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WO1992003160A1 - Virus t-lymphotrope humain de papouasie-nouvelle-guinee - Google Patents

Virus t-lymphotrope humain de papouasie-nouvelle-guinee Download PDF

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Publication number
WO1992003160A1
WO1992003160A1 PCT/US1991/005896 US9105896W WO9203160A1 WO 1992003160 A1 WO1992003160 A1 WO 1992003160A1 US 9105896 W US9105896 W US 9105896W WO 9203160 A1 WO9203160 A1 WO 9203160A1
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WIPO (PCT)
Prior art keywords
htlv
png
variant
cell
infection
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PCT/US1991/005896
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English (en)
Inventor
Richard Yanagihara
Ralph Garruto
Marc Miller
Vivek Nerurkar
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The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce
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Publication of WO1992003160A1 publication Critical patent/WO1992003160A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
    • C12N2740/14021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
    • C12N2740/14022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to Papua New Guinea variants of HTLV-I.
  • the present invention relates to Papua New Guinea variants of HTLV-I.
  • the present invention relates to Papua New Guinea variants of HTLV-I.
  • the present invention relates to Papua New Guinea variants of HTLV-I.
  • the present invention relates to Papua New Guinea variants of HTLV-I.
  • the present invention relates to Papua New Guinea variants of HTLV-I.
  • the present invention relates to a human T-cell line persistently infected with a Papua New Guinea HTLV-I variant.
  • the present invention further relates to bioassays and kits for the diagnosis of HTLV-I infections.
  • HTLV human T-cell leukemia lymphoma viruses
  • HTLV-I a member of this group, is the causative agent of adult T-cell leukemia/lymphoma
  • a cell line persistently infected with an HTLV-I variant derived from a healthy New Guinean, would facilitate testing in Melanesia, where high prevalences of HTLV-I infection have been found. Such a cell line would also have important application in testing populations elsewhere in the world and in the development of a vaccine for the prevention of infection with and of diseases caused by HTLV-I and related viruses.
  • methods and diagnostic kits which detect Melanesian HTLV-I variants may obviate serodiagnostic problems encountered in Melanesia and in other geographical regions where serological tests employing cosmoplitan prototypes of HTLV-I yield high frequencies of indeterminate results.
  • the present invention relates to a cell line, designated Papua New Guinea-1 (PNG-1) comprising an HTLV-I variant, for example, (ATCC
  • the present invention relates to a purified antibody specific for a PNG-1 viral protein.
  • the present invention relates to a vaccine for humans against infection with and diseases caused by HTLV-I and related viruses comprising a non-infectious antigenic portion of the PNG-1 variant, in an amount sufficient to induce immunity against said infection and disease, and a pharmaceutically acceptable carrier.
  • the present invention relates to bioassays for the diagnosis of infection with the PNG-1 variant.
  • PNG-1 cells are fixed on a solid support. The cells are then contacted with a biological sample from a human
  • a solid support is coated with viral protein and contacted with a biological sample from a human suspected of being infected, under
  • a further bioassay to which the present invention relates involves preparing a lysate from PNG-1 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 infection with the PNG-1 variant by the detection of PNG-1 specific genomic sequences.
  • the presence or absence of PNG-1 sequences can be detected by amplifying RNA in a biological sample using reverse transcriptase-directed polymerase chain reaction.
  • the present invention also relates to bioassays utilizing antibodies specific for PNG-1 viral proteins.
  • 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 PNG-1 viral proteins within the sample. The presence or absence of the complex is then detected.
  • the present invention relates to a diagnostic kit comprising variant-specific peptides for the Papua New Guinea HTLV-I variant and ancillary reagents suitable for use in detecting the presence or absence of antibody-peptide complexes.
  • the present invention relates to a diagnostic kit comprising variant-specific oligonucleotide primers for the Papua New Guinea HTLV-I variant and ancillary reagents suitable for use in DNA amplification and detection.
  • Figure 1 shows virus-specific fluorescence in PNG-1 cells by double-label immunofluorescence test, using sera from (upper) a Colombian patient with
  • Figure 2 show a thin-section electron micrograph of PNG-1 cells demonstrating a solitary mature virus particle resembling HTLV-I (arrow). (Original
  • Figure 3 shows the sequence analysis of amplified, cloned DNA.
  • DNA from virus infected cell lines was amplified and (A) pX, (B) pol, (C) gp21 and (D) gp46 regions were sequenced. Fractions above a nucleotide change indicate the frequency of that mutation seen in different clones from an individual patient.
  • sequences of the corresponding regions of the HTLV-I-infected cell line, HS-35, derived from a Caribbean patient, and the STLV-I-infected cell line, PtM3, from a pig-tailed macaque (Macaca nemestrina) originally imported from Indonesia are included where data was available. Sites of insertion are as
  • Figure 4 shows the nucleotide sequence alignment of the 522-base pair, gp21-encoding region of the env gene amplified from DNA from six Melanesians (HTLV-I
  • the arrow indicates the cleavage site between the carboxy terminus of gp46 and the amino terminus of gp21. There were no deletions or
  • Figure 5 shows the comparison of deduced amino acid sequences of the env gene region from a Japanese
  • Blanks indicate homologous sequence with prototype HTLV-I MT2 . Note shared amino acids between the Melanesian HTLV-I variants and HTLV-II (and STLV-I) at positions 305, 328, 330 and 372. The single letter amino acid code was used.
  • Figure 6 shows a hydropathy analysis of the deduced amino acid sequence of the env protein.
  • the plot shows a large hydrophobic region and alternating hydrophobic and hydrophilic domains typical of membrane proteins.
  • the positions of the amino acid residues and the values of the hydrophobic indices are shown on the x and y axes, respectively.
  • the cleavage site between the C-terminus of the major envelope glycoprotein gp46 and the N-terminus of the transmembrane protein gp21 is indicated by an arrow.
  • Figure 7 shows dendrograms evolutionary trees for the HTLV/STLV family of retroviruses.
  • A Relationship based on the regions sequenced from pol , env (gp21, gp46) and tax.
  • B Relationship based on sequences from env (gp21, pg46) and tax.
  • the present invention relates to a cell line, preferably a human T-cell line, persistently infected with a Papua New Guinea (PNG) HTLV-I variant.
  • Cells of the present invention express viral antigens, type C particles and have a low level of reverse transcriptase activity.
  • the inventors have established a human T-cell line, designated PNG-1, derived from peripheral blood mononuclear cells of a healthy New Guinean with the above described characteristics.
  • PNG-1 a CD8 + T-cell line
  • PNG-1 variant indigenous to Papua New Guinea
  • the present invention further relates to the virus infecting PNG-1 cells.
  • a substantially pure virus infecting PNG-1 cells A substantially pure virus infecting PNG-1 cells.
  • the preparation of the infecting PNG-1 variant can easily be isolated from the cell line or a lysate thereof by one skilled in the art without undue experimentation.
  • the PNG-1 variant is only about 92% identical to a Japanese prototype HTLV-I (ATK-1) (Seiki et al., PNAS USA 1983; 80: 3618-3622) and to HTLV-I strains isolated from Japanese patients with HTLV-I-associated myelopathy (Kinoshita et al., Int. J.
  • the PNG-1 variant lacks close sequence homology with a prototype strain (C344/Mo) of HTLV-II (Shimotohno et al., PNAS USA 1985; 82: 3101-3105) and an Asian subtype of STLV-I (Watanabe et al., Virology 1985; 144: 59-65), it is somewhat more closely related to HTLV-II than are cosmopolitan prototypes of HTLV-I.
  • the present invention relates to antibodies specific for the PNG-1 variant or viral proteins expressed by PNG-1.
  • PNG-1 antibodies specific for the PNG-1 variant or viral proteins expressed by PNG-1.
  • One skilled in the art using standard methodology can raise monoclonal and/or polyclonal antibodies to the variant or viral proteins expressed by the cells of the present invention without undue experimentation.
  • the present invention also relates to a vaccine for use in humans to prevent infection with and diseases caused by HTLV-I and related viruses.
  • Diseases to which the present invention relates include, adult T-cell leukemia/lymphoma and tropical spastic
  • a non-infectious antigenic portion of the PNG-1 variant can be delivered to a human in a pharmacologically
  • Antigen preparations for use in the vaccine can take the form of inactivated/attenuated whole virus concentrates, for example, PNG-1 cell lysate, or viral proteins (or fragments thereof).
  • the viral proteins and protein fragments can be produced, for example, by recombinant DNA techniques.
  • Vaccines of the present invention can also include effective amounts of immunological adjuvants known to enhance an immune response.
  • immunological adjuvants known to enhance an immune response.
  • antigenic portion of PNG-1 variant is in the vaccine in an amount sufficient to induce an immune response against the antigenic portion and thus to protect against infection with and diseases caused by HTLV-I and related viruses.
  • the vaccines can be administered via the intradermal, subcutaneous or intra-muscular route.
  • the vaccination may consist of a single
  • administration or a series of administrations. This will vary depending on several factors, such as the patient's age and condition and the route of
  • PNG-1 and variant-specific peptides thereof can be used in a variety of serological test systems,
  • the present invention relates to bioassays for use in human medicine. For diagnosis of adult T- cell leukemia/lymphoma, tropical spastic
  • the presence of antibodies to PNG-1 proteins or the presence of the viral proteins in a biological sample such as, for example, serum or culture fluid, can be determined.
  • antibodies against Papua New Guinea HTLV-I variants are detected with the use of variant-specific peptides.
  • the variant-specific peptides can be
  • Suitable peptides include those encoded by variant- specific regions of the env gene sequences, such as, gp46 aa 17-28 and gp21 aa 324-335.
  • Preferred peptides include, ProIleLeuSerPheTyrSerProSerCysCysThr (amino acids 17-28) for the major envelope glycoprotein gp46 and LeuAlalleGlyThrGlylleAlaGlyGlylleThr (amino acids 324-335) for the transmembrane glycoprotein gp21.
  • the peptides are purified such as, by preparative high- performance liquid chromatography. Peptide sequence and purity can be confirmed by amino acid composition and sequence studies.
  • the variant-specific peptides are used to detect IgG, IgM or IgA antibodies in a biological sample (such as serum or cerebrospinal fluid) using immunoassays.
  • a biological sample such as serum or cerebrospinal fluid
  • Wells of plates such as polyvinyl chloride plates, are coated with the peptides.
  • the wells are then coated with an agent to block excess reactive sites, such as 3% bovine serum albumin.
  • the biological sample is then diluted (for example, 1:20) and added to the wells.
  • the antibody-antigen complexes are detected by labelled antibody against human IgG, IgM or IgA.
  • the antibody can be labelled with alkaline phosphatase which causes a change in color detectable by an ELISA reader.
  • PNG-1 cells are fixed on a surface and then their membranes are permeabilized, such as with acetone.
  • the fixed cells are contacted with serum from a patient and the presence or absence of the viral protein-antibody complex is then detected using methods well known in the art.
  • a surface i.e., a solid support
  • a nitrocellulose membrane used in Western blots on which PNG-1 cell lysates or purified virus or variant specific recombinant proteins have been
  • electrotransferred is contacted with a sample, such as serum, from a patient suspected of having disease or infection.
  • a sample such as serum
  • the presence of a resulting complex formed between the viral protein(s) and antibodies specific therefor in the serum can be detected by any of the known methods common in the art, such as biotinylated or enzyme-labeled secondary antibodies.
  • the PNG-1 protein or variant- specific peptide thereof can be bound to an inert particle of, for example, bentonite or polystyrene latex.
  • the particles are mixed with serum from a patient in, for example, a well of a plastic
  • agglutination tray The presence or absence of antibodies in the patient's serum is determined by observing the settling pattern of the particles in the well.
  • the presence or absence of viral nucleic acid in a serum sample is detected.
  • Viral genomic sequences can be amplified (for example, polymerase chain reaction) and detected by, for example, ethidium bromide staining or Southern blot analysis. Confirmation of the
  • Suitable variant-specific primers for env gene amplification include 5'-CCGGCCTCACAATCCCGTTCCCGC-3' and 5'-TGGCGGTCTGGCTAGTCTCC-3' (sense primers) and 5'-AAACGTGGGAATTAGTGATGTTTA-3' and 5'- CTTGTAGCGCCTTGCATAATCC-3' (antisense primers).
  • the amplified sequences can be detected with an oligoprobe, such as 5'-CAGACGAGGCCTTGATCTCC-3'.
  • the presence or absence of PNG-1 variant-specific protein in a serum sample is detected with antibodies.
  • Antibodies of the present invention specific for a virus protein thereof can be coated onto a solid surface such as a plastic and contacted with the serum sample. After washing, the presence or absence of the virus protein from the serum bound to the fixed
  • antibodies is detected such as by addition of a labeled (e.g. enzyme-labeled) antibody specific for the virus.
  • a labeled antibody e.g. enzyme-labeled
  • kits for the diagnosis of HTLV-I infections, particularly PNG-I infections provide an easy and safe means of diagnosing infections.
  • kits of the present invention includes variant-specific peptides from the Papua New Guinea variant virus, such as
  • the kit also includes ancillary reagents suitable for use in
  • Another diagnostic kit of the present invention contains oligonucleotide primers specific for the Papua New Guinea variant virus and ancillary reagents
  • Suitable primers include, 5'-CCGGCCTCACAATCCCGTTCCCGC-3' and 5'-TGGCGGTCTGGCTAGTCTCC-3' (sense primers) and 5'-AAACGTGGGAATTAGTGATGTTTA-3' and 5'-CTTGTAGCGCCTTGCATAATCC-3' (antisense primers).
  • one such kit contains PCR reaction mix (Tris HCl at pH 8.3, KCl, MgCl 2 , dNTPs and AmpliTaq DNA polymerase), and primers for routine PCR and for nested PCR. The PCR reaction is carried out at 94° C for 5 min., followed by 35 cycles of 94o C for 1 min., 55° C for 1 min. and 72° C for 3 min. PCR is then continued at 72° C for 7 minutes and cooled to 4o C until
  • the amplified product can be detected using the standard methods. For example, agarose gel electrophoresis and ethidium bromide staining can be employed. Alternatively, the amplified product can be detected using Southern blot analysis with a full- length HTLV-I probe or internal oligonucleotide probes, such as, 5'-CAGACGAGGCCTTGATCTCC-3', labeled with 32 P and high stringency wash conditions.
  • the human T-cell line PNG-1 was deposited on August 14, 1990 at the American Type Culture Collection (Rockville, MD), in accord with the requirements of the Budapest Treaty.
  • the cell line PNG-1 has been assigned the ATCC accession number CRL 10528.
  • the Hagahai lack the HLA-A2 antigen associated with recent years
  • Bellona (population 650), known also as Mu Ngiki (or "small island”), is, along with Rennell, Tikopia, Anuta (Cherry Island), Sikaiana
  • IL-2 interleukin 2
  • transcriptase activity and for viral particles by electron microscopy.
  • Re-isolation attempts were conducted in an HTLV-I-free laboratory on lymphocytes from 15 Hagahai. Lymphocytes, preserved in 10% DMSO and stored in liquid nitrogen, were rapidly thawed in a 37oC water bath and were stimulated with PHA, as described above. Cells were then co-cultivated with approximately 2 ⁇ 10 6 PHA-stimulated umbilical cord blood mononuclear cells obtained from healthy Caucasian neonates (Advanced Biotechnologies, Inc.), who lacked evidence of HTLV-I infection as determined by the polymerase chain reaction. Cultures were maintained with growth medium supplemented with IL-2. Fresh PHA-stimulated cord mononuclear cells were added, as needed, to maintain the cell density at 10 6 per ml.
  • lymphocytes spotted onto 10-well slides (Cell-line Associates, Newfield, NJ) and fixed with cold acetone for 10 min, were examined for the expression of HTLV-I antigens by the indirect immunofluorescent antibody technique, using monoclonal antibodies against HTLV-I pl9 (Pan-Data Systems, Inc., and Cambridge Biotech Corp., Rockville, Md) and p24 (Cambridge Biotech
  • rabbit antiserum prepared against native p24 protein and against synthetic peptides of the C- terminus of gp46 (generously provided by Steve S.
  • 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, Penn). Incubations were performed in a humidified chamber at 37 °C for 30 min, and slides were washed with 0.01 M phosphate buffered saline (pH 7.2).
  • HTLV- I infected mouse, rabbit and human negative control sera and HTLV- I infected (MT-2 cells) (Miyoshi et al., Nature 1981; 294: 770-771) and uninfected cells (MOLT-3) (American Type Culture Collection, Rockville, Md) were included in each test. Fluorescence was observed using a Leitz epifluorescence microscope.
  • Cell lysates were prepared by gently mixing 50 ⁇ 10 6 cells in 2 ml 0.1 M Tris-HCl (pH 7.4) containing 0.5% sodium deoxycholate (Sigma Chemical Co., St. Louis, Mo), 0.5% Triton X100 and 0.05% sodium dodecyl sulfate at 4°C for 30 min.
  • Lysates were clarified by centrifugation at 35,000 rpm (100,000 g) in a Beckman 50.2 Ti rotor for 1 hr. The supernatant was then mixed with sample buffer, and viral proteins were separated by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels (Laemmili, Nature 1970;227:680-685) Proteins were transferred electrophoretically to nitrocellulose membranes
  • oligonucleotide primers synthesized on a PCR-Mate DNA synthesizer (Applied Biosystems), which were specific for env, gag and tax sequences of ATK-1, a prototype Japanese strain of HTLV-I (Seiki et al., PNAS USA 1983;80:3618-3622).
  • the reaction mixture consisted of 50 mM KC1, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , 0.01% gelatin, 0.05% Nonidet P-40, 0.2 mM each dATP, dCTP, dTTP and dGTP, 1 ⁇ M each oligonucleotide primer, and 2.5 U of TaqDNA polymerase (Perkins-Elmer Cetus, Norwalk, Ct). Following denaturation at 93°C for 4 min, the reaction mixtures were cycled 35 times at 93°C for 1 min, 55°C for 2 min and 72°C for 3 min. After one round of amplification with env primers, products were further amplified using "nested" primers.
  • Amplified DNA was size-fractionated by agarose gel electrophoresis and transferred to nylon membranes for hybridization using oligoprobes or a full-length HTLV-I probe labeled with 32 P.
  • Genomic DNA extracted from PNG-1, MT-2 and MOLT-3 cells, was digested with several restriction endonucleases (EcoRI, Pstl, Sad, Hindlll) .
  • EcoRI restriction endonucleases
  • the digested DNA was separated on a 0.8% agarose gel, transferred onto Nylon membrane (Schleicher & Schuell) and hybridized with a full- length HTLV-I genomic probe labeled with 32 P.
  • Electron Microscopy Cells were centrifuged at 1000 rpm for 10 min, and pellets were fixed in 2% glutaraldehyde for 2 hrs. at 4oC, postfixed in 1% osmium tetroxide for 2 hrs., dehydrated through a graded series of ethanol and propylene oxide and embedded in Embed (Electron Microscopy Sciences, Fort Washington, Penn). Ultrathin sections, stained with lead citrate and uranyl acetate, were examined using a Hitachi H7000 transmission electron microscope at 75 kV.
  • PNG-1 One culture, designated PNG-1, derived from a 20- year old Hagahai man, who had IgG antibodies against HTLV-I gag and env-encoded proteins by Western
  • HTLV-I-infected T-cell lines Like some HTLV-I-infected T-cell lines, mature viral particles resembling HTLV-I were found only rarely in extracellular spaces of PNG-1 cells, by thin- section electron microscopy ( Figure 2) . However, lysates of PNG-1 cells, analyzed by Western immunoblot, exhibited virus-specific bands at 15, 19, 24, 46 and 53 kilodaltons, using sera from Colombian and Chilean patients with virologically confirmed HTLV-I
  • HTLV-I sequences were detected in DNA extracts from PNG-1 cells by polymerase chain reaction (PCR), using oligonucleotide primers specific for gag, env and tax sequences of ATK-1, a prototype strain of HTLV-I.
  • PCR polymerase chain reaction
  • PNG-l was more extensively compared with other HTLV-I and HTLV-II isolates to determine the variability of PNG-1 from cosmopolitans prototype strains of HTLV-I (see Table I below).
  • DNA from the HTLV-I-infected cell lines PNG-1 and HSC-CTCL-11B was amplified and sequenced in specific regions of the pol , pX, and env (gp21 and gp46) genes.
  • DNA from the cell line MoT and from two HTLV-II-infected patients (RW and FF) was amplified in equivalent regions of the pol and pX genes.
  • Oligonucleotides were named by a two letter initial for HTLV (HT) followed by the number of the designated virus (I or II), then by an initial for the gene or region of the indicated virus with the numbered
  • PNG-1, HSC-CTCL-llB, HUT 102B2, and MT-2 are cell lines containing HTLV-I isolates from a Papua New Guinean, a Liberian of American slave descent, an
  • PNG-1 belongs to the HTLV-I subgroup, it varies considerably from
  • a protein-coding region in the tax gene (ORF pX-II) of HTLV-I was sequenced for PNG-1 and HSC-CTCL-llB and the corresponding HTLV-II region for MoT (Kalyanaraman et al., Science 1982; 218: 571-573; and Shimotohno et al., PNAS USA 1985; 82: 3101-3105) and two other HTLV- II isolates (RW and FF) (Fig. 3A) .
  • Published sequences for a prototype Japanese HTLV-I isolate (ATK-1) (Seiki et al., PNAS USA 1983; 80: 3618-3622) (EMBL numbers are identical to the sequence of Seiki et al.
  • a Caribbean HTLV-I (HS-35) (Malik et al., J. Gen. Virol. 1988; 69: 1695-1710), and STLV-I (Watanabe et al., Virology 1985; 144: 59-64) were also included for comparison. Very little sequence variation was found among the isolates in this pX region. The HTLV-II isolates vary only 15% from the Japanese isolate as compared to 40% for the entire proviral DNA sequence.
  • sequencing error of the original ATK-1 clone may account for the discrepancy.
  • a deletion in 6 independent env (gp21) clones of PNG-1 resulted in an altered reading fraire of the transmembrane protein.
  • One PNG-1 env (gp21) clone of 6 contained a deoxyguanidine that is not present in the others. Since the env gene (Fig. 3C and 3D) is more variable overall than the tax or pol genes, quasispecies (Shaw et al., PNAS USA 1984; 81: 4544-4548) may exist as defined by gp21 and gp46. In fact, two distinct gp46 clones for PNG-1 were found, indicating the presence of quasispecies.
  • Figure 3D shows the sequence comparison of the HTLV variants from the 5' region of the env gene which encodes the extracellular membrane protein gp46. It was expected that portions of this gene region would be highly variable since the protein it encodes is under continuous selective pressure of the patient's immune system (Paquette et al., PNAS USA 1989; 86: 3896-3900) and provides a target for neutralizing antibodies and antibody-dependent cellular cytotoxicity, while other portions would be strongly conserved since
  • PNG-1 contained a deletion near the 5' end of gp46 (as indicated in Fig. 3D1) which changed the reading frame, but an insertion occurred shortly thereafter that restored the protein to the consensus frame.
  • This specific region of the gp46 (EMBL No. 5250-5265) exhibited considerable variation in the STLV-I isolate and HTLV-II isolates, as well as the Caribbean and Liberian HTLV-I isolates.
  • This nucleotide sequence and its corresponding peptide may be valuable in typing virus variants and for diagnosis of infection by creating specific oligonucleotide primers for PCR or specific peptides for ELISA and Western blot
  • Oligonucleotide primer pairs derived from highly conserved regions of the HTLV-I env gene (sense strand, 5'-TTTGAGCGGCCGCTCAAGCTATAGTCTCCTCCCCTG-3'; anti-sense strand, 5'-ACTTAGAATTCGGAGGTGTCGTAGCTGACGGAGG-3 ' ) and containing NotI and EcoRI restriction sites
  • the 522- base pair amplified region which corresponded to bases 6046 to 6567 (equivalent to EMBL no. 6068 to 6589 of prototype HTLV-I ATK-1 ), encompassed the cleavage site of the envelope precursor protein and included nearly the entire coding region for the transmembrane glycoprotein gp21.
  • Amplified DNA was cloned into the NotI and EcoRI restriction sites of the Bluescript vector, then transformed into HB101 competent cells.
  • Recombinant clones were screened by hybridization, under high stringency conditions, with a 32 P-end-labeled internal oligonucleotide probe (5'-CAGACGAGGCCTTGATCTCC-3', corresponding to bases 6313 to 6332). Nucleotide sequences of one to three clones from each DNA sample were determined by the dideoxynucleotide termination method, and sequence analysis was facilitated by using the Microgenie program (Beckman).
  • HTLV-I env gene region of the HTLV-I env gene was amplified and sequenced.
  • STLV-I Asian subtype of simian T-lymphotropic virus type I isolated from a pig-tailed macaque (Macaca nemestrina) originally imported from Indonesia (Watanabe et al., Virology 1985; 144: 59-65) (Table III).
  • the nucleotide changes identified in the HTLV-I variants from Melanesia corresponded primarily to single base substitutions within a given codon, the vast majority (85%) occurring at the third position, resulting in no amino acid change (see Fig. 5).
  • the Melanesian HTLV-I variants differed by 2.3% to 4.0% (4 to 7 amino acids in 174 residues) from the prototype Japanese HTLV-I MT-2 .
  • HSC-CTCL-llB HSC-CTCL-llB
  • Fig. 7A the four sequenced regions (pol, pX, gp21 and gp46) were compared and the divergence pattern showed that PNG-1 diverged from a common ancestor of HTLV-I prior to strains from Africa (EL) (Paine et al., Virology 1991; 182: 111-123), the Caribbean (HS-35) (Malik et al., J. Gen. Virol. 1988; 69: 1695-1710), Liberia (HSC-CTCL-llB) (Ehrlich et al., Am. J. Hematol.
  • HTLV-I may have originated at the same time or prior to the time when the ancestors of these ancient Hagahai people of Papua, New Guinea became isolated.
  • FIG. 7B A dendrogram, constructed exclusive of the pol region (Fig. 7B permits inclusion of STLV-I (Watanabe et al., Virology 1985; 144: 59-64) and an isolate from a Japanese HTLV-I-associated myelopathy patient (H5) (Tsujimoto et al., Mol. Biol. Med. 1988; 5: 29-42).
  • the degree of divergence for PNG-1 decreased slightly relative to other HTLV-I isolates, but now an Asian subtype of STLV-I can be seen branching from HTLV-II prior to PNG-1.
  • the Asian subtype of STLV-I varied from prototype HTLV-I by 10% and at the same time the African subtype of STLV-I varies by only 5% (Watanabe et al., Virology 1986; 143: 385-388). These estimates are based on comparisons of a highly variable region of HTLV, the LTR. When more conservative regions are analyzed, the Asian subtype of STLV-I varied by almost 10% and PNG-1 varied by approximately 6.5%.
  • dendrograms seem to reflect the entire genome, as the divergence pattern for the isolates other than PNG-1 is in complete agreement with dendrograms created for the HTLV family based on sequences of full-length clones and of clones of several kilobases in length.

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Abstract

La présente invention concerne une lignée de lymphocytes T humains (PNG-1) infectée de manière persistante avec une variante du HTLV-I de Papouasie-Nouvelle-Guinée (PNG), ainsi que le virus contaminant (variante de PNG-1). Les cellules décrites expriment des antigènes viraux, des particules du type C et présentent un faible niveau d'activité de transcriptase inverse. La mise en évidence de cette lignée cellulaire, la première de son genre provenant d'un individu de Papouasie-Nouvelle-Guinée, rend possible le dépistage des populations mélanésiennes au moyen d'une souche virale locale. La présente invention concerne également des vaccins s'utilisant chez des humains contre l'infection et les maladies provoquées par le HTLV-I et les virus apparentés. L'invention concerne par ailleurs une grande variété de titrages biologiques et de trousses pour la détection et le diagnostic de l'infection et des maladies causées par le HTLV-I et les virus apparentés.
PCT/US1991/005896 1990-08-24 1991-08-23 Virus t-lymphotrope humain de papouasie-nouvelle-guinee WO1992003160A1 (fr)

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
THE JOURNAL OF INFECTIOUS DISEASES, Volume 155, No. 16, issued June 1987, KAZURA et al., "Epidemiology of Human T Cell Leukemia Virus Type 1 infection in Fast Sepik Provine, Papua New Guinea", pages 1100-1107. *
THE JOURNAL OF INFECTIOUS DISEASES, Volume 159, No. 6, issued June 1989, WEBER et al., "HTLV-I infection in papua New Guinea: Evidence for Serologic False Positivity", pages 1025-1028. *
THE JOURNAL OF MEDICAL VIROLOGY, Volume 30, issued 1991, PATERLINI et al., "Polymerase Chain Reaction for studies of Mother to child Transmission of HIV-I in Africa", pages 53-57. *
THE LANCET, issued 12 November 1988, RABONA et al., "HTLV-1 antibodies in Papua New Guinea", page 1148, see entire document. *
THE NEW ENGLAND JOURNAL OF MEDICINE, Volume 321, No. 9, issued August 1989, AJDUKIEWICZ et al., "HTLV-T Myelo-neuropathy in the Solomon Islands", pages 615-616. *

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