WO1995029240A1

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

Info

Publication number: WO1995029240A1
Application number: PCT/US1995/004910
Authority: WO
Inventors: Genoveffa Franchini; Robert C. Gallo; Phillip Markham; Adriana Giri
Original assignee: US Department of Health and Human Services; Government of the United States of America
Current assignee: US Department of Health and Human Services; Government of the United States of America
Priority date: 1994-04-22
Filing date: 1995-04-21
Publication date: 1995-11-02
Anticipated expiration: 1996-10-22
Also published as: AU2392795A

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 STLV_pan-p.

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

invention to provide synthetic nucleic acid sequences, especially nucleic acid sequences capable of directing production of recombinant STLV_pan-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 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

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 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

expression of STLV_pan-p genes.

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. Alternatively, STLV_pan-p viral particles may be used as diagnostic agents and as immunogens in the

production of anti-STLV_pan-p antibodies.

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.

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 STLV_pan-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 STLV_pan-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 STLV_pan-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 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 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 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.

DETAILED DESCRIPTION OF INVENTION

The present invention relates to the isolation and characterization of a novel primate T-cell

lymphotropic virus designated STLV_pan-p. As used throughout the specification and claims, "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. 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. STLV_pan-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).

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,

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 STLV_pan-p by

expanding the cultures and then subjecting the culture supernatants to ultracentrifugation in a sucrose density gradient. (Poiesz et al. (1980). The STLV_pan-p viral particles are found at a density of about 1.170 g/ml.

During this isolation procedure, 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. For detection of STLV_pan-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 STLV_pan-p virus may be further characterized via the isolation of RNA from purified STLV_pan-p viral particles. Alternatively, 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

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 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.

For example, 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.

Alternatively, 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. 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 STLV_pan-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 STLV_pan-p or from the sera or tissues obtained from STLV_pan-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 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

T-cell lymphotropic viruses. A partial nucleic acid sequence of STLV_pan-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 STLV_pan-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 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. 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 STLV_pan-p. Thus, the information provided herein makes it possible to clone and sequence the STLV_pan-p genome. The sequence of the 5' region of STLV_pan-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 STLV_pan-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 STLV_pan-p or parts thereof as antigens.

In another embodiment, nucleic acid sequences of viral homologues of STLV_pan-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 STLV_pan-p variants in other primates may be derived from the proviral DNA of STLV_pan-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 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. In addition, 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).

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 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. 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 STLV_pan-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 STLV_pan-p viruses in primates.

In one embodiment of the invention, 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.

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 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.,

(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 STLV_pan-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 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

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 STLV_pan-p-infected cells is described in

Example 4.

In a second preferred embodiment, the DNA is analyzed for the presence of STLV_pan-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 STLV_pan-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 STLV_pan-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 STLV_pan-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 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. In one embodiment, 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. 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 STLV_pan-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

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.

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 STLV_pan-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 STLV_pan-p nucleic acid sequences.

These primers can be labelled as described earlier and the RT-PCR products can be analyzed for the presence of

STLV_pan-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 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.

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 STLV_pan-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 STLV_pan-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 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. 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 STLV_pan-p genome so long as the requisite activity is maintained.

In one embodiment, the overlapping DNA sequences encoding the STLV_pan-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 STLV_pan-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 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

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

STLV_pan-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 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

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 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

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 STLV_pan-p proteins in immunoassays for diagnosing or prognosing STLV_pan-p infection in a primate. In a preferred embodiment, the immunoassay is useful in diagnosing

STLV_pan-p infection in humans. Immunoassays using the

STLV_pan-p proteins, particularly the envelope proteins, provide a highly specific and sensitive method for

diagnosing STLV_pan-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

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

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, 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.

Alternatively, 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.

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 STLV_pan-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 STLV_pan-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-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. When provided prophylactically, the immunogen is provided in advance of any exposure to

STLV_pan-p or in advance of any symptom due to such

infection. The prophylactic administration of the

immunogen serves to prevent or attenuate any subsequent STLV_pan-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 STLV_pan-p viruses. The therapeutic administration of the immunogen serves to attenuate the infection or disease.

In one embodiment, the vaccine comprises an STLV_pan-p envelope protein substantially purified from an isolated STLV_pan-p viral particle.

In addition to use as a vaccine, the compositions can be used to prepare antibodies to STLV_pan-p virus-like particles. When used to prepare antibodies, 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. 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 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.

Alternatively, anti-STLV_pan-p antibodies can be induced by administering anti-idiotype antibodies as immunogens. Conveniently, a purified anti-STLV_pan-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-STLV_pan-p antibodies, or by affinity chromatography using anti-STLV_pan-p antibodies bound to the affinity matrix. The 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.

When used as a means of inducing anti-STLV_pan-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 STLV_pan-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 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

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 STLV_pan-p proteins as described herein. The present invention also relates to the production of cell lines expressing STLV_pan-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 STLV_pan-p or alternatively, that

peripheral blood mononuclear cells (PBMCs) 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.

Infection of cell lines with STLV_pan-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 STLV_pan-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 STLV_pan-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 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.

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 STLV_pan-p produced by the present invention may be used to screen for antiviral agents which inhibit

STLV_pan-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 STLV_pan-p antigens and STLV_pan-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 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.

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 STLV_pan-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 STLV_pan-p From the Peripheral

Blood Mononuclear Cells of Pigmy Chimpanzees

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. 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 STLV_pan-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

STLV_pan-p Genome Present In The CoCulture Isolates

To genetically characterize the STLV_pan-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 STLV_pan-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 STLV_pan-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 STLV_pan-p, HTLV-I_atk (Seiki, M. et al. (1983) Proc. Natl. Acad. Sci. U.S.A., 80:3618-3622), HTLV-I_mel5 (Gessain, A. et al. (1993) J. Virol., 67:1015-1023) and HTLV-II_mo (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 STLV_pan-p is a novel primate T-cell lymphotropic virus.

Example 5

Isolation Of Nucleic Acid Sequence Of A Human Variant

Of STLV_pan-p Using Polymerase Chain Reaction Nucleotide sequences of PCR primers useful in screening human samples for variants of STLV_pan-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 STLV_pan-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 STLV_pan-p.

Example 6

Use Of STLV_pan-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 STLV_pan-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 STLV_pan-p.

Example 7

Infection Of Human T And B Lymphocytic Cell Lines With

STLV_pan-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 STLV_pan-p by co-cultivation with infected, primary, human cord leukocytes or irradiated (6000 rad) STLV_pan-p transformed cord blood leukocyte cultures. For this purpose, donor cells (STLV_pan-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 STLV_pan-p Viral Particles Or Purified STLV_pan-p Viral Structural Proteins As Antigens

To detect serum antibodies to STLV_pan-p or STLV_pan-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 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).

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 STLV_pan-p Or Related Virus

Antigens Or RNA In Serum Or Plasma Samples.

The detection of STLV_pan-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 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

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

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

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

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 995 amino acids

(B) TYPE: amino acids

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(xi) SEQUENCE DESCRIPTION: 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 60C)

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

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 399 amino acids

(B) TYPE: amino acids

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(xi) SEQUENCE DESCRIPTION: 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 Pro 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

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

TGTTTAGCGA TTGTGTTCAA GCCGA 25

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(c) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

CGGATACCCA GTCTACGTGT 20

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

GAGCCGATAA CGCGTCCATC G 21

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

ACGCCCTACT GGCCACCTGT CCAGAGCATC AGATCACCTG 40 (2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

GAGAAGGGCG TTCCGGTGCA GGCGGGTGGA ACAAAGCCAA 40

Claims

1. A cell line L93-79C containing a primate T-cell lymphotropic virus having the characteristics of ATCC Deposit No.

.

2. An isolated and purified T-cell

lymphotropic viral particle characterized by having a genomic nucleic acid sequence, wherein said genomic nucleic acid sequence includes a sequence as shown in SEQ ID NO: 7.

3. A substantially purified and isolated DNA, wherein the DNA has a nucleic acid sequence which includes a sequence as shown in SEQ ID NO : 7.

4. A nucleic acid sequence capable of directing host organism synthesis of at least one complete open-reading frame protein, wherein said sequence

comprises a portion of the genome of the T-cell

lymphotropic viral particle of claim 2.

5. A nucleic acid sequence of claim 4, wherein said sequence encodes a viral structural protein.

6. The nucleic acid sequence of claim 5, wherein said structural protein is an envelope protein.

7. A nucleic acid sequence of claim 4, wherein said sequence encodes a viral non-structural protein.

8. A recombinant protein derived from the nucleic acid sequence of claim 4.

9. A recombinant protein of claim 8, wherein said protein is a viral structural protein.

10. The recombinant protein of claim 9 wherein said viral structural protein is an envelope protein.

11. A recombinant protein of claim 8, wherein said protein is a viral non-structural protein.

12. A purified and substantially isolated protein encoded by a nucleic acid sequence included within the genome of a T-cell lymphotropic viral particle

according to claim 2.

13. A protein according to claim 12 wherein said protein is a viral structural protein.

14. The protein of claim 13 wherein said viral structural protein is an envelope protein.

15. A protein according to claim 12 wherein said protein is a viral non-structural protein.

16. A protein according to claim 12, wherein said protein is substantially isolated from the T-cell lymphotropic viral particle.

17. A protein according to claim 16, wherein said protein is a viral structural protein.

18. The protein of claim 17, wherein said viral structural protein is an envelope protein.

19. A protein according to claim 16, wherein said protein is a viral non-structural protein.

20. A protein according to claim 12, wherein said protein is substantially isolated from a tissue culture cell line infected with the T-cell lymphotropic viral particle.

21. A protein according to claim 20, wherein said protein is a viral structural protein.

22. The protein of claim 21, wherein said structural protein is a envelope protein.

23. A protein according to claim 20, wherein said protein is a viral non-structural protein.

24. An immunoassay for detecting antibody against STLV_pan-p comprising:

(a) providing a protein according to claim 12;

(b) incubating a biological sample with said protein under conditions that allow for the formation of antibody-protein complex; and (c) detecting said antibody-protein

complex.

25. An immunoassay for detecting antibody against STLV_pan-p comprising:

(a) providing a viral particle according to claim 2;

(b) incubating a biological sample with said particle under conditions that allow for the formation of a complex between antibody and said viral particle; and

(c) detecting said antibody-viral particle complex.

26. A tissue culture cell line infected with a T-cell lymphotropic virus characterized by having a genomic nucleic acid sequence, wherein said genomic nucleic acid sequence includes a sequence as shown in SEQ ID NO: 7.

27. The tissue culture cell line of claim 26, wherein the cell line is a human B-cell line.

28. The tissue culture cell line of claim 26, wherein the cell line is derived from peripheral blood mononuclear cells obtained from an STLV_pan-p-infected individual.

29. A recombinant expression vector comprising a nucleic acid sequence according to claim 4.

30. An anti-STLV_pan-p antibody composition comprising antibodies that bind the T-cell lymphotropic viral particle according to claim 2 or antigenic parts thereof.

31. The antibody composition of claim 30, wherein said composition is produced by administering to an animal an isolated and purified T-cell lymphotropic viral particle characterized by having a genomic nucleic acid sequence, wherein said genomic nucleic acid sequence includes a sequence as shown in SEQ ID NO.: 7.

32. The antibody composition of claim 30, wherein said composition is produced by administering to an animal a purified and substantially isolated protein encoded by a nucleic acid sequence included within the genome of a T-cell lymphotropic virus, said genome having a nucleic acid sequence which includes a sequence as shown in SEQ ID NO: 7.

33. The antibody composition of claim 30, wherein said composition is produced by administering to an animal a cell line having the characteristics of ATCC Deposit No. .

34. The antibody composition according to claim

30 wherein the anti-STLV_pan-p antibodies are fixed to a solid phase.

35. An immunoassay method for detecting STLV_pan-p in a biological sample comprising;

(a) providing an anti-STLV_pan-p antibody composition according to claim 30;

(b) incubating said biological sample with said anti-STLV_pan-p antibody composition under conditions that allow for the formation of an antibody-antigen complex; and

(c) detecting said antibody-antigen

complex comprising the anti-STLV_pan-p antibody.

36. A pharmaceutical composition comprising a protein according to claim 12 and a pharmaceutically acceptable carrier or diluent.

37. A method of preventing STLV_pan-p viral infection in a primate, comprising administering the pharmaceutical composition of claim 36 to said primate in an amount effective to stimulate the production of protective antibody.

38. A vaccine for immunizing a primate against STLV_pan-p viral infection comprising the pharmaceutical composition of claim 36.

39. A pharmaceutical composition comprising a recombinant expression vector according to claim 29.

40. A method of preventing STLV_pan-p viral infection in a primate, comprising administering the pharmaceutical composition of claim 39 to said primate in an amount effective to stimulate the production of protective antibody.

41. A vaccine for immunizing a primate against STLV_pan-p viral infection comprising the pharmaceutical composition of claim 39.

42. A method for detecting STLV_pan-p in a biological sample comprising: analyzing DNA of a subject for the presence of STLV_pan-p nucleic acid sequence.

43. The method of claim 42, wherein said step of analyzing comprises Southern blot analysis.

44. The method of claim 42, wherein said step of analyzing is carried out by polymerase chain reaction.

45. A method for detecting STLV_pan-p in a biological sample comprising: analyzing RNA of a subject for the presence of STLV_pan-p nucleic acid sequence.

46. The method of claim 45, wherein said step of analyzing is carried out by RT-PCR.