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WO1999067429A1 - Analyse a base de cellules pour determiner l'infectivite et la sensibilite du virus vih - Google Patents

Analyse a base de cellules pour determiner l'infectivite et la sensibilite du virus vih Download PDF

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Publication number
WO1999067429A1
WO1999067429A1 PCT/US1999/014104 US9914104W WO9967429A1 WO 1999067429 A1 WO1999067429 A1 WO 1999067429A1 US 9914104 W US9914104 W US 9914104W WO 9967429 A1 WO9967429 A1 WO 9967429A1
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Prior art keywords
hiv
cell line
primary
virus
marker gene
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PCT/US1999/014104
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English (en)
Inventor
John C. Kappes
Xiaoyun Wu
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Uab Research Foundation
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Publication date
Application filed by Uab Research Foundation filed Critical Uab Research Foundation
Priority to CA002331760A priority Critical patent/CA2331760A1/fr
Priority to AU48278/99A priority patent/AU4827899A/en
Priority to EP99931859A priority patent/EP1088108A4/fr
Priority to US09/719,340 priority patent/US6797462B1/en
Publication of WO1999067429A1 publication Critical patent/WO1999067429A1/fr

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    • 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

Definitions

  • the present invention relates genetically modified cells, to an assay and methods and the usage thereof to measure the infectivity and viral resistant/sensitivity of isolate from peripheral blood mononuclear cells (PBMC) and plasma of an immunodeficiency virus.
  • PBMC peripheral blood mononuclear cells
  • the present invention has utility in determining the HIV co-receptor usage, discovery of new drugs effective against HTV and monitoring a drug therapy protocol in order to enhance the effectiveness of drug treatment regimes against HIV-l infection.
  • RT reverse transcriptase
  • ELISA based assays for the detection of HTV/SlV core antigen
  • direct quantitation of infectious virus by syncytial focus plaque assays or limiting dilution titration in susceptible host cells visualization of virions by electron microscopy, in situ hybridization, and various nucleic acid-based assays.
  • genetic reporter-based assays have been created to detect HIV/SIV infection.
  • mammalian cells are genetically modified to express a reporter gene such as ⁇ -galactosidase ( ⁇ -gal), green fluorescent protein (GFP) or chloramphenicol acetyltransferase (CAT) in response to infection and Tat protein expression.
  • a reporter gene such as ⁇ -galactosidase ( ⁇ -gal), green fluorescent protein (GFP) or chloramphenicol acetyltransferase (CAT) in response to infection and Tat protein expression.
  • ⁇ -gal ⁇ -galactosidase
  • GFP green fluorescent protein
  • CAT chloramphenicol acetyltransferase
  • CAT chloramphenicol acetyltransferase
  • the sensitive detection of the virus quasispecies that comprise primary HIV isolates has proved difficult using immortalized CD4 positive cell lines. At least in part, this has been due to the lack of expression of the CCR5 chemokine co-receptor on the surface of such cell lines.
  • the failure to detect infection of primary virus isolates (T-cell and macrophage tropic viruses) using immortalized cell lines has greatly impeded the development of useful approaches for detecting, quantifying and analyzing HIV infection of primary virus isolates.
  • the present invention largely overcomes the prior art limitations.
  • Figure 1 is a schematic block diagram illustrating a generalized sequence of steps in creating an assay for detecting and analyzing primary HIV.
  • Figures 2A-2E are schematics illustrating the construction of various gene transfer expression plasmids of the present invention.
  • FIG. 26 is a schematic illustrating the production of lentiviral transduction vectors for the delivery of marker genes of the present invention.
  • a gene transfer plasmid representatively including those shown in Figure 2 are separately transfected into a host cell together with viral based packaging and envelope plasmids.
  • Figures 4A and 4B are graphs illustrating the relationship between the concentration of vector and infectious units as determined with ⁇ -gal, GFP and luciferase activity.
  • Figure 5 is a graph illustrating a nearly linear relationship between HIV-1 infectious units and luciferase activity for a cell line of the present invention.
  • Figure 6 is a graph illustrating the relationship between infectious virus units and luciferase activity for viruses: TIVI, WIMI, KIWE and YU2, using a cell line of the present invention.
  • Figure 7 is a graph illustrating a correlation between infectious virus units and luciferase activity.
  • Figures 8A-8C are graphs illustrating the effect that different concentrations of 3TC, AZT, and Nevaripine, respectively, have on virus replication relative to non-drug treated viruses as determined by luciferase activity according to the present invention.
  • Figures 9A-9C are graphs illustrating drug sensitivity to AZT for 100, 500, and 2500 virus infectious units respectively, according to the present invention.
  • Figure 10 is a schematic illustrating the construction of a Tat transduction plasmid.
  • Figures 1 1A and 1 IB are graphs illustrating the viral amplification two days following infection with equal quantities of YU2 HIV (a) for infectivity and (b) p24 antigen.
  • the present invention pertains to a cell-based assay for analyzing primary
  • HIV including an immortalized cell line that expresses the CCR5, CXCR4 and
  • CD4 receptors and a marker gene.
  • the CCR5 or CD4 receptors enable binding and entry of HIV wherein marker gene expression correlates the magnitude of virus infection.
  • An immortalized cell line is disclosed capable of allowing efficient amplification of primary HIV.
  • a method is further disclosed wherein the cell line contains a gene that can be expressed in response to infection of the virus.
  • a method for producing an immunodeficiency virus infection sensitive clonal cells including selecting a cell line expressing CCR5,
  • the present invention finds utility as a method for detecting, isolating and analyzing primary HIV by infecting a cell line of the present invention with a quantity of virus and after some time measuring marker gene expression.
  • Practicing the method of the present invention for determining immunodeficiency virus titer and conducting the presence of a drug candidate indicates the sensitivity of a given strain, type, species or genus of virus to the given drug candidate.
  • the present invention affords the ability to test virus derived from blood plasma as well as cell culture. Description of the Invention
  • an immortalized cell line to detect and analyze primary HIV other than PBMC offers numerous advantages which are exploited to develop a novel assay.
  • immortalized cell lines are refractory to primary HIV.
  • the expression of CCR5 in an immortalized cell line significantly enhances the detection of primary HIV-1.
  • the ability to detect primary isolates of HIV-1 with greater sensitivity than currently possible is an aspect of the present invention.
  • the present invention provides for: (1) the sampling and analysis of a representative population of viruses that comprise primary HIV-1; (2) the analysis of a significantly greater proportion of virus; and (3) high throughput testing, via miniaturization and sampling of small sample volumes.
  • T-trophic virus is intended herein to define a phenotype of an immunodeficiency virus capable of infecting a T-cell by binding the CD4 receptor on the T-cell.
  • macrophage trophic virus is intended to mean a phenotype of an immunodeficiency virus capable of infecting a macrophage by binding the CCR5 co-receptor on the macrophage.
  • the SG3 (S.K. Ghosh et al. 1993, Virology 194:858-864) and NL43 (W. Paxton et al. 1993, J. Virol. 67:7229-7237) strains of HIV-1 are derived by extensive passage in tissue culture. They represent T-cell tropic viruses and do not infect monocytes and macrophages. These viruses are not representative of the complex mixtures of viruses that exit in infected individuals.
  • Primary HIV-1 represent virus that is derived directly from the blood of an HIV infected individual. Primary HIV can also be derived by short term culture in vitro culture in primary peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • Primary HIV can also be derived using the cell line of this invention.
  • Such isolates are complex mixtures and may contain macrophage- and/or T-tropic viruses.
  • T-cell tropic viruses are able to infect cells that express CD4 and CXCR4, while macrophage tropic (M-tropic) viruses also require expression of the CCR5 chemokine co-receptor.
  • M-tropic macrophage tropic
  • Most HIV-2 and SIV viruses also require the CCR5.
  • Several groups have produced cell lines that express CD4, CXCR4 and CCR5 in attempts to render them sensitive to infection with primary HIV-1 (both T-cell and macrophage tropic viruses).
  • primary HIV is defined as HTV derived directly from an infected host organism from sources such as blood, plasma, PBMC, CSF and other tissues.
  • immunodeficiency virus is defined as various strains and stocks of HIV-1, HIV-2, SIV and lentiviruses.
  • minor population is defined as a titer of a given viral strain, type or species or genus that constitutes less than 10% of the total quantity of virus present obtained from a host culture or organism.
  • major population is defined as the numerically dominant viral strain, type, species or genus of a viral titer obtained from a host culture or organism.
  • drug sensitivity is defined as the effectiveness of a drug to inhibit HIV replication and/or expression with a host cell, the term is used synonymously with “drug resistivity.”
  • drug resistivity By making genetic modifications to a CCR5 or CD4 expressing cell line, the present invention represents is an efficient method for analyzing the drug sensitivity properties of primary HIV, such as HIV-1.
  • FIG. 1 is a schematic block diagram illustrating a generalized sequence of steps in creating an assay for measuring HIV-1 drug sensitivity according to the present invention.
  • the creation of a cell based assay of the present invention involves a series of steps. Initially, a vector is constructed for the purpose of transducing mammalian cells with a marker gene. Such a marker gene transduction plasmids bring the marker gene expression under the regulation of an immunodeficiency virus 10.
  • the marker gene vector is placed under the control of HIV-1 or HIV-2 long terminal repeats (LTRs) and the Rev responsive element (RRE).
  • the marker genes illustratively including ⁇ -gal, luciferase, GFP, CAT and other fluorescent proteins.
  • the marker gene is luciferase.
  • CD4 positive cells are then transduced with the vector in order to confirm appropriate marker gene expression from the transduction vectors 20.
  • the cells are CD4, CCR5, CXCR4 positive.
  • an amplicon gene is readily substituted for a marker gene to induce amplification of viral stocks (not shown).
  • the amplicon gene is Tat. Clones of the stable CD4 positive cell line are established 30. A stable
  • CD4 positive cell line is selected for low marker background expression levels 40.
  • the marker gene is luciferase.
  • An immortalized cell line 15 is positive for CCR5, CD4 and CXCR4 receptors and optionally other receptors illustratively including CCR3, CCR2B and T-lymphocyte expressed 7 transmembrane domain receptor.
  • the origin cell line is HeLa. More preferably, the cell line is J53 (Oregon Health Sciences University) or a cell line that naturally expresses CD4, CCR5 and CXCR4.
  • the cells of immortalized cell line 15 are then tested for sensitivity to HIV-1 infection 25. Expansion of highly sensitive cells to HIV-1 infection 35. The clone of 40 is used to transduce 50 the highly sensitive immortalized cell line of 35.
  • the receptor is selected to create a cell which is highly sensitive to infection by HIV-1 isolates.
  • Clones established from this second transduction are both highly sensitive to infection with primary HIV- 1 isolates and express low background levels of the marker gene product in the absence of HIV-1 60.
  • Those clones which are positive for the marker gene are identified 70.
  • Such positive clones 70 have utility to promote HIV production upon transduction with Tat 75. HIV primary virus stock production is exploited herein to selectively enrich drug resistant minor HIV strains infecting a host 85.
  • Infection of the clones expressing low background levels of at least two markers such as ⁇ -gal and luciferase with HIV-1 confirms the relationship between infectious viral units and marker gene (luciferase) activity 80, although in practice expression of a single marker gene is operative herein.
  • Clones that relate infectious virus units, such as ⁇ -gal, with a second marker gene activity such as luciferase find utility in the measurement of HIV co-receptor utilization (not shown).
  • the cells capable of expressing marker genes in response to HIV infection are optionally used to measure viral sensitivity in the presence of a drug 90. Drug resistance to various pharmaceutical during viral life cycle events such as envelop formation 92, reverse transcription 94 and proteo lysis 96 is optionally determined.
  • the measurement of viral sensitivity finds utility in HIV viral target resistance analysis 98.
  • the resulting composition of clones in a suitable medium is amenable for use to quantify HIV-1 titer.
  • the present invention also finds utility in quantifying the drug sensitivity of particular HIV-1 phenotypes. It is appreciated that the present invention is applicable to use with immunodeficiency viruses other than the representative HIV-1. By transducing a cell line to express co-receptors adapted for binding an immunodeficiency virus, a variety of viruses may be assayed with the present invention.
  • the present invention pertains to cell lines that are capable of detecting sensitivity of a given strain of HIV to inhibitors that act upon various stages of the virus life cycle by monitoring the effect various drugs have on early viral life cycle stages such as reverse transcription, integration and envelop mediated receptor binding, envelope fusion, as well as the late life cycle stages complexes such as proteolysis and Gag complex formation.
  • a method and a kit are provided for monitoring the major and minor virus populations infecting a given host. Through the enrichment and detection of minor drug resistant virus populations and the sensitivity of those populations to viral inhibitors, the assay is well suited for determining specific anti-retroviral drugs suited to contain replication of the various HIV strains infecting a given host.
  • Such a tailored therapeutic protocol is more effective in inhibiting viral amplification and/or reduces pharmacological side effects.
  • the J53 Tat cell line is well suited to detect sensitivity of a host's particular viral infection to inhibit or affect the various stages of the virus life cycle.
  • the present invention provides more rapid viral amplification as compared to conventional PBMC cells thereby allowing more rapid amplification, with fewer cycles of reverse transcription. Further applications of the present invention include measurement of HIV attributes of co-receptor utilization, antibody neutralization, isolation, titration, gene sequencing, and CTL assays.
  • the present invention also provides a method for detection of primary HTV from plasma, including noninfectious HIV-1 found in plasma.
  • VSV-G serves to mediate infection of HIV-1 particles and thus, in the absence of VSV-G the virus remains noninfectious, whereas in its presence infectivity is complemented.
  • VSV-G in addition to VSV-G other infectivity complements are also operative herein including adenovirus, liposome, monoclonal antibody and other vectors to complement noninfectious HIV.
  • the methods and indicator cell lines of the present invention are operative to analyze drug sensitivity of primary HIV which has been purified and taken directly from infected host plasma.
  • luciferase expression To directly analyze the relationship between luciferase expression and infectious virus units, a series of gene transfer plasmids are constructed to express luciferase, ⁇ -galactosidase ( ⁇ -gal), and green fluorescence protein (GFP), respectively, ⁇ -gal, GFP, and luciferase (luf) are placed under control of the HIV-
  • LTR long terminal repeats
  • RRE Rev Responsive Element
  • Figures 2A through 2E illustrate the different gene transfer expression plasmids that are constructed.
  • the ⁇ -gal and GFP markers allow for direct enumeration of the number of infectious virus units as infected cells by counting under a microscope.
  • the luciferase marker allows for sensitive and high throughput quantitation of HIV infection. In the present invention the requirement of Tat and
  • Rev for marker gene expression is different from previous work in that it allows for highly regulated and decreased background level expression of the marker gene. This is particularly important for luciferase.
  • the J53BL cell line or its functional equivalents are operative in accordance with the present invention. It is appreciated that the nucleic acid sequences coding for CCR5, CXCR4, CD4, luciferase, ⁇ -galactosidase, GFP, CAT, Tat and the J53 cell line as a whole can be altered by substitutions, additions or deletions that provide for functionally equivalent cells.
  • "functional equivalency” is defined to mean a nucleic acid sequence which encodes for a product that performs operationally within the present invention with at least half the effectiveness of the product derived from the unaltered nucleic acid sequence of a receptor, amplicon, marker gene or cell line.
  • nucleotide coding sequences which encode substantially the same receptor amino acid sequences, cell line amino acid sequences, marker gene sequences and amplicon sequences may be used in the practice of the present invention. These include but are not limited to nucleotide sequences which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence thus producing a silent change.
  • receptor, marker, amplicon and cell lines proteins or fragments or derivatives thereof of the present invention include, but are not limited to, those containing as a primary amino acid sequence all or part of the amino acid sequence of the sequences for CCR5, CXCR4, CD4, luciferase, ⁇ - galactosidase, GFP, CAT, Tat and J53 sequences including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • one or more amino acid residues within a sequence are optionally substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within a sequence may be selected from other members of a class to which the amino acid belongs.
  • non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present invention are proteins or fragments or derivatives associated with J53BL which are differentially modified during or after translation by operations such as glycosilation, proteolytic cleavage and linkage to an antibody or other cellular ligand.
  • Example 1 Generation of transduction vectors for the delivery of marker genes.
  • the ⁇ -gal, GFP and luciferase gene transfer plasmids are separately transfected into cultures of 293T cell together with a lentiviral-based packaging plasmid
  • the vector-containing culture supernatants are harvested, clarified by low- speed centrifugation, filtered through 0.45 micron filters, analyzed for HIV-1 p24 core antigen concentration by ELISA, aliquoted, and cryopreserved as stocks.
  • Four serial five- fold dilutions (normalized for p24 antigen concentration) of the stocks are prepared and used to infect replica cultures of HIV-HeLa cell.
  • the HIV-HeLa cells contained an integrated HIV-1 pro virus that is defective in vpr and env, and produces the Tat and Rev protein for transactivating marker gene expression. Two days after infection of the HIV-HeLa cells with the different
  • vector stocks, ⁇ -gal and GFP expression is quantified using a microscope to count
  • FIGS. 4A and 4B show the relationship between concentration (HIV-1 p24 antigen, Coulter Inc.) of the
  • Example 2 Generation of ⁇ -gal, luciferase and GFP indicator cell lines to quantify HIV/SIV infection.
  • the following pairs of vector stocks (derived as described above) are used
  • microscopy are expanded into replica cultures.
  • One replica culture set is infected
  • HIV-1 infection provided Tat and Rev to activate marker gene expression
  • luciferase activity levels 36 non-HIV-1 infected, luciferase expression- positive clonal cultures (derived from HeLa-CD4 cells transduced with pluf + p ⁇ - gal) are analyzed for luciferase activity to determine basal background expression levels.
  • the HeLa-CD4 cells being obtained from the AIDS Research and Reference Reagent Repository of NTH.
  • luciferase activity ranged from 15 to 250 units.
  • Analysis for ⁇ -gal expression in response to HIV-1 infection indicated approximately 70% of the clones expressed both ⁇ - gal and luciferase.
  • the two clones (referred to as HeLa- ⁇ -gal-lufl, and HeLa- ⁇ - gal-luf2) that exhibited the lowest background levels of luciferase expression and are positive for ⁇ -gal expression are used to directly analyze the relationship between HIV-1 infectious units and luciferase activity.
  • Serial dilutions of two different HIV-1 strains (HIV-1/SG3 and HIV-1/NL43) are normalized for p24 antigen concentration and used to infect replica cultures of HeLa- ⁇ -gal-lufl, and HeLa- ⁇ -gal-luf2. After 48 hours, one set of cultures is analyzed for luciferase activity and the other was analyzed for ⁇ -gal.
  • Figure 5 shows the relationship between HIV-1 infectious units ( ⁇ -gal positive cells) and luciferase activity for the HeLa- ⁇ -gal-lufl cell line.
  • the HeLa- ⁇ -gal-luf2 cell line gave nearly identical results with slightly higher luciferase activity levels at the lower virus inoculums. Between approximately 10 and 10,000 virus infectious units.
  • a near- linear relationship to luciferase activity is shown in Figure 5.
  • the linear range of detection using the luciferase marker in Figure 5 is approximately 3 orders of magnitude, and as few as 10-20 infected cells out of approximately 100,000 can generate a virus-positive (above background) result.
  • This dynamic range allows for quantitative analysis of virus infection from approximately 10 to 10000 infectious units, thereby reducing the necessity of dilution of virus in order to generate quantitative
  • Example 3 Sensitive detection of HIV-1 primary viruses using ⁇ -gal and luciferase reporter genes.
  • the present invention utilizes a combination of a reporter assay system for
  • Table 1 shows that all viruses, including the macrophage tropic
  • YU2 clone included as a control, are highly infectious in the J53 ⁇ -gal/luf cell
  • viruses are prepared and analyzed: TIVI, WIMI, KIWE and YU2. Between approximately 100 to 10,000 infectious units, the data show a linear relationship with luciferase activity (Figure 6). Background levels of luciferase are between 100 and 150.
  • the J53 ⁇ -gal/luf cell line represents a transduced population of cells since integration of the transduction vector into the genome of the J53 cells can occur differently in each cell.
  • cultures of single cell clones are derived from the J53 ⁇ -gal/luf cell line as described above and characterized for luf and ⁇ -gal expression in response to HIV-1 infection. Ten clones expressing between 17 and 750 luf activity are selected for analysis. Clone number 13, termed J53-C13, is confirmed to express both luciferase and ⁇ -gal, and is used for subsequent analysis as described below. Stocks of twenty different HIV-1 isolates are obtained from HIV-1 infected individuals by standard coculture techniques.
  • the J53-C13 cell line is sensitive to HIV-1 infection to a degree similar to PBMC.
  • the JC11 cell line is analyzed for comparison.
  • JC11 is the parental cell line to J53-C13. It expresses equal amounts of CD4 and CXCR4 but is negative for CCR5.
  • JC11 is transduced to express b-gal and luciferase, and positive cells are biologically cloned exactly as described above for J53-C13.
  • Jl 1-C5 A clone designated Jl 1-C5, which is capable of expressing both b-gal and designated Jl 1-C5, which is capable of expressing both b-gal and luciferase, is selected for comparison with J53-C13. Both cell lines are infected with primary virus isolates and molecularly cloned virus including YU2, SG3, and 89.6 (a dual tropic clone).
  • Table 4 shows the titer of each virus in the J53-C13 and Jl 1-C5 cell lines.
  • results show a marked reduction in virus titer in the Jl 1-C5 cell line, indicating that the CCR5 co-receptor is necessary for efficient infection/detection of primary
  • HIV-1 isolates are derived by PBMC coculture from two different HIV-1
  • YU2 (included as a control) virus stocks are used to infect the J53-C13 reporter
  • viruses - as determined by luciferase activity as an indicator.
  • a major problem with existing methods for evaluating HIV-1 drug sensitivity is that differences in virus inoculum can have significant effects on the
  • IC50 for a given drug That is, as the infectious dose of virus is increased, the
  • J53-C13 cells are infected with 100, 500, and 2500 infectious units of virus and analyzed for drug sensitivity as described above.
  • Figure 9 shows the results for drug sensitivity to AZT. There is no significant
  • Example 5 Generation of a Tat expressing cell line to rapidly amplify virus production from infected cells. The amplification of primary virus from infected individuals is required
  • the present invention confirms that the JC53 and the J53-C13 cell lines are highly sensitive to infection of primary virus isolates.
  • cell lines may be utilized to amplify the primary virus isolate instead of PBMC.
  • JC53 cells are transduced with the HIV Tat gene under control of the CMV, or LTR promoter, as shown in Figure 10.
  • Tat is constructed into a self-deleting U3 transduction vector, Figure 10. Three days after transduction, single cells are cloned and 33 are identified to be Tat expression positive, 10 containing LTR-2 as a promoter and 23 containing CMV as a promoter for Tat expression.
  • HIV-1 p24 antigen ELISA is measured by HIV-1 p24 antigen ELISA and the highest HIV-1 producing lines from each are selected for further analysis.
  • J53-CMVtat is infected with the YU2 clone and the KEWI virus isolate
  • the JC53 cell line is analyzed in a
  • Tat expressing cell lines causes a 4-6-fold increase in HIV-1 replication.
  • Example 6 The use of CD4/CCR5/CXCR4 + Tat expressing cell line to capture and amplify primary virus.
  • the J53tat cell line is compared with PBMC for primary virus
  • PBMC and J53tat are each infected with 2.5E5 infective particles of YU2. Two days later the concentration of progeny virus is analyzed for
  • the J53tat cell line amplifies primary virus to
  • Tat facilitates the rapid generation of high titered primary virus stocks for resistance testing without selection of longer term culture, such as PBMC culture for virus amplification.
  • Example 7 Detection of drug resistance/sensitivity that effect various stages of virus life cycle.
  • the J53tat cell line is used to produce virus and thereby enable viral testing
  • viral drug resistance mutations in early stage targets such as reverse transcriptase (RT), integrase (IN) and env
  • late stage targets such as protease and Gag
  • the J53tat cells are infected
  • HIV YU2 MOI of either 0.2 or 0.04
  • protease inhibitor infavir
  • protease inhibitor with increasing concentrations causing greater inhibition.
  • Example 8 Detection of noninfectious cultured virus.
  • the pellet is resuspended in 100 ul DMEM.
  • the infectivity is then determined using J53BL cells. The infectivity is determined to be 7.5E4.
  • YU2 virus containing wild-type envelope is pelleted through sucrose by
  • the resuspended (100 ul) virus is mixed with and without VSG-G (1:1) and repelleted by ultracentrifugation (150,000g, 2 hours, 4°C).
  • the pellets are resuspended in 100 ul DMEM, and the infectious units are determined using J53BL cell summarized as in Table 7. Virus pelleted through sucrose is noninfectious. Virus pelleted
  • the recovery in infectivity is approximately 20% compared with the
  • VSV-G 2500 infectious particles are detected as summarized in
  • Plasma from patients infected with HIV-1 is tested for the presence of
  • HIV is incubated with J53BL cell line for four hours to allow binding and entry into J53BL cells, reverse transcription proceeds and the viral cDNA is
  • HIV replication is suppressed through expression of an inhibitor of viral gene expression, such as the rev inhibitor, rev mlO by conventional techniques.
  • the HIV genome is expanded as J53BL cells divide and increase in number, without further rounds of reverse transcription.
  • the increased copy numbers of the viral genome are purified and sequenced. By relieving the inhibitory effect on rev, viral gene expression will return to normal in the expanded cells, and virus can be analyzed.
  • HIV-1 isolates were derived by coculture (7-10 days) of HIV-1 infected patient PBMC with PHA stimulated normal donor BPMC.
  • Virus titer was determined by counting the # of beta-gal positive cells. Results indicate infection positive cells per ml of stock virus. Neg. (negative) titers were undetectable below 40 infectious units per ml.
  • Nos. represent pg of p24 antigen per ml.
  • TCIU tissue culture infectious units

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Abstract

L'invention concerne des méthodes et des réactifs permettant de détecter une primo-infection à VIH. Une lignée cellulaire exprimant les récepteurs CCR5, CXCR4, et CD4 est liée et infectée par le VIH primaire. La lignée cellulaire contient une séquence de gènes marqueurs, la séquence de gènes marqueurs exprimée en quantités quasi-linéaires sur au moins deux ordres de grandeur en réaction à une infection à VIH. Le VIH primaire est amplifié pour créer un stock de virus primaires par insertion d'un gène amplicon dans le récepteur exprimant la lignée cellulaire. L'amplification du VIH, qui se produit rapidement, peut être réalisée avec un VIH non-infectieux par amplification en présence d'un complément d'infectivité. La présente invention sert à déterminer le titre du VIH hôte, la sensibilité aux médicament, l'amplification du VIH, le séquençage des gènes et l'utilisation des co-récepteurs.
PCT/US1999/014104 1998-06-23 1999-06-23 Analyse a base de cellules pour determiner l'infectivite et la sensibilite du virus vih WO1999067429A1 (fr)

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Application Number Priority Date Filing Date Title
CA002331760A CA2331760A1 (fr) 1998-06-23 1999-06-23 Analyse a base de cellules pour determiner l'infectivite et la sensibilite du virus vih
AU48278/99A AU4827899A (en) 1998-06-23 1999-06-23 Cell-based assay for immunodeficiency virus infectivity and sensitivity
EP99931859A EP1088108A4 (fr) 1998-06-23 1999-06-23 Analyse a base de cellules pour determiner l'infectivite et la sensibilite du virus vih
US09/719,340 US6797462B1 (en) 1998-06-23 1999-06-23 Cell-based assay for immunodeficiency virus infectivity and sensitivity

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US9031798P 1998-06-23 1998-06-23
US60/090,317 1998-06-23

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US10/936,037 Continuation US20050032046A1 (en) 1998-06-23 2004-09-08 Cell-based method and assay for measuring the infectivity and drug sensitivity of immunodeficiency virus

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US6800729B2 (en) 1995-06-06 2004-10-05 Human Genome Sciences, Inc. Human G-Protein chemokine receptor HDGNR10 (CCR5 receptor)
US7160546B2 (en) 1995-06-06 2007-01-09 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
US6461809B1 (en) 1996-10-15 2002-10-08 Bio-Tech Imaging, Inc Methods of improving infectivity of cells for viruses
WO2000065356A1 (fr) * 1999-04-27 2000-11-02 Bio-Tech Imaging, Inc. Mesure de l'infectiosite du vih1
US7294458B2 (en) 2000-09-26 2007-11-13 Health Research Inc. Analysis of HIV-1 coreceptor use in the clinical care of HIV-1-infected patients
US7718356B2 (en) 2000-09-26 2010-05-18 Health Research Inc. Heteroduplex tracking assay
WO2002027321A3 (fr) * 2000-09-26 2003-08-07 Health Research Inc Analyse de l'utilisation du co-recepteur du vhi-1 dans le suivi clinique de patients infectes par le vhi-1
US8119339B2 (en) 2000-09-26 2012-02-21 Health Research Inc. Heteroduplex tracking assay
WO2002027321A2 (fr) * 2000-09-26 2002-04-04 Health Research Incorporated Analyse de l'utilisation du co-recepteur du vhi-1 dans le suivi clinique de patients infectes par le vhi-1
US7344830B2 (en) 2000-09-26 2008-03-18 Health Research Inc. Heteroduplex tracking assay
EP2333543A1 (fr) 2000-09-26 2011-06-15 Health Research, Incorporated Analyse de l'utilisation du co-recepteur du vih-1 pour le suivi clinique de patients infectes par le vih-1
US7943297B2 (en) 2000-09-26 2011-05-17 Health Research Inc. Analysis of HIV-1 coreceptor use in the clinical care of HIV-1-infected patients
US6727060B2 (en) 2000-09-26 2004-04-27 Health Research, Inc. Analysis of HIV-1 coreceptor use in the clinical care of HIV-1-infected patients
US7709191B2 (en) 2000-09-26 2010-05-04 Health Research, Inc. Heteroduplex tracking assay
US7175988B2 (en) 2001-02-09 2007-02-13 Human Genome Sciences, Inc. Human G-protein Chemokine Receptor (CCR5) HDGNR10
US7393934B2 (en) 2001-12-21 2008-07-01 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
US7501123B2 (en) 2004-03-12 2009-03-10 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
WO2007084568A3 (fr) * 2006-01-17 2008-10-02 Health Research Inc Bio-essai de suivi en hétéroduplex

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CA2331760A1 (fr) 1999-12-29
AU4827899A (en) 2000-01-10
EP1088108A1 (fr) 2001-04-04

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