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WO1993005185A1 - Procede de detection de souches de virus infectieuses - Google Patents

Procede de detection de souches de virus infectieuses Download PDF

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Publication number
WO1993005185A1
WO1993005185A1 PCT/US1992/007386 US9207386W WO9305185A1 WO 1993005185 A1 WO1993005185 A1 WO 1993005185A1 US 9207386 W US9207386 W US 9207386W WO 9305185 A1 WO9305185 A1 WO 9305185A1
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WIPO (PCT)
Prior art keywords
cells
hiv
virus
present
assay
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Ceased
Application number
PCT/US1992/007386
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English (en)
Inventor
Marvin S. Reitz
David J. Waters
William Blattner
Carolyn A. Wilson
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National Institutes of Health NIH
US Department of Health and Human Services
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National Institutes of Health NIH
US Department of Health and Human Services
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Publication of WO1993005185A1 publication Critical patent/WO1993005185A1/fr
Anticipated expiration legal-status Critical
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    • 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
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • 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

Definitions

  • the present invention relates to a new assay system which can be used to screen for and to quantitate infectious retroviral strains.
  • the present invention relates to a simple and rapid colori etric transactivation assay for the detection of HIV-1, HIV-2 and SIV using HeLa cells containing CD4 and a reporter gene, for example, ⁇ -galactosidase, that is dependent on transactivation by the infecting virus for expression.
  • Rocancourt et al reported the construction of an expression plasmid and the use of that plasmid in a detection system based on the trans-activation of the HIV-1, LTR and expression of ⁇ -galactosidase J. Virology 64:2660-2668 (1990).
  • This expression plasmid contained the early SV40 promoter and enhancer sequences internal to the HIV-1 LTR. For this reason, Rocancourt et al reported low basal levels of ⁇ -galactosidase activity in exponentially growing cells, although this disappeared in their confluent cultures.
  • the adherent CD4 containing cells preferably HeLaT4 cells
  • the adherent CD4 containing cells used to express the plasmid HXB2 Flacz of the present invention have been subcloned and selected on the basis of having no basal activity under any circumstances. This makes it possible to use the assay system of the present invention to titer or detect low levels of viruses. Similarly, this selection distinguishes the cell line of the present invention from the rabbit cell line described by Roberts and Blair (Antiviral Chemistry and ChsWMXhe apy 1: 129-148 (1990)).
  • Roberts and Blair developed a rabbit cell line expressing a construct analogous to HXB2Flacz (HIV-1 LTR and ⁇ - galactosidase) , but these authors screened for a cell line that had detectable levels of basal ⁇ - galactosidase activity so that basal levels could be compared to those observed after activation by HIV- 1 tat or other trans-activator of the HIV-1 LTR.
  • the present invention provides for such an assay.
  • transfecting an expression plasmid HxB2- LaczF into HeLa cells with the CD4 receptor and selecting out those cells capable of expressing ⁇ -galactosidase following transactivation by HIV the present invention makes it possible to detect infection of a cell by a single virion of HIV-l or HIV-2 where the infected cells can be detected as blue cell clusters or syncytium.
  • Figure 1 is a schematic representation of the expression plasmid HXB2Flacz (Spe-Xho) .
  • the plasmid sequences, derived from pSP65 gpt are represented as jagged black lines; the HIV LTR's as rectangles with hash marks; HIV coding regions as rectangles with dots, and the ⁇ -galactosidase coding region an empty rectangle.
  • SUBSTITUTE SHEET reporter gene for example the ⁇ -galactosidase (Lacz) gene.
  • the present invention relates to an assay that may be simply performed by utilizing adherent CD4 containing HeLa cells (preferably, HeLaT4 cells) that are transfected with the expression plasmid HxB2-Lac2F.
  • Expression of the fusion protein ⁇ -galactosidase by the transfected HeLaT4-cells is dependent on transactivation by the infecting virus, for example by the tat gene.
  • the infected cells can be detected and quantified, for example, as clusters of blue cells or syncytia.
  • a variety of substrates are available for the detection of ⁇ - galactosidase, microscopically as fixed blue cells, by fluorescent excitation and measurement in a fluorometer, or viably sorted by FACS.
  • the present invention relates to the transactivated expression of ⁇ -galactosidase, detected by the 5-bromo-4- chloro-3-indolyl- ⁇ -D-glucopyranoside (X-Gal) histochemical assay which is scored macroscopically and microscopically at low magnification.
  • Virus titers obtained using the HeLaT4-Lacz cells are comparable to those obtained by endpoint dilution using HuBPL's and indirect assays. Wild-type nonfusogenic HIV-1 isolates may by titered with the assay of the present invention.
  • the assay system provided by the present invention results in the elimination of the need for an assay using endpoint dilutions to yield quantitative results where the virus is detected with time consuming and expensive indirect assays such as reverse transcriptase or p-24 antigen capture.
  • the present invention provides for an assay system that exhibits a sensitivity similar to that obtained using peripheral blood lymphocytes (PBL's) and will speed and simplify a variety of studies requiring the assay of live virus, agents or molecular constructs capable of activating the HIV-1 LTR.
  • PBL's peripheral blood lymphocytes
  • This adherent cell, nonradioactive, 72 hour assay system can be done in 96 micro-well plates.
  • the present invention relates to a DNA construct, expression plasmid HxB2-LaczF, comprising a DNA segment encoding a HIV gag- ⁇ -galactosidase fusion protein and a vector.
  • the HxB2-LaczF construct contains both the 5* and 3• HIV LTRs where expression of the fusion protein is dependent upon activation of the HIV-LTR by a transactivator, for example, tat.
  • Yet another embodiment of the present invention relates to a detection assay suitable for use in an infectious center assay that quanti tes the number of virus infected cells from patient or animals. For example, the effectiveness of a drug or vaccine in eliminating or reducing the number of circulating cells carrying the virus in AIDS patients can be ascertained using the assay of the present invention.
  • the present invention relates to a virus neutralization assay to quantitate virus neutralizing antibody in serum obtained from patients, vaccine recipients or animals.
  • virus strain variations can be checked and peptide competition neutralization assays can be made rapidly and easily.
  • a detection method using the transactivation assay of the present invention may provide a rapid and sensitive means for the large scale screening and detection of compounds that either prevent virus infection or suppress its replication following infection.
  • the present invention relates to a diagnostic method using the transactivation bioassay.
  • Specific drug resistant viruses can be selected from patients using the assay of the present invention followed by FACS to retrieve the cells containing the virus.
  • Another embodiment of the present invention relates to a diagnostic method to identify any cell transfected with molecular constructs which may transactivate HIV-1.
  • the transactivation bioassay provides a simple and rapid method to identify and separate by FACS any cells transtecte ⁇ with molecular constructs which can transactivate the HIV-l LTR, thus eliminating the need for drug selection.
  • the present invention relates to a novel method to isolate new or wild type lentivirus from clinical samples.
  • the HxB2-LaczF construct-containing HeLa cells provided in the present invention can be used in the transactivation assay, also provided in the present invention, to activate expression of the reporter gene, ⁇ -galactosidase when infected with a new or wild type lentivirus.
  • Transactivated cells expressing ⁇ -galactosidase can be directly removed from the monolayer by pipette, cloned and sequenced by PCR.
  • the cells were rinsed once with cold PBS after removal of the cover and superstructure of the chamber slide.
  • the cells were fixed with a cold 2% glutaraldehyde and 2% formaldehyde, PBS solution for 5 minutes followed by a 5 minute cold PBS wash.
  • the annealed linker fragment when ligated correctly to the other two fragment carries the palindromes recognized by three restriction enzymes: Spel, NotI and BamHI; it also maintains an open reading frame between the HIV-l ⁇ aq gene and lacz.
  • the resulting plasmids were screened first by restriction analysis (Spel and Clal-shown in Figure 1) , and the presence of the linker was confirmed by dideoxy sequencing.
  • the expression plasmid was transfected into HeLaT4 cells by calcium phosphate mediated DNA transfection. Twenty-four hours post-transfection the medium was changed. Forty-eight hours post- transfection the cells were seeded at approximately 5 X 10 ! cells/10 cm plate. After 24 hours the media was changed to selective media (DMEM supplemented with 250 ⁇ l/ml mycophenolic acid and 250 ⁇ g/ml xanthine) in order to select for cells which had taken up and expressed the rjpt marker on the plasmid. After three weeks of selection, colonies were transferred to 12-well plates using cloning cylinders.
  • ⁇ a - ⁇ - ⁇ alactosidase fusion protein was assessed after transfection with pCV-1 (HIV-l tat) .
  • Transfected and untransfected clones were histochemically stained for expression of ⁇ -galactosidase.
  • One of these clones with one in 500 cells positive for expression of ⁇ -galactosidase was subcloned in 96-well plates. Wells containing single cells were identified and marked. After the cell in each marked well had formed a colony they were split between two 96-well plates with the cells in one plate challenged by HIV-l/IIIB.
  • Twelve-wells containing cells exhibiting the greatest expression of ⁇ -galactosidase following virus challenge were identified and the corresponding uninfected cells from the sister plate were single cell cloned a second time. Cells from each of these twelve wells were seeded into 96-well plates at a cell concentration to yield a single cell in each well. Wells containing single cells were identified, marked and handled as described above. Twenty clones derived from single cells were identified which possessed high levels of virus inducible ⁇ - galactosidase activity. The point of optimum HIV virus induced ⁇ -galactosidase production and titer was 72 hours post infection.
  • Figure 2 is a representative clone of the .HeLaT4 lacz cells challenged with HIV-1/IIIB. ⁇ -galactosidase positive (blue) cell clusters can still be seen in the 10 4 virus dilution. Virus stocks of HIV-l strains IIIB,RF and MN were titered by ⁇ - galactosidase induction using the HeLaT4-Lacz cells. The resulting titers were comparable to these titers previously obtained using human peripheral blood lymphocytes (HuPBL's). The HeLaT4-Lacz cells can be used to determine the amount of virus neutralizing antibody in serum samples in virus neutralization assays.

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  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention se rapporte à un nouveau système de dosage pouvant être utilisé pour détecter et quantifier des souches rétrovirales infectieuses. En particulier la présente invention se rapporte à une méthode rapide de détection, par transactivation colorimétrique, des virus HIV-1, HIV-2 et SIV au moyen de cellules HeLa contenant CD4 et un gène reporteur, tel que la β-galactosidase qui dépend de la transactivation par le virus infectieux pour son expression.
PCT/US1992/007386 1991-09-06 1992-09-08 Procede de detection de souches de virus infectieuses Ceased WO1993005185A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US75498791A 1991-09-06 1991-09-06
US754,987 1991-09-06

Publications (1)

Publication Number Publication Date
WO1993005185A1 true WO1993005185A1 (fr) 1993-03-18

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PCT/US1992/007386 Ceased WO1993005185A1 (fr) 1991-09-06 1992-09-08 Procede de detection de souches de virus infectieuses

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AU (1) AU2578692A (fr)
WO (1) WO1993005185A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000043515A3 (fr) * 1999-01-25 2001-10-04 Musc Found For Res Dev Procede de surveillance de la resistance aux medicaments du vih
WO2001081608A3 (fr) * 2000-04-26 2002-02-21 Musc Found For Res Dev Vecteurs viraux permettant de controler la resistance aux medicaments du vih
WO2002018624A3 (fr) * 2000-09-01 2003-05-22 Immunoclin Lab Ltd Methode d'analyse
US6900010B2 (en) 1999-01-25 2005-05-31 Musc Foundation For Research Development Compositions and methods for detecting human immunodeficiency virus

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF VIROLOGY, Volume 64, Number 6, issued June 1990, D. ROCANCOURT et al., "Activation of Beta-Galactosidase Recombinant Provirus: Application to Titration of Human Immunodeficiency Virus (HIV) and HIV-Infected Cells", pages 2660-2668. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000043515A3 (fr) * 1999-01-25 2001-10-04 Musc Found For Res Dev Procede de surveillance de la resistance aux medicaments du vih
US6406911B1 (en) 1999-01-25 2002-06-18 Musc Foundation For Research Development Compositions and methods for sensitive detection of HIV infection and monitoring of drug resistance
US6410013B1 (en) 1999-01-25 2002-06-25 Musc Foundation For Research Development Viral vectors for use in monitoring HIV drug resistance
JP2002534979A (ja) * 1999-01-25 2002-10-22 マスク ファウンデーション フォー リサーチ デヴェロップメント Ｈｉｖ薬剤抵抗性のモニタリング方法
US6884576B2 (en) 1999-01-25 2005-04-26 Musc Foundation For Research Development Methods of monitoring HIV drug resistance
US6900010B2 (en) 1999-01-25 2005-05-31 Musc Foundation For Research Development Compositions and methods for detecting human immunodeficiency virus
US6967076B2 (en) 1999-01-25 2005-11-22 Musc Foundation For Research Development Method for producing recombinant cells for detecting HIV
WO2001081608A3 (fr) * 2000-04-26 2002-02-21 Musc Found For Res Dev Vecteurs viraux permettant de controler la resistance aux medicaments du vih
WO2002018624A3 (fr) * 2000-09-01 2003-05-22 Immunoclin Lab Ltd Methode d'analyse

Also Published As

Publication number Publication date
AU2578692A (en) 1993-04-05

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