A novel single-cell-level phenotypic assay is described, which can simultaneously analyze HIV-1 drug susceptibility and intrinsic replication capacity. This allows quantitative dissection of the functions of antiretroviral drugs into suppression of viral replication and selection of resistant viruses with diminished replication capacities. The disclosed assay provides a tool for the rational evaluation of treatment decisions for patients failing antiretroviral therapy and is expected to be an important part in clinical management of HIV.

Description of the Invention

SUMMARY OF THE INVENTION

The invention addresses one of the more frustrating problems in clinical treatment of AIDS patients, which frequently arise because use of HAART, currently a widely used therapy, becomes less effective in controlling viral load and maintenance of near normal CD4+ cell levels. In particular, a novel method of determining the replicative capacity of drug-resistant HIV in HAART-treated patients has been developed. Using a series of in vitro measurements of drug resistance of mutant virus from the patient, compared with resistance of the wildtype virus originally treated, a clinical test has been designed. The measurements obtained from the disclosed assay will permit the physician to make rational decisions relating to continuing, adjusting or discontinuing multiple drug regimens.

An important and novel feature of the invention is the design of HIV vectors that are capable of infecting a competent host cell; i.e. a viral replicating cells, and which deliver a detectable protein to the endoplasmic reticulum (ER) of the host cell. The protein is a tagged fluorescent protein such as green fluorescent protein (GFP), red fluorescent protein (DsRed) and the like, which after viral replication remains associated with the ER of the host cell because of a fused signaling sequence. A preferred fluorescent protein is green fluorescent protein, but other fluorescent proteins are available; for example, red fluorescent proteins such as DsRed (available in a color series from Clontech, Palo Alto, CA), reef coral fluorescent proteins available with emission manima in ranges 489-539 nm and several color modifications of green fluorescent protein with emission maxima ranges from 476-529 nm. The ER targeting feature is due to fusion of a short C-terminal signal protein such as KDEL (SEQ ID NO: 14) or HIEL (SEQ ID NO: 15) to the fluorescent protein.

The targeting and subsequent association of the fluorescent protein to the host cell ER is accomplished by engineering a fluorescent protein incorporating a signal sequence, which is expressed as a fusion polypeptide in the host cell and remains anchored in the ER of the host cell, while not affecting the normal expression of the HIV. This allows an accurate count of the number of infected cells, in turn allowing a determination of replicative capacity of drug-resistant mutant virus in the patient.

An exemplary signal sequence is KDEL (SEQ ID NO: 14), as illustrated in the model construct described herein; however similar targeting sequences known in the art may be employed; for example, HIEL (SEQ ID NO: 15). It is important that the expressed fusion protein remain in the host cell ER; otherwise, an accurate measurement of HIV-replicative capacity cannot be determined.

The HIV vector is designed so that the fluorescent protein inserts into the HIV genome at a deletion position in the env polynucleotide sequence. The insertion contains the coding signal for the tag protein in frame with the ER targeting polypeptide so that a targeting polypeptide tag, preferably a fluorescent protein, is expressed. While the designed HIV vector preferably incorporates a fluorescent protein, other detectable tags are envisioned, such that the constructs may be engineered with radiolabels or colorimetric labels, so long as single cell separation/detection means are employed.

The invention thus in one aspect is a single-cell-level phenotypic assay that allows analytical comparison of the contributions of residual susceptibility and reduced replication capacity, thereby addressing the need to provide a rational basis for treatment decisions in the setting of virologic failure.

As discussed, a novel HIV vector designed to express a fluorescent protein in the endoplasmic reticulum of an infected cell forms the basis of the new phenotypic assay. The env region of HIV is modified by deleting a region beginning about 125 bp or so downstream from the N-terminus and inserting a coding sequence for a fluorescent protein fused with a signal sequence that causes the expressed fluorescent protein to be retained in the endoplasmic reticulum of an infected cell.

An exemplary fusion sequence is GFP in frame with KDEL (SEQ ID NO: 14) and a stop codon. The fusion protein coding sequence is preferably inserted near the N-terminus of the env gene, about 125 bp downstream, or in such a position that the N-terminus signal region responsible for importing the protein into the endoplasmic reticulum of the infected cell is retained. While exemplified with GFP, other fluorescent proteins may be used and engineered in frame with a stop codon, exemplified with TAA. It is believed that there are several suitable deletions that could be used in place of deleted 6351-7260. Variations of the vector are possible, all of which can be readily constructed by those of skill in the art.

In an exemplary HIV vector embodiment, a GFPKDEL (SEQ ID NO: 16) fusion protein in frame with TAA is inserted within deleted positions 6351 to 7260 of the HIV-1 env gene. The gag-pol sequence may be wildtype or heterologous; that is, gag-pol obtained HIV-1 or from a variant or mutant HIV. Mutant HIV-1 is typically detected in human patients undergoing retroviral drug treatment. While the originally infecting "wildtype" HIV-1 may be present, the mutant(s) begin to predominate and exhibit increased resistance to drug therapies.

In an exemplary HIV vector embodiment, a GFPKDEL fusion protein in frame with TAA is inserted within deleted positions 6351 to 7260 of the HIV-1 env gene. The gag-pol sequence may be wildtype or heterologous; that is, gag-pol obtained HIV-1 or from a variant or mutant HIV. Mutant HIV-1 is typically detected in human patients undergoing retroviral drug treatment. While the originally infecting "wildtype" HIV-1 may be present, the mutant(s) begin to predominate and exhibit increased resistance to drug therapies.

Recombinant HIV-1 vectors containing patient-derived gag-pol sequences are prepared by replacing the 1.5 ApaI/AgeI fragment of pNF4-3-DE-GFP (see FIG. 8 (see Original Patent)) with the patient-derived gag-pol sequences. Of course other deletion/insertion modifications could be used, so long as replicative capacity is not significantly altered compared with in vivo replicative capacity.

A particularly important source of heterologous gag-pol sequences are from HIV samples from human patients who are undergoing highly active anti-retroviral therapy (HAART) and who show indications of development of drug resistance. The resistance usually develops because of virus mutation; however, because HAART utilizes a combination of several drugs, often three or four, it is often not immediately clear which of the drugs has become ineffective. There are only about 20 drugs currently used to formulate the most appropriate mixtures, yet the drug combinations are several thousand. Some of the more commonly used drugs for HAART combinations are zalcitabine (ddC), didanosine (ddI) amprenavir (AVP), Ritonavir (RTV), abacavir (ABC), tenofovir disoproxil fumarate (TDF), nelfinavir (NFV), saquinavir (SQV), lopinavir (LPV) and indinavir (IDV).

Yet another aspect of the invention includes HIV pseudotypes. These are particularly useful for in vitro assays to measure viral replication or, as used herein, viral replicative capacity. Pseudotyped viruses are well known and generally are the replacement of part of a viral coat protein with a heterologous protein. In an exemplary embodiment, vesicular stomatitis virus glycoprotein (VSV-G) was pseudotyped with HIV-1, by transfecting competent cells with pVSV-G and wt or recombinant pNL4-3-DE-GFP (described above). Other pseudotypes could be employed; for example, heterologous HIV env protein.

The present invention takes advantage of pseudotyped HIV to prepare pseudotyped HIV stocks from patient HIV. This is accomplished by coinfecting or cotransfecting VSV-G and the disclosed HIV vector into a cell and preparing pseudotyped stocks of HIV. The stocks can be "normalized" by measuring the number of cells in an aliquot expressing a detectable protein, such as fluorescent protein GFP.

A particularly novel aspect of the invention is the use of normalized pseudotyped HIV stocks to determine HIV replicative capacity of the virus from AIDS patient samples. The method includes the steps of transfecting a selected host cell with the described pseudotyped HIV, culturing the transfected cell to obtain a stock of pseudotyped HIV; normalizing said stock by determining the number of transfected cells expressing fluorescent protein in an aliquot of the stock; and infecting a population of target cells with an amount of stock supernatant containing a determined number of transfected cells. This provides the number of infected target cells is indicative of HIV replication capacity. The number of infected target cells will fluoresce when GFP or other fluorescent protein is encoded in the HIV env and can be quantified by methods such as flow cytometry.

The target cell is preferably a T-cell and most preferably a CD4+ cell because those are the cells infected by HIV. Host cells can be selected from a range of suitable cells that are capable of supporting HIV replication, including Jurkat cells, 293T cells and CD4+ cells.

In medical practice, the novel assay can be used to assess drug susceptibility of HIV-1 infected patients. The method involves the steps of first preparing a normalized HIV pseudovirus stock as described where the gag-pol is from an HIV-1 infected patient. A second normalized HIV pseudovirus stock is also prepared where the gag-pol is from wildtype HIV. Preferably, the wildtype HIV will be the same HIV that infected the patient when originally treated. The next step is to infect different selected target virus-producing cell samples in vitro, one with an aliquot of the first normalized pseudovirus stock and the other with the second normalized pseudovirus stock. The pseudovirus in each sample is then replicated. The whole process is repeated with each for the infection steps, except that infection is conducted in the presence of the drugs being used to treat the patient. The relative differences between replication capacity of the patient's (mutant) pseudovirus and the wildtype virus are compared. This comparison is a measure of drug susceptibility of the HIV-1 infected patient.

Susceptibility measure can be used to evaluate selection of a drug regimen for AIDS patients resistant to HAART therapy. Drug susceptibility of the AIDS patient is determined as described by calculating the replicative capacity ratio of patient mutant HIV/patient wildtype HIV, the repeating the measure and ratio determination in the presence of drugs employed in the HAART. The two ratios are compared and used to provide an indication of whether or not to continue current HAART, modify HAART or discontinue altogether the particular drugs used. The ratio of the two ratios is a "replication capacity index" (RCI) and has been assessed for several HAART regimens (see Table 2, see Original Patent). Where the RCI is less than 1, and the wildtype is strongly inhibited by the drug regimen while the drug-resistant isolate only partially inhibited, there is indication that HAART may control viremia, suggesting continuation of HAART.

Where the RCI is greater than 1 relative to wildtype and drug resistant HIV-1 has a high replication capacity and minimal drug susceptibility, the resistant isolate is highly fit despite its mutations and was only minimally suppressed by HAART. Such comparisons indicate that at least for the isolate examined, HAART is of little use.

In another instance, the RCI is greater than or almost equal to 1 at both minimal and maximal drug concentrations indicating resistance, yet the replication capacity compared to wildtype was diminished. This suggests that if a patient has a wildtype virus similar to the standard wildtype incorporated in pNL4-3, HAART may still benefit by selecting for a resistant variant with reduced replication capacity.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a novel phenotypic assay that can simultaneously measure, on the same scale, HIV-1 susceptibility to drug combinations and changes in replication capacity relative to reference or patient-specific wild-type sequences. This provides a quantitative tool for analyzing the efficacy of antiretroviral therapy, especially the mechanism of the clinical benefit of HAART in the setting of virologic failure.

While no in vitro assay can fully duplicate the in vivo conditions under which the antiretroviral drugs mediate suppression of viral replication; nevertheless, in vitro phenotypic assays of drug resistance have potential clinical utility (Katzenstein, et al., 2003; Shulman, et al., 2002). Results from single-cycle assays of replication capacity generally parallel results of virus culture assays for fitness (Resch, et al., 2002), although the correlation is not always perfect.

In the assay described here, several steps have been taken to ensure that the cultures mimic in vivo conditions as closely as possible. First, the protein binding properties of some antiretroviral drugs have been accounted for by supplementing the culture medium with 50% normal human serum.

Second, the prodrug activation required for the function of all NRTIs has also been taken into account. Because all NRTIs require multiple steps of intracellular phosphorylation to be converted to active nucleoside triphosphate analogues, CD4.sup.+ T cells were pretreated with NRTIs 16 h prior to infection. This time is sufficient for intracellular levels of the active forms of these drugs to reach a steady state, as evidenced by the fact that pretreatment for longer times does not increase inhibition.

Third, drugs were tested at their Cmin and Cmax values under the conditions described above. This effectively circumvents issues related to drug absorption and metabolism and exposes target cells to concentrations of drugs that bracket the concentrations that should be experienced by cells in vivo.

Finally, and most significantly, the drugs were tested in the same combinations that are used in vivo. Because many combinations of antiretroviral drugs produce a profound synergistic inhibition of wild-type virus, quantitative analysis of drug inhibition is only possible with assays that have a wide dynamic range. The flow cytometric assay described here has a dynamic range of up to 4 logs, allowing quantification of the synergistic inhibitory effects of drug combinations as well as of individual components of the regimen. The assay faithfully reproduced reported drug interactions that occur at the level of target cells. For example, the reported antagonism between AZT and d4T caused by competition at the step of prodrug activation was readily observed with this assay (Table 1, see Original Patent). Taken together, these results suggest that the disclosed phenotypic assay provides a reasonable first approximation of drug inhibitory effects in vivo.

Using this assay, the potencies of available antiretroviral drugs were compared by examining the ratio of the C.sub.min and Cmax values to the IC.sub.50 determined in this system. The data highlight the extraordinary potency of the NNRTI EFV, which has a Cmin/IC.sub.50 ratio of >1,000 in the disclosed system. In contrast, the commonly used NRTIs d4T and ddI are relatively inefficient at inhibiting viral replication in this system. C.sub.max values for these drugs are actually below the IC.sub.50 and IC.sub.90 values, respectively. Because the actual IC.sub.50 depends on the viral strain, the target cell type, the culture medium, and the multiplicity of infection in specific phenotypic assay systems, direct comparison of Cmin IC.sub.50 and Cmax/IC.sub.50 ratios between different assay systems is not possible.

Drug susceptibility measured in the disclosed system was also dependent on the properties of the virus-producing cells, 293T cells, and the target cells, the Jurkat CD4.sup.+-T-cell line. These cells may differ from primary CD4.sup.+ T cells in the absorption and metabolism of antiretroviral drugs. They may differ from primary cells in the expression of transporters, such as the P glycoprotein, that can export PIs from the cytoplasm. The in vivo efficacy of a drug is dependent upon more than its potency in inhibiting a single round of viral replication. Additional factors such as genetic barriers to resistance, tolerability, and pharmacokinetics may have an effect and these also contribute to contribute to inaccuracy in methods that utilize a single round of replication as basis for measurement.

The heterogeneity of replication capacities of wild-type HIV-1 isolates relative to that of a reference sequence, NL4-3, will have some effect on the accuracy of the results. The replication capacities of wildtype HIV-1 clones from patients vary up to 2.5-fold from NL4-3 capacity measured in the disclosed system. Mean replication capacity index relative to NL4-3 was 0.81.+-.0.34 (n=7) in the examples reported herein. These results indicate that in order to most accurately assess changes in viral fitness in vivo, it is necessary to compare the replication capacity of the patient's drug-resistant virus to that of the drug-sensitive virus obtained from the same patient. This is readily done in the system described here, provided that the wild-type sequence is available.

In compliant patients who are failing therapy and have drug-resistant viruses, wild-type viruses are typically not found in the plasma but do persist in the latent reservoir in resting memory CD4.sup.+ T cells. Viral clones with different mutations are likely to be present in each patient with drug resistance and so that results of this type of analysis may be different for each clone. Ideally, a large number of distinct clones representing the full range of variation in pol should be analyzed, although this may not be practical as a routine clinical test. Alternatively, analysis of selected clones that represent extremes on the spectrum of wild-type to fully resistant viruses should provide meaningful data. Another factor affecting viral fitness are the compensatory mutations outside of the gag-pol region; however, the construct employed in the present invention includes the Gag p7/p1 and p1/p6 cleavage sites that frequently accumulate compensatory mutations in response to PIs.

The ability of the novel assay to simultaneously measure HIV-1 drug susceptibility and replication capacity permits an assessment of the mechanisms of the apparent clinical benefit of HAART in the setting of virologic failure. The data presented here show that the benefit of nonsuppressive HAART can be quantitatively deconstructed into two additive effects. This is illustrated graphically in FIG. 7 (see Original Patent) and numerically in Table 2 (see Original Patent). One effect is the residual suppression of replication of the resistant variants by antiretroviral drugs. This is a benefit that operates in real time, reflecting direct inhibition of viral enzymes by the drugs.

An additional beneficial feature of the invention is the assessment of whether or not the drug regimen will allow selection for drug-resistant variants with a diminished replication capacity. The importance of selection for such variants becomes apparent if the drugs are stopped and archived drug-sensitive variants with higher replication capacities emerge. A recent study by Ruff et al. (2002) demonstrated the persistence of archival wild-type HIV-1 in the latent reservoir in resting memory CD4.sup.+ T cells even after years of selection for drug-resistant variants by failing drug regimens. Further evidence for the persistence of wild-type viruses in the setting of failure comes from the work of Deeks et al. (14) demonstrating simultaneous loss of all drug-resistant variants accompanied with the appearance of wild-type HIV-1 in patients with multidrug resistance who interrupt therapy. These data suggest that the selection pressure exerted by drugs in failing regimens can prevent drug-sensitive variants with potentially higher replication capacities from emerging.

The analysis of HAART therapy and the assay method for residual drug susceptibility and reduced replication capacity of drug resistant HIV-1 provides the basis for the rational management of antiretroviral therapy in the problematic setting of virologic failure. For example, in circumstances in which the clinical benefit of the drug combination is solely due to selection for resistant variants with diminished replication capacities, as shown in FIG. 5C (see Original Patent), the drug regimen can be simplified, retaining the minimum number of drugs needed to provide selection pressure favoring the resistant variants over the wild-type virus. On the other hand, in cases in which the HAART regimen exerts little suppression on viral replication and the evolved resistant virus has achieved a replication capacity equivalent to that of the archived wild-type viruses present in the latent reservoir (FIG. 5B (see Original Patent)), continued treatment with the same regimen provides no obvious benefit.

Human immunodeficiency virus type 1 (HIV-1)-infected individuals who develop drug-resistant virus during antiretroviral therapy may derive benefit from continued treatment for two reasons. First, drug-resistant viruses can retain partial susceptibility to the drug combination. Second, therapy selects for drug-resistant viruses that may have reduced replication capacities relative to archived, drug-sensitive viruses.

The present invention is a novel single-cell-level phenotypic assay that allows these two effects to be distinguished and compared quantitatively. Patient-derived gag-pol sequences were cloned into an HIV-1 reporter virus that expresses an endoplasmic reticulum-retained Env-green fluorescent protein fusion. Flow cytometric analysis of single-round infections allowed a quantitative analysis of viral replication over a 4-log dynamic range. The assay faithfully reproduced known in vivo drug interactions occurring at the level of target cells. Simultaneous analysis of single-round infections by wild-type and resistant viruses in the presence and absence of the relevant drug combination divided the benefit of continued nonsuppressive treatment into two additive components, residual virus susceptibility to the drug combination and selection for drug-resistant variants with diminished replication capacities.

In some patients with drug resistance, the dominant circulating viruses retained significant susceptibility to the combination. However, in other cases, the dominant drug-resistant viruses showed no residual susceptibility to the combination but had a reduced replication capacity relative to the wild-type virus. Thus, simplification of the regimen may still allow adequate suppression of the wild-type virus. In a third pattern, the resistant viruses had no residual susceptibility to the relevant drug regimen but nevertheless had a replication capacity equivalent to that of wild-type virus. In such cases, there is no benefit to continued treatment.

The ability to simultaneously analyze residual susceptibility and reduced replication capacity of drug-resistant viruses may provide a basis for rational therapeutic decisions in the setting of treatment failure.

Treatment of human immunodeficiency virus type 1 (HIV 1)-infected patients with highly active antiretroviral therapy (HAART) can reduce plasma virus levels to below the detection limit (Perelson, et al., 1997) and can allow a significant degree of immune reconstitution when control of viremia is maintained (Lederman, et al., 2000). However, eradication of HIV-1 infection has not been achieved despite suppression of viremia to below detection limits for as long as 7 years (Siliciano, et al., 2003). A viral reservoir in latently infected resting memory CD4.sup.+ T cells has shown remarkable stability and can support life-long persistence of replication competent HIV-1 (for a review, see Blankson, et al., 2002). This reservoir in resting CD4.sup.+ T cells can serve as a permanent archive for all major forms of the virus present during the entire course of infection, including the original drug-sensitive forms as well as drug-resistant viruses that arise due to inadequate suppression of viral replication by antiretroviral drugs (Ruff, et al., 2002).

Although HAART can effectively suppress viremia to below the limit of detection for prolonged periods in some infected individuals, virologic failure, as evidenced by consistently detectable viremia, is also common (Lucas, et al., 1999). Failure is frequently associated with the development of resistance to one or more of the drugs in the regimen, and drug resistance has emerged as a major problem in the management of HIV-1 infection.
 

Claim 1 of 21 Claims

1. A human immunodeficiency virus (HIV) vector comprising in frame gag-pol and an endoplasmic reticulum (ER) retained fluorescent protein/C-terminal signal sequence in frame with a stop codon inserted into the HIV env at a position about 125 bp from the env N-terminus replacing an env restriction fragment deletion.

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