AGAGCTCCTC GCCCTCCTTT 4000
CAGGACTCCG GGCGGCCAAG TCATGGCCAT CCCTAAATAT 4040
TTTTCTTGAC TCCAAGTACT TAATTAAATA TCTCCATTCC 4080
CTTGCCACTG GAGCCTTTCT TGGGACTTCG ACTCACCAGT 4120
CCCTCTATGC CCATTTGCCT GCCCTATTGC ATAACAAAGT 4160
TATCTACCTC CACCACATTC GTAGCCACAC CAATCTCCCC 4200 GACCCCATCT CCACTCTCAA CGAATACACA GACTCGCTTA 4240
TTATTATAGC CCCCCTCATT CCCCTAACGC CTCAGGATCT 4280
GCACAGACTT ACCCATTGCA ACTCCAGGGG CCCTTGTTTC 4320
TTCCGGGCCA CCCCACAGCA GAGCCAAGTC GCTCTTGAAA 4360
GCCGTGTTAC ACCTGCAACA TCACTAAACT CACAACATCA 4400
CATGCCTCAA GGGCACATCC GTCGGGGGTT GCTGCCCAAC 4440
CACATATGGC AAGGAGATGT AACCCACTAC AAATACAAGC 4480
GGCACCGATA CTGCCTGCAC GTCTGGGTTG ATACCTTCTC 4520
CAACGCAGTC TCCATTACCT GCAAAACAAA AGAGACTAGC 4560
TCTGAGACTG TCAGCGCCCT CTTGCACGCT ATCACTATCT 4600
TAGGCAAGCC CCTTTCTATA AACACTGACA ATGGTTCTGC 4640
CTTCCTATCT CAAGAATTCC AGGCCTTTTG TACCTCATGG 4680
CACATCAGAC ATTCCACCCA TGTGCCCTAC AACCCTACCA 4720
GCTCCGGGCT GGTAGAGAGA ACCAATGGCA TTGTCAAGGC 4760
CCTCCTCAAC AAATATCTCC TAGACAGCCC GAACCTCCCC 4800
CTGGACAATG CCATCAGTAA ATCCCTATGG ACCCTAAACC 4840
AACTCAATGT CATGTCCCCC AGTGGAAAAA CCCGCTGGCA 4880
ACTCCATCAT GGCCCCCGGT TGCCCCCATC CCTGCAAATA 4920
CCTCAGCCCT CTAAGGCACC CGCGAACTGG TACTATGTAC 4960
TAACTCCTGG TCTTACCAAT CAGCGGTGGA AAGGACCTTT 5000
ACATCTCTCC AGGAAGCTGC AGGAGCGCTT ACTTTCGATA 5040
GACGGCTCCC CTCAGTGGAT CCCGTGGCGG CTCCTGAAAA 5080
AGACTGTATG CCCAAGGCCA GACGGCAGCG AACTCGCCGC 5120
GGACAACGCA GCAACAGACC ACCAACACCA TGG 5153 and sequence from the 3' region (STL tax LTR) encompassing the tax/rex gene and 3' LTR is shown below as SEQ ID NO: 9
CCCACTTTCC AGGTTTTGGA CAGAGCCTCC TCTATGGATA 40
CCCAGTCTAC GTGTTTGGCG ATTGTGTTCA AGCCGATTGG 80
TGTCCCGTTT CAGGTGGGCT TTGTTCCACC CGCCTGCACC 120
GGAACGCCCT TCTCGCCACC TGCCCAGAGC ACCAGCTCAC 160
CTGGGACCCC ATCGATGGAC GGGTTGTCGG CTCTCGTCTC 200
CAATACCTTA TCCCTCGACT CCCCTCCTTC CCCACCCAGA 240
GAACCTCCAA GACCCTTAAG GTCCTCACTC CCCCTACCAC 280
TCCTGTCTCC CCCAAGATTC CACCGCCTTC TTCCCAATCA 320
ATGCGGAGGC TCTCCCCTTA CCGGAACGGT TGCCTGCATC 360
CAACTCTCGG AGATCAGCTC CCCTCCCTTT CCTTCCCCGA 400 CCCCGGTGTC CGACCCCAAA ACATCTACAC CACCTGGGGA 440
AGAACTGTAG TTTGCTTGTA CCTCTACCAA CTATCCCCCC 480 CGATGACCTG GCCCCTAATA CCTCATGTCA TATTCTGCCA 520
TCCCAAGCAG TTAGGAACCT TCCTGACCAA CGTACCTCTA 560
AAACGCCTGG AAGAATTACT ATACAAAATA TTCCTACACA 600
CCGGGGCCAT CATAGTTCTC CCCGAAGACA GGGTGGGTAC 640
TACATTGTTC CAACCTGTTA GAGCCCCCTG CGTCCAGACC 680
GCCTGGGATA CAGGGCTTCT ACCCTACCAC TCTCTCATAA 720
CTACTCCGGG CCTAATATGG ACATTTAATG ACGGTTCACC 760
CATGATTTCC GGCCCCTGCC CCAAAACAGG GCAGCCATCT 800
TTCCTAGTAC AGTCCTCCCT GTTAATCTTT GAAAAATTCC 840
AGACCAAGGC CTTTCATCCT TCCTTCCTAC TGTCCCATCA 880
ACTCATCCAG TACTCTTCAT TCCAGTACTC TTCATTCCAT 920
AACCTCCACC CTTCCTTCGA GGAATACACT AACATCCCAG 960
TTTCCTATTT CTTTAACGAA AAACAGGCAG ATGACAGTGA 1000
CTCTGACCCT GGGCCTTCTA GCCTCGGGGC CCCCAGGGCG 1040
AGTCATCAGC TTAAAGGTCA CGCTGTCTCA CACAAACAAT 1080
CCCGGGAACA GGCTCTGACG TTTCCCCCTG CAGACCATTT 1120
GAGGAACCAG GAACCAGTCT CCAGAAAAAT AACCTCACCC 1160
TTACCCACTT CCCCTTGCCT TGAAAAACAA AGGCTCTGAC 1200
GACTACCCCC CCCACCCATA AAATTTGCCT ACTCAATAAA 1240
GCCCAGGCCT ATAAAAGCGC AAGGACGGTT CAGGAGGGGG 1280
TCACCTTCTT TCCACCTGCC CGCTGTGCCT ACCTTGGAGA 1320
TCCATTCCGC CCGGGGCCTC GGTCGAGACG TCTCGAGAAA 1360
AGCTCCGTCT CCCGTTCCAG ACACTCTGAA CCGCGCCTTT 1400
CAGGGTAAGT CTCCCCCCGG TTGAGCTGGG CTACGACTCC 1440
CGTAGTCGCT CCCGCAGTCG GTTGAGGCTC CCTGACCCTC 1480
CGGCCGCGCA CGTTAGGCTC TGGTTTGTAA CCCTACTTCC 1520
GCGTTCTTGT CTTATTCTGC GCCGAAACCG AAAGCACAGC 1560
GCCTCTGGGT GCCACGCTGG CCCGGGGCCA GCATCCTGTC 1600
CAGGGACGCG CGCCTACTGA ACCCGAAGGC GCCTCAGGCC 1640
TCTCCGGGAG GGGCACTCGG CTCGGGCCCT TGTTCTCTCC 1680
GGGAGAGACA AACA 1694 where nucleotides 55-172 of SEQ ID NO: 9 correspond to the nucleotide sequence shown in SEQ ID NO: 7.
The information provided in the present application also enables the isolation of viral homologues of STLVpan-p from other primates such as humans. For example, a radioimmunoprecipitation assay may be used to immunoprecipitate virus or viral proteins from sera obtained from human patients exhibiting HTLV-I/HTLV-II indeterminant seropositivity. HTLV-I/HTLV-II
indeterminate seropositivity can be determined by Western blotting to detect antigens specific to HTLV-I and/or HTLV-II. Alternatively, the patient's sera can be screened using immunoassays containing STLVpan-p or parts thereof as antigens.
In another embodiment, nucleic acid sequences of viral homologues of STLVpan-p may be isolated from tissues or serum obtained from infected individuals via
amplification by methods such as polymerase chain
reaction. Examples of tissues from which the virus may be isolated include, but are not limited to, peripheral blood leukocytes, lymph nodes and spleen. Primers which may be used in PCR amplification of STLVpan-p variants in other primates may be derived from the proviral DNA of STLVpan-p. Alternatively, the primers may be derived from regions of conserved nucleic acid sequences found within the family of T-cell lymphotropic viruses.
In yet another embodiment, viral homologues of STLVpan-p may be isolated from infected individuals using antibodies raised to purified STLVpan-p viral particles, to proteins purified from these particles, to recombinant proteins produced from STLVpan-p nucleic acid sequences, or to chemically synthesized STLVpan-p proteins.
The nucleic acid sequence information derived from STLVpan-p and its viral homologues can be aligned with sequences from known T-cell lymphotropic viruses to determine areas of homology and heterogeneity within the viral genome (s) which could indicate the presence of different strains of the genome. In addition, computer alignment of the STLVpan-p sequences with sequences of known T-cell lymphotropic viruses such as HTLV-I, HTLV-II and STLV-I can be used to identify regions of inter-strain homology and non-homology which would be useful in the design of strain-common and strain-specific
oligonucleotide and peptide probes useful in detecting STLVpan-p antigens or nucleic acids in biological samples. An example of a computer program useful for carrying out such sequence alignments is GENALIGN (Intelligenetics, Inc., Mountainview, CA).
Once obtained, the nucleic acid sequences of the present invention may be inserted into a suitable
expression vector by methods known to those skilled in the art. By suitable expression vector is meant a vector that is capable of carrying and expressing a nucleic acid sequence coding for protein. In a preferred embodiment, the nucleic acid sequence encodes at least a single open-reading frame of a known T-cell lymphotropic gene. Such genes include, but are not limited to, gag, pol, env, tax and rex.
Suitable expression vectors for the present invention include any vectors into which a nucleic acid sequence as described above can be inserted, along with any preferred or required operational elements, and which vector can then be subsequently transferred into a host organism and replicated in such organism. Preferred vectors are those whose restriction sites have been well documented and which contain the operational elements preferred or required for transcription of the nucleic acid sequence.
The "operational elements" as discussed herein include at least one promoter, at least one operator, at least one leader sequence, at least one determinant, at least one terminator codon, and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the inserted nucleic acid sequence. In particular, it is contemplated that such vectors will contain at least one origin of replication recognized by the host organism along with at least one selectable marker and at least one promoter sequence capable of initiating transcription of the nucleic acid sequence.
To construct the cloning vector of the present invention, it should additionally be noted that multiple copies of the nucleic acid sequence encoding STLVpan-p protein(s) and its attendant operational elements may be inserted into each vector. In such an embodiment, the host organism would produce greater amounts per vector of the desired protein(s). In a similar fashion, multiple different proteins may be expressed from a single vector by inserting into the vector a copy (or copies) of nucleic acid sequence encoding each protein and its attendant operational elements. In yet another embodiment, a polycistronic vector in which multiple proteins (either identical in sequence or different) may be expressed from a single vector is created by placing expression of each protein under the control of an internal ribosomal entry site (IRES) (Molla A. et al. Nature, 356:255-257 (1992); Jang S.K. et al. J. Virol., 263:1651-1660(1989)). The number of multiple copies of the nucleic acid sequence encoding protein which can be inserted into the vector is limited only by the ability of the resultant vector, due to its size, to be transferred into, and replicated and transcribed in, an appropriate host organism.
Expression vectors suitable for the present method include those vectors capable of producing high efficiency gene transfer in vivo. Such vectors include but are not limited to retroviral, adenoviral and vaccinia viral vectors. Operational elements of such expression vectors are disclosed previously in the present
specification and are known to those skilled in the art. Preferred vectors are attenuated vaccinia or pox virus vectors. An expression vector containing nucleic acid sequence capable of directing host cell synthesis of STLVpan-p protein can be administered in a pure or
substantially pure form or as a complex with a substance having affinity for nucleic acid and an internalizing factor bound to the substance having affinity for nucleic acid. (Wu G. et al. J. Biol. Chem. 262:4429-4432 (1987); Wagner E. et al. Proc. Natl. Acad. Sci. USA. 87:3655-3659
(1990)). A preferred substance having affinity for nucleic acid is a polycation such as polylysine.
Internalizing factors include, but are not limited to, antibodies to T-cell markers.
The present invention also relates to methods for detecting the presence of STLVpan-p viruses in primates.
In one embodiment of the invention, the method for detecting the presence of STLVpan-p viruses comprises analyzing the DNA of a primate subject for the presence of a STLVpan-p nucleic acid sequence. For analysis of the DNA, a biological specimen is obtained from the subjects.
Examples of biological specimens that can be obtained for use in the present method include, but are not limited to, whole or heparinized blood, tissue biopsies such as lymph nodes and spleen or other samples normally tested in the diagnosis of T-cell lymphotropic virus infection.
Preferred biological specimens are peripheral blood leukocytes and lymphoid tissues.
Although it is not always required, it is preferable to at least partially purify DNA from the biological specimen prior to analysis. For example, after disruption of cells in a specimen, nucleic acid can be extracted from contaminating cell debris and other protein substances by extraction of the sample with phenol. In phenol extraction, the aqueous sample is mixed with an approximately equal volume of redistilled phenol and centrifuged to separate the two phases. The aqueous containing nucleic acid is removed and precipitated with ethanol to yield nucleic acid free of phenol. Alternative methods of purifying DNA from biological samples are disclosed in Sambrook et al. ((1989) in "Molecular
Cloning, A Laboratory Manual", Cold Spring Harbor Press, Plainview, NY).
Methods for analyzing the DNA for the presence of STLVpan-p viral nucleic acid sequences include, but are not limited to, Southern blotting after digestion with the appropriate restriction enzymes (restriction fragment length polymorphism, RFLP) (Botstein, D., Amer. J. Hum. Genet., (1980) 69:201-205), oligonucleotide hybridization (Conner, R. et al., EMBO J., (1984) 3:13321-1326), RNase A digestion of a duplex between a probe RNA and the target DNA (Winter, E. et al., Proc. Natl. Acad. Sci. U.S.A.,
(1985) 82:7575-7579), polymerase chain reaction (PCR) (Saiki, P.K. et al., Science, (1988) 239:487-491; U.S.
Patents 4,683,195 and 4,683,202) and ligase chain reaction (LCR) (European Patent Application Nos. 0,320,308 and 0,439,182).
In one embodiment, DNA is analyzed by Southern blotting following digestion with one or more restriction enzymes. The restriction enzymes to be used in the present invention are those enzymes for whom the presence or absence of their recognition site is linked to STLVpan-p virus expression. Preferred restriction enzyme include, but are not limited to, Pst-1. Following restriction digestion, resultant DNA fragments are separated by gel electrophoresis and the fragments are detected by
hybridization with a labelled nucleic acid probe
(Southern, E.M. J. Mol. Biol., (1975) 98:503-517).
The nucleic acid sequence used as a probe in Southern analysis can be labelled in single-stranded or double-stranded form. Labelling of the nucleic acid sequence can be carried out by techniques known to one skilled in the art. Such labelling techniques can include radiolabels and enzymes (Sambrook, J. et al. (1989) in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, Plainview, New York). In addition, there are known non-radioactive techniques for signal
amplification including methods for attaching chemical moieties to pyrimidine and purine rings (Dale, R.N.K. et al. (1973) Proc. Natl. Acad. Sci., 70:2238-2242; Heck, R.F. 1968) S. Am. Chem. Soc., 90:5518-5523), methods which allow detection by chemiluminescence (Barton, S.K. et al. (1992) J. Am. Chem. Soc., 114:8736-8740) and methods utilizing biotinylated nucleic acid probes (Johnson, T. K. et al. (1983) Anal. Biochem., 133:126-131; Erickson, P.F. et al. (1982) J. of Immunology Methods, 51:241-249;
Matthaei, F.S. et al. (1986) Anal. Biochem., 157:123-128) and methods which allow detection by fluorescence using commercially available products. The size of the probe can range from about 50 nucleotides to about several kilobases. The nucleic acid sequences used as a probe in Southern analysis are derived from STLVpan-p genomic nucleic acid sequence. Once the separated DNA fragments are hybridized to the labelled nucleic acid probes, the restriction digest pattern can be visualized by
autoradiography and examined for the presence of a T-cell lymphotropic virus. Amounts of DNA which can be analyzed by Southern blot range from about 5 micrograms to about 50 micrograms. Southern blot analysis of 20 μg of DNA prepared from STLVpan-p-infected cells is described in
Example 4.
In a second preferred embodiment, the DNA is analyzed for the presence of STLVpan-p nucleic acid
sequences by PCR analysis. In this method, each of the pairs of primers selected for use in PCR are designed to hybridize with sequences in the STLVpan-p genome which are an appropriate distance apart (at least about 50
nucleotides) to permit amplification and subsequent detection of the presence of T-cell lymphotropic viruses. Primer pairs which can specifically hybridize to such viral sequences can be derived from a T-cell lymphotropic virus genome containing a region of nucleic acid sequence substantially homologous to the DNA sequence shown in SEQ ID NO: 7. Each primer of a pair is a single-stranded oligonucleotide of about 15 to about 50 bases in length which is complementary to a sequence at the 3' end of one of the strands of a double-stranded target sequence. Each pair comprises two such primers, one of which is
complementary 3' end and the other of which is
complementary to the other 3' end of the target sequence. The target sequence is generally about 100 to about 400 base pairs long but can be as large as 1,000 base pairs. Optimization of the amplification reaction to obtain sufficiently specific hybridization to STLVpan-p virus genomes is well within the skill in the art and is preferably achieved by adjusting the annealing
temperature. The present invention also provides purified and isolated pairs of primers for use in analysis of DNA for the presence of STLVpan-p viruses. Examples of such primers include, but are not limited to, the primers set forth in SEQ ID NOS: 10-12.
The primers of the present invention can be synthesized using any of the known methods of
oligonucleotide synthesis (e.g., the phosphodiester method of Agarwal et al. (1972) Agnew. Chem. Int. Ed. Engl., 11:451, the phosphodiester method of Hsiung et al. (1979) Nucleic Acids Res., 6:1371, or the automated
diethylphosphoramidite method of Beuacage et al. 1981. Tetrahedron Letters, 22:1859-1862), or they can be
isolated fragments of naturally occurring or cloned DNA. In addition, those skilled in the art would be aware that oligonucleotides can be synthesized by automated
instruments sold by a variety of manufacturers or can be commercially custom ordered and prepared. In one
embodiment, the primers can be derivatized to include a detectable label suitable for detecting and/or identifying the primer extension products (e.g., biotin, avidin, or radiolabeled dNTP's), or with a substance which aids in the isolation of the products of amplification (e.g.
biotin or avidin). In a preferred embodiment, the primers are synthetic oligonucleotides.
In an alternative embodiment, primer pairs can be selected to hybridize selectively to STLVpan-p nucleic acid sequences present in primates. The selected primer pairs will hybridize sufficiently specifically to the STLVpan-p sequences such that non-specific hybridization to HTLV-I, HTLV-II and STLV-I sequences will not occur.
Primer pairs which hybridize selectively to STLVpan-p nucleic acid sequences can be used to amplify such sequences present in the DNA of a biological sample. In one embodiment, such primers are derived from STLVpan-p nucleic acid sequences encoding the env genes.
The amplification products of PCR can be detected either directly or indirectly. In one embodiment, direct detection of the amplification products is carried out via labelling of primer pairs. Labels suitable for labelling the primers of the present
invention are known to one skilled in the art and include radioactive labels, biotin, avidin, enzymes and
fluorescent molecules. The derived labels can be
incorporated into the primers prior to performing the amplification reaction. A preferred labelling procedure utilizes radiolabeled ATP and T4 polynucleotide kinase (Sambrook, J. et al. (1989) in "Molecular Cloning, A
Laboratory Manual", Cold Spring Harbor Press, Plainview, NY). Alternatively, the desired label can be incorporated into the primer extension products during the
amplification reaction in the form of one or more labelled dNTPs. In the present invention, the labelled amplified PCR products can be analyzed for the presence of STLVpan-p virus sequences via separating the PCR products by
polyacrylamide gel electrophoresis or via direct
sequencing of the PCR-products.
In yet another embodiment, unlabelled amplification products can be analyzed for the presence of virus nucleic acid sequences via hybridization with
STLVpan-p nucleic acid probes radioactively labelled or, labelled with biotin, in Southern blots or dot blots.
Nucleic acid probes useful in this embodiment are those described earlier for Southern analysis.
The present invention also encompasses methods for detecting the presence of STLVpan-p viruses comprising analyzing the RNA of a primate subject.
For the analysis of RNA by this method, RNA is isolated from blood or tissue biopsies such as lymph nodes and spleen obtained from the subject. The RNA to be analyzed can be isolated by methods known to those skilled in the art. Such methods include extraction of RNA by differential precipitation (Birnboim, H.C. (1988) Nucleic Acids Res., 16:1487-1497), extraction of RNA by organic solvents (Chomczynski, P. et al. (1987) Anal. Biochem.,
162:156-159) and extraction of RNA with strong denaturants (Chirgwin, J.M. et al. (1979) Biochemistry, 18:5294-5299). A preferred method of isolating RNA utilizes guanidine thiocyanate.
The methods for analyzing the RNA for STLVpan-p virus-specific mRNA expression include Northern blotting (Alwine, J.C. et al. (1977) Proc. Natl. Acad. Sci.,
74:5350-5354), dot and slot hybridization (Kafatos, F.C. et al. (1979) Nucleic Acids Res., 7:1541-1522), filter hybridization (Hollander, M.C. et al. (1990)
Biotechniques, 9:174-179), RNase protection (Sambrook, J. et al. (1989) in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, Plainview, NY) and reverse-transcription polymerase chain reaction (RT-PCR) (Watson, J.D. et al. (1992) in "Recombinant DNA" Second Edition, W.H. Freeman and Company, New York).
One preferred method is reverse transcription-polymerase chain reaction (RT-PCR) where the primers used to amplify the cDNA produced via reverse transcription of RNA are derived from STLVpan-p nucleic acid sequences.
These primers can be labelled as described earlier and the RT-PCR products can be analyzed for the presence of
STLVpan-p sequences via polyacrylamide gel electrophoresis of the RT-PCR products or via direct sequencing of the RT-PCR products.
The invention also relates to isolated and substantially purified STLVpan-p proteins and analogs thereof where such proteins are derived from T-cell lymphotropic virus genomes which encode the amino acid sequences shown in SEQ ID NO: 10.
Val Phe Ser Asp Cys Val Gln Ala Asp Trp Cys Pro
Val Ser Gly Gly Leu Cys Ser Thr Arg Leu His Arg
Asn Ala Leu Leu Ala Thr Cys Pro Glu His Gln Leu
Thr Trp Asp Pro;
SEQ ID NO: 11
Cys Leu Ala Ile Val Phe Lys Pro Ile Gly Val Pro
Phe Gln Val Gly Phe Val Pro Pro Ala Cys Thr Gly
Thr Pro Phe Ser Pro Pro Ala Asn Ser Thr Ser Ser
Pro Gly Thr; SEQ ID NO: 12
Met Pro Lys Ala Arg Arg Gln Arg Thr Arg Arg Gly Gln Arg
1 5 10
Ser Asn Arg Pro Pro Thr Pro Trp Pro Thr Phe Gln Val Leu
15 20 25
Asp Arg Ala Ser Ser Met Asp Thr Gln Ser Thr Cys Leu Ala
30 35 40
Ile Val Phe Lys Pro Ile Gly Val Pro Phe Gln Val Gly Phe
45 50 55
Val Pro Pro Ala Cys Thr Gly Thr Pro Phe Ser Pro Pro Ala
60 65 70 Gln Ser Thr Ser Ser Pro Gly Thr Pro Ser Met Asp Gly Leu
75 80
Ser Ala Leu Val Ser Asn Thr Leu Ser Leu Asp Ser Pro Pro
85 90 95
Ser Pro Pro Arg Glu Pro Pro Arg Pro Leu Arg Ser Ser Leu 100 105 100
Pro Leu Pro Leu Leu Ser Pro Pro Arg Phe His Arg Leu Leu
115 120 125
Pro Asn Gln Cys Gly Gly Ser Pro Leu Thr Gly Thr Val Ala
130 135 140
Cys Ile Gln Leu Ser Glu Ile Ser Ser Pro Pro Phe Pro Ser
145 150
Pro Thr Pro Val Ser Asp Pro Lys Thr Ser Thr Pro Pro Gly
155 160 165
Glu Glu Leu;
170
SEQ ID NO: 13
Met Gly Gln Thr Tyr Gly Leu Ser Pro Ser Pro Ile Pro Lys
1 5 10
Ala Pro Arg Gly Leu Ser Thr His His Trp Leu Asn Phe Leu
15 20 25
Gln Ala Ser Tyr Arg Leu Gln Pro Gly Pro Ser Asp Phe Asp
30 35 40
Phe Gln Gln Leu Arg Arg Phe Leu Lys Leu Ala Leu Lys Thr
45 50 55 Pro Ile Trp Leu Asn Pro Ile Asp Tyr Ser Leu Leu Ala Ser
60 65 70
Leu Ile Pro Lys Gly Tyr Pro Gly Arg Thr Ser Glu Ile Ile
75 80
Asn Val Leu Ile Arg Asn Gln Ala Ser Pro Thr Pro Pro Pro
85 90 95
Ala Pro Ser Leu Pro Glu Pro Ala Asn Pro Pro Pro Leu Gln
100 105 110
Gln Pro Ser Ala Pro Pro Glu Pro His Thr Pro Pro Pro Tyr
115 120 125 Ile Glu Pro Pro Ala Thr His Cys Leu Pro Ile Leu His Pro
130 135 140
His Gly Ala Pro Ser Ala His Arg Pro Trp Gln Met Lys Asp
145 150
Leu Gln Ala Ile Lys Gln Glu Val Asn Thr Ser Ala Pro Gly 155 160 165
Ser Pro Gln Phe Met Gln Thr Val Arg Leu Ala Ile Gln Gln
170 175 180
Phe Asp Pro Thr Ala Lys Asp Leu Gln Asp Leu Leu Gln Tyr
185 190 195
Leu Cys Ser Ser Leu Val Val Ser Leu His His Gln Gln Leu
200 205 210
His Thr Leu Ile Thr Glu Ala Glu Thr Arg Gly Met Thr Gly
215 220
Tyr Asn Pro Met Ala Gly Pro Leu Arg Met Gln Ala Asn Asn 225 230 235
Pro Ala Gln Glu Gly Leu Arg Arg Glu Tyr Gln Asn Leu Trp
240 245 250
Leu Ala Ala Phe Ser Ala Leu Pro Gly Asn Thr Arg Asp Pro
255 260 265
Ser Trp Ala Ala Ile Leu Gln Gly Leu Glu Glu Pro Tyr Cys
270 275 280
Ala Leu Leu Glu Arg Leu Asn Val Ala Leu Asp Asn Gly Leu
285 290
Pro Glu Gly Thr Pro Lys Glu Pro Ile Leu Arg Ser Leu Ala 295 300 305
Tyr Ser Asn Ala Asn Lys Asp Cys Gln Lys Leu Leu Gln Ala 310 315 320 Arg Gly His Thr Asn Ser Pro Leu Gly Asp Met Leu Arg Ala
325 330 335
Cys Gln Ala Trp Thr Pro Lys Asp Lys Ala Arg Val Leu Val
340 345 350
Val Gln Ser Arg Lys Pro Pro Pro Thr Gln Pro Leu Phe Arg
355 360
Cys Gly Lys Ala Gly His Trp Ser Arg Asp Cys Thr Leu Pro
365 370 375
Arg Pro Pro Pro Gly Pro Cys Pro Leu Cys Lys Asp Pro Ser 380 385 390
His Trp Lys Arg Asp Cys Pro Gln Leu Lys Pro Pro Pro Thr
395 400 405
Glu Glu Glu Pro Leu Leu Leu Asp Leu Pro Ser Asp Ala Val
410 415 420
Ala Thr Glu Glu Lys Asn Ser Leu Gly Gly Glu Ile,
425 430
SEQ ID NO: 14
Thr Gln Gly Leu Ile Ser Pro Ser Cys Pro Arg Pro Leu Pro
1 5 10
Pro Ser Gln Lys Val Ser Asp Thr Thr Val Leu Gly Ala Gly
15 20 25
Gly Gln Thr Ser Ser Gln Phe Lys Leu Leu Arg Ser Pro Leu
30 35 40
Cys Val Tyr Leu Pro Phe Arg Lys Ala Pro Val Thr Leu Pro
45 50 55
Ser Cys Leu Leu Asp Thr Asp Asn Lys Trp Ala Ile Ile Gly
60 65 70
Arg Asp Ile Leu Gln Gln Cys Gln Ser Val Leu Tyr Leu Pro
75 80
Glu Asp Asn Leu Cys Glu Gly Thr Pro Arg Pro Ser Gly Arg
85 90 95
Met Asn Ser Pro Arg Leu Leu Pro Val Ala Thr Pro Ser Val
100 105 110
Ile Gly Leu Glu His Phe Pro Pro Pro Pro Gln Ile Asp Gln
115 120 125 Phe Pro Phe Lys Pro Glu Arg Leu Gln Ala Leu Thr Asp Leu 130 135 140
Val Ser Lys Ala Leu Glu Ala Ser Tyr Ile Glu Pro Tyr Ser
145 150
Gly Pro Gly Asn Asn Pro Val Phe Pro Val Lys Lys Pro Asn
155 160 165
Gly Lys Trp Arg Phe Ile His Asp Leu Arg Ala Thr Asn Ala
170 175 180
Ile Thr Thr Thr Leu Ala Ser Pro Ser Pro Gly Pro Pro Asp
185 190 195
Leu Thr Ser Leu Ser Thr Ala Leu Pro Tyr Val Gln Thr Ile
200 205 210
Asp Leu Ala Asp Ala Phe Phe Gln Ile Pro Leu Pro Lys Gln
215 220
Phe Gln Pro Tyr Phe Ala Phe Thr Ile Pro Gln Pro Cys Asn
225 230 235
Tyr Gly Pro Gly Ala Arg Tyr Ala Trp Thr Val Leu Pro Gln
240 245 250
Gly Phe Lys Asn Ser Pro Thr Leu Phe Glu Gln Gln Leu Ala
255 260 265
Ala Ile Leu Ser Pro Ile Arg Lys Ala Phe Pro Thr Ser Thr
270 275 280 Ile Ile Gln Tyr Met Asp Asp Ile Leu Leu Ala Ser Pro Ala
285 290
Gln Gly Glu Leu Gln Gln Leu Ser Lys Met Thr Leu Gln Ala
295 300 305
Leu Val Thr His Gly Leu Pro Val Ser Gln Ala Pro Arg Glu
310 315 320
Gln Thr Pro Gly Gln Ile Arg Phe Leu Gly Gln Val Ile Ser
325 330 335
Pro Asp His Ile Thr Tyr Glu Thr Thr Pro Thr Ile Pro Met
340 345 350
Lys Ser Gln Trp Thr Leu Ala Glu Leu Gln Thr Val Leu Gly
355 360
Glu Ile Gln Trp Val Ser Lys Gly Thr Pro Ile Leu Arg Lys
365 370 375 His Leu Gln Cys Leu Tyr Ser Ala Leu Arg Gly Tyr Gln Asp 380 385 390
Pro Arg Ala His Leu Leu Leu Gln Lys Gln Gln Leu His Ala
395 400 405
Leu His Ala Ile Gln Gln Ala Leu Gln His Asn Cys Arg Ser
410 415 420
Arg Leu Asn Pro Ala Leu Pro Ile Leu Gly Leu Ile Ser Leu
425 430
Ser Ser Ser Gly Thr Thr Ser Val Leu Phe Gln Ala Arg Gln
435 440 445
Arg Trp Pro Leu Val Trp Leu His Thr Pro His Pro Pro Thr 450 455 460
Ser Leu Gly Pro Trp Gly His Leu Leu Ala Cys Thr Ile Leu
465 470 475
Thr Leu Asp Lys Tyr Ser Leu Gln His Tyr Gly Arg Leu Cys
480 485 490 Gln Ser Leu Glu Asp Asn Met Ser Asn Thr Ala Leu His Asp
495 500
Phe Val Lys Asn Ser Pro His Pro Arg Val Gly Ile Leu Ile
505 510 515
His His Met Ser Arg Phe His Asn Leu Gly Ser Gln Pro Ser 520 525 530
Gly Pro Trp Lys Ala Leu Leu His Leu Pro Ala Leu Leu Gln
535 540 545
Ala Ala Arg Leu Leu Arg Pro Leu Phe Thr Leu Ser Pro Val
550 555 560
Val Leu Thr Thr His Pro Val Ser Ser Pro Thr Gly Leu Pro
565 570
Lys Lys Ala Ala Tyr Val Leu Trp Asp Gln Thr Ile Leu Arg
575 580 585
His Asp Ser Ile Thr Leu Pro Pro His Gly Ser Asn Ser Ala
590 595 600
Gln Arg Gly Glu Leu Leu Ala Leu Leu Ser Gly Leu Arg Ala
605 610 615
Ala Lys Ser Trp Pro Ser Leu Asn Ile Phe Leu Asp Ser Lys
620 625 630
Tyr Leu Ile Lys Tyr Leu His Ser Leu Ala Thr Gly Ala Phe
635 640 Leu Gly Thr Ser Thr His Gln Ser Leu Tyr Ala His Leu Pro
645 650 655
Ala Leu Leu His Asn Lys Val Ile Tyr Leu His His Ile Arg
660 675 670
Ser His Thr Asn Leu Pro Asp Pro Ile Ser Thr Leu Asn Glu
675 680 685
Tyr Thr Asp Ser Leu Ile Ile Ile Ala Pro Leu Ile Pro Leu
690 695 700
Thr Pro Gln Asp Leu His Arg Leu Thr His Cys Asn Ser Arg
705 710
Gly Pro Cys Phe Phe Arg Ala Thr Pro Gln Gln Ser Gln Val 715 720 725
Ala Leu Glu Ser Arg Val Thr Pro Ala Thr Ser Leu Asn Ser
730 735 740
Gln His His Met Pro Gln Gly His Ile Arg Arg Gly Leu Leu
745 750 755
Pro Asn His Ile Trp Gln Gly Asp Val Thr His Tyr Lys Tyr
760 765 770
Lys Arg His Arg Tyr Cys Leu His Val Trp Val Asp Thr Phe
775 780
Ser Asn Ala Val Ser Ile Thr Cys Lys Thr Lys Glu Thr Ser 785 790 795
Ser Glu Thr Val Ser Ala Leu Leu His Ala Ile Thr Ile Leu
800 805 810
Gly Lys Pro Leu Ser Ile Asn Thr Asp Asn Gly Ser Ala Phe
815 820 825
Leu Ser Gln Glu Phe Gln Ala Phe Cys Thr Ser Trp His Ile
830 835 840
Arg His Ser Thr His Val Pro Tyr Asn Pro Thr Ser Ser Gly
845 850
Leu Val Glu Arg Thr Asn Gly Ile Val Lys Ala Leu Leu Asn 855 860 865
Lys Tyr Leu Leu Asp Ser Pro Asn Leu Pro Leu Asp Asn Ala
870 875 880
Ile Ser Lys Ser Leu Trp Thr Leu Asn Gln Leu Asn Val Met
885 890 895 ser Pro Ser Gly Lys Thr Arg Trp Gln Leu His His Gly Pro
900 905 910 Arg Leu Pro Pro Ser Leu Gln Ile Pro Gln Pro Ser Lys Ala
915 920
Pro Ala Asn Trp Tyr Tyr Val Leu Thr Pro Gly Leu Thr Asn 925 930 935
Gln Arg Trp Lys Gly Pro Leu His Leu Ser Arg Lys Leu Gln
940 945 950
Glu Arg Leu Leu Ser Ile Asp Gly Ser Pro Gln Trp Ile Pro
955 960 965
Trp Arg Leu Leu Lys Lys Thr Val Cys Pro Arg Pro Asp Gly
970 975 980
Ser Glu Leu Ala Ala Thr Thr Gln Gln Gln Thr Thr Asn Thr
985 990
Met
995 and SEQ ID NO: 15
Met His Phe Pro Gly Phe Gly Gln Ser Leu Leu Tyr Gly Tyr
1 5 10
Pro Val Tyr Val Phe Gly Asp Cys Val Gln Ala Asp Trp Cys
15 20 25
Pro Val Ser Gly Gly Leu Cys Ser Thr Arg Leu His Arg Asn
30 35 40
Ala Leu Leu Ala Thr Cys Pro Glu His Gln Leu Thr Trp Asp
45 50 55
Pro Ile Asp Gly Arg Val Val Gly Ser Arg Leu Gln Tyr Leu
60 65 70 Ile Pro Arg Leu Pro Ser Phe Pro Thr Gln Arg Thr Ser Lys
75 80
Thr Leu Lys Val Leu Thr Pro Pro Thr Thr Pro Val Ser Pro 85 90 95
Lys Ile Pro Pro Pro Ser Ser Gln Ser Met Arg Arg Leu Ser
100 105 110
Pro Tyr Arg Asn Gly Cys Leu His Pro Thr Leu Gly Asp Gln
115 120 125
Leu Pro Ser Leu Ser Phe Pro Asp Pro Gly Val Arg Pre Gln
130 135 140
Asn Ile Tyr Thr Thr Trp Gly Arg Thr Val Val Cys Leu Tyr
145 150 Leu Tyr Gln Leu Ser Pro Pro Met Thr Trp Pro Leu Ile Pro
155 160 165
His Val Ile Phe Cys His Pro Lys Gln Leu Gly Thr Phe Leu
170 175 180
Thr Asn Val Pro Leu Lys Arg Leu Glu Glu Leu Leu Tyr Lys
185 190 195
Ile Phe Leu His Thr Gly Ala Ile Ile Val Leu Pro Glu Asp
200 205 210
Arg Val Gly Thr Thr Leu Phe Gln Pro Val Arg Ala Pro Cys
215 220
Val Gln Thr Ala Trp Asp Thr Gly Leu Leu Pro Tyr His Ser 225 230 235
Leu Ile Thr Thr Pro Gly Leu Ile Trp Thr Phe Asn Asp Gly
240 245 250
Ser Pro Met Ile Ser Gly Pro Cys Pro Lys Thr Gly Gln Pro
255 260 265
Ser Phe Leu Val Gln Ser Ser Leu Leu Ile Phe Glu Lys Phe
270 275 280 Gln Thr Lys Ala Phe His Pro Ser Phe Leu Leu Ser His Gln
285 290
Leu Ile Gln Tyr Ser Ser Phe Gln Tyr Ser Ser Phe His Asn 295 300 305
Leu His Pro Ser Phe Glu Glu Tyr Thr Asn Ile Pro Val Ser
310 315 320
Tyr Phe Phe Asn Glu Lys Gln Ala Asp Asp Ser Asp Ser Asp
325 330 335
Pro Gly Pro Ser Ser Leu Gly Ala Pro Arg Ala Ser His Gln
340 345 350
Leu Lys Gly His Ala Val Ser His Lys Gln Ser Arg Glu Gln
355 360
Ala Leu Thr Phe Pro Pro Ala Asp His Leu Arg Asn Gln Glu 365 370 375
Pro Val Ser Arg Lys Ile Thr Ser Pro Leu Pro Thr Ser Pro
380 385 390
Cys Leu Glu Lys Gln Arg Leu
395
where amino acids 18-57 of SEQ ID NO: 15 correspond to the amino acid sequence shown in SEQ ID NO: 10 (the single amino acid difference between SEQ ID NO: 10 and the
corresponding forty amino acid stretch of SEQ ID NO: 15 is believed to be insignificant and may be due to
microheterogeneity within the viral population) and amino acids 40-78 of SEQ ID NO: 12 correspond to the amino acid sequence shown in SEQ ID NO: 11.
SEQ ID NO: 12 shows the amino acid sequence of rex where this sequence is encoded starting at nucleotide 5088 of SEQ ID NO: 8, continuing at nucleotide 1 of SEQ ID NO: 9, and ending at nucleotide 447 of SEQ ID NO: 9; SEQ ID NO: 13 shows the amino acid sequence of gag where this sequence is encoded by nucleotides 748 to 2043 of SEQ ID NO: 8; SEQ ID NO: 14 shows the amino acid sequence of pol where this sequence is encoded by nucleotides 2167 to 5151 of SEQ ID NO: 8 and SEQ ID NO: 15 shows the amino acid sequence of tax where this sequence is encoded by
nucleotides 3 to 1196 of SEQ ID NO: 9.
It is therefore understood that nucleic acid sequences capable of directing production of proteins encoded by the STLVpan-p genome are intended to be
encompassed within the present invention. Such proteins include, but are not limited, to, gag (SEQ ID NO:13), env, pol (SEQ ID NO:14), tax (SEQ ID NO:15) and rex (SEQ ID NO: 12). Due to the degeneracy of the genetic code, it is to be understood that numerous choices of nucleotides may be made that will lead to a DNA sequence capable of directing production of the instant STLVpan-p proteins or their analogs.
The term analog as used throughout the specification and claims refers to a protein having an amino acid sequence substantially identical to a sequence encoded by the STLVpan-p genome in which one or more
residues have been conservatively substituted with a functionally similar residue and which displays the functional aspects of the STLVpan-p proteins. Examples of conservative substitutions include, for example, the substitution of non-polar (i.e. hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (i.e. hydrophilic) residue for another, such as a substitution between arginine and lysine, between glutamine and asparagine, or between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
The phrase conservative substitution may also include the use of a chemically derivatized residue in place of a non-derivatized residue provided that the resulting protein or polypeptide displays the requisite functional activity.
Chemical derivative refers to a protein or polypeptide having one or more residues chemically derivatized by reaction of a functional side group.
Examples of such derivatized molecules include, but are not limited to, those molecules in which free amino groups have been derivatized to form, for example, amine
hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-imbenzylhistidine. Also included as chemical
derivatives are those proteins or peptides which contain one or more naturally-occurring amino acid derivatives of the twenty standard amino acids. For examples, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine;
homoserine may be substituted for serine; and ornithine may be substituted for lysine. A protein or polypeptide of the present invention also includes any protein or polypeptide having one or more additions and/or deletions of residues relative to the sequence of a protein or polypeptide whose sequence is encoded by the STLVpan-p genome so long as the requisite activity is maintained.
In one embodiment, the overlapping DNA sequences encoding the STLVpan-p genome obtained by the methods
described earlier in the specification may be used to create expression vectors for the production of
polypeptides derived from the STLVpan-p genome. Such
nucleic acid sequences may be natural or synthetic. The recombinant protein can be composed of one open-reading frame (ORF) protein or a combination of the same or different ORF proteins.
In an alternative embodiment, the recombinant protein may be a fusion protein produced by ligating together STLVpan-p nucleic acid sequence encoding at least one ORF and a nucleic acid sequence encoding a protein capable of targeting the recombinant fusion protein to STLVpan-p infected cells. Proteins capable of targeting the recombinant fusion protein to virus-infected cells
include, but are not limited to antibodies to T-cell markers. The production of such recombinant fusion proteins may be accomplished by techniques known to those skilled in the art. The nucleic acid sequence capable of directing host organism synthesis of the recombinant
STLVpan-p protein, whether fused or unfused, may be cloned into a suitable expression vector capable of being
transferred into, and replicated in, a host organism.
Operational elements of suitable expression vector are disclosed previously in the present specification and are known to one skilled in the art. Suitable expression vectors can function in prokaryotic or eukaryotic cells. Preferred expression vectors are those that function in eukaryotic cells. Examples of such vectors include but are not limited to retroviral vectors, vaccinia virus, adenovirus, and adeno-associated virus.
Once a nucleic acid sequence encoding the protein is present in a suitable expression vector, the expression vector may then be used for purposes of expressing the protein in a suitable eukaryotic cell system. Such eukaryotic cell systems include, but are not limited to cell lines such as SF9 insect cells, Chinese Hamster Ovary (CHO) cells, CEM, H7 and HeLa cells.
Preferred eukaryotic cell systems are SF9 insect cells. The expressed protein may be detected by methods known in the art such as metabolic radiolabelling or Western blotting.
In a further embodiment, the protein expressed by the cells may be obtained as crude lysate or it may be purified by standard protein purification procedures known in the art which may include differential precipitation, molecular sieve chromatography, ion-exchange
chromatography, isoelectric focusing, gel electrophoresis, affinity, and immunoaffinity chromatography and the like. In the case of immunoaffinity chromatography, the protein may be purified by passage through a column containing a resin which has bound thereto antibodies specific for the protein.
In yet another embodiment, the STLVpan-p proteins of the present invention may be purified from STLVpan-p viral particles. Methods for purifying viral particles and for detecting them during the purification procedure have been previously described. Purified proteins may be obtained from the viral particles following disruption of the particles by treatment with, for example, detergents in the presence of chaotropic agents. The lysates produced by the disruption of the viral particles may then be subjected to the various protein purification
procedures described above. In a preferred embodiment, selected proteins present in the lysate such as the envelope proteins, may be purified via immunoaffinity chromatography by passage through a column containing a resin which has bound thereto antibody specific for the envelope protein.
In an alternative embodiment, the proteins present in the lysate produced from the disrupted viral particles may be separated via gel electrophoresis.
Comparison of the migration of the electrophoretically separated proteins with the migration pattern of known markers for T-cell lymphotropic viral proteins (for example: p24, env, gag and pol) permits those skilled in the art to identify the location of specific proteins within the electrophoretic pattern. The gel slice
containing the protein of interest may then be excised from the gel and the protein electroeluted via techniques known to one skilled in the art.
In yet another embodiment, the STLVpan-p proteins may be synthetic peptides derived from STLVpan-p nucleic acid sequences where those skilled in the art would be aware that the peptides of the present invention or analogs thereof can be synthesized by automated
instruments sold by a variety of manufactures or can be commercially custom ordered and prepared. The term analog has been previously described in the specification and for purposes of describing peptides of the present invention, analogs can further include branched or non-linear
peptides.
The present invention therefore relates to the use of STLVpan-p proteins in immunoassays for diagnosing or prognosing STLVpan-p infection in a primate. In a preferred embodiment, the immunoassay is useful in diagnosing
STLVpan-p infection in humans. Immunoassays using the
STLVpan-p proteins, particularly the envelope proteins, provide a highly specific and sensitive method for
diagnosing STLVpan-p infection.
Immunoassays of the present invention may be a radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, immunohistochemical assay and the like. Standard
techniques known in the art for ELISA are described in
Methods in Immunodiagnosis, 2nd Edition, Rose and Bigazzi, eds., John Wiley and Sons, 1980 and Campbell et al.,
Methods of Immunology, W.A. Benjamin, Inc., 1964. Such assays may be a direct, indirect, competitive, or
noncompetitive immunoassay as described in the art
(Oellerich, M. 1984. J.Clin. Chem. Clin. BioChem. 22:
895-904). Biological samples appropriate for such detection assays include, but are not limited to, whole or heparinized blood, lymph nodes and spleen.
In one embodiment, test serum is reacted with a solid phase reagent having surface-bound recombinant
STLVpan-p protein as an antigen, preferably an envelope protein or a combination of an envelope protein with different proteins. The solid surface reagent can be prepared by known techniques for attaching protein to solid support material. These attachment methods include non-specific adsorption of the protein to the support or covalent attachment of the protein to a reactive group on the support. After reaction of the antigen with anti-STLVpan-p antibody, unbound serum components are removed by washing and the antigen-antibody complex is reacted with a
secondary antibody such as labelled anti-human antibody. The label may be an enzyme which is detected by incubating the solid support in the presence of a suitable
fluorimetric or calorimetric reagent. Other detectable labels may also be used, such as radiolabels or colloidal gold, and the like.
The STLV proteins and analogs thereof may be prepared in the form of a kit, alone, or in combinations with other reagents such as secondary antibodies, for use in immunoassays.
In an alternative embodiment, STLVpan-p viral particles may be substantially isolated by previously described methods and used as an antigen in immunoassays of the present invention.
The STLVpan-p proteins and analogs thereof can also be used as a vaccine to protect mammals against challenge with STLVpan-p viruses. The vaccine, which acts as an immunogen, may be a cell lysate prepared from cells transfected with a recombinant expression vector or a cell culture supernatant containing the expressed protein.
Alternatively, the immunogen is a partially or
substantially purified recombinant protein, a protein substantially purified from STLVpan-p viral particles or a synthetic peptide derived from STLVpan-p nucleic acid sequence.
While it is possible for the immunogen to be administered in a pure or substantially pure form, it is preferable to present it as a pharmaceutical composition, formulation or preparation.
The formulations of the present invention, both for veterinary and for human use, comprise an immunogen as described above, together with one or more
pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The carrier (s) must be
"acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The formulations may
conveniently be presented in unit dosage form and may be prepared by any method well-known in the pharmaceutical art.
All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In
general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation.
Formulations suitable for intravenous intramuscular, subcutaneous, or intraperitoneal
administration conveniently comprise sterile aqueous solutions of the active ingredient with solutions which are preferably isotonic with the blood of the recipient. Such formulations may be conveniently prepared by
dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride (e.g. 0.1-2.0M), glycine, and the like, and having a buffered pH compatible with physiological 1- 1,000ug conditions to produce an aqueous solution, and rendering said solution sterile. These may be present in unit or multi-dose containers, for example, sealed ampoules or vials. The formulations of the present invention may incorporate a stabilizer. Illustrative stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids which may be used either on their own or as admixtures. These stabilizers are preferably incorporated in an amount of 0.11-10,000 parts by weight per part by weight of immunogen. If two or more stabilizers are to be used, their total amount is preferably within the range specified above. These stabilizers are used in aqueous solutions at the
appropriate concentration and pH. The specific osmotic pressure of such aqueous solutions is generally in the range of 0.1-3.0 osmoles, preferably in the range of 0.8-1.2. The Ph of the aqueous solution is adjusted to be within the range of 5.0-9.0, preferably within the range of 6-8. In formulating the immunogen of the present invention, anti-adsorption agent may be used.
Additional pharmaceutical methods may be
employed to control the duration of action. Controlled release preparations may be achieved through the use of polymer to complex or absorb the proteins or their
derivatives. The controlled delivery may be exercised by selecting appropriate macromolecules (for example
polyester, polyamino acids, polyvinylpyrrolidone,
ethylenevinylacetate, methylcellulose,
carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled-release preparations is to incorporate the proteins, protein analogs or their functional derivatives, into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxy methylcellulose or gelatin-microcapsules and
poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
When oral preparations are desired, the compositions may be combined with typical carriers, such as lactose, sucrose, starch, talc magnesium stearate,
crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate or gum arabic among others.
When administered as immunogens, the STLVpan-p viral particles or viral proteins may be administered at a range from about 1 to about 1,000 ug of protein.
In another embodiment, the immunogen may be an expression vector containing nucleic acid sequences capable of directing expression of STLVpan-p protein(s).
Such recombinant expression vectors have been previously described in the present specification.
The immunogens of the present invention may be supplied in the form of a pharmaceutical composition as described above.
Vaccination can be conducted by conventional methods. For example, the immunogen can be used in a suitable diluent such as saline or water, or complete or incomplete adjuvants. Further, the immunogen may or may not be bound to a carrier to make it more immunogenic. Examples of such carrier molecules include but are not limited to bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid, and the like. The immunogen can be administered by any route appropriate for antibody production such as intravenous, intraperitoneal, intramuscular, subcutaneous, and the like. The immunogen may be administered once or at periodic intervals until a significant titer of anti-STLVpan-p antibody is produced. The antibody may be detected in the serum using any of the immunoassays described earlier.
The administration of the immunogen of the present invention may be for either a prophylactic or therapeutic purpose. When provided prophylactically, the immunogen is provided in advance of any exposure to
STLVpan-p or in advance of any symptom due to such
infection. The prophylactic administration of the
immunogen serves to prevent or attenuate any subsequent STLVpan-p infection in a primate. When provided therapeutically, the immunogen is provided at (or shortly after) the onset of the infection or at the onset of any symptom of infection or disease caused by STLVpan-p viruses. The therapeutic administration of the immunogen serves to attenuate the infection or disease.
In one embodiment, the vaccine comprises an STLVpan-p envelope protein substantially purified from an isolated STLVpan-p viral particle.
In addition to use as a vaccine, the compositions can be used to prepare antibodies to STLVpan-p virus-like particles. When used to prepare antibodies, the immunogens of the present invention may include purified STLVpan-p viral particles, cells infected with STLVpan-p, or STLVpan-p proteins. The antibodies produced in response to these immunogens can be used directly as antiviral agents. To prepare antibodies, a host animal is immunized using the virus particles or, as appropriate, non-particle antigens native to the virus particle are bound to a carrier as described above for vaccines. The host serum or plasma is collected following an appropriate time interval to provide a composition comprising antibodies reactive with the virus particle. The gamma globulin fraction or the IgG antibodies can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art. The antibodies are substantially free of many of the adverse side effects which may be associated with other anti-viral agents such as drugs.
The antibody compositions can be made even more compatible with the host system by minimizing potential adverse immune system responses. This is accomplished by removing all or a portion of the Fc portion of a foreign species antibody or using an antibody of the same species as the host animal, for example, the use of antibodies from human/human hybridomas. Humanized antibodies (i.e., nonimmunogenic in a human) may be produced, for example, by replacing an immunogenic portion of an antibody with a corresponding, but nonimmunogenic portion (i.e., chimeric antibodies). Such chimeric antibodies may contain the reactive or antigen binding portion of an antibody from one species and the Fc portion of an antibody
(nonimmunogenic) from a different species. Examples of chimeric antibodies, include but are not limited to, non-human mammal-human chimeras, rodent-human chimeras, murine-human and rat-human chimeras (Robinson et
al., International Patent Application 184,187; Taniguchi M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al., (1987) Proc.
Natl. Acad. Sci. USA, 84:3439; Nishimura et al., (1987) Cane. Res., 47:999; Wood et al., (1985) Nature, 314:446; Shaw et al., (1988) J. Natl. Cancer Inst., 80: 15553, all incorporated herein by reference).
General reviews of "humanized" chimeric antibodies are provided by Morrison S., 1985 Science
229:1202 and by Oi et al., 1986 BioTechniques 4:214.
Suitable "humanized" antibodies can be alternatively produced by CDR or CEA substitution (Jones et al., (1986) Nature, 321:552; Verhoeyan et al., 1988 Science 239:1534; Biedleret al. (1988) J. Immunol.,
141:4053, all incorporated herein by reference).
The antibodies or antigen binding fragments may also be produced by genetic engineering. The technology for expression of both heavy and light chain genes in E. coli is the subject the PCT patent applications;
publication number WO 901443, WO901443, and WO 9014424 and in Huse et al., 1989 Science 246:1275-1281.
The antibodies can also be used as a means of enhancing the immune response. The antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody. For example, pooled gamma globulin is administered at 0.02-0.1 ml/lb body weight during the early incubation period of other viral diseases such as rabies, measles and hepatitis B to interfere with viral entry into cells. Thus, antibodies reactive with the STLVpan-p virus particle can be passively administered alone or in conjunction with another anti-viral agent to a host infected with a STLVpan-p viruses to enhance the immune response and/or the effectiveness of an antiviral drug.
Alternatively, anti-STLVpan-p antibodies can be induced by administering anti-idiotype antibodies as immunogens. Conveniently, a purified anti-STLVpan-p
antibody preparation prepared as described above is used to induce anti-idiotype antibody in a host animal. The composition is administered to the host animal in a suitable diluent. Following administration, usually repeated administration, the host produces anti-idiotype antibody. To eliminate an immunogenic response to the Fc region, antibodies produced by the same species as the host animal can be used or the FC region of the
administered antibodies can be removed. Following
induction of anti-idiotype antibody in the host animal, serum or plasma is removed to provide an antibody
composition. The composition can be purified as described above for anti-STLVpan-p antibodies, or by affinity chromatography using anti-STLVpan-p antibodies bound to the affinity matrix. The anti-idiotype antibodies produced are similar in conformation to the authentic STLVpan-p-antigen and may be used to prepare an STLVpan-p vaccine rather than using an STLVpan-p particle antigen.
When used as a means of inducing anti-STLVpan-p virus antibodies in an animal, the manner of injecting the antibody is the same as for vaccination purposes, namely intramuscularly, intraperitoneally, subcutaneously or the like in an effective concentration in a physiologically suitable diluent with or without adjuvant. One or more booster injections may be desirable.
For both in vivo use of anti-idiotype antibodies and antibodies to STLVpan-p virus-like particles and
proteins and diagnostic use, it may be preferable to use monoclonal antibodies. Monoclonal anti-virus particle antibodies or anti-idiotype antibodies can be produced as follows. The spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art. (Goding, J.W. 1983. Monoclonal Antibodies: Principles and Practice, Pladermic Press, Inc., NY, NY, pp. 56-97). To produce a human-human hybridoma, a human lymphocyte donor is selected. A donor known to be infected with STLVpan-p (where infection has been shown for example by the presence of anti-virus antibodies in the blood or by virus culture) may serve as a suitable lymphocyte donor.
Lymphocytes can be isolated from a peripheral blood sample or spleen cells may be used if the donor is subject to splenectomy. Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a human fusion partner can be used to produce human-human hybridomas. Primary in vitro immunization with peptides can also be used in the generation of human monoclonal antibodies.
Antibodies secreted by the immortalized cells are screened to determine the clones that secrete
antibodies of the desired specificity. For monoclonal anti-virus particle antibodies, the antibodies must bind to STLV virus particles. For monoclonal anti-idiotype antibodies, the antibodies must bind to anti-virus
particle antibodies. Cells producing antibodies of the desired specificity are selected.
The above described antibodies and antigen binding fragments thereof may be supplied in kit form alone, or as a pharmaceutical composition for in vivo use. The antibodies may be used for therapeutic uses,
diagnostic use in immunoassays or as an immunoaffinity agent to purify recombinant STLVpan-p proteins as described herein. The present invention also relates to the production of cell lines expressing STLVpan-p viruses.
Methods for culturing T-cell lymphotropic viruses are known to those skilled in the art (Markham, P.D. et al.
(1983) Int. J. Cancer, 31:413-420; Markham, P.D. et al.
(1984) Int. J. Cancer, 33:13-17). Generally, suitable cells or cell lines for culturing T-cell lymphotropic viruses may include those known to support lymphotropic virus replication. Such cell lines include, but are not limited to, B cell lines such as A-2 and B-JAB and T-cell lines such HUT-78 and SUP-T1. It is possible that
peripheral blood mononuclear cells can be cultured and then infected with STLVpan-p or alternatively, that
peripheral blood mononuclear cells (PBMCs) can be derived from STLVpan-p infected individuals (e.g., human or
chimpanzees). The latter case is an example of the cell which is infected in vivo being passaged in vitro.
Immortalization of such primary PBMCs may be achieved by cocultivating the virus-infected PBMCs with cells such as human blood cord leukocytes as described in Example 2. Also, primary cultures of infected PBMCs may be infected with transforming viruses or transfected with transforming genes in order to create permanent or semi-permanent cell lines.
Infection of cell lines with STLVpan-p viruses may be accomplished by techniques known in the art for
infecting cells with T-cell lymphotropic viruses (Markham et al. (1983) and (1984)). For example, infection of cells with STLVpan-p viruses in vitro can be accomplished by cocultivation of the cells to be transformed with virus-infected cells. Examples of cells to be transformed include, but are not limited to, peripheral blood or bone narrow leukocytes. In vitro transformation of human peripheral blood cord blood lymphocytes typically results primarily in transformation of CD4 cells. However, CD8+ cells can also be transformed by STLVpan-p viruses in vitro and immature CD4- CD8- cells from bone marrow can also be transformed. In addition, primate B cell lines such as B- JAB and AA2 may be infected by STLVpan-p viruses. It is understood by one skilled in the art that cells
transformed in vitro may actively transcribe STLVpan-p RNA to produce virions and that they themselves may be used to transform other normal cells.
In addition to cultured cells, animal model systems may also be used to promote viral replication.
Animal systems in which T-cell lymphotropic viruses are known to replicate include chimpanzees or other non human primates, rats, rabbits and cattle.
In one embodiment, cell culture and animal model systems for STLVpan-p produced by the present invention may be used to screen for antiviral agents which inhibit
STLVpan-p replication, and particularly for those agents which preferentially allow for cell growth and
multiplication while inhibiting viral replication.
Generally, the antiviral agents are tested at a variety of concentrations for their effect on preventing viral replication in cell culture systems and then for an inhibition of infectivity of viral pathogenicity and a low level of toxicity in an appropriate animal model system. The methods provided herein for detecting STLVpan-p antigens and STLVpan-p nucleic acid sequences are useful for
screening of anti-viral agents in that they provide a sensitive means for detecting the effect of the agents on viral replication. For example, the STLVpan-p nucleic acid probes described herein may be used to quantitate the amount of viral nucleic acid produced in a cell culture via a method such as PCR amplification.
Any citation, including articles or patents etc. referenced herein, are expressly incorporated herein in total, by reference. The following examples illustrate various aspects of the invention but are in no way
intended to limit the scope thereof.
Materials and Methods
Materials:
The Pigmy Chimpanzees used in the following examples were from a colony housed at the Yerkes Regional Primate Center (Atlanta, GA). This Pigmy chimpanzee breeding colony was initiated in the mid-seventies with two founder females, LI-1954 and MA-1970, both wild born, with estimated birth dates of 1954 and 1970, which were obtained from the San Diego Zoo and Zaire, respectively. Another female, KI-1950, the oldest Pigmy chimpanzee
(estimated birth date 1950) housed at Yerkes, was wild caught and did not generate progeny. The two male
founders, KI-1974 and BO-1971 (both wild born), were obtained from Wisconsin and Zaire, respectively. Breeding of these animals yielded 12 living offspring whose
designation and birth date are shown in Figure 2. None of the Pigmy chimpanzees at the Yerkes Primate Center were inoculated with human or other animals tissues.
Example 1
Evaluation of Sera From Pigmy Chimpanzees
For HTLV- I/HTLV- II Indeterminant Seropositivity
Sera from 12 Pigmy chimpanzees were evaluated by Western blot analysis for the presence antibodies against HTLV-I and -II proteins. In brief, sera from each animal and from two positive controls, strong reactive HTLV-I and -II (Cellular Products, Buffalo, NY), were tested at a 1:100 dilution using commercially available Western blot strips from Cellular Products (Buffalo). The antigens present on the Western blot strips are indicated on the righthand side of Figure 1 where MTA-1 and K55 are
peptides specific for the gp46 protein of the HTLV-I and II strains respectively; p24 is a major gag protein of both HTLV strains; rgp21 is a major envelope protein of both HTLV strains and pl9 is an HTLV-I-specific antigen. The designation SC is a serum control introduced to each strip by Cellular Products which serves as a positive control to ensure that serum has been added to each strip. The animal designations for the 12 chimpanzees and the designations for the two positive controls are shown at the top of Figure 1. The results of this Western blot analysis demonstrated that eight (numbers 1-8 at the bottom of Figure 1) of the 12 Pigmy chimpanzees tested for HTLV-I and -II antigens scored positive for the recombinant
HTLV-I gp21 envelope protein and the p24 major Gag protein (both proteins are common for both HTLV-I and HTLV-II) but not for the gp46 protein of HTLV-I or -II (MTA-1 and K55, respectively). In addition, three (number 1-3 at the bottom Figure 1) of the 8 positive sera also had
antibodies against the HTLV-I pl9 gag antigen suggesting that STLVpan-p is cross-reactive to HTLV-I. The serological profile generated by the Western blot analysis is
represented in the genealogical tree shown in Figure 2. Figure 2 show that the founder female (LI-1954) was seropositive (blackened symbol) for HTLV-I and -II
antigens and produced three female offspring with two different males (the identity of one is unknown). All three female offspring tested were also seropositive. Two of the female offspring (LO-1969 and LA-1967) conceived, with the same seronegative (open symbol) male founder (BO-1971), 4 and 2 offspring. In both cases at least 50% of the offspring were seropositive for viral antigens. As expected, none of the offspring from the seronegative founder female (MA-1970) scored positive for viral
antigens. Taken together, the data presented in Figures 1 and 2 suggested the presence of an infectious virus in the colony, related to, but distinct from both HTLV-I and -II, which was transmitted exclusively from mother to
offspring.
Example 2
Isolation of STLVpan-p From the Peripheral
Blood Mononuclear Cells of Pigmy Chimpanzees
STLVpan-p was isolated by coculturing peripheral blood mononuclear cells (PBMCs) prepared from heparinized blood obtained from Pigmy Chimpanzees diagnosed as of HTLV- I/HTLV- II indeterminate seropositivity based on the Western blot analysis shown in Figure 1. In particular, PBMCs from three seropositive female chimpanzees, LI 1954
(grandmother), LA 1967 (mother) and JI 1985 (daughter) were cocultured with human cord blood by well established techniques (Popovic, M. et al. (1983) Science, 219:856-859). Each of the resulting three independent cocultures (one from each chimpanzee) expressed virus as detected by measuring the presence of HTLV-I and -II p24-related antigen in the supernatant fluid of the cocultures as determined by p24 antigen capture assay. (Eisner, R.
(1989) Diag. Clin. Testing 27:9-10; Lee, M. et al. (1986) N. Engl. J. Med., 315: 1610-1611; Dimitrov, D. et al.
(1990) J. Clin. Micro, 28:734-737).
The presence of a retrovirus in these cocultures was verified by electron microscopic analysis as shown in Figures 3A-C with the size and morphology of this type C retrovirus resembling that of HTLV-I and -II (Figs. 3A-C). In addition, virus budding was observed, albeit rarely, on the surface of infected cells (Fig. 3C). These three cocultures of Pigmy chimpanzee PBMC and human cord blood cells designated L93-79A, L93-79B and L93-79C, have developed into continuous cell lines suggesting a viral immortalization event analogous to the immortalization of human peripheral blood mononuclear cells by HTLVs. The cell line L93-79C was deposited with the American Type Culture Collection (ATCC, Rockville, MD) on April 13, 1994.
Indirect immunofluorescence on each coculture using the following antibodies: LEU5(CD2), LEU4(CD3), LEU3a(CD4), LEU2a(CD8), 2A3(CD25), L234(DR), LEU12(CD19), LEU16(CD20) (Becton Dickinson, San Jose, CA);
ALB11 (CD45RA) and VCHL1 (CD45RO) (Biodesign, Kennebunkport, ME). BW242/412 (TCRαβ) and TS/1(TCRδγ) : (T-cell Diagnostic, Cambridge, MA) followed by flow cytometric assay of the immunolabelled cells using a FACScan™ (Becton Dickinson), demonstrated that each of these three cell lines displayed a clear phenotype of activated T-cells as evidenced by the expression of both CD4 and CD8 markers on these cells.
The results of these immunofluorescence assays are presented in Table 1.
TABLE 1
T-CELL
MARKERS CD2 CD3 CD4 CD8 CD25 DR TCRδγ TCRαβ CD19 CD20 CD45RA CD45R0
L93-79A 95 95 86 89.4 49.2 88.4 0.5 95.8 0.25 0.39 71.2 84.6 (LI-1954)
L93-79B 96.3 97.3 47.9 98.4 97.2 94.8 0.5 78.2 ND 0.7 80.7 82.1 (LA-1967)
L93-79C 97.2 97 26.2 93 76.8 91.5 0.3 95.4 0.29 0.36 61.3 91.7 (JI-1985)
In two instances (culture A and B), a high percent of cells co-expressed both markers. Furthermore, all the cultures were positive for the memory T-cell marker CD45RO as well as CD45RA (Table 1). These data indicate that like HTLV-I and -II, the target cell population for these primate viruses is a mature T-cell.
Example 3
Detection Of Viral Proteins In CoCultures Of STLVpan-p- Infected Cells The presence of viral proteins in the infected cocultures was detected by radioimmunoprecipitation assays. Approximately 106 cells from the co-culture
L93-79C, HTLV-II-infected MO-T (Kalyanaraman, V.S. et al. (1982) Science, 218:571-573), HTLV-I-infected MT-2
(Popovic, M. et al. (1983) and uninfected H9 cell lines were metabolically labeled with 100uci/ml 35S-methionine and cysteine (New England Nuclear). Lysates of the radiolabelled cells were immunoprecipitated using 1:100 dilution of sera from two donors seropositive with HTLV-I (lane 4) and -II (lane 5) (Cellular Products), as well as sera from two seropositive Pigmy chimpanzees (LI 1954, lane 1, and ZA 1984, lane 2) and a seronegative orangutan (lane 3). All the sera tested, with the exception of the orangutan serum, recognized 24 kilodalton (Kd) major gag proteins from HTLV-I, -II and the L93-79C cells as shown in Figure 4. These results again suggested cross-reactivity among the major Gag proteins of these three viruses as previously demonstrated by the Western blot analysis shown in Figure 1.
Example 4
Genetic Characterization of The
STLVpan-p Genome Present In The CoCulture Isolates
To genetically characterize the STLVpan-p culture L93-79C isolates, cellular proviral DNA prepared from the L93-79A, B and C-infected cultures was analyzed by
Southern blot using the HTLV-I and -II genomic probes, p1711 (Cardoso, E.A. et al. (1989) Int. J. Cancer. 43:195-200) and pMO-4 (Gelmann, E. et al. (1984) Proc. Natl.
Acad. Sci. U.S.A., 81:993-997), respectively. Twenty micrograms of DNA from the cell lines L93-79A, B and C, the HTLV-I- and -II-infected cell lines C91/PL (Popovic, M. et al. (1983) and MO-T, respectively, and the
uninfected T-cell line H9, were cleaved with the Pst-1 endonuclease, transferred to nitrocellulose and duplicate filters hybridized with probes to HTLV-I (Figure 5B) or HTLV-II (Figure 5A). The sizes of the internal Pst-1 fragments for HTLV-I and -II are indicated in kilobases (kb) by the arrows on the right-hand side of Figure 5A.
The results of this Southern analysis show that Pst-1 cleavage of the cell line DNA yielded a single fragment of 3 kb (arrow on left-hand side of Figure 5) under low stringency hybridization (0.25M Na2HPO4 (pH7.2) 1mM EDTA (pH8) 7% SDS at 65°C; wash 4 times with 2X SSC at 65°C) with the HTLV-II probe. The same probe detected several fragments of different size in the DNA of the HTLV-II-infected MO-T-cell line. Conversely, the HTLV-I probe did not hybridize to DNAs from the L93-79A, B, and C cultures but readily hybridized to the DNA of the
HTLV-I-infected C91/PL cell line used as positive control. Neither probe hybridized to the DNA of the uninfected H9 cells. These results suggested a distant genetic
cross-reactivity between HTLV-II and the STLVpan-p viral genomic DNA.
Polymerase chain reaction analysis of the DNA prepared from the cocultures was also carried out using envl and env2 primers derived from HTLV-I. The sequences of these primers were previously shown as SEQ ID NO:1 and SEQ ID NO:2, respectively. These primers were
demonstrated to amplify STLV-I from 12 different species of nonhuman primates (Koralnik et al. (1994) in press), as well as all three of the HTLV-I clades (cosmopolitan, Melanesian and Zairian) known to date (Gessain, A. et al. (1992) J. Virol., 66:2288-2295) but they failed to recognize STLVpan-p. Similarly, commercially available oligonucleotides primers (Perkin-Elmer) shown previously as SEQ ID NOS: 3 and 4, and designed to discriminate between HTLV-I and -II failed to amplify viral sequence.
Finally, partial sequence data on proviral DNA, presented herein as SEQ ID NO: 7, indicated that
approximately 80% of the nucleotides present in this sequence are shared in a short stretch of DNA from the pX regions of STLVpan-p, HTLV-Iatk (Seiki, M. et al. (1983) Proc. Natl. Acad. Sci. U.S.A., 80:3618-3622), HTLV-Imel5 (Gessain, A. et al. (1993) J. Virol., 67:1015-1023) and HTLV-IImo (Gelmann, E. et al. (1984) and Shimotono, K. et al. (1985) Proc. Natl. Acad. Sci. U.S.A.. 82:3101-3105). Taken together, these results demonstrate that STLVpan-p is a novel primate T-cell lymphotropic virus.
Example 5
Isolation Of Nucleic Acid Sequence Of A Human Variant
Of STLVpan-p Using Polymerase Chain Reaction Nucleotide sequences of PCR primers useful in screening human samples for variants of STLVpan-p are shown below as SEQ ID NOS: 16-18 where SEQ ID NO: 16
TGTTTAGCGA TTGTGTTCAA GCCGA
and
SEQ ID NO: 17
CGGATACCCA GTCTACGTGT
represent one pair of primers and SEQ ID NO: 11 and
SEQ ID NO: 18
GAGCCGATAA CGCGTCCATC G
represent a second pair of primers.
PCR is performed with both primer pairs to amplify fragments of DNA from the tax region of STLVpan-p related viruses. In this assay, 1 μg of DNA is mixed with PCR reaction mix containing 50 mM KCl, 10 mMTris-HCl pH 8.3, 1.6 mM MgCl2, 50 pmol of each primer, 200 μg dATP, dGTP, dCTP, dTTP, and 2.5 unite of taq DNA polymerase (Perkin Elmer Cetus), in 2 final volumes of 100 μl.
Enzymatic DNA amplification is performed in 2 DNA Thermal Cycler (Perkin Elmer) with the following thermal parameters: denaturation step (5 min at 95°C), 35 cycles of denaturation (1 min 94°C) and annealing (2 min 55°C) and at least synthesis step (72°C 7 min.).
After amplification, DNA is hybridized in solution with SEQ ID NO: 19
ACGCCCTACT GGCCACCTGT CCAGAGCATC AGATCACCTG
and/or SEQ ID NO: 20
GAGAAGGGCG TTCCGGTGCA GGCGGGTGGA ACAAAGCCAA
labeled by T4 polynucleotide kinase. 1.5 × 105 cpm of probe are mixed with 30 μl of amplified products in a final volume of 40 μl. The mixture is heated at 95°C for 5 min and then at 56°C for 15 min to allow hybridization to occur. Six μl of 0.01% bromophenol blue and 30 μl chloroform are then added and loaded onto a 10%
polyacrylamide gel. Electrophoresis was carried out in a 1x TBE buffer at 200V for 42 min. The gel was enclosed with a plastic wrap and exposed to Amersham Hyperfilm at -70°C overnight to allow visualization of the labelled nucleic acid sequences of human variants of STLVpan-p.
Example 6
Use Of STLVpan-p Infected Cells In A
Radioimmunoprecipitation Assay To Screen Human Sera.
Cells from the cultures L93-79A, L93-79C and HTLV-II-infected MO-T and HTLV-I infected C91/PL cells cultured in cysteine-free RPMI 1640 plus 15% dialyzed FBS, 10% XL-2, 1% glutamine and 1% pen-strap can be labeled with 35S-methionine and 35S-cysteine. Both cell lysates and supernatants are brought to IX RIPA buffer after addition of 2X RIPA buffer (1% Triton, ) .1% SDS, 1% deoxycholate, 0.05M Tris pH 7.5, 0.25M NaCl) to reach a volume of 500 μL. Samples are precleared with 100 μL of Protein A agarose beads (Boehringer Mannheim Co.) plus 10 μL of normal rabbit serum by incubation at 4 degrees for 5 hours. Sera from STLVpan-p sero-positive Pigmy (positive control) chimpanzees and from humans are added at a 1:10 dilution (50 μL) and incubated overnight at 4 degrees shaking. Samples are then immunoprecipitated with 30 μL of Protein A agarose beads rocking at 4 degrees for 45 minutes. Following 4 washes with IX RIPA buffer (15 min at 4 degrees) beads are resuspended in 20 μL of sample buffer with β-mercaptoethanol, then heated at 100 degrees for 10 min and loaded onto a 12% SDS-polyacrylamide gel . Gels are fixed and treated with Enlightening (NEN) and exposed to Kodak XAR-5 film at -70 degrees, to detect human antibodies that cross-react with STLVpan-p.
Example 7
Infection Of Human T And B Lymphocytic Cell Lines With
STLVpan-p And Resulting Cytopathic Effects.
Cells from established human B-cell lines, e.g., AA-2, B-JAB, RAJI, and T-cell lines, e.g., Sup T1, can be infected with STLVpan-p by co-cultivation with infected, primary, human cord leukocytes or irradiated (6000 rad) STLVpan-p transformed cord blood leukocyte cultures. For this purpose, donor cells (STLVpan-p-infected) are mixed with target cells at a ratio of ~1:2 and incubated in growth media required by the target cells, e.g. RPMI 1640 supplemented with 10% fetal calf serum, glutamine, pen./strep., at 37°C, 5% CO2. Cultures are monitored for morphological changes and for release of virus as detected by antigen capture assays for antigens cross reacting with HTLV-I/II p24. A unique characteristic of cultures treated as described is the frequent induction of multiple syncytia or giant cells similar, but not identical to those observed in some HIV-1-infected cells, but clearly distinguishable from the multi-lobulated nuclei-containing giant cells often observed in HTLV-I-infected cells.
Example 8
Immunoassay Based On STLVpan-p Viral Particles Or Purified STLVpan-p Viral Structural Proteins As Antigens
To detect serum antibodies to STLVpan-p or STLVpan-p viral antigens, the ELISA (Sarngadharan, M.G. et al.
(1984) Science 224:506-508) is performed as follows:
The wells in polystyrene microtiter plates are coated with STLVpan-p prepared from culture supernatant fluids by density centrifugation or with purified viral structural proteins. This is done by incubating the appropriate concentrations of the antigens in the wells overnight at 4°C in a humid chamber. Following overnight incubation, the unbound protein is poured off, and the wells are coated with 1% BSA for two hours to block any open binding sites for proteins. The contents of the wells are then poured off, and the wells are washed with PBS-Tween (PBS containing 0.05% Tween-20).
The coated wells are filled with the test sera expected to contain the antibodies, and the plates are incubated for 3 hours at room temperature in a humid chamber or overnight at 4°C. The unreacted material is washed off as before. Any antibody in the unknown sample remains attached to the antigen molecules on the well surface.
The conjugate, consisting of enzyme-linked specific antibody reactive against the test antibody
(e.g., enzyme-linked goat anti-rabbit IgG if the test antibody is in a rabbit serum), is then added to the wells and allowed to react with the antibody already on the plate as a result of complexing with the surface-bound antigen. The excess conjugate is washed out.
Finally, the amount of the enzyme-linked specific antibody on the plate is determined as a measure of the amount of the test antibody in the immune complex. This is achieved by adding the appropriate substrate to the plate and following the rate of the enzyme reaction.
The most commonly utilized enzymes for conjugation to an antigen or antibody can be used such as horseradish peroxidase and alkaline phosphatase although other enzymes known to those skilled in the art can also be used. Example 9
Detection And Quantitation Of STLVpan-p Or Related Virus
Antigens Or RNA In Serum Or Plasma Samples.
The detection of STLVpan-p viral antigens in human serum or plasma is accomplished using a variation of the ELISA technology described in Example 8. For this
purpose, specific monoclonal or polyclonal antibodies directed against the viral antigen(s) of interest are first immobilized onto the multi well plates followed by incubation with the test serum or plasma and subsequent processing (Eisner, R. (1989); Lee, M. et al. (1988) and Dimitrov, D. et al. (1990).
Viral load can also be assessed at the level of STLVpan-p viral RNA present in human serum, plasma or cells using one or more molecular amplification-detection systems such as, but not limited to, RT-PCR (Saksela, K. et al. (1994) Proc. Natl. Acad. Sci. U.S. A., 91:1104-1108 and NASBA (Kievits, T. et al. (1991) J. Virol. Methods. 35:273-286; Bruisten, S. et al. (1993) AIDS Res. Human Retroviruses, 9:259-265; van Gemen, B. et al. (1993) J. Virol. Methods, 43:177-188). Both procedures are
dependent on the preparation of sequence-specific nucleic acid primers. RT-PCR involves use of a preliminary reverse transcriptase reaction followed by amplification at high temperatures by conventional PCR procedures.
NASBA is a specific, isothermal amplification method which uses the coordinated activities of reverse transcriptase, RNase H, and T7 polymerase.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANTS: The Government of the United
States of America as represented by the Secretary, Department of Health and Human Services
(ii) TITLE OF INVENTION: ISOLATION AND
CHARACTERIZATION OF A NOVEL PRIMATE T-CELL LYMPHOTROPIC VIRUS AND THE USE OF THIS VIRUS OR COMPONENTS THEREOF IN DIAGNOSTIC ASSAYS AND VACCINES
(iii) NUMBER OF SEQUENCES: 20
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: MORGAN & FINNEGAN
(B) STREET: 345 PARK AVENUE
(C) CITY: NEW YORK
(D) STATE: NEW YORK
(E) COUNTRY: USA
(F) ZIP: 10154
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: FLOPPY DISK
(B) COMPUTER: IBM PC COMPATIBLE
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: WORDPERFECT 5.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 21-APR-1995
(vii) PRIORITY APPLICATION DATA:
(A) APPLICATION NUMBER: US08/231,526
(B) FILING DATE: 22-APR-1994
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: WILLIAM S. FEILER
(B) REGISTRATION NUMBER: 26,728
(C) REFERENCE/DOCKET NUMBER: 2026-4125PCT (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 758-4800
(B) TELEFAX: (212) 751-6849
(C) TELEX: 421792
(2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TTTGAGCGGC CGCTCAAGCT ATAGTCTCCT CCCCCTG 37
(2) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
ACTTAGAATT CGGGAGGTGT CGTAGCTGAC GGAGG 35
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
CCCTACAATC CCACCAGCTC AG 22
(2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
GTACTTTACT GACAAAACCC GACCTAC 27 (2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :
GTGGTGGATT TGCCATCGGG TTTT 24 (2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
TCATGAACCC CAGTGGTAA 19
(2) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 122 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
ATGGGGTCCC AGGTGAGCTG GTGCTCTGGG CAGGTGGCGA 40
GAAGGGCGTT CCGGTGCAGG CGGGTGGAAC AAAGCCCACC 80
TGAAACGGGA CACCAATCGG CTTGAACACA ATCGCTAAAC 120
AC 122
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5153 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
TGACAGTGAC TCTGACCCTG GGCCTTCTAG CCTCGGGGCC 40
CCCAGGGCGA GTCATCAGCT TAAAGGTCAC GCTGTCTCAC 80
ACAAACAATC CCGGGAACAG GCTCTGACGT TTCCCCCTGC 120
AGACCATTTG AGGAACCAGG AACCAGTCTC CAGAAAAATA 160
ACCTCACCCT TACCCACTTC CCCTTGCCTT GAAAAACAAA 200
GGCTCTGACG ACTACCCCCC CCACCCATAA AATTTGCCTA 240
CTCAATAAAG CCCAGGCCTA TAAAAGCGCA AGGACGGTTC 280 AGGAGGGGGT CACCTTCTTT CCACCTGCCC GCTGTGCCTA 320
CCTTGGAGAT CCATTCCGCC CGGGGCCTCG GTCGAGACGT 360
CTCGAGAAAA GCTCCGTCTC CCGTTCCAGA CACTCTGAAC 400
CGCGCCTTTC AGGGTAAGTC TCCCCCCGGT TGAGCTGGGC 440 TACGACTCCC GTAGTCGCTC CCGCAGTCGG TTGAGGCTCC 480
CTGACCCTCC GGCCGCGCAC GTTAGGCTCT GGTTTGTAAC 520
CCTACTTCCG CGTTCTTGTC TTATTCTGCG CCGAAACCGA 560
AAGCACAGCG CCTCTGGGTG CCACGCTGGC CCGGGGCCAG 600 CATCCTGTCC AGGGACGCGC GCCTACTGAA CCCGAAGGCG 640
CCTCAGGCCT CTCCGGGAGG GGCACTCGGC TCGGGCCCTT 680
GTTCTCTCCG GGAGAGACAA ACAAGTGGGG GCTCGTCCGG 720
GGATACCTAC CCCTGCCCTG TCGCATTATG GGACAAACCT 760
ACGGCCTCTC GCCTAGCCCA ATCCCCAAGG CCCCCAGAGG 800
TTTATCAACC CACCACTGGT TAAATTTCCT CCAAGCCTCC 840
TACCGGCTAC AACCTGGGCC CTCCGACTTT GATTTTCAGC 880
AACTACGTCG TTTCCTGAAA CTAGCTCTTA AAACACCTAT 920
TTGGTTAAAT CCAATCGATT ACTCCCTCCT GGCCAGCCTC 960
ATCCCCAAGG GTTACCCCGG GCGGACAAGC GAGATTATTA 1000
ATGTGTTAAT TAGAAATCAA GCGTCCCCCA CCCCGCCTCC 1040
TGCCCCGTCT CTACCCGAAC CGGCTAACCC ACCGCCCCTC 1080
CAGCAGCCCT CGGCTCCTCC GGAACCCCAT ACGCCCCCCC 1120
CCTATATAGA GCCTCCCGCT ACCCATTGCC TTCCCATACT 1160
ACATCCACAT GGGGCTCCCT CGGCTCACAG GCCATGGCAA 1200 ATGAAAGACC TGCAGGCCAT CAAACAGGAG GTCAATACCT 1240
CGGCCCCTGG GAGTCCCCAG TTCATGCAAA CAGTCCGGCT 1280
CGCAATTCAG CAATTCGACC CCACGGCCAA AGACTTACAA 1320
GATCTCTTGC AGTACCTCTG CTCCTCCCTA GTCGTCTCCC 1360
TTCACCATCA ACAACTCCAT ACCCTAATTA CCGAGGCTGA 1400
AACCAGGGGA ATGACAGGTT ATAATCCCAT GGCCGGACCC 1440 CTAAGGATGC AGGCCAACAA CCCCGCCCAG GAAGGACTCC 1480
GGAGGGAATA CCAAAACCTC TGGCTGGCAG CCTTTTCGGC 1520
TCTGCCAGGG AACACCCGCG ACCCATCTTG GGCGGCAATC 1560
TTGCAAGGGC TAGAAGAACC CTATTGCGCC TTGCTAGAGC 1600 GCCTTAATGT TGCTCTTGAC AACGGTCTCC CCGAGGGAAC 1640
CCCAAAGGAG CCCATCTTGC GCTCCCTGGC TTACTCCAAC 1680
GCCAACAAAG ACTGCCAAAA ACTGCTGCAG GCTCGGGGCC 1720
ATACTAATAG CCCCTTGGGA GACATGCTCC GAGCCTGTCA 1760 AGCATGGACA CCCAAGGACA AAGCCCGGGT CCTCGTCGTC 1800
CAATCACGAA AGCCCCCGCC CACGCAGCCC CTGTTCCGCT 1840
GTGGAAAGGC AGGGCATTGG AGCCGAGACT GCACCCTCCC 1880
ACGCCCCCCC CCTGGTCCAT GCCCCTTGTG CAAAGACCCT 1920
TCCCATTGGA AACGAGATTG TCCACAGCTC AAACCCCCCC 1960
CGACGGAGGA GGAACCCCTC CTGCTGGACC TGCCCTCAGA 2000
TGCCGTTGCC ACCGAGGAAA AAAACTCCCT AGGGGGGGAG 2040
ATCTAATCTC CCCCCAACAA ATCTCACTGC TCCCTCTCAT 2080
TCCTTTAGAG CAACAGCAGC AGCCCCTTCT AGACGTTCAG 2120
GTCTCCATTG CAGGCGCCCC CCCTCGACCT ACCCAGGCAC 2160
TCTTAGACAC AGGGGCTGAT CTCACCGTCC TGCCCCAGGC 2200
CCTTGCCCCC GAGTCAGAAG GTCTCTGACA CAACGGTTCT 2240
AGGCGCTGGC GGTCAGACCA GCTCCCAGTT CAAACTCCTC 2280
CGATCCCCCC TGTGTGTCTA CCTGCCCTTC CGGAAGGCCC 2320
CCGTTACTCT CCCGTCATGC CTTCTAGACA CCGATAATAA 2360 ATGGGCCATT ATTGGCCGTG ATATCCTCCA GCAATGCCAG 2400
AGTGTCCTGT ACCTCCCGGA GGACAATCTC TGCGAAGGTA 2440
CCCCCCGGCC CTCCGGCAGA ATGAATTCCC CCCGACTATT 2480
ACCCGTGGCC ACCCCCAGTG TCATCGGCCT TGAGCACTTC 2520
CCACCACCCC CACAGATAGA TCAGTTCCCC TTTAAACCTG 2560
AGCGCCTCCA GGCCTTGACT GACCTGGTCT CCAAGGCCCT 2600 GGAGGCTAGC TACATTGAAC CTTACTCTGG ACCAGGCAAC 2640
AACCCTGTCT TTCCGGTCAA AAAACCAAAC GGCAAGTGGA 2680
GATTTATTCA TGACCTGAGG GCCACCAATG CCATCACGAC 2720
CACCTTGGCC TCCCCGTCCC CCGGACCTCC CGATCTCACC 2760 AGCTTATCAA CAGCCCTCCC ATATGTGCAA ACTATAGACC 2800
TTGCTGACGC CTTCTTCCAA ATTCCCCTTC CAAAACAGTT 2840
CCAGCCATAC TTCGCCTTCA CTATCCCCCA GCCATGCAAT 2880
TATGGCCCCG GGGCTCGGTA TGCCTGGACT GTCCTTCCAC 2920 AAGGATTCAA AAATAGTCCC ACCCTGTTTG AGCAACAGCT 2960
GGCTGCCATC CTTTCCCCTA TCAGGAAAGC CTTCCCTACG 3000
TCCACTATCA TCCAATACAT GGATGATATA CTCTTGGCCA 3040
GTCCTGCGCA GGGGGAACTG CAACAACTCT CCAAGATGAC 3080
CCTCCAAGCG CTAGTCACCC ACGGCCTTCC AGTCTCCCAG 3120
GCCCCCCGGG AACAAACCCC TGGGCAAATA CGTTTCTTAG 3160
GTCAAGTTAT ATCTCCTGAC CATATTACCT ATGAAACCAC 3200
CCCCACCATT CCTATGAAAT CCCAGTGGAC TCTCGCTGAA 3240
CTACAGACTG TGCTAGGAGA GATCCAATGG GTCTCAAAAG 3280
GAACCCCCAT CCTCCGCAAA CACCTGCAGT GTTTATACTC 3320
CGCCCTTCGC GGATATCAGG ACCCCAGGGC ACACCTCCTC 3360
CTCCAGAAAC AACAGCTCCA TGCCCTCCAT GCCATCCAAC 3400
AGGCCTTACA GCACAATTGC CGCAGCCGTC TCAATCCTGC 3440
CTTGCCCATC CTGGGACTTA TCTCCTTGAG CTCGTCTGGT 3480
ACAACCTCCG TCCTCTTCCA AGCCCGGCAG CGATGGCCCC 3520 TGGTTTGGTT GCACACCCCC CATCCACCAA CCAGCCTTGG 3560
CCCCTGGGGT CACTTACTGG CCTGCACTAT CCTGACCTTA 3600
GACAAATACT CCCTTCAGCA CTATGGCCGG CTGTGCCAGT 3640
CCTTGGAGGA CAACATGTCC AACACAGCCC TCCACGATTT 3680 TGTGAAAAAC TCCCCCCACC CGAGGGTAGG CATCCTTATC 3720
CATCACATGA GCCGCTTTCA TAACCTCGGT AGTCAGCCAT 3760 CTGGCCCTTG GAAGGCTCTC TTACACCTTC CCGCTCTCCT 3800
CCAAGCGGCA CGGCTCCTCC GACCTCTCTT CACGCTATCT 3840
CCCGTGGTCT TAACAACGCA CCCTGTCTCT TCTCCGACGG 3880
GTCTTCCCAA AAAGGCGGCA TACGTACTCT GGGACCAGAC 3920 CATCCTCCGC CATGACTCTA TCACATTGCC CCCCCATGGC 3960
AGCAACTCAG CTCAAAGAGG AGAGCTCCTC GCCCTCCTTT 4000
CAGGACTCCG GGCGGCCAAG TCATGGCCAT CCCTAAATAT 4040
TTTTCTTGAC TCCAAGTACT TAATTAAATA TCTCCATTCC 4080 CTTGCCACTG GAGCCTTTCT TGGGACTTCG ACTCACCAGT 4120
CCCTCTATGC CCATTTGCCT GCCCTATTGC ATAACAAAGT 4160
TATCTACCTC CACCACATTC GTAGCCACAC CAATCTCCCC 4200
GACCCCATCT CCACTCTCAA CGAATACACA GACTCGCTTA 4240
TTATTATAGC CCCCCTCATT CCCCTAACGC CTCAGGATCT 4280
GCACAGACTT ACCCATTGCA ACTCCAGGGG CCCTTGTTTC 4320
TTCCGGGCCA CCCCACAGCA GAGCCAAGTC GCTCTTGAAA 4360
GCCGTGTTAC ACCTGCAACA TCACTAAACT CACAACATCA 4400
CATGCCTCAA GGGCACATCC GTCGGGGGTT GCTGCCCAAC 4440
CACATATGGC AAGGAGATGT AACCCACTAC AAATACAAGC 4480
GGCACCGATA CTGCCTGCAC GTCTGGGTTG ATACCTTCTC 4520
CAACGCAGTC TCCATTACCT GCAAAACAAA AGAGACTAGC 4560
TCTGAGACTG TCAGCGCCCT CTTGCACGCT ATCACTATCT 4600
TAGGCAAGCC CCTTTCTATA AACACTGACA ATGGTTCTGC 4640
CTTCCTATCT CAAGAATTCC AGGCCTTTTG TACCTCATGG 4680 CACATCAGAC ATTCCACCCA TGTGCCCTAC AACCCTACCA 4720
GCTCCGGGCT GGTAGAGAGA ACCAATGGCA TTGTCAAGGC 4760
CCTCCTCAAC AAATATCTCC TAGACAGCCC GAACCTCCCC 4800
CTGGACAATG CCATCAGTAA ATCCCTATGG ACCCTAAACC 4840
AACTCAATGT CATGTCCCCC AGTGGAAAAA CCCGCTGGCA 4880
ACTCCATCAT GGCCCCCGGT TGCCCCCATC CCTGCAAATA 4920 CCTCAGCCCT CTAAGGCACC CGCGAACTGG TACTATGTAC 4960
TAACTCCTGG TCTTACCAAT CAGCGGTGGA AAGGACCTTT 5000
ACATCTCTCC AGGAAGCTGC AGGAGCGCTT ACTTTCGATA 5040
GACGGCTCCC CTCAGTGGAT CCCGTGGCGG CTCCTGAAAA 5080 AGACTGTATG CCCAAGGCCA GACGGCAGCG AACTCGCCGC 5120
GGACAACGCA GCAACAGACC ACCAACACCA TGG 5153
(2) INFORMATION FOR SEQ ID NO : 9 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1694 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :
CCCACTTTCC AGGTTTTGGA CAGAGCCTCC TCTATGGATA 40
CCCAGTCTAC GTGTTTGGCG ATTGTGTTCA AGCCGATTGG 80
TGTCCCGTTT CAGGTGGGCT TTGTTCCACC CGCCTGCACC 120
GGAACGCCCT TCTCGCCACC TGCCCAGAGC ACCAGCTCAC 160 CTGGGACCCC ATCGATGGAC GGGTTGTCGG CTCTCGTCTC 200
CAATACCTTA TCCCTCGACT CCCCTCCTTC CCCACCCAGA 240
GAACCTCCAA GACCCTTAAG GTCCTCACTC CCCCTACCAC 280
TCCTGTCTCC CCCAAGATTC CACCGCCTTC TTCCCAATCA 320
ATGCGGAGGC TCTCCCCTTA CCGGAACGGT TGCCTGCATC 360
CAACTCTCGG AGATCAGCTC CCCTCCCTTT CCTTCCCCGA 400
CCCCGGTGTC CGACCCCAAA ACATCTACAC CACCTGGGGA 440
AGAACTGTAG TTTGCTTGTA CCTCTACCAA CTATCCCCCC 480
CGATGACCTG GCCCCTAATA CCTCATGTCA TATTCTGCCA 520
TCCCAAGCAG TTAGGAACCT TCCTGACCAA CGTACCTCTA 560
AAACGCCTGG AAGAATTACT ATACAAAATA TTCCTACACA 600
CCGGGGCCAT CATAGTTCTC CCCGAAGACA GGGTGGGTAC 640
TACATTGTTC CAACCTGTTA GAGCCCCCTG CGTCCAGACC 680 GCCTGGGATA CAGGGCTTCT ACCCTACCAC TCTCTCATAA 720
CTACTCCGGG CCTAATATGG ACATTTAATG ACGGTTCACC 760
CATGATTTCC GGCCCCTGCC CCAAAACAGG GCAGCCATCT 800
TTCCTAGTAC AGTCCTCCCT GTTAATCTTT GAAAAATTCC 840 AGACCAAGGC CTTTCATCCT TCCTTCCTAC TGTCCCATCA 880
ACTCATCCAG TACTCTTCAT TCCAGTACTC TTCATTCCAT 920
AACCTCCACC CTTCCTTCGA GGAATACACT AACATCCCAG 960
TTTCCTATTT CTTTAACGAA AAACAGGCAG ATGACAGTGA 1000 CTCTGACCCT GGGCCTTCTA GCCTCGGGGC CCCCAGGGCG 1040
AGTCATCAGC TTAAAGGTCA CGCTGTCTCA CACAAACAAT 1080
CCCGGGAACA GGCTCTGACG TTTCCCCCTG CAGACCATTT 1120
GAGGAACCAG GAACCAGTCT CCAGAAAAAT AACCTCACCC 1160
TTACCCACTT CCCCTTGCCT TGAAAAACAA AGGCTCTGAC 1200
GACTACCCCC CCCACCCATA AAATTTGCCT ACTCAATAAA 1240
GCCCAGGCCT ATAAAAGCGC AAGGACGGTT CAGGAGGGGG 1280
TCACCTTCTT TCCACCTGCC CGCTGTGCCT ACCTTGGAGA 1320
TCCATTCCGC CCGGGGCCTC GGTCGAGACG TCTCGAGAAA 1360
AGCTCCGTCT CCCGTTCCAG ACACTCTGAA CCGCGCCTTT 1400
CAGGGTAAGT CTCCCCCCGG TTGAGCTGGG CTACGACTCC 1440
CGTAGTCGCT CCCGCAGTCG GTTGAGGCTC CCTGACCCTC 1480
CGGCCGCGCA CGTTAGGCTC TGGTTTGTAA CCCTACTTCC 1520
GCGTTCTTGT CTTATTCTGC GCCGAAACCG AAAGCACAGC 1560
GCCTCTGGGT GCCACGCTGG CCCGGGGCCA GCATCCTGTC 1600 CAGGGACGCG CGCCTACTGA ACCCGAAGGC GCCTCAGGCC 1640
TCTCCGGGAG GGGCACTCGG CTCGGGCCCT TGTTCTCTCC 1680
GGGAGAGACA AACA 1694
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 amino acids (B) TYPE: amino acids
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Val Phe Ser Asp Cys Val Gln Ala Asp Trp Cys Pro
1 5 10
Val Ser Gly Gly Leu Cys Ser Thr Arg Leu His Arg
15 20
Asn Ala Leu Leu Ala Thr Cys Pro Glu His Gln Leu
25 30 35
Thr Trp Asp Pro
40 (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 amino acids
(B) TYPE: amino acids
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Cys Leu Ala Ile Val Phe Lys Pro Ile Gly Val Pro
1 5 10
Phe Gln Val Gly Phe Val Pro Pro Ala Cys Thr Gly
15 20
Thr Pro Phe Ser Pro Pro Ala Asn Ser Thr Ser Ser
25 30 35
Pro Gly Thr
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acids
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Met Pro Lys Ala Arg Arg Gln Arg Thr Arg Arg Gly Gln Arg 1 5 10
Ser Asn Arg Pro Pro Thr Pro Trp Pro Thr Phe Gln Val Leu
15 20 25
Asp Arg Ala Ser Ser Met Asp Thr Gln Ser Thr Cys Leu Ala
30 35 40
Ile Val Phe Lys Pro Ile Gly Val Pro Phe Gln Val Gly Phe
45 50 55
Val Pro Pro Ala Cys Thr Gly Thr Pro Phe Ser Pro Pro Ala
60 65 70 Gln Ser Thr Ser Ser Pro Gly Thr Pro Ser Met Asp Gly Leu
75 80 Ser Ala Leu Val Ser Asn Thr Leu Ser Leu Asp Ser Pro Pro
85 90 95
Ser Pro Pro Arg Glu Pro Pro Arg Pro Leu Arg Ser Ser Leu
100 105 100
Pro Leu Pro Leu Leu Ser Pro Pro Arg Phe His Arg Leu Leu
115 120 125
Pro Asn Gln Cys Gly Gly Ser Pro Leu Thr Gly Thr Val Ala
130 135 140
Cys Ile Gln Leu Ser Glu Ile Ser Ser Pro Pro Phe Pro Ser
145 150
Pro Thr Pro Val Ser Asp Pro Lys Thr Ser Thr Pro Pro Gly 155 160 165
Glu Glu Leu
170 (2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 432 amino acids
(B) TYPE: amino acids
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Met Gly Gln Thr Tyr Gly Leu Ser Pro Ser Pro Ile Pro Lys
1 5 10
Ala Pro Arg Gly Leu Ser Thr His His Trp Leu Asn Phe Leu
15 20 25
Gln Ala Ser Tyr Arg Leu Gln Pro Gly Pro Ser Asp Phe Asp 30 35 40
Phe Gln Gln Leu Arg Arg Phe Leu Lys Leu Ala Leu Lys Thr
45 50 55
Pro Ile Trp Leu Asn Pro Ile Asp Tyr Ser Leu Leu Ala Ser
60 65 70
Leu Ile Pro Lys Gly Tyr Pro Gly Arg Thr Ser Glu Ile Ile
75 80
Asn Val Leu Ile Arg Asn Gln Ala Ser Pro Thr Pro Pro Pro
85 90 95
Ala Pro Ser Leu Pro Glu Pro Ala Asn Pro Pro Pro Leu Gln
100 105 110
Gln Pro Ser Ala Pro Pro Glu Pro His Thr Pro Pro Pro Tyr
115 120 125 Ile Glu Pro Pro Ala Thr His Cys Leu Pro Ile Leu His Pro
130 135 140 His Gly Ala Pro Ser Ala His Arg Pro Trp Gln Met Lys Asp
145 150
Leu Gln Ala Ile Lys Gln Glu Val Asn Thr Ser Ala Pro Gly 155 160 165
Ser Pro Gln Phe Met Gln Thr Val Arg Leu Ala Ile Gln Gln
170 175 180