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WO1992015325A1 - Solomon islands variants of human t-lymphotropic virus - Google Patents

Solomon islands variants of human t-lymphotropic virus Download PDF

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Publication number
WO1992015325A1
WO1992015325A1 PCT/US1992/001413 US9201413W WO9215325A1 WO 1992015325 A1 WO1992015325 A1 WO 1992015325A1 US 9201413 W US9201413 W US 9201413W WO 9215325 A1 WO9215325 A1 WO 9215325A1
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Prior art keywords
htlv
virus
cell line
cell
variant
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PCT/US1992/001413
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French (fr)
Inventor
Richard Yanagihara
Ralph M. Garruto
Andrew Ajdukewicz
D. Carleton Gajdusek
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The United States Of America, Represented By The Secretary, United States Department Of Commerce
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Publication of WO1992015325A1 publication Critical patent/WO1992015325A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
    • C12N2740/14021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
    • C12N2740/14034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a cell line infected with a variant of human T-lymphotropic virus type I (HTLV-I) derived from a native Solomon Islander, desig ⁇ nated SI-5, and to the infecting virus, designated HTLV-I SI variant.
  • HTLV-I human T-lymphotropic virus type I
  • the present invention further relates to bioassays for the detection of the virus, viral proteins or antibodies thereto in a biological sample.
  • the retrovirus HTLV-I is the causative agent of adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-I-associated myelopathy.
  • the prevalence of HTLV-I infection in Melanesia has been controversial since different testing methods have produced conflicting data.
  • the present invention relates to a eucaryotic cell line comprising an HTLV-I variant virus isolated from the
  • the invention further relates to antibodies specific for the infecting virus.
  • the present invention also relates to a vaccine for humans against infection with and diseases caused by HTLV-I and related viruses.
  • the vaccine comprises a non- infectious antigenic portion of the variant virus produced by the cell line of the present invention, administered in an amount sufficient to induce immunity against infection and to prevent disease.
  • the present invention also relates to bioassays for the diagnosis of infection caused by variants of HTLV-I.
  • bioassays for the diagnosis of infection caused by variants of HTLV-I.
  • cells of the present invention or protein from the virus infecting the cells are fixed on a solid support and contacted with a biologi ⁇ cal sample from a human suspected of being infected. The presence or absence of a complex formed between protein of the cell and antibodies specific therefor present in the sample is then detected.
  • the HTLV-I-SI variant virus nucleic acid is detected in a biological sample.
  • the viral genomic seguences are amplified by polymerase chain reaction and the presence or absence of the amplified product are detected by restriction enzyme mapping and hybridization, and confirmed by DNA sequencing.
  • Figure 1 shows intracytoplasmic virus-specific fluorescence in SI-5, a T-cell line derived from a 58-year old man with hepatosplenomegaly, using a monoclonal antibody against HTLV-I gagr-encoded protein pl9. (Origi ⁇ nal magnification, 500X) .
  • Figure 2 shows extracellular virus particles resembling HTLV-I and budding forms in SI-5, a T-cell line derived from a native Solomon Islander. (Original magni- fication, 90,000X).
  • Figure 3 shows a Western analysis of (lanes 1 and 2) HTLV-I isolates, SI-1 and SI-3, from two Solomon Islanders and of (lane 3) a prototype strain of HTLV-I (MT-2) , using a serum from a rabbit immunized with an 18- residue synthetic peptide spanning the extreme C-terminus of the major envelope glyc protein gp46 [Lillehog et al., Clin. Biotechnol.. 1:27-41 (1989)] (Ab gp46) and a serum from a rabbit experimentally infected with HTLV-I (Ab HTLV-I) .
  • SI-5 gave identical results.
  • Figures 4A and 4B show (A) ethidium bromide- stained agarose gel showing the 119-bp HTLV-I pol sequence amplified by PCR and (B) Southern analysis of the ampli ⁇ fied product hybridized, under high-stringency conditions, with a 32 P-labelled HTLV-I pol-specific 35-mer oligo probe
  • PCR was performed on PNA isolated from (lanes 1-3) SI-1, SI-2 and SI-3, three T-cell lines derived from two Solomon Islanders, (lane 4) MOLT-3, an uninfected T-cell line derived from a patient with acute lymphoblastic leukemia and (lane 5) MT-2, an HTLV-I- infected T-cell line derived from a Japanese patient with adult T-cell leukemia [Miyoshi et al.. Nature, 294:770- 771(1981)]. SI-5 gave identical results.
  • Figure 5 shows the partial sequence data (SEQ. IP. NO: 1) of the env gene of the HTLV-I-SI variant virus from the SI-5 cell line.
  • the present invention relates to a human T-cell line persistently infected with an HTLV-I variant virus isolated from the Solomon Islands and the infecting virus.
  • the present inventors have isolated HTLV-I variant viruses from peripheral blood mononuclear cells of asymptomatic carriers in the Solomon Islands.
  • Human T-cell lines infected with an HTLV-I variant virus have been estab ⁇ lished, one such example is the SI-5 cell line.
  • the cell lines of the present invention grow more rapidly and produce more virus than PNG-1, a T-cell line infected with an HTLV-I variant established from a New Guinean.
  • the cell lines of the present invention constitute a major improvement over the only other Melane ⁇ sian T-cell line infected with an HTLV-I variant virus from this area.
  • the improved characteristics should greatly facilitate antigen production for use in diagnos ⁇ tic tests and for use in vaccines.
  • the present invention further relates to the HTLV-I variant virus infecting the above described cell lines.
  • the HTLV-I-related viruses of the present invention variants of prototype HTLV-I, are referred to as HTLV-I-SI variants.
  • the env gene nucleotide sequences of the viruses of the present invention diverge signifi ⁇ cantly (by approximately 8%) from prototype HTLV-I.
  • the virus isolated from the SI-5 cell line differs by 7.9% in its nucleotide sequence of the env gene from that of prototype HTLV-I.
  • a substantially pure preparation of the viruses of the present invention can easily be isolated from the cell line of the present invention or a lysate thereof by one skilled in the art without undue experimen ⁇ tation.
  • Unique peptides of the viruses make possible the accurate diagnosis of HTLV-I infection in Melanesia and in other geographical regions where tests employing prototype HTLV-I yield high frequencies of indeterminate results.
  • the present invention relates to antibodies specific for the HTLV-I Si-variant or viral proteins expressed by the SI-5 cell line.
  • One skilled in the art using standard methodology can raise monoclonal and/or polyclonal antibodies to the virus or viral pro ⁇ teins expressed by the cells of the present invention without undue experimentation.
  • the present invention further relates to vaccines for use in humans to prevent infection with and diseases caused by HTLV-I and variants thereof.
  • diseases include, adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-I-associated myelopathy.
  • the vaccine can further comprise adjuvant.
  • the attenuated or inactivated HTLV-I-SI variant virus or a non-infections antigenic portion thereof can be administered in an amount sufficient to effect protection.
  • Viral proteins may also be produced by recombinant DNA techniques or protein chemical methods.
  • the vaccine can be administered via the intramuscular, subcutaneous or intradermal routes.
  • the HTLV-I-SI variant can be used in a variety of serological test systems, including, but not limited to, enzyme immunoassay gel particle agglutination, immunofluo- rescence, Western immunoblot, radioimmunoprecipitation and antigen-capture assays.
  • Antigen preparations for use in such systems can take the form of whole virus concentrates or viral subunit proteins/peptides produced by, for example, recombinant PNA techniques or protein chemical methods .
  • the present invention further relates to bioassays for the diagnosis of infection with an HTLV-I variant virus.
  • SI-5 cells are fixed on a solid support.
  • the cells are then contacted with a biological sample from a human suspected of being infected and the presence or absence of a complex formed between proteins of cell and antibodies specific therefor present in said sample is detected.
  • the solid support can be coated with viral protein to detect antibodies in a biological sample.
  • the presence or absence of protein- antibody complexed can be detected by any of the known methods common in the art, such as colorimetry.
  • a further bioassay to which the present invention relates involves preparing a lysate from SI cells and contacting the lysate with a biological sample from a human suspected of being infected, under conditions such that a complex is formed between protein of the lysate and antibodies specific therefor present in the sample. The presence or absence of the formed complex is then detected.
  • the present invention also relates to bioassays for the diagnosis of HTLV-I variant viral infection whereby specific viral genomic sequences are detected.
  • the presence or absence of sequences can be detected by amplifying RNA in a biological sample using reverse transcriptase-directed polymerase chain reaction and detecting the PNA by restriction enzyme mapping and hybridization, or PNA sequencing.
  • the present invention also relates to bioassays utilizing antibodies specific for HTLV-I variant viral proteins. In one bioassay, a solid support is coated with such antibodies and then contacted with a biological sample from a human suspected of having the infection under conditions such that the antibody forms a complex with viral proteins within the sample. The presence or absence of the complex is then detected.
  • the present invention also relates to diagnostic - 1 - kits, such as immunoassay kits, PCR kits and hybridization kits.
  • the kits of the present invention contain the cell line of the present invention, the infecting virus of the present invention or a unique peptide of the virus. Unique peptides are used herein refer to peptides of at least 5 or 6 amino acids which are unique to the HTLV-I SI variant.
  • the kits may further comprise a solid support such as a plastic tray. The solid support may be coated with the virus or viral peptide, for example.
  • the kits of the present invention also contain ancillary reagents suitable for detection of antibody-protein complexes.
  • Another diagnostic kit of the present invention contains peptide primers of at least 20 bases of a unique sequence from the HTLV-I SI variant and ancillary reagents suitable for use in amplifying a PNA sequence and detect ⁇ ing, the presence or absence of the amplified sequence in a biological sample.
  • the primers are used to amplify viral sequences in the biological sample to detect the presence of the virus in the sample.
  • HTLV-I variant virus was deposited on February 8, 1991 at the American Type Culture Collection (ATCC) , 12301 Parklawn Prive, Rockville, Maryland 20852, U.S.A., in accord with the requirements of the Budapest Treaty.
  • the cell line designated SI-5 has been assigned the ATCC accession number CRL 10683, and is further described in the following examples.
  • Peripheral blood mononuclear cells were separated by Ficoll-Hypaque gradient centrifugation 36 hours after collection in a laboratory in which HTLV-I and other human or animal retroviruses had not been handled. Cells were stimulated for 3 days with phytohemagglutinin (PHA) (Wellcome Diagnostics, Dartford, England) at 2 ⁇ g per 10 6 cells, after which they were cultured independently, as well as co-cultivated with an equal number of PHA-stimu- lated umbilical cord blood mononuclear cells obtained from healthy, HTLV-I-seronegative Caucasian neonates (Advanced Biotechnologies, Inc., Columbia, Md) .
  • PHA phytohemagglutinin
  • Cultures were maintained at 37°C under 5% C0 2 atmosphere with biweekly media changes of RPMI 1640 supplemented with 20% heat-inactivated fetal bovine serum, 10% interleukin 2 (IL-2) (Advanced Biotechnologies) , 2 mM 1-glutamine and 50 ⁇ g of gentamicin per ml.
  • IL-2 interleukin 2
  • cultured lymphocytes were spotted onto 10-well slides (Cell-line Associates, Newfield, NJ) , then fixed with acetone for 10 min. , and examined for expression of HTLV-I antigens by the indirect immunofluorescent antibody technique, using monoclonal antibodies against HTLV-I pl9 and p24 (Cambridge Biotech Corp.
  • Virus-specific antibodies were then detected using either rhodamine-labeled goat antibodies against mouse or rabbit IgG F(ab')2 (Accurate Chemical & Scientific Corp., Westbury, NY) , or fluorescein isothiocyanate-labeled goat antibodies against human IgG (Cappel Laboratories, Inc., Cochranville, Pa) .
  • SI-1, SI-2, SI-5 and SI-6 were established from patients 1 and 2. All T-cell lines grew as clumpy suspensions, and one (SI-5 from patient 2) has been maintained in continuous culture for nearly 6 months. Of the four T-cell lines, only one (SI-1 from patient l) expressed C04 at high density (80%) , while CP8 expression was predominant (80% to 95%) in the other three.
  • high molecular weight PNA was extracted from approximately 10 7 cells from each T-cell line, using a non-organic method (Oncor, Gaithersburg, Md) , and PNA was subjected to PCR analysis.
  • Oligonucleo- tide primer pairs specific for gag (bases 863-886 and 1375-1397 for pl9; bases 1423-1444 and 1537-1560 for p24) [Reddy et al.. Science. 243:529-533 (1989)], pol (SK54, bases 3365-3384; SK55, bases 3465-3483) [Kwok et al., J. Infect. Pis..
  • the reaction mixture consisted of 1 ⁇ M each oligonucleotide primer pair, 1 ⁇ g of PNA, 2.5 units of Thermus aquaticus PNA polymerase (Perkin-Elmer Cetus, .Norwalk, Ct) , 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , 0.01% gelatin, 0.05% Nonidet P-40, and 0.2 mM each of dATP, dCTP, dGTP and dTTP. Following denaturation at 93°C for 4 min. , the reaction mixtures were cycled 35 times at 93°C for 1 in., 55°C for 2 min. and 72°C for 3 in.
  • Amplified PNA was size-fractionated by agarose gel electrophoresis and transferred to nylon membranes (Nytran, Schleicher & Schuell, Passel, Germany) for hybridization, under high stringency, with oligoprobes end-labeled with ⁇ 32 P-dCTP or with a full-length HTLV-I probe labeled with 32 P by nick translation (Oncor) .
  • HTLV-I gag and env sequences were detected in PNA from all four cell lines derived from patients 1 and 2.
  • HTLV-I-specific pol gene sequences (Fig. 4A and Fig. 4B) .
  • the inventors have maintained that the prevalence of HTLV-I infection in Melanesia is due to a variant of HTLV-I rather than to prototypical strains of HTLV-I.
  • the viral isolates from native Solomon Islanders resemble prototype HTLV-I by immunofluorescence, Western immunoblot and PCR sequence analysis of the 3 ' end of the env gene indicate that the HTLV-I variants from Solomon Islanders diverge significantly from prototype HTLV-I.

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Abstract

The present invention relates to a human T-cell line persistently infected with a Solomon Island HTLV-I-related virus, for example, the cell line, and to the infecting virus, for example, the HTLV-I-SI variant. The cell lines of the present invention grow better and produce more virus than the other known T-cell line persistently infected with an HTLV-I variant from that area. The present invention also relates to vaccines for use in humans against infection with and diseases caused by HTLV-I and variants thereof. The invention further relates to a variety of bioassays for the detection of infection by HTLV-I and variants thereof.

Description

SOLOMON ISLANDS VARIANTS OF HUMAN T-LYMPHOTROPIC VIRUS
BACKGROUND OF THE INVENTION
1. Field of the Invention The present invention relates to a cell line infected with a variant of human T-lymphotropic virus type I (HTLV-I) derived from a native Solomon Islander, desig¬ nated SI-5, and to the infecting virus, designated HTLV-I SI variant. The present invention further relates to bioassays for the detection of the virus, viral proteins or antibodies thereto in a biological sample.
2. Background Information
The retrovirus HTLV-I is the causative agent of adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-I-associated myelopathy. The prevalence of HTLV-I infection in Melanesia has been controversial since different testing methods have produced conflicting data.
High prevalences of antibodies against HTLV-I have been reported for several remote population groups in Melanesia, the ethnogeographic region comprised of Papua New Guinea, West New Guinea, Solomon Islands, Vanvuatu and New Caledonia, using enzyme immunoassay and gelatin particle agglutination [Goudsmit et al., Abstracts of the 16th Pacific Science Congress. Seoul, Korea, 73 (1987) ; Kazura et al., J. Infect. Pis.. 155: 1100-11007 (1987); Asher et al., J. Med. Virol.. 26:339-351 (1988); Brindle et al., Epidemiol. Infect.. 100: 153-156 (1988); Babona et al., Lancet, ii: 1148 (1988); Brabin et al., Int. J. Cancer. 44: 59-62 (1989) Garruto et al. , Retrovirus Humanos. Cali, Colombia, 95-108 (1989); Garruto et al. Am. J. Hum. Biol.. 2:439-447 (1990)]. However, the claims of HTLV-I hyperendemicity in Melanesia have been contested because of the inability of verifying screening results by confirmatory tests and the failure of Melanesian sera to neutralize a prototype strain of HTLV-I [Babona et al., Lancet. ii: 1148 (1988); and Weber et al., J. Infect.
Pis.. 159: 1025-1028 (1989)]. Though strains of HTLV-I isolated from different geographical regions exhibit slight genetic variation, serological diagnosis of infection for most populations can be accomplished by using tests which employ prototype HTLV-I. By contrast, most of the high sero-reactivity to prototype HTLV-I antigens in Melanesian populations cannot be confirmed with these standard tests.
The isolation of variants of HTLV-I (which diverge significantly from prototype strains of HTLV-I isolated elsewhere in the world) from T-cell lines derived from
Melanesian natives of the Solomon Islands would greatly facilitate accurate serological testing not only in the
Solomon Islands but elsewhere in Melanesia. Such a cell line would also have important application in testing other populations suspected of being infected with HTLV-I variants and in the development of a vaccine for the prevention of infection with and of diseases caused by
HTLV-I and related viruses.
SUMMARY OF THE INVENTION It is an object of the present invention to provide a eucaryotic human T-cell line, designated SI-5, persistently infected with a Solomon Islands HTLV-I variant virus.
The present invention relates to a eucaryotic cell line comprising an HTLV-I variant virus isolated from the
Solomon Islands (HTLV-I-SI variant) . The invention further relates to antibodies specific for the infecting virus.
The present invention also relates to a vaccine for humans against infection with and diseases caused by HTLV-I and related viruses. The vaccine comprises a non- infectious antigenic portion of the variant virus produced by the cell line of the present invention, administered in an amount sufficient to induce immunity against infection and to prevent disease.
The present invention also relates to bioassays for the diagnosis of infection caused by variants of HTLV-I. In one such bioassay, cells of the present invention or protein from the virus infecting the cells are fixed on a solid support and contacted with a biologi¬ cal sample from a human suspected of being infected. The presence or absence of a complex formed between protein of the cell and antibodies specific therefor present in the sample is then detected.
In another bioassay, the HTLV-I-SI variant virus nucleic acid is detected in a biological sample. The viral genomic seguences are amplified by polymerase chain reaction and the presence or absence of the amplified product are detected by restriction enzyme mapping and hybridization, and confirmed by DNA sequencing.
Various other objects and advantages of the present invention will become apparent from the figures and the following description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows intracytoplasmic virus-specific fluorescence in SI-5, a T-cell line derived from a 58-year old man with hepatosplenomegaly, using a monoclonal antibody against HTLV-I gagr-encoded protein pl9. (Origi¬ nal magnification, 500X) .
Figure 2 shows extracellular virus particles resembling HTLV-I and budding forms in SI-5, a T-cell line derived from a native Solomon Islander. (Original magni- fication, 90,000X).
Figure 3 shows a Western analysis of (lanes 1 and 2) HTLV-I isolates, SI-1 and SI-3, from two Solomon Islanders and of (lane 3) a prototype strain of HTLV-I (MT-2) , using a serum from a rabbit immunized with an 18- residue synthetic peptide spanning the extreme C-terminus of the major envelope glyc protein gp46 [Lillehog et al., Clin. Biotechnol.. 1:27-41 (1989)] (Ab gp46) and a serum from a rabbit experimentally infected with HTLV-I (Ab HTLV-I) . SI-5 gave identical results. Figures 4A and 4B show (A) ethidium bromide- stained agarose gel showing the 119-bp HTLV-I pol sequence amplified by PCR and (B) Southern analysis of the ampli¬ fied product hybridized, under high-stringency conditions, with a 32P-labelled HTLV-I pol-specific 35-mer oligo probe
(SK56, bases 3426-3460) [Kwok et al. , J. Infect. Pis.,
158:1193-1197 (1988)]. PCR was performed on PNA isolated from (lanes 1-3) SI-1, SI-2 and SI-3, three T-cell lines derived from two Solomon Islanders, (lane 4) MOLT-3, an uninfected T-cell line derived from a patient with acute lymphoblastic leukemia and (lane 5) MT-2, an HTLV-I- infected T-cell line derived from a Japanese patient with adult T-cell leukemia [Miyoshi et al.. Nature, 294:770- 771(1981)]. SI-5 gave identical results.
Figure 5 shows the partial sequence data (SEQ. IP. NO: 1) of the env gene of the HTLV-I-SI variant virus from the SI-5 cell line.
DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a human T-cell line persistently infected with an HTLV-I variant virus isolated from the Solomon Islands and the infecting virus. The present inventors have isolated HTLV-I variant viruses from peripheral blood mononuclear cells of asymptomatic carriers in the Solomon Islands. Human T-cell lines infected with an HTLV-I variant virus have been estab¬ lished, one such example is the SI-5 cell line.
The cell lines of the present invention grow more rapidly and produce more virus than PNG-1, a T-cell line infected with an HTLV-I variant established from a New Guinean. Thus, the cell lines of the present invention constitute a major improvement over the only other Melane¬ sian T-cell line infected with an HTLV-I variant virus from this area. The improved characteristics should greatly facilitate antigen production for use in diagnos¬ tic tests and for use in vaccines.
The present invention further relates to the HTLV-I variant virus infecting the above described cell lines. The HTLV-I-related viruses of the present invention, variants of prototype HTLV-I, are referred to as HTLV-I-SI variants. The env gene nucleotide sequences of the viruses of the present invention diverge signifi¬ cantly (by approximately 8%) from prototype HTLV-I. In particular, the virus isolated from the SI-5 cell line differs by 7.9% in its nucleotide sequence of the env gene from that of prototype HTLV-I.
A substantially pure preparation of the viruses of the present invention can easily be isolated from the cell line of the present invention or a lysate thereof by one skilled in the art without undue experimen¬ tation. Unique peptides of the viruses make possible the accurate diagnosis of HTLV-I infection in Melanesia and in other geographical regions where tests employing prototype HTLV-I yield high frequencies of indeterminate results.
In addition, the present invention relates to antibodies specific for the HTLV-I Si-variant or viral proteins expressed by the SI-5 cell line. One skilled in the art using standard methodology can raise monoclonal and/or polyclonal antibodies to the virus or viral pro¬ teins expressed by the cells of the present invention without undue experimentation.
The present invention further relates to vaccines for use in humans to prevent infection with and diseases caused by HTLV-I and variants thereof. Such diseases include, adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-I-associated myelopathy. The vaccine can further comprise adjuvant. The attenuated or inactivated HTLV-I-SI variant virus or a non-infections antigenic portion thereof can be administered in an amount sufficient to effect protection. Viral proteins may also be produced by recombinant DNA techniques or protein chemical methods. The vaccine can be administered via the intramuscular, subcutaneous or intradermal routes.
The HTLV-I-SI variant can be used in a variety of serological test systems, including, but not limited to, enzyme immunoassay gel particle agglutination, immunofluo- rescence, Western immunoblot, radioimmunoprecipitation and antigen-capture assays. Antigen preparations for use in such systems can take the form of whole virus concentrates or viral subunit proteins/peptides produced by, for example, recombinant PNA techniques or protein chemical methods .
Accordingly, the present invention further relates to bioassays for the diagnosis of infection with an HTLV-I variant virus. In one such bioassay SI-5 cells are fixed on a solid support. The cells are then contacted with a biological sample from a human suspected of being infected and the presence or absence of a complex formed between proteins of cell and antibodies specific therefor present in said sample is detected. Further, the solid support can be coated with viral protein to detect antibodies in a biological sample. The presence or absence of protein- antibody complexed can be detected by any of the known methods common in the art, such as colorimetry.
A further bioassay to which the present invention relates involves preparing a lysate from SI cells and contacting the lysate with a biological sample from a human suspected of being infected, under conditions such that a complex is formed between protein of the lysate and antibodies specific therefor present in the sample. The presence or absence of the formed complex is then detected.
The present invention also relates to bioassays for the diagnosis of HTLV-I variant viral infection whereby specific viral genomic sequences are detected. The presence or absence of sequences can be detected by amplifying RNA in a biological sample using reverse transcriptase-directed polymerase chain reaction and detecting the PNA by restriction enzyme mapping and hybridization, or PNA sequencing. The present invention also relates to bioassays utilizing antibodies specific for HTLV-I variant viral proteins. In one bioassay, a solid support is coated with such antibodies and then contacted with a biological sample from a human suspected of having the infection under conditions such that the antibody forms a complex with viral proteins within the sample. The presence or absence of the complex is then detected.
The present invention also relates to diagnostic - 1 - kits, such as immunoassay kits, PCR kits and hybridization kits. The kits of the present invention contain the cell line of the present invention, the infecting virus of the present invention or a unique peptide of the virus. Unique peptides are used herein refer to peptides of at least 5 or 6 amino acids which are unique to the HTLV-I SI variant. The kits may further comprise a solid support such as a plastic tray. The solid support may be coated with the virus or viral peptide, for example. The kits of the present invention also contain ancillary reagents suitable for detection of antibody-protein complexes.
Another diagnostic kit of the present invention contains peptide primers of at least 20 bases of a unique sequence from the HTLV-I SI variant and ancillary reagents suitable for use in amplifying a PNA sequence and detect¬ ing, the presence or absence of the amplified sequence in a biological sample. The primers are used to amplify viral sequences in the biological sample to detect the presence of the virus in the sample. EXAMPLES
The following examples are given to further illustrate the present invention and are not deemed limitative thereof.
Statement of Deposit A human T-cell line infected with a Solomon
Islands HTLV-I variant virus was deposited on February 8, 1991 at the American Type Culture Collection (ATCC) , 12301 Parklawn Prive, Rockville, Maryland 20852, U.S.A., in accord with the requirements of the Budapest Treaty. The cell line designated SI-5 has been assigned the ATCC accession number CRL 10683, and is further described in the following examples. Virus Isolation
In March and August 1990, blood specimens were collected, after obtaining prior informed consent, from two native Solomon Islanders, who were previously identi¬ fied as being HTLV-I seropositive by immunofluorescence and Western analysis. Patient 1 was a 40-year old woman with a history of transfusion-acquired hepatitis from Marovo Lagoon in Western Province and patient 2 was a 58-year old man with hepatosplenomegaly from Guadacanal in Guadacanal Province. The SI-5 cell line was derived from patient 2 and the SI-1 and the SI-2 cell lines were derived from patient 1.
Peripheral blood mononuclear cells were separated by Ficoll-Hypaque gradient centrifugation 36 hours after collection in a laboratory in which HTLV-I and other human or animal retroviruses had not been handled. Cells were stimulated for 3 days with phytohemagglutinin (PHA) (Wellcome Diagnostics, Dartford, England) at 2 μg per 106 cells, after which they were cultured independently, as well as co-cultivated with an equal number of PHA-stimu- lated umbilical cord blood mononuclear cells obtained from healthy, HTLV-I-seronegative Caucasian neonates (Advanced Biotechnologies, Inc., Columbia, Md) .
Cultures were maintained at 37°C under 5% C02 atmosphere with biweekly media changes of RPMI 1640 supplemented with 20% heat-inactivated fetal bovine serum, 10% interleukin 2 (IL-2) (Advanced Biotechnologies) , 2 mM 1-glutamine and 50 μg of gentamicin per ml. At weekly intervals, cultured lymphocytes were spotted onto 10-well slides (Cell-line Associates, Newfield, NJ) , then fixed with acetone for 10 min. , and examined for expression of HTLV-I antigens by the indirect immunofluorescent antibody technique, using monoclonal antibodies against HTLV-I pl9 and p24 (Cambridge Biotech Corp. , Rockville, Md) , sera from rabbits experimentally infected with a Colombian strain of HTLV-I, and sera from patient with HTLV-I myeloneuropathy [Ajdukiewicz et al., N. Enσl. J♦ Med.. 321:615-616 (1989); McKhann et al., J. Infect. Pis.. 160:371-379 (1989)].
Virus-specific antibodies were then detected using either rhodamine-labeled goat antibodies against mouse or rabbit IgG F(ab')2 (Accurate Chemical & Scientific Corp., Westbury, NY) , or fluorescein isothiocyanate-labeled goat antibodies against human IgG (Cappel Laboratories, Inc., Cochranville, Pa) . Appropriate dilutions of HTLV-I antibody-negative sera, as well as HTLV-I-infected (MT-2) [Miyoshi et al.. Nature. 294:770-771 (1981)] and unin- fected T-cells (MOLT-3) (American Type Culture Collection, Rockville, Md) served as controls. Cell Lines
Four IL-2-dependent T-Cell lines, designated SI-1, SI-2, SI-5 and SI-6 were established from patients 1 and 2. All T-cell lines grew as clumpy suspensions, and one (SI-5 from patient 2) has been maintained in continuous culture for nearly 6 months. Of the four T-cell lines, only one (SI-1 from patient l) expressed C04 at high density (80%) , while CP8 expression was predominant (80% to 95%) in the other three. By immunofluorescence, viral antigen was evident in more than 50% of cells, using autologous sera, sera from patients with HTLV-I myeloneuropathy, sera from rabbits experimentally infected with HTLV-I, as well as monoclonal antibodies against HTLV-I gag proteins p24 and pl9 (Fig. 1) . No staining was observed with HTLV-I antibody-negative sera or with a monoclonal antibody against HTLV-II gag-encoded protein p24. Mature and immature viral particles resembling HTLV-I were readily observed by thin-section electron microscopy in all four T-cell lines (Fig. 2) .
Western analysis of viral pellets, prepared by centrifuging culture medium for 1 hour at 35,000 rpm (100,000 g) in a Beckman 50.2 Ti rotor, revealed virus- specific proteins with molecular weights of 15, 19, 24, 46 and 53 kilodaltons (Fig. 3) , using monoclonal and poly- clonal antibodies against HTLV-I structural proteins, including a rabbit antiserum prepared against the C-terminus of the major envelope glycoprotein gp46 [Lillehoj et al., Clin. Biotechnol.. 1:27-41 (1989)]. HTLV-I Verification
To further verify that the viral isolates were HTLV-I rather than HTLV-II, high molecular weight PNA was extracted from approximately 107 cells from each T-cell line, using a non-organic method (Oncor, Gaithersburg, Md) , and PNA was subjected to PCR analysis. Oligonucleo- tide primer pairs, specific for gag (bases 863-886 and 1375-1397 for pl9; bases 1423-1444 and 1537-1560 for p24) [Reddy et al.. Science. 243:529-533 (1989)], pol (SK54, bases 3365-3384; SK55, bases 3465-3483) [Kwok et al., J. Infect. Pis.. 158:1193-1197 (1988)] and env (bases 5684- 5707 and 6128-6151) [Reddy et al.. Science, 243:529-533 (1989)] sequences of a prototype strain of HTLV-I [Seiki et al., Proc. Natl. Acad. Sci. USA 80:3618-3622 (1983)], were synthesized on a PCR-Mate PNA synthesizer (Applied Biosystems, Foster City, Ca) .
The reaction mixture consisted of 1 μM each oligonucleotide primer pair, 1 μg of PNA, 2.5 units of Thermus aquaticus PNA polymerase (Perkin-Elmer Cetus, .Norwalk, Ct) , 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.01% gelatin, 0.05% Nonidet P-40, and 0.2 mM each of dATP, dCTP, dGTP and dTTP. Following denaturation at 93°C for 4 min. , the reaction mixtures were cycled 35 times at 93°C for 1 in., 55°C for 2 min. and 72°C for 3 in. Amplified PNA was size-fractionated by agarose gel electrophoresis and transferred to nylon membranes (Nytran, Schleicher & Schuell, Passel, Germany) for hybridization, under high stringency, with oligoprobes end-labeled with α32P-dCTP or with a full-length HTLV-I probe labeled with 32P by nick translation (Oncor) .
HTLV-I gag and env sequences were detected in PNA from all four cell lines derived from patients 1 and 2.
As verification that the isolates were HTLV-I, PNA from the T-cell lines derived from patients 1 and 2 contained
HTLV-I-specific pol gene sequences (Fig. 4A and Fig. 4B) .
Earlier studies of more than 3500 sera, collected between 1956 and 1988 from 34 Melanesian populations, for antibodies against HTLV-I indicate high prevalence of infection, as verified by strict Western immunoblot criteria, in several remote population groups having no contact with Japanese or Africans and minimal or no contact with Europeans prior to their bleeding [Goudsmit et al.. Abstracts of the 16th Pacific Science Congress. Seoul, Korea, 73 (1987); Asher et al., J. Med. Virol.. 26:339-351 (1988); Garruto et al., Retrovirus Humanos. Cali, Colombia, 95-108 (1989); Garruto et al.. Am. J. Hum. Biol.. 2:439-447 (1990); Yanagihara et al., J. Infect. Pis.. 162:649-654 (1990)].
By contrast, some Micronesian populations having intense contact with Japanese for more than five decades have no evidence of infection. [Brindle et al., Epidemiol. Infect.. 100:153-156 (1988) and Garruto et al.. Am. J. Hum. Biol.. 2:439-447 (1990)]. These data argue against the dissemination of HTLV-I in the Pacific basin by the Japanese. The detection of HTLV-I genomic sequences by PCR and the successful isolation of HTLV-I from unrelated individuals from widely separated provinces in the Solomon Islands, reported here, adds further credibility to the serological data.
The inventors have maintained that the prevalence of HTLV-I infection in Melanesia is due to a variant of HTLV-I rather than to prototypical strains of HTLV-I. Although the viral isolates from native Solomon Islanders resemble prototype HTLV-I by immunofluorescence, Western immunoblot and PCR sequence analysis of the 3 ' end of the env gene indicate that the HTLV-I variants from Solomon Islanders diverge significantly from prototype HTLV-I.
* * * * * *
All publications mentioned hereinabove are hereby incorporated by reference.
While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: YANAGIHARA, RICHARD
AJDUKIEWICZ, ANDREW B GARRUTO, RALPH M GAJDUSEK, D. CARLETON
(ii) TITLE OF INVENTION: SOLOMON ISLANDS VARIANT OF HUMAN T-LYMPHOTROPIC VIRUS
(ϋi) NUMBER OF SEQUENCES: 1
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: CUSHMAN, DARBY & CUSHMAN
(B) STREET: Eleventh Floor, 1615 L. Street, N.W.
(C) CITY: Washington
(D) STATE: D.C.
(E) COUNTRY: U.S.A.
(F) ZIP: 20036-5601
(V) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25
( i) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Scott, Watson T.
(B) REGISTRATION NUMBER: 26,581
(C) REFERENCE/DOCKET NUMBER: WTS/5683/83619/SLO
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 861-3000
(B) TELEFAX: (202) 822-0944
(C) TELEX: 6714627 CUSH
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 522 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TCATAACTCC CTCATCCTGC CCCCCTTTTC CCTGTCACCC GTTCCGGCCC 50
CACGATCCCG TTCCCGCCGA GCGGTGCCGG TGGCGGTCTG GCTAGTCTCC 100
GCCCTGGCCA TGGGAACCGG GATTGCTGGC AGGATTACCG GCTCCATGTC 150
CCTCGCCTCA GGAAAGAGTC TCCTACATGA GGTAGACAAA GATATTTCCC 200
AATTAACCCA AGCAATAGTC AAAAATCACA AAAATCTACT CAAAATTGCA 250
CAATATGCTG CCCAGAACAG ACGACGCCTT GATCTCCTGT TCTGGGAACA 300
AGGAGGATTA TGCAAGGCGC TACAAGAACA GTGCTGTTTT CTAAACATTA 350
CTAATTCCCA CGTTTCCATA CTACAAGAAA GACCACCCCT TGAGAATCGA 400
GTCCTGACTG GCTGGGGTCT TAACTGGGAC CTTGGCCTCT CACAATGGGC 450
CCGAGAGGCC CTACAAACTG GCATCACCCT TGTTGCGCTA CTCCTTCTTG 500 TTATCCTTGC AGGACCATGC AT 522

Claims

WHAT IS CLAIMED IS:
1. A eucaryotic cell line comprising an HTLV-I variant virus isolated from the Solomon Islands (HTLV-I-SI variant virus) .
2. The cell line according to claim 1 which is a
T-cell.
3. The cell line according to claim 2 which is a human T-cell.
4. The cell line according to claim 1 wherein said virus has the partial env sequence given in SEQ. ID.
NO.: 1, or an allelic variation thereof.
5. The cell line according to claim 4 which is designated SI-5 having the accession number ATCC CRL 10683.
6. A purified antibody specific for an HTLV-I-SI variant virus.
7. A vaccine for humans against infection with and diseases caused by HTLV-I and related viruses compris¬ ing a non-infectious antigenic portion of the virus produced by said cell according to claim 1, administered in an amount sufficient to induce immunity against said infection and disease, and a pharmaceutically acceptable carrier.
8. The vaccine according to claim 8 which further comprises an adjuvant.
9. A bioassay for the diagnosis of infection with an HTLV-I-SI variant virus comprising the steps of: i)fixing said cell according to claim 1 to a solid support; ii) contacting said cell with a biological sample from a human suspected of being infected; and iii)detecting the presence or absence of a complex formed between protein of said cell and antibodies specific therefor present in said sample.
10. The bioassay according to claim 9 further comprising permeabilizing said fixed cell prior to con¬ tacting said cell with a biological sample.
11. A bioassay for the diagnosis of infection with an HTLV-I-SI variant virus comprising the steps of: i) coating a solid support with protein from the virus infecting said cells according to claim 1; ii) contacting said coated support with a biological sample from a human suspected of being infected, under conditions such that a complex is formed between said protein and antibodies specific therefor present in said sample; and iii) detecting the presence or absence of said complex.
12. A bioassay for the detection of HTLV-I-SI variant virus nucleic acid in a biological sample compris¬ ing the steps of: i) amplifying viral genomic sequences by polymerase chain reaction; and ii) detecting the presence or absence of the amplified product by restriction enzyme mapping and hybridization, and DNA sequencing.
13. A bioassay for the diagnosis of infection with an HTLV-I-SI variant virus comprising the steps of: i) coating a solid support with said antibody according to claim 6; ii) contacting said coated support with a biological sample from a human suspected of having said infection under conditions such that said antibody forms a complex with said virus or viral proteins within said sample; and ii) detecting the presence or absence of said complex.
14. The HTLV-I variant virus infecting cell line
SI-5 having the accession number ATCC CRL 10683.
15. A diagnostic kit comprising the cell line according to claim 1 and ancillary reagents suitable for use in detecting the presence of antibodies to the virus in said cell line in a biological sample.
16. A diagnostic kit comprising the virus accord¬ ing to claim 14 or a peptide of at least 6 amino acids from said virus, and ancillary reagents suitable for use in detecting the presence of antibodies to said virus in a biological sample.
17. A diagnostic kit comprising oligonucleotide primers of at least 20 bases from the virus according to claim 14 and ancillary reagents suitable for use in amplifying a DNA sequence and detecting the presence or absence of the amplified sequence in a biological sample.
18. A composition comprising a non-infectious antigenic portion of the virus produced by the cell line according to claim 1 for use in the preparation of a vaccine for the treatment of humans against infection with disease caused by HTLV-I and related viruses.
19. A composition according to claim 18, which further comprises an adjuvant.
PCT/US1992/001413 1991-02-28 1992-02-28 Solomon islands variants of human t-lymphotropic virus WO1992015325A1 (en)

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CA2089761A1 (en) * 1990-08-29 1992-03-01 Elaine Defreitas Method and compositions for diagnosing and treating chronic fatique immunodysfunction syndrome

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
THE JOURNAL OF INFECTIOUS DISEASES, Vol. 155, No. 6, issued June 1987, KAZURA et al., "Epidemiology of Human T Cell Leukemia Virus Type I Infection in East Sepik Province, Papua New Guinea", pages 1100-1107. *
THE JOURNAL OF INFECTIOUS DISEASES, Vol. 158, No. 6, issued December 1988, KWOK et al., "Characterization of a Sequence of Human T Cell Leukemia Virus Type I from a Patient with Chronic Progressive Myelopathy", pages 1193-1197. *
THE LANCET, issued 12 November 1988, BABONA et al., "HTLV-I Antibodies In Papua New Guinea", page 1148. *
THE NEW ENGLAND JOURNAL OF MEDICINE, Vol. 321, No. 9, issued 31 August 1989, AJDUKIEWICZ et al., "HTLV-I Myeloneuropathy In The Solomon Islands", pages 615-616. *

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