[go: up one dir, main page]

WO1993010222A1 - Nouveau retrovirus humain intracisternal associe a la deficience et au dysfonctionnement des cellules t cd4 + - Google Patents

Nouveau retrovirus humain intracisternal associe a la deficience et au dysfonctionnement des cellules t cd4 + Download PDF

Info

Publication number
WO1993010222A1
WO1993010222A1 PCT/US1992/010238 US9210238W WO9310222A1 WO 1993010222 A1 WO1993010222 A1 WO 1993010222A1 US 9210238 W US9210238 W US 9210238W WO 9310222 A1 WO9310222 A1 WO 9310222A1
Authority
WO
WIPO (PCT)
Prior art keywords
hicrv
cells
antigen
complex
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1992/010238
Other languages
English (en)
Inventor
Sudhir Gupta
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California Berkeley
University of California San Diego UCSD
Original Assignee
University of California Berkeley
University of California San Diego UCSD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of California Berkeley, University of California San Diego UCSD filed Critical University of California Berkeley
Publication of WO1993010222A1 publication Critical patent/WO1993010222A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • 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
    • C12N2740/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences

Definitions

  • the present invention relates to the discovery of a new human intracisternal retrovirus (HICRV) and to assays and therapy related to that virus.
  • HCRV human intracisternal retrovirus
  • Human retroviruses are associated with several human diseases that involve disturbances of the growth of CD4 + T lymphocytes (Wong-Staal F. and R. Gallo, Nature 317: 395, 1985).
  • Human T lymphotropic virus type I (HT V I) is associated with malignant expansion of CD4+ T cells (Poiesz B.J. et al. Proc. Natl. Acad. Sci.. USA. 77: 7415, 1980)
  • human immunodeficiency virus (HIV-1 ) is associated with depletion of CD4+ T cells, resulting in the acquired immunodeficiency syndrome (AIDS) (Barre-Sinoussi F. et al. Science 220: 868, 1983; Popovic M. et al.
  • HIV-1 acts by attacking lymphocytes and macrophages which display the CD4 antigen on their cellular surface. These cells are responsible for profound immunity against foreign invaders. HIV-1 defeats the hosts' ability to combat disease by killing these cell of the immune system.
  • Retroviruses (including HIV-1 , HIV-2, HTLV I, HTLV II) act by carrying within their protein coat an RNA message that is transcribed into DNA by the viral protein reverse transcriptase. This viral DNA is then incorporated into the host DNA through homologous recombination and used as a template for future messenger RNA production. The protein sequences coded by the viral nucleotides instruct the cell to make more viral particles.
  • the virus causes lysis of the cell, releasing new viral particles into the body (Ho D. D. et al. N. Eno. J. Med. 317: 278, 1987; Green W.C. N. En ⁇ . J. Med. 324: 308, 1991 ).
  • PCP Pneumocystis carinii pneumonia
  • AIDS Fauci A.S. Science 239:617, 1988.
  • PCP occurred primarily in immunocompromised patients, particularly patients with conditions such as lymphoma, leukemia, organ transplantation, hypogammaglobulinemia, or therapy with cytotoxic agents and corticosteroids.
  • Rare cases of Pneumocystis carinii without predisposing conditions have been reported (Anderson CD. et at. Amer. J. Clin. Path.
  • PCP has also been reported in normal immunocompetent children (Pifer L.L. et al. Pediatrics. 61 (1 ): 35, 1978). More recently a number of patients with PCP and T cell deficiency, and in particular CD4+ T cell deficiency, have been described (Jacobs J.L. et al. N. En ⁇ . J. Med. 324: 246, 1991 ; Gaut ⁇ erV. et al. Clin. EXD. Allergy 21 : 63, 1991 ). All these patients were negative for HIV-1 infection.
  • One aspect of the invention provides cell lines in continuous culture infected with HICRV. Another aspect of the invention comprises substantially purified HICRV. Still another embodiment of the invention is monoclonal antibody against HICRV.
  • the invention may include recombinant DNA, such as plasmid DNA, coding for HICRV viral protein.
  • the present invention also includes a method for determining the presence or absence of HICRV antibody or HICRV antigen in a biological sample, comprising the steps of contacting the biological sample with a reagent adapted to immunologically and preferentially bind with the antibody or antigen to form an immunological complex, and detecting the presence or absence of the immunological complex.
  • the reagent is HICRV antigen.
  • the complex may be detected through a colorimetric change.
  • the complex radioactive and the detection comprises identifying or measuring the radioactivity.
  • Still another alternative involves detecting the complex through fluorescence.
  • kits for detecting the presence or absence of HICRV antigen or anti-HICRV antibody in a sample comprising means for forming an immunological complex with the HICRV antigen or antibody in the sample, and means for indicating the presence or absence of the complex.
  • the indicating means can be a detectable label for labeling the complex.
  • the means for forming the complex can be anti-HICRV antibody adapted to bind with HICRV antigen in the sample, or HICRV antigen adapted to bind with anti-HICRV antibody in the sample.
  • Figure 1 is a series of transmission electron micrographs of mononuclear cells from the patient (photographs A and B) and the daughter (C). Viral particles are indicated by arrows.
  • Figure 2 is a bar graph illustrating the results of a reverse transcriptase assay from supernatants of PHA stimulated, IL-2 activated patient mononuclear cells.
  • Figure 3 illustrates the results from a PCR amplification of genomic DNA
  • DNA using primers for HIV and HTLV tax gene regions. Amplified nucleic acid was separated by agarose gel eiectrophoresis and stained with ethidium bromide.
  • Figure 4 illustrates the results from a PCR amplification of genomic DNA using primers for HTLV I or HTLV II pol gene regions. Amplified nucleic acid was separated by agarose gel eiectrophoresis and stained with ethidium bromide.
  • Figure 5 is a histogram illustrating the presence of anti-HICRV in the patients serum as compared with normal control serum.
  • Figure 6 is a Western Blot analysis of HICRV infected H9 and control cultures separated by one-dimensional SDS polyacrylamide gel eiectrophoresis. Arrows illustrate protein bands not found in control cultures.
  • This invention relates to the discovery of a novel human intracisternal retrovirus (HICRV).
  • HIV human intracisternal retrovirus
  • HICRV Ultrastructurally, enzymatically, serologically and by polymerase chain reaction (PCR), HICRV is distinct from HIV-1 , HIV-2, HTLV I, HTLV II and the recently described A type intracisternal retroviral particle (Garry R.F. et al. Science 250: 1127, 1990.
  • CD4 + cells appear to be the primary target for HICRV infection. This observation is based on the severe quantitative and qualitative deficiency of CD4+ cells in the patient, along with in vitro transmissibility of the virus to CD4+ H9 cells
  • the patient reported here is different from any previously reported. This patient was asymptomatic at the time of diagnosis and has remained asymptomatic for 18 months following the diagnosis of PCP. No evidence of infection with HIV-1, HIV-2, HTLV I or HTLV II was observed as determined by serological, enzymatic, and PCR techniques. Furthermore, it has been demonstrated that the patient's cells are infected with HICRV. The patient had a blood transfusion in 1949-50 following bleeding from a spontaneous abortion. It is probable that the patient was infected with the virus early in life via blood transfusion and developed immunodeficiency over several decades. HICRV poses a potential long term threat to the safety of the blood supply.
  • H9 cells have been grown in continuous culture in H9 cells, as reported in Example 4.
  • Cell lines in continuous culture infected with the virus comprise one aspect of the invention. Such cell lines are infected as in Example 4, and are maintained and expanded using techniques well known to those or ordinary skill in the HIV and other retrovirus arts.
  • H9/200 HICRV-infected H9 cell line
  • Dr. Gupta Dr. Gupta, in his laboratory at the University of California, Irvine. This cell line is available to the U.S.
  • Patent and Trademark Office during the pendency of this application and will be publicly available after issuance of the patent. If properly required by the Patent and Trademark Office, it will be deposited with a commercial depository contractually obligated to maintain the deposit for a length of time and under such conditions of public availability as are acceptable to the Patent and Trademark Office.
  • a number of other cell lines are currently been used to propagate HICRV. These cell lines include: AA-2 (Chaffee S. et al. J. EXP. Med. 168: 605, 1988; CEM-SS (Foley G.E. et al. Cancer 18: 522, 1965; Nara P.L. et al. AIDS Res. Hum. Retroviruses 3: 283, 1987), HUT78 (Gazdar et al. Blood 55: 409,
  • One aspect of the invention relates to immunoassays for detection of HICRV.
  • One assay using serum from infected individuals is reported in Example 8.
  • a Western Blot assay is also reported in Example 8.
  • Monoclonal antibody and purified antigen based assays are also contemplated, as illustrated in Examples 11, 12, and 20. Of course, the exemplified assays are only illustrative.
  • Polyclonal or monoclonal antibody against HICRV antigen, and purified viral antigen can be used in radioimmunoassays (RIA), enzyme linked immunosorbent assays (ELISA), and immunofiuorescence assays, using those reagents in direct and indirect competitive assays, sandwich assays, flow cytometry or other conventional assays. These assays can be used to determine the presence of viral antigen or to detect antibodies against HICRV antigen. Also contemplated are DNA probes directed against the proviral DNA in infected cells, and reverse transcriptase assays.
  • the cell free virus in the supernatants of HICRV infected cell lines activated with phorbol myristate acetate may be purified, the nucleic acid can be isolated, and cloned and then be used in well known manners to determine the amino acid sequence of specific viral antigens, or to make nucleic acid probes to identify and/or isolate the RNA and proviral DNA of HICRV, and ultimately to use those viral genes to make cDNA piasmid constructs which can express viral proteins in vitro.
  • Example 1 Lymphocyte Subsets Peripheral blood lymphocyte subsets were analyzed with monoclonal antibodies, anti-CD2 (Leu 5), anti-CD3 (Leu 4), anti-CD4 (Leu 3), anti-CD8 (Leu 2), anti-CD25 (IL-2 receptor), and anti-CD20 (Leu 16) using FACScan (Becton Dickinson, San Jose, CA). Analysis was done on whole blood samples using lysing solution. Mononuclear cells were mixed with appropriate dilutions of monoclonal antibodies for 30 minutes at 4°C, washed three times with phosphate buffer saline (PBS) and resuspended in PBS. Mouse Ig of the same isotypes as that of monoclonal antibodies were used as controls. Ten thousand cells were analyzed using FACScan. Data are presented for both percentage and the absolute number of subsets in Table 1 below.
  • CD2 + T cells 61 (1041 ) 65-85 (1467-3003) CD3 + T cells 52 (857) 59-76 (1277-2929) CD4+ T cells 10 (1 71 ) 32-47 (782-1 626) CD8+ T cells 50 (700) 24-36 (463-1369) CD4 + /CD8 + ratio 0.2 0.9-2.1 CD20+ B cells 17 (290) 4.6-16.8 (163-41 1 ) CD25 + T cells 72 54-78
  • the data presented show a marked decrease in proportions and numbers of CD4+ and increase in the proportions and numbers of CD8+ T cells in the patient as compared with the controls.
  • Example 2 Lymphocyte DNA Synthesis Mononuclear cells isolated on a Ficoll-Hypaque density gradient were stimulated with soluble antigens and mitogens in 96 well microtiter plates. One hundred thousand cells/well (in triplicates) were incubated with optimum concentrations of soluble antigens (mumps, Candid albicans, tetanus toxoid, and PPD) and mitogens (phytohemagglutinin, concanavalin A and pokeweek mitogen) for 5 and 3 days respectively. Cells were pulsed with 1 mCi/well of 3 H thymidine and cultured for the final 18 hours. Cells were harvested and 3 H thymidine incorporation was measured using a scintillation counter. Data shown in Table 2 below are expressed as mean net counts per minute of triplicate cultures. Table 2: DNA S n he is C un s Per Minute
  • Example 3 Serological Tests Serum immunoglobulins were measured by nephelometry. Serum immunoglobulins were measured by nephelometry. Serum immunoglobulins were measured by nephelometry. Serum immunoglobulins were measured by nephelometry.
  • HIV-1 antibodies were measured by ELISA (DuPont) and Western blot, and P24 antigen by antigen capture ELISA technique (DuPont).
  • Antibodies against HTLV I and HTLV II were examined by immunofluorescence assay (Dr. Jay Levy, University of California, San Francisco) and no activity was observed. All of these test showed no cross activity between HICRV and HIV-1 , HTLV I or HTLV II, indicating that HICRV is distinct from these viruses.
  • Mononuclear cells (2 X 10°7ml) in 12 X 75 mm falcon test tubes were suspended in medium containing 10% fetal calf serum and antibodies, and cultured with 10mg/ml of PHA for 72 hours. Medium was changed and cells were cultured in the presence of 10 unit/ml of recombinant interleukin 2 (AMGEM, Thousand Oaks, CA). At day 7, cells were co-cultured with H9 cells at a ratio of 1 :3 (patient : H9 cells), and culture medium (RPMI-1640 and 10% fetal calf serum) was changed every 3 days. After 3 weeks in culture, cells were analyzed for virus particles by electron microscopy.
  • AGEM recombinant interleukin 2
  • Intracisternal viral particles were regularly seen in the cytoplasmic vesicles of the H9 cells ( Figure 1A and IB [higher magnification].
  • the HICRV particles appeared uniform, approximately 80 nm in diameter with two concentric rings of electron dense material and a clear center. Twenty to thirty particles in each cell were seen per this section.
  • similar viral particles were seen H9 cells co-cultured with the mononuclear cells taken from the patient's daughter ( Figure 1C).
  • Example 6 Reverse Transcriptase activity Culture supernatants from the patient's mononuclear cells stimulated for 3 days with 10mg/ml PHA plus 10U/ml rL-2 were examined for precipitable reverse transcriptase (RT) activity in the presence or absence of divalent cations Mg ++ or Mn ++ , using dTrA and dGrC as synthetic primers. Supernatants were centrifuged at 10,000 x g for 15 minutes and treated with polyethylene glycol 8000 (30% W/V with 0.4 M NaCl). The mixture was maintained overnight at 0°C. All specimen were centrifuged at 40,000 x g for 45 minutes at 4 ⁇ C.
  • RT precipitable reverse transcriptase
  • genomic DNA was isolated from (the patient's) PHA stimulated and IL-2 activated mononuclear cells, HIV-1 infected and HTLV I infected MT-2 cell lines, and examined for HIV and HTLV proviruses by polymerase chain reaction, using S 145 and SK 431 primers to amplify both HIV-1 and HIV-2 provirus (gag), SK 1 10 and SK 1 1 1 primers to amplify HTLV-I and HTLV-II provirus (pol), and SK 43 and SK 44 to amplify HTLV I and HTLV II provirus (tax).
  • Figure 3A shows lack of HIV-1 or HIV-2 (gag) proviruses in mononuclear cells from patient (200), healthy normal control or with MT-2/HTLV-1 but a positive band of with U1 /HIV-1 .
  • Figure 3B shows a lack of HTLV I and HTLV II (tax) and
  • Figure 4 shows a lack of HTLV I or HTLV II (pol) proviruses in control mononuclear cells, patient's cells (200) or U1 /HIV-1 .
  • a positive reaction was however, seen with MT-2/HTLV 1.
  • CD4+ T cell deficiency in the present patient is not due to infection with HIV-1 , HIV-2, HTLV-I or HTLV-II.
  • Lack of HIV-1 provirus in the patient's mononuclear cells and negative serology for HIV-1 distinguishes HICRV from A-type intracisternal retroviral particle.
  • H9/HICRV HICRV-infected H9 cells
  • Figure 6A shows two (2) distinct bands of approximately 24kd in H9/200 (H9/HICRV) and not in H9 cells. Normal serum showed no bands at 24kd either in H9 or H9/200 cells ( Figure 6B). This further confirmed the presence of antibodies against HICRV.
  • Example 9 HICRV Infection exists in the general population
  • Example 6 as did serum from the wife of one of those three individuals (Table 1 ).
  • the p value is .027 (after excluding the wife of the HICRV positive individual).
  • HICRV is indigenous in the general population and that it can be transmitted from infected individuals to other family members. Furthermore, HICRV can produce CD4 + T cell dysfunction/deficiency.
  • mice The lysates of HICRV infected cell lines and cell free virus are used to immunize mice. Subsequently, the mice are sacrificed and homogenized spleens from those animals are used to prepare hybridomas
  • the monoclonal antibodies are purified by ammonium sulfate precipitation and size exclusion chromatography followed by DE52 ion exchange chromatography (Colligan et al. Current Protocols in Immunology pp. 2.71 , 1991 ). All column fractions are retained until SDS-PAGE and antibody activity with ELISA and immunofluorescence assays have been done. Antibodies are also conjugated with various fluorochromes for direct immunofluorescence assay.
  • Example 11 (a) ELISA for HICRV
  • Monoclonal antibodies produced in accordance with Example 10 is adsorbed to a nylon membrane.
  • Bove serum albumin (0.1 % in phosphate buffer saline [PBS]) is then added to block nonspecific binding sites on the membrane. After washing and drying, the membrane is contacted with a sample suspected of containing HICRV antigen. The membrane is washed with PBS and contacted with monoclonal antibody from Example 10 which has been conjugated with horseradish perox ⁇ dase or alkaline phosphatase enzyme.
  • the membrane is then incubated with a substrate solution, 0.4 mM 2,2 r -azo-di(3-ethyl- benzothiazoline sulfonate) ⁇ n 0.05 M citric acid (pH 4.0) containing 0.3% H 2 O 2 (ABTS, Kirkegaard and Perry, Gaithesburg, MD).
  • a substrate solution 0.4 mM 2,2 r -azo-di(3-ethyl- benzothiazoline sulfonate) ⁇ n 0.05 M citric acid (pH 4.0) containing 0.3% H 2 O 2 (ABTS, Kirkegaard and Perry, Gaithesburg, MD).
  • ABTS Kirkegaard and Perry, Gaithesburg, MD
  • HICRV provirus in the mononuclear cells is detected by PCR.
  • Genomic DNA from the mononuclear cells from subject in question and HICRV infected cell lines is isolated by the proteinase K/phenol extraction method (Strauss W.M. in: Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York pp. 2.2.1 , 1989). DNA from normal cells is used as a negative control. 0.5mg of each DNA is used as template for PCR. Primers are designed to amplify the pol, the gag and the tax regions of HICRV are used. PCR is done with a thermal cycler at 35-45 cycles for 1 to 3 minutes. Amplified DNA is confirmed by Southern hybridization analysis with probes.
  • This method is utilized for the detection of HICRV in the biological sample of cells by direct or indirect immunofluorescence assay.
  • Cells are incubated with unconjugated or direct fluorochrome conjugated (Example 10) monoclonal antibodies against various viral antigens.
  • Cells are washed with PBS and in the case of unconjugated antibodies, are counterstained with FITC/PE/TR-conjugated anti-mouse Ig. After 30 minutes of incubation, cells are washed with PBS and examined for positive fluorescence, using microscope to flow cytometry.
  • Cells from healthy subjects and HICRV infected cells serve as negative and positive controls.
  • Mouse Ig of the same isotypes as that of the monoclonal antibodies are used as negative controls.
  • Virus-containing supernatants from HICRV infected cell lines are passed through 0.45m filter. Supernatants are centrifuged at 17,000 rpm for 3 hours at 4°C to pellet virions. Viral pellets are resuspended in a total volume of 1 ml TNE buffer and kept overnight at 4°C. Virus is further purified by column chromatography, using 1 10 ml column of sepharose 4B at room temperature with TNE as the column buffer. The eluted virus is placed in microcentrifuge tubes and pelleted at full speed for 1 hour and 15 minutes in a microcentrifuge at 4°C.
  • virus pellets are resuspended in 150ml of buffer, such as JD.5% Triton X-100 in CMF-PBS.
  • the virus is approximately x 10,000 concentrated and can be used for ELISA or Western blotting.
  • Monoclonal antibodies from Example 10 are bound to immunochromatography column.
  • Virus from supernatants of HICRV infected cell lines (Example 4) are purified by sepharose 4B column chromatography, are directed through monoclonal antibody affinity column.
  • Viral antigens are eluted with Glycine/HCI buffer (0.1 M adjusted to pH 2.5 with 0.2M HCI).
  • the amino acid sequence of viral antigen protein is determined using repeated cycles of the Edman degradation reactors as described (G. Allen. In “Seouencing of Protein and Peptides” [T.S. Work and R.H. Burdon, edsJ, Amsterdam, New York: Elsevier, 1981 ). Each degradation cycle consists of three steps. The N-terminal amino acid is modified with phenyl isothiocyanate, cleaved from protein, and finally modified amino acid will be determined by an automatic amino acid sequencer.
  • HICRV probe is prepared for screening a genomic library by three different methods.
  • Genomic RNA is isolated from purified ' viral particles and used as a template to synthesize cDNA probe for RT. It is possible that purified RNA is lost during cDNA synthesis, this method may not be practical for repeated preparation of cDNA probe.
  • double stranded cDNA is synthesized, using purified viral RNA as template and commercially available cDNA kit (Invitrogen, San Diego, CA). The cDNA is digested with restriction endonucleases. The restricted DNA fragments are cloned in E. coli, using pUC plasmids and will be used as probes.
  • proviral DNA library and a genomic library of virus infected cells are constructed. Unintegrated proviral DNA is isolated from the cytoplasm of virus-infected cells and chromosomal
  • DNA is isolated from virus-infected cells (Hirt B. J. Mol. Biol. 26: 365, 1967) .
  • Expression libraries are made with lambda bacteriophage vectors (Maniatis T. et al. Molecular Cloning. A Laboratory Manual, pp.270, 1987. Cold Spring Harbor Lab. New York), including lambda gt 1 1 .
  • the libraries are screened with monoclonal antibodies raised against viral antigens and viral DNA probes. The clones expressing viral antigens will be isolated and viral antigen genes will be subcloned in M13-derivative plasmids for DNA sequencing.
  • Example 18 Expression of viral antigen genes
  • the genes encoding viral antigens are cloned in both prokaryotic and eukaryotic expression plasmids, and expressed in E. coli and eukaryotic cell lines. We will stably transfect DNA including viral gene expression system into eukaryotic cell lines (Ausubel F.M. et al. (eds.) in: Current Protocols in Molecular Biology, pp. 16.0.1 , 1989. John Wiley & Sons, Inc. New York). Viral antigen gene is fused to a strong partner of bacteriophages (e.g. T3, T7, lambda etc.) and E.coli (LacZ, trpE etc.) and transformed into E.
  • bacteriophages e.g. T3, T7, lambda etc.
  • E.coli LacZ, trpE etc.
  • viral antigen gene is expressed using a eukaryotic expression system, such as the Baculoviral expression system and the vaccinia virus expression system.
  • a eukaryotic expression system such as the Baculoviral expression system and the vaccinia virus expression system.
  • fusion plasmids of constitutive eukaryotic promotor and viral antigen gene are made and transfected to mammalian cell lines, including COS and CHO, for viral antigen expression.
  • Example 19 Purification of recombinant viral antigens
  • Expressed viral antigens are purified by an affinity chromatography, using CNBR-activated sepharose 4B column conjugated with monoclonal antibodies against viral antigens.
  • Cell extracts containing expressed viral antigens are applied to the affinity column.
  • Viral antigens are bound to the column and the remaining cell components are washed with PBS.
  • Viral antigens are eluted from the column by glycine/HCI buffer (0.1M adjusted to pH 2.5 with 0.2M HCI).
  • Purified viral antigens from Example 19 are immobilized on a solid styrene support.
  • the antigen-labeled support is incubated with test sample from subject suspected of HICRV infection, washed with PBS and incubated with FITC-conjugated goat-anti-human IgG. Complexes are washed and anti-HICRV antibody is measured by fluorescence of the labeled antibody remaining on the support.
  • Supernatants of cell lysates of normal and HICRV infected cells or purified antigen are mixed with sample loading buffer, and 20ml sample is applied to 10% SDS-PAGE. Separated proteins are transferred to nitrocellulose paper. A Western blot is probed with normal serum or patient's serum preabsorbed with normal mononuclear cells. Proteins bound to serum antibodies are visualized with alkaline phosphate-conjugated goat-anti-human Ig and a Blot detection kit.
  • the RIPA is carried out in five steps (Aldovini A. and Walker B.D. in: Techniques in HIV Research (Stockton Press), New York, pp. 1 1 ,
  • ELISA Assay HICRV purified virus from Example 13 is lysed in PBS with 10% non-idet P-4 (Sigma, St. Louis, MO.) Dilutions of virus are prepared in PBS. Dilutions of antigen (1 :200 to 1 :2000) are bound to microtiter wells by incubation at 4°C overnight. After washing with PBS, nonspecific binding sites are blocked with 1 % Bovine Serum Albumen (BSA) and 2% human control serum in PBS for 1 hour at 37°C. Blocking is followed by successive washes of PBS Tween 20.
  • BSA Bovine Serum Albumen
  • the cell line containing HICRV was deposited under the Budapest Treaty on September 11 , 1992 with the ATCC and it was ad warded ATCC Accession CRL No. 11121.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Biophysics (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un nouveau rétrovirus humain provoquant et associé à la déficience et au dysfonctionnement des cellules T CD4 +, denommé le rétrovirus humain intracisternal (HICRV) en cultures cellulaires continues, l'invention concerne également le HICRV sous forme purifié; des anticorps contre le virus, des antigènes viraux purifiés, des dosages immuno-logiques à base de polynucléotides pour détecter le virus, ainsi que des plasmides recombinés et des microorganismes pour exprimer les protéines virales du HICRV.
PCT/US1992/010238 1991-11-18 1992-11-18 Nouveau retrovirus humain intracisternal associe a la deficience et au dysfonctionnement des cellules t cd4 + Ceased WO1993010222A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US79384691A 1991-11-18 1991-11-18
US793,846 1991-11-18

Publications (1)

Publication Number Publication Date
WO1993010222A1 true WO1993010222A1 (fr) 1993-05-27

Family

ID=25160966

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/010238 Ceased WO1993010222A1 (fr) 1991-11-18 1992-11-18 Nouveau retrovirus humain intracisternal associe a la deficience et au dysfonctionnement des cellules t cd4 +

Country Status (2)

Country Link
AU (1) AU3149193A (fr)
WO (1) WO1993010222A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6177081B1 (en) 1994-03-09 2001-01-23 Pacific Biotech International, Inc. Human and marmoset activating viruses

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106834243B (zh) * 2017-03-20 2020-12-29 汉恒生物科技（上海）有限公司 原代t细胞感染用重组逆转录病毒、感染方法及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PEDIATRICS, Volume 61, No. 1, issued January 1978, L.L. PIFER et al., "Pseudomonas Carinii Infection: Evidence for High Prevalence in Normal and Immunosuppressed Children", pages 35-41. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6177081B1 (en) 1994-03-09 2001-01-23 Pacific Biotech International, Inc. Human and marmoset activating viruses

Also Published As

Publication number Publication date
AU3149193A (en) 1993-06-15

Similar Documents

Publication Publication Date Title
KR920003788B1 (ko) 임파선증과 후천성 면역 결핍증후군의 실험관내 진단방법에 사용되는 레트로 바이러스 추출물의 제조방법 및 진단용 키트의 제조방법
US5087557A (en) Human monoclonal antibody to lymphadenopathy-associated virus
US5637453A (en) Whole blood method and kit for the detection of antibodies against human immunodeficiency virus (HIV) in HIV-seronegative individuals
Gupta et al. Detection of a human intracisternal retroviral particle associated with CD4+ T-cell deficiency.
US5135864A (en) Human Immunodeficiency Virus (HIV) associated with Acquired Immunual Deficiency Syndrome (AIDS), a diagnostic method for aids and pre-aids, and a kit therefor
IE63109B1 (en) Human monoclonal antibody to lymphadenopathy-associated virus
US6395495B1 (en) Methods and kits for detecting antibodies against an HIV variant
US6627395B1 (en) Methods for the propagation, isolation, identification, and preparation of human immunodeficiency virus type 1 (HIV-1)
Hayes et al. Antibodies to human T lymphotropic virus type I in a population from the Philippines: evidence for cross-reactivity with Plasmodium falciparum
US7122188B1 (en) Antibodies which bind with proteins of human immunodeficiency virus type 1 (HIV-1), and immune complexes comprising proteins of HIV-1
US6428952B1 (en) Methods and kits employing LAV antigens for the detection of HIV-1-specific antibodies
Srivastava et al. Human T-Cell Leukemia Virus Type I or a Related Retrovirus in Patients with Mycosis Fungoides/Sézary Syndrome and Kaposi's Sacroma
EP0643835A1 (fr) Titrage de htlv-i/htlv-ii et procede
WO1993010222A1 (fr) Nouveau retrovirus humain intracisternal associe a la deficience et au dysfonctionnement des cellules t cd4 +
US6352826B1 (en) Method and kit for the detection of retroviral specific antibodies in seronegative individuals
De Stasio et al. Screening for anti-human T-lymphotropic virus antibody in blood donors and polytransfused patients in Apulia (Italy)
US5514542A (en) Methods for propagating retrovirus for use in antibody assays
Verdier et al. Comparison of immunofluorescence, particle agglutination, and enzyme immunoassays for detection of human T-cell leukemia virus type I antibody in African sera
Bastian et al. HTLV‐I among Northern Territory blood donors
Grant et al. Comparison of enzyme-linked immunosorbent and indirect immunofluorescence assays for the detection of human T-cell lymphotropic virus type-I antibodies in sera from rural Haiti
US5580772A (en) Association between a novel human intracisternal A-type retroviral particle-type II (HIAP-II) and idiopathic CD4+ T-lymphocytopenia (ICL)
US6824977B2 (en) Assays for human T cell lymphotrophic virus types I and II
a Reduced Peripheral Blood Mononuclear Cells from
Nishimura et al. Human T Lymphotropic Virus Type I (Htlv-I)-Specific T Helper Cell Responses from Htlv-I Seronegative Patients with Chronic Myelopathy and Ms in Japan
Murphy et al. Human T-Cell Lymphotropic Virus Type

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR CA CH CS DE DK ES FI GB HU JP KP KR LK LU MG MN MW NL NO PL RO RU SD SE

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL SE BF BJ CF CG CI CM GA GN ML MR SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA