WO2001040447A1 - Hiv-1 protease inhibitor resistance assay - Google Patents

Abstract

Methods and reagents for the capture of primary HIV are provided, as are assays for testing HIV drug resistance and sensitivity. The assays make use of a virus-amplication step that entails the use of an amplicon, followed by an indicator or reporting step in which an indicator cell line is infected with amplified virus from the first step. The base system is also useful for determining host titer, sequencing, CTL production, and co-receptor utilization.

Description

HIV-1 PROTEASE INHIBITOR RESISTANCE ASSAY

Background of the Invention

Familiarity with principles of retroviral biology, e.g., as described in Coffin et al., Retroviruses, Cold Spring Harbor Library Press (1997), is assumed. The detection, isolation, propagation and study of whole, natural retroviruses including HIV has been difficult due to the natural isolates not infecting and propagating well in cell cultures. This has posed a problem for developing "virus isolate-based" assays for measuring the susceptibility of HIV to anti-retroviral compounds. Alternative approaches have focused on the analysis of specific viral components such as reverse transcriptase and protease. These components have been engineered away from their natural genetic and physiological mileau and into an artificial one. For example, the genes encoding these components are amplified by PCR and cloned into a laboratory proviral construct, which is then introduced back into a mammalian cell culture system for study. Such constructs may include a pseudotype env gene to mediate infection. For maximum clinical significance and context, study of the natural isolate, unencumbered by artifacts deriving from the generation of artificial recombinant virus, would be preferred.

Recently, light has been shed on why infection and propagation of natural HIV may be inefficient in vitro using continuous cell lines. Besides CD4, other cellular factors have been shown to be needed for efficient infection. These include but are not limited to the chemokine receptors CCR5 and CXCR4. Platt et al. (1998) J. Virol. 72:4, pp. 2855- 2864, for example, teach the effects of CCR5, CXCR4, and CD4 cell surface co-receptor concentrations on HIV infectivity. .

Ferguson et al. (1991) US Patent 5,026,635 teach an HIV reporter system wherein a single mammalian cell line is engineered to possess two different heterologous coding sequences, one that constitutes a reporter gene, and another that encodes a trans-activating regulatory protein, i.e., Tat, that serves to specifically activate the reporter. Ferguson et al. employed this system to screen for compounds that specifically interfered with HIV tat function. Neither Platt nor Ferguson teach or suggest the use of an amplicon such as Tat for propagating, i.e., amplifying, isolating, titering, and/or further characterizing or manipulating whole virus. We previously described such a system in PCT application US99/14104, a copy of which is appended hereto. Our system allows for greater infectivity to primary HIV and amplification of those HIV. Using cell type(s) employing these infectivity and amplifying features in tandem enables primary virus isolates (whole virus) to be detected without significant bias or selection. This system solves long-felt needs by providing for the efficient capture of whole virus (of all variants) and by further providing for improved yields of whole virus. This has the benefit of aiding many types of clinically useful applications, including drug testing and research aimed at further elucidating complicated retroviral biology.

We herein describe how our system may be employed to effectively screen anti- retroviral drug candidates and/or to compare the effect of various known inhibitors on different retroviral strains. Our approach is applicable to drugs and drug candidates of all types, and targeting any aspect or feature of retroviral biology.

Summary of the Invention

The invention advantageously exploits our previously described amplicon system for use in drug testing. This derives from the ability of our originally described system to amplify, detect, isolate, and characterize primary virus.

In a first aspect, the invention features a cell for amplifying virus gene expression. The cell preferably expresses CCR5, CXCR4, and CD4 or equivalents that allow for infection by a retrovirus of interest, e.g., HIV. The cell further contains an expressible nucleic acid sequence within that encodes an amplicon for amplifying the retrovirus of interest, e.g., Tat. The cell can be part of a larger system that also includes a second cell type or "indicator cell" for measuring or determining viral amplification or presence once the virus has been amplified in the amplicon-bearing cell line. The indicator cell preferably also expresses factors that allow for good infectivity and further possesses within a marker gene that allows for the phenotypic determination of the presence and/or amount of virus within. Anti-retroviral drugs or drug candidates may be employed at various times in the media in which the amplifying and/or indicating cells grow to determine the effect of that drug on the particular virus being testing, or else the susceptibility or resistivity of that virus to the drug In preferred embodiments, the marker gene is selected from the group of marker genes consisting of β-galactosidase, luciferase, fluorescent protein, and antibiotic resistance and is driven by a promoter which is measurably or detectably responsive to the presence of the virus being tested (e.g., in the instance of HIV-1, an LTR promoter). The promoter used to express the amplicon may be either constitutive or inducible. Representative promoters include but are not limited to CMV and Tet. One of skill in the art is familiar with many others that can be conveniently employed. Drug or drug candidates may be added to the media system at any time during amplification/propagation of the retrovirus of interest. One of skill is familiar with a existing antiviral drugs, some of which are later detailed herein.

In another aspect, the invention features a method that employs the features of the first aspect for determining retroviral susceptibility to a drug or drug candidate of interest, such as an inhibitor of a retroviral gene or gene product, e.g., reverse transcriptase, integrase, protease, T-20, etc. The method includes amplifying or propagating a virus of interest in a cell that expresses an amplicon suitable for amplifying the virus. A drug of interest may be added at a given time during the propagation/amplification of the virus. The amplified virus is then harvested and used to infect an indicator cell line. The same and/or different drug may be included at various times during the indicator cell step to survey effect on the retrovirus life cycle. Thus, the invention also features a method of assaying primary HIV for susceptibility to a drug or drug candidate that includes the steps of amplifying a primary HIV using an amplifying cell line and indicating the presence or amount of that amplified primary HIV using an indicator cell line. One or more drugs or drug candidates are added to one or more of the amplifying and indicator cell lines to determine effect on the primary virus being tested.

High throughput drug screening and other applications will be appreciated by one of skill. The invention is especially advantageous for its superior sensitivity over existing systems, and allows for enhanced and more accurate clinical testing and evaluation of both known and prospective anti-retroviral drugs. The invention is further useful for isolating and characterizing rare, minority or mutant viral populations within a greater population. Other advantages, aspects, and embodiments will be apparent from the figures, the detailed description of the preferred embodiments, and the claims. Brief Description of the Figures

Figure 1 shows an embodiment for whole virus amplification and/or drug resistance/sensitivity testing. Although drug in this embodiment is indicated to be added at the amplifying step, it will be appreciated that drug may be added at any or all steps in the procedure, including at the indicator cell line step.

Figures 2A-2E are schematics illustrating various indicator constructs useful for constructing indicator cell lines of the present invention.

Figure 3 is a schematic illustrating the production of lentiviral transduction particles for establishing indicator cell lines for use in the invention. This is accomplished, e.g., by transfecting a host cell with the thee individual constructs shown, establishing retroviral particles, transfecting those particles into a new host and monitoring for stable intregration of the marker constuct in the new host, e.g., by PCR or inducing expression of the marker. The same approach can be used to establish the amplifier cell line, except that an amplicon construct as shown in Figure 10 is employed in lieu of, or in addition to, the marker gene construct shown here.

Figure 4A and 4B are graphs illustrating the relationship between the concentration of vector and infectious units as determined with β-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 11A and 11B are graphs illustrating the viral amplification two days following infection with equal quantities of YU2 HIV (a) for infectivity and (b) p24 antigen. Figure 12 shows various HIV-1 life cycle events that are possible targets for inhibitory drugs.

Figure 13 shows method step embodiments for assaying for HIV-1 phenotype resistance to drugs that are active in early stage replication events.

Figures 14-16 show the viral infectivity observed in whole virus assays for AZT, NVP and 3TC, respectively, and as obtained using the methodology detailed in Example 12.

Figures 17-20 show the methodology of Example 12 as applied against the phenotypes of Table 2 and strains DK 32499, TZ 0572, ML 32399 and JD 5928 upon exposure to AZT, NVP, 3TC and Delaviridine, respectively. Figure 21 shows an illustrative method of assaying for HIV-l phenotype resistance to drugs that are active against late stage replication events

Figures 22-24 show the viral infectivity observed in whole virus phenotype assays for the protease inhibiting drug, indinavir.

Figure 25 shows a J53BL indicator cell line (derived from HeLa) and its advantages and properties.

Detailed Description of the Preferred Embodiments

There is currently no cost effective, "high throughput" method for analyzing the drug resistant phenotype of primary virus isolates derived from individuals receiving antiretroviral treatment. Various in vitro biologic and immunologic techniques have been developed to detect human and simian immunodeficiency viruses (HIV and SIV, respectively). These include assays that detect the enzymatic activity of the reverse transcriptase (RT) protein, ELIS A based assays for the detection of HIV/SIV core antigen (HIV-1 p24 or HIV-2/SrV p27), direct quantitation of infectious virus by synctial 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, e.g.,

PCR. Recently, genetic reporter-based assays have been created to detect HIV/SIV infection. In this approach, mammalian cells are genetically modified to express a reporter gene such as β-galactosidase (β-gal), green fluorescent protein (GFP) or chloramphenicol acetyltransferase (CAT) in response to infection and Tat protein expression. These detection systems require enumeration of the number of infection-positive cells by flow cytometry or fluorescence microscopy (GFP), microscopy (β-gal), or the utilization of radioisotopes (CAT). The firefly luciferase gene, under control of the HIV-1 LTR promoter, has been used as a reporter gene for HIV-1 infection. Luciferase is a sensitive marker gene for HIV-1 infection, since expression of a relatively few number of luciferase molecules can result in appreciable activity levels using standard luciferase detection assays. Prior to the invention, the sensitive detection of 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 co-receptor expression, e.g., the CCR5 chemokine co-receptor on the surface of such cells. 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 these prior art limitations.

The invention relates to cell-based assays and components thereof for amplifying and/or analyzing primary virus populations. The assays preferably include the use of two different cell lines, one an ampifying cell line, and the other an indicator cell line. Each cell line is capable of infection with the retrovirus of interest, here HIV-1 by virtue of expression of CCR5, CXCR4 and CD4 receptors on the host cell surface. It is anticipated that other co-receptors will be identified and/or engineered that can supplement or supplant one or more of these receptors to further modulate infectivity. The amplifier cell line will additionally possess an amplicon gene whereas the indicator cell line will additionally possess a reporter/marker.

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. Immunodeficiency virus infection- sensitive clonal cells are preferably produced by selecting CCR5, CXCR4 and CD4, expressing cells and thereafter transducing them with a marker gene vector such as luciferase that is engineered to be responsive the magnitude of immunodeficiency virus infection.

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. Determining immunodeficiency virus titer or drug sensitivity for a given strain, type, species or genus of virus is also rendered practical by the invention., which also affords the ability to test virus derived from blood plasma as well as cell culture.

The invention is further useful for other purposes, e.g., determining co-receptor usage for further modulating infectivity. The invention is especially useful for the discovery of new anti-retrovral drugs and monitoring drug therapy protocols to enhance the effectiveness of drug treatment regimes against retrovirus infection.

The invention is illustratively described herein as applied to HIV. However, one of skill will appreciate that the general system may be applied to other types of retroviruses as well.

The ability to detect primary isolates of HIV-1 with greater sensitivity than currently possible is an aspect of the present invention. Unlike previous assays, 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.

Most prior art molecular clones of HIV-1 have been derived by tissue culture methods that select for viruses that do not require CCR5 co-receptor for infection, herein defined as T-tropic viruses. Such clones are not able to infect monocytes and macrophages. The term "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. The term "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. This difference in tropism has been mapped to the viral env gene. 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 are virus that are derived directly from the blood of an HIV infected individual. Primary HIV can also be derived by short term culture in vitro culture of primary peripheral blood mononuclear cells (PBMC). Primary HIV can also embody complex mixtures that may contain macrophage- and/or T-tropic viruses. During the natural history/progression of HIV-1 infection there is generally a shift from a population of macrophage-tropic toward one of 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. 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). Only recently have such cell lines been derived which appear to be susceptible to infection with diverse HIV-1 isolates (Platt et al., J. Virol. 72:2855, 1988; Overbaugh et al, J. Virol. 71:3932, 1997).

As used herein, "primary HIV" is defined as HIV derived directly from an infected host organism from sources such as blood, plasma, PBMC, CSF and other tissues.

As used herein, "immunodeficiency virus" is defined as various strains and stocks of HIV-1, HIV-2, SIV and lentiviruses. As used herein, "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.

As used herein, "major population" is defined as the numerically dominant viral strain, type, species or genes of a viral titer obtained from a host culture or organism. As used herein, "drug sensitivity" is defined as the effectiveness of a drug to inhibit HIV replication and/or expression within a host cell.

By making genetic modifications to a CCR5 or CD4 expressing cell line, an efficient method for analyzing drug sensitivity properties of primary HIV, such as HIV-1, is provided. 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. Further applications of the present invention include measurement of HIV attributes of co-receptor utilization, antibody neutralization, isolation, titration, gene sequencing, and CTL assays. One of skill will readily appreciate and employ such applications without undue experimentation, and using routine methods known in the art. 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. To directly analyze the relationship between luciferase expression and infectious virus units, a series of gene transfer plasmids are constructed to express luciferase, β-galactosidase (β-gal), and green fluorescence protein (GFP), respectively, β-gal, GFP, and luciferase (luf) are placed under control of the HIV-1 or HIV-2 long terminal repeats (LTR), and the Rev Responsive Element (RRE). Figures 2A through 2E illustrate the different gene transfer expression plasmids that can be constructed. The β- 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. 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 equivalent is a good indicator cell line for use in the invention and readily created by transfecting suitable constructs into HeLa cells and monitoring for low basal expression and good inducible of the marker. It is appreciated that the nucleic acid sequences coding for CCR5, CXCR4, CD4, luciferase, β-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. As used herein, "functionally equivalent" is defined as performing with at least half the effectiveness of the standard system. Due to the degeneracy of the genetic code, different DNA sequences can encode essentially the same product. Also, conservative amino acid substitutions as known in the art are also within the scope of the invention. For example, 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.

The utility of the invention for drug testing is demonstrated using an HIV-1 phenotype assay to measure sensitivity/resistance to various drugs. Figures 1A and B are illustrative embodiments. These drugs can target various stages of viral replication including early and late stage events. Various HIV-1 life cycle events that are possible targets for inhibitory drugs are shown schematically in Figure 12. Illustrative steps for assaying HIV-1 resistance to drugs that are active in early stage replication are shown in Figure 13. Some of the HIV-1 reverse transcriptase (RT) inhibitor resistant phenotypes are summarized in Table 10 with the drug relevant to the specific mutation. Figures 14-16 show the viral infectivity observed in whole virus assays for AZT, NVP and 3TC, respectively, and as obtained using the methodology detailed in Example 12. The methodology is applied against the phenotypes of Table 2 and strains DK 32499, TZ 0572, ML 32399 and JD 5928 upon exposure to AZT, NVP, 3TC and Delaviridine in Figures 17-20, respectively. Some of the HIV-1 protease (PR) inhibitor resistant phenotypes are summarized in Table 11 with the drug relevant to the mutation. The method steps of assaying for HIV-1 phenotype resistance to drugs that are active in late stage replication events are shown schematically in Figure 21. Figures 22-24 show the viral infectivity observed in whole virus phenotype assays for indinavir. It is appreciated that the present invention is also operative in assaying for peptide based drugs such as T20, which targets fusion events with the host cell membrane.

The following Examples nonexhaustively illustrate the utility of the invention. Example 1 - Generation of transduction vectors for the delivery of marker genes

In order to generate vector stocks for transduction with the different reporter genes, the β-gal, GFP and luciferase gene transfer plasmids, including those containing the HIV-2 LTR (shown in Figure 2), are separately transfected into cultures of 293T cell together with a lentiviral-based packaging plasmid (pCMV-GPl), and the pCMV-VSV-G env plasmid (Figure 3). Forty-eight hours later, 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 provirus 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, β-gal and

GFP expression is quantified using a microscope to count the number of positive cells/well. Luciferase expression is measured using standard assay methods (Promega) and a luminometer. Figures 4A and 4B show the relationship between concentration

(HIV-1 p24 antigen, Coulter Inc.) of the vector stocks and infectious units as determined with β-gal and GFP (virus infectious units) or luciferase activity.

Example 2 - Generation of β-gal, luciferase and GFP indicator cell lines to quantify HIV/SIV Infection.

The following pairs of vector stocks (derived as described above) are used to co-transduce cultures of HeLa-CD4 cells: (a) pluf + pβ-gal, (b) pluf + pLTR2-B-gal, (c) pluf + pGFP, (d) pluf + pLTR2-GFP, (e) pLTR2-luf + pβ -gal, (f) pLTR2-luf + pLTR-2β-gal, (g) pLTR2-luf + pGFP, (h) pLTR2-luf + pLTR2-GFP. Three days later, the cells are biologically cloned by limiting dilution in 48 well plates. Wells containing clonal cells (confirmed after initial plating by microscopy) are expanded into replica cultures. One replica culture set is infected with HIV-1/SG3 and analyzed for marker gene expression (HIV-1 infection provided Tat and Rev to activate marker gene expression) as described above. Expression positive cells cultures are identified, expanded and cryopreserved.

Since the expression or relatively few molecules of luciferase produces substantial luciferase activity levels, 36 non-HIV-1 infected, luciferase expression-positive clonal cultures (derived from HeLA-CD4 cells transduced with pluf + pβ-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 NIH. Of the 36 clones analyzed, luciferase activity ranged from 15 to 250 units. Analysis for β-gal expression in response to HIV-1 infection indicated approximately 70% of the clones expressed both β-gal and luciferase. The two clones (referred to as HeLa-β-gal-lufl , and HeLa-β-gal-luf2) that exhibited the lowest background levels of luciferase expression and are positive for β-gal expression are used to directly analyze the relationship between HIV-1 infections 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-β-gal-lufl, and HeLa-β-gal-luf2. After 48 hours, one set of cultures is analyzed for luciferase activity and the other was analyzed for β-gal. Figure 5 shows the relationship between HIV-1 infectious units (β-gal positive cells) and luciferase activity for the HeLa-β-gal-lufl cell line. The HeLa-β-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 infections 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. As referred to herein "near linear" is intended to mean an increase in marker activity, A proportional to an increase in the surrounding virus infectious unit concentration, IU such that A=n(IU)^{1 ±x} + b where n is a real number; x is a real number between 0 and 0.5; b is the measured background level of marker expression in the absence of virus; for at least 2 orders of magnitude of IU. 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 data.

Example 3 - Sensitive detection of HIV-1 primary viruses using β-gal and luciferase reporter genes.

The present invention utilizes a combination of a reporter assay system for sensitively and rapidly quantifying infections HIV-1 over a wide linear range with a cell line which is highly sensitive to infection with both M-tropic and T-cell tropic viruses.

Transduction of the CD4-CCR5 positive J53 cell clone (Dr. David Kabat, Oregon Health

Sciences University, Portland, Oregon) with the pluf and pβ-gal expression vectors as described above. The pluf and pβ-gal transduced J53 cells (termed J53-βgal/luf) are infected with six different virus isolates (using four five-fold serial dilations) that were unable to efficiently infect other CD4, CXCR4 expressing cell lines (P4 or Hi5) or a CD4,

CXCR4 expressing cell line (MAGI) (see Tables 2 and 9). Table 1 shows that all viruses, including the macrophage tropic YU2 clone, included as a control, are highly infectious in the J53β-gal/luf cell line. To assess the relationship between infectious virus units and luciferase activity in the J53β-gal/luf cell line, four serial five-fold dilutions of the following viruses are prepared and analyzed: TIVI, WLMI, 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 J53β-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. To minimize luciferase background levels of non HIV induced expression and thus maximize sensitivity using luciferase as a reporter for HIV infection, cultures of single cell clones are derived from the J53β-gal/luf cell line as described above and characterized for luf and β-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 β-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. Each stock is analyzed for HTV-1 p24 antigen concentration, SI and NSI phenotype, and infectivity in HeLa-CD4 (MAGI), HeLa-CD4-CCR5 (P4), H9 CD4-CCR5 (Hi5), and HeLa-CD4-CCR5 (J53-C13) cells. These results are summarized in Table 2.

These results show that the J53-C13 cell line is sensitive to primary HIV-1. Importantly, the J53-C13 cell line is sensitive to HIV-1 infection to a degree similar to PBMC. To confirm the importance of the CCR5 co-receptor for this level of sensitivity, 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. JCl l is transduced to express B-gal and luciferase, and positive cells are biologically cloned exactly as described above for J53-C13. A clone designated J11-C5, which is capable of expressing both b-gal and designated J11-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. The results show a marked reduction in virus titer in the J11-C5 cell line, indicating that the CCR5 co-receptor is necessary for efficient infection/detection of primary virus isolates. To analyze the relationship between luciferase and β-gal expression over a range of different virus concentrations, 5-fold serial dilutions are prepared from seven different virus stocks and used to infect J53-C13 cells. After two days the number of β-gal positive cells and luciferase activity is determined. Figure 7 shows a strong correlation (r=0.92) between β-gal expression (infectious virus units) and luciferase activity over 2 orders of magnitude.

Example 4 - Evaluation of primary HIV-1 isolates for drug sensitivity/resistance using the J53β-gal/luf (J51-C13) cell line

HIV-1 isolates are derived by PBMC coculture from two different HIV-1 infected patients (LEMI and SARO) receiving anti -retroviral treatment. The RT sequence of each isolate is analyzed for nucleic acid sequence using ABI sequencing methods. Known drug resistance conferring mutations found in the LEMI and SARO RT sequences are shown in Table 3. The LEMI and SARO and YU2 (included as a control) virus stocks are used to infect the J53-C13 reporter cell line in the presence of AZT, 3TC and Nevaripine (NVP), respectively. Two days after infection the cells are lysed and the clarified lysates are examined for luciferase activity using standard methods (Promega). Figure 8 shows the effect of different concentrations of drug on virus replication relative to non-drug treated 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 concentration of drug that inhibits virus replication by 50% is increased. This factor has made drug sensitivity testing extremely difficult to standardize among independent laboratories. 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 shift in the IC50 among the different drug concentrations tested.

Analysis of 3TC and Nevaripine showed similar results (data not shown). 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 for phenotypic resistance in assays that test whole virus. Currently, the only effective means by which this can be accomplished is by culture of infected tissue with donor PBMC. The present invention confirms that the JC53 and the J53-C13 cell lines are highly sensitive to infection of primary virus isolates. Thus, these cell lines may be utilized to amplify the primary virus isolate instead of PBMC. To this end, JC53 cells are transduced with the HIV Tat gene under control of the CMV, or LTR promoter, as shown in Figure 10. To eliminate Tat transactivation of the lentivirus vector LTR, 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. To identify which of these clones could most efficiently promote HIV-1 replication, HIV-1 YU2 is used for infection at an MOI=0.01. After 40 hrs. virus production is measured by HIV-1 p24 antigen ELISA and the highest HIV-1 producing lines from each are selected for further analysis. The highest HIV-1 producer, designated J53-CMVtat is infected with the YU2 clone and the KEWI virus isolate at MOIs of approximately 0.1. As a control, the JC53 cell line is analyzed in a parallel experiment. 40 hrs. later culture supernatants are analyzed for HIV-1 production by p24 antigen ELISA. The results, shown in Table 5, indicate that the 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 amplification.

PBMC and J53tat are each infected with 2.5E5 infective particles of YU2. Two days later the concentration of progeny virus is analyzed for infectivity in J53BL indicator cells as shown in Figure 11(a) and by p24 antigen ELISA, as shown in Figure 11(b). The J53tat cell line amplifies primary virus to higher titers and more rapidly than PBMC. Since the parental J53BL cell line is highly sensitive to primary virus, 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 of drug candidates that affect various stages of the virus life cycle. Thus, viral drug resistance mutations in early stage targets such as reverse transcriptase (RT), integrase (IN) and env; and late stage targets such as protease and Gag are analyzed by the methods of the present invention. The J53tat cells are infected with HIV YU2 (MOI of either 0.2 or 0.04), and protease inhibitor (indinavir) is added to the cultures at various concentrations. Forty hours after infection the culture supernatant is collected and used to infect the J53BL cell line in the presence of the same drug concentrations. Table 6 shows that YU2 is sensitive to protease inhibitor, with increasing concentrations causing greater inhibition. Example 8 - Detection of noninfectious cultured virus.

To test how to recover noninfectious virus, a molecular clone is generated to produce env minus HIV-1 (pSG3-env). SG3-env virus, derived by transfection, is mixed (1:2, v:v) with VSV-G derived from the supernatant of pDm transfected 293T cell cultures. The mixture is ultracentrifuged for 1.5 hours at 115,000g at 4°C. The pellet is resuspended in 100 ul DMEM. The infectivity is then determined using J53BL cells. The infectivity is determined to be 7.5E4. Without mixing of VSV-G the infectivity is 0. YU2 virus containing wild-type envelope is pelleted through sucrose by ultracentrifugation to strip away the gpl20 glycoprotein (SU). 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 through sucrose, mixed with VSV-G and repelleted had a marked increase in infectivity. The recovery in infectivity is approximately 20% compared with the original virus stock. Example 9 - Detection of noninfectious plasma virus

Patient plasma (GADA) is mixed with and without VSV-G, pelleted through sucrose, resuspended in 100 ul DMEM as per Example 10. Infectivity is measured using

J53BL cells. Without VSV-G 1500 infectious particles are detected. With VSV-G 2500 infectious particles are detected as summarized in Table 7.

Example 10 - Detection of plasma Virus using CD4/CCR5/CXCR4 or

CD4/CCR5/CXCR4 + Tat expressing cell line to capture and amplify primary virus

Plasma from patients infected with HIV-1 is tested for the presence of infectious virus in the plasma towards J53BL cells. Three serial dilutions of plasma are incubated with J53BL cell line for 4 hours. Three days later the cells are stained for β-gal and infectious units are counted by microscopy as summarized in Table 8. Example 11 - Integrated HIV genome expansion with limited rounds of reverse transcription.

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 integrated into the chromosomes of J53BL cells. Thereafter, 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. Example 12 - An HIV-1 Protease Inhibitor Resistance Assay

Day 1. Late in the afternoon (3-5:00 p.m.), seed the JC53Δ3 Tat cells that have been well maintained in DMEM with 10% FBS in 6-well plates, 250,000 cells/well, 3 wells for each virus/drug.

Day 2. Infection of the O/N seeded JC53Δ3 Tat cells (an ampifying or amplifier cell line step)

At 1 :00 p.m. infect the cells with the viruses with a MOI between 0.01 to 0.2 (Note: 0.2 MOI = 100,000 IU of the virus, considering now the cell is 500,000/well).

Infection solution (per well): 1.0 ml of DMEM containing 1% FBS, 40 ug /ml of DEAE-dextrin and the virus.

Infection is carried out for 4 hours at 37 C with gentle shaking of the plates every 20-30 min. Then, at 5:00 p.m., add 2.0 ml of DMEM containing 5,% FBS to each well.

Let the culture go for 16 hours.

Day 3. Splitting the infected Tat cells into 96-well plates

At 8:00 a.m., to a set of 6 wells in a 96-well plate, place 75 ul of DMEM that contains 5% FBS and 2X of a HIV protease inhibitor (Indiviar) in a concentration of 0.0.016. 0.08, 0.4, 2.0, and 10.0 uM. Set four repeats for each viral infection.

At 9:00 am, remove the culture medium from the O N infected Tat cells. Trypsinize to collect the cells with 3,750 ml/well of DMEM that contains 5% FBS into a 15 -ml conical tube. Moderately vortex the tube for half minutes to disperse the cells. Aliquot the cell suspension to six 1.2-ml dilution tubes (0.5 ml per tube). Use a multiple channel pipet to transfer 75 ul of the cell suspensions to each of the wells in the 96-well plates prepared above (plates that contain 2x drugs).

Let the culture go for 24 hours.

Late in the afternoon (3-5:00 p.m.), seed the JC53 BL in 96-well plates, 5,000 cells/well, 24 wells for each virus/drug.

Day 4. Infection of JC53 BL cells (and indicator cell line)

At 10:00 a.m., remove the culture medium from the O/N seeded BL cells. Immediately add 20 ul of DMEM containing 1% FBS and 90 ug/ml of DEAE-dextrin to each well.

Transfer 40 ul of the culture medium from each of the infected Tat cell wells to the BL cell wells. Injection is carried out for 4 hours at 37 C with gentle shaking the plates every 20-30 min. Then at 2:00 p.m., add 120 ul of DMEM containing 5% FBS to each well. Let the culture go for 48 hours.

Day 6. Luciferase assay

At 2:00 p.m., carefully remove, as much as possible, the culture medium from the infected BL cell plates. Add 75 ul of lx cell lysis buffer (lx lysis buffer = 1:5 dilution with water of the original 5X reporter buffer). Keep the plates -70 C for one hour. Then, put the plates onto a horizontal shaker and shake the plates for 45 to 60 minutes at speed setting of 2.5 to completely lyse the cells. Repeat the freeze/thaw process one more time.

Transfer 20 ul of the cell lysate from each well to a well of 96-well luciferase assay plate. Carry out the Luciferase assay in a LUCIFERASE OPERATION SYSTEM. All publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent. The methods and systems described herein are exemplary and not intended as limitations on the scope of the invention. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other groups.

Thus, additional embodiments are within the scope of the invention and within the claims of our previous applications referenced herein. Table 1 . Efficient HIV- 1 in fection of a CD4 CCR5 expressing I le a cell line.

HIV- 1 isolates were derived by coculture (7- 10 days) of I IIV- 1 infected patient PBMC with PI IA stimulated normal donor BPMC.

Virus liter - determined by endpoint dilution titration in PBMC, calculated by the Spearman-Karber Formula (TCID 50/ml)

Virus titer - determined by counting the number of virus infected (blue) I IeLa-CD4-CCR5-[ ga! indicator cells (,153-C 16).

Table 3. RT resistance conferring mutations

Table 4. CCR5 facilitates infection of primary isolates of 11 IV-

Virus Slock J53-C13 titer 1-C5 titer

DBP D-7 6.30E+04 Neg

DBPdl40 1.20E+04 4.00E-I-01

I-IVI I D-7 6.10E-I-04 1.1 E+04

HVI-ID140 6.80E+04 1.00E+03

SHLD-7 ^ή.00E+04 Neg.

SHLD140 7.70E+04 Neg.

TED D 127 1.3ΩE+05 Neg.

TED 211 3.20E+04 Neg.

XHB2 1.75E+05 Neg.

YU2 4.85E+05 7.50E+04

89.6 1.40E+05 Neg.

Vims titer was determined by counting the // of beta-gal positive cells. Results indicate infection positive cells per ml of stock virus. Neg. (negative) tilers were tindelectable below 40 infectious units per ml.

Table 5. Trans Tat expression enhances I IIV- 1 production

Virus JC53 JC53-CMVtai

Exp 1 . Exp. 2 Exp. 1 Exp. 2

YU2 1240 920 6430 4910 KEWI 3120 2760 9590 7590

Nos. represent pg of p24 antigen per ml

Table 6. Number of colony formed in the presence of protease Inhibitor Indinavir

Table 7

Tabl S. Detection/Isolation of IIIV-1 from human plasma using J53BL cells

Plasma virus TCIU/PBMC TCIU/J53 BL

LEMI 3.47 x 10 m 4.40 x 107ml

ALPI 3.47 x lOVm 1.20 x 10 ml

GADA 7.81 x lOVml 1.20 x 107ml

TCRJ = tissue culture infectious units

8

-.,Zε/00SflΛLDd Z-tΦOfr/IO OΛΛ HIV RT Inhibitor Resistant Viruses

Viruses Resistance mutations Drug

ΛZT S NIIΪ Isolate pre- AZT AZT

ΛZT R NIH Isolate post-AZT

ΛZT I 305 NIH T215Y

ΛZT 1495 Nil! K.70R.T215Y

πvπ D-7

IIVII 1) MO

V-2 K70R.T215Y

3TC R NIH Ml 84V 3TC

V-2 Ml 84 V

NVP R Mil 1 YI8IC NVP

NVP Pv U I7 Y181C

IIVII n MO Y 181 C

TAΠLR IO

Claims

Claims

1. A method of assaying primary HIV for susceptibility or resistance to a drug or drug candidate, comprising the steps of: amplifying a primary HIV using an amplifying cell line; and indicating the presence or amount of said amplified primary HIV by infecting an indicator cell line with said amplified primary HIV; wherein said method further comprises contacting one or more drugs or drug candidates with one or more of said amplifying and indicator cell lines to determine the effect on said primary HIV.

2. The method of claim 1 wherein said contacting is performed with a known antiretroviral drug selected from one or more of the group consisting of reverse transcriptase inhibitors, fusion inhibitors, protease inhibitors, and integrase inhibitors.

3. The method of claim 1 wherein said contacting is performed with a drug candidate.