A change in viral tropism occurs in many HIV positive individuals over time and can be indicated by a shift in coreceptor use from CCR5 to CXCR4. The shift in coreceptor use to CXCR4 has been shown to correlate with increased disease progression. In patients undergoing HAART, the predominant populations of virus can be shifted back to CCR5-mediated entry after the CXCR4-specific strains have emerged. The present invention relates to a diagnostic method to monitor coreceptor use in the treatment of human immunodeficiency virus (HIV) infection. The present invention further relates to a diagnostic method applied to HIV-positive individuals undergoing HAART to monitor the suppression of CXCR4 specific strains. The diagnostic methods can be used to assist in selecting antiretroviral therapy and to improve predictions of disease prognosis over time.

OBJECTS AND SUMMARY OF THE INVENTION

Unexpectedly, it has now been shown that in patients undergoing HAART, the predominant populations of virus can be shifted back to CCR5-mediated entry after the CXCR4-specific strains have emerged. Thus, a diagnostic method for use in monitoring shifts in coreceptor use would be beneficial for measuring the therapeutic efficacy of various HIV treatment regimes, such as HAART.

The correlation between CXCR4-specific strains and rapid disease progression indicates that a diagnostic method would be useful to monitor the presence of CXCR4-specific strains and shifts in coreceptor use associated with HIV disease progression. Application of the diagnostic method allows more accurate predictions of disease prognosis over time.

The effect of HAART on coreceptor use by populations of virus has not heretofore been quantitatively studied. Herein, it is shown that in patients undergoing combination antiretroviral therapy, including HAART, the predominant populations of virus can be shifted back to CCR5-mediated entry once the CXCR4-specific strains have emerged.

Therefore, a diagnostic method is also useful to monitor the presence of CXCR4-specific strains and shifts in coreceptor use in patients undergoing antiretroviral therapy. Application of the diagnostic method allows the effectiveness of antiretroviral therapy to be more closely monitored.

The present invention relates to a diagnostic method to determine whether CXCR4 or CCR5 isolates are present in a patient comprising assaying for coreceptor use.

The present invention further relates to a diagnostic method to determine whether CXCR4 or CCR5 strains are present in a patient comprising transforming cells with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line expressing an HIV envelope-compatible coreceptor, and assaying for fusion.

The present invention further relates to a diagnostic method to determine whether CXCR4 or CCR5 strains are present in a patient comprising obtaining patient-derived virus and assaying the isolates for coreceptor use.

The present invention further relates to a diagnostic method to monitor shifts in coreceptor use associated with changes in HIV disease progression.

The present invention further relates to a diagnostic method to monitor shifts in coreceptor use associated with changes in HIV disease progression comprising transforming cells with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line expressing an HIV envelope-compatible coreceptor, and assaying for fusion.

The present invention further relates to a diagnostic method to monitor shifts in coreceptor use associated with changes in HIV disease progression comprising transforming cells containing an HIV Tat-activatable reporter gene construct with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line containing a constitutively active tat gene and an HIV envelope-compatible coreceptor, and assaying for fusion by detection of reporter gene expression.

The present invention further relates to a diagnostic method to monitor shifts in coreceptor use associated with changes in HIV disease progression comprising determining CXCR4 coreceptor use, CCR5 coreceptor use, and a ratio of HIV using the CXCR4 coreceptor compared to HIV using the CCR5 coreceptor.

The present invention further relates to a diagnostic method to monitor shifts in coreceptor use associated with changes in HIV disease progression by obtaining patient-derived virus and deriving biological clones therefrom, assaying the clones for coreceptor use, and applying a method of quantitating the proportion of virus that uses each coreceptor.

The present invention further relates to a diagnostic method to determine whether CXCR4 or CCR5 strains are present in patients infected with HIV undergoing antiretroviral therapy.

The present invention further relates to a diagnostic method to determine whether CXCR4 or CCR5 strains are present in patients infected with HIV undergoing antiretroviral therapy comprising transforming cells with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line expressing an HIV envelope-compatible coreceptor, and assaying for fusion before and/or after initiating antiretroviral therapy.

The present invention further relates to a diagnostic method to determine whether CXCR4 or CCR5 strains are present in patients infected with HIV undergoing antiretroviral therapy comprising transforming cells containing an HIV Tat-activatable reporter gene construct with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line containing a constitutively active tat gene and an HIV envelope-compatible coreceptor, and assaying for fusion by detection of reporter gene expression before and/or after initiating antiretroviral therapy.

The present invention further relates to a diagnostic method to determine whether CXCR4 or CCR5 strains are present in patients infected with HIV undergoing antiretroviral therapy comprising obtaining patient-derived virus and assaying the virus for coreceptor use before and/or after initiating antiretroviral therapy.

The present invention further relates to a diagnostic method to monitor the suppression of CXCR4-specific strains in patients infected with HIV undergoing antiretroviral therapy.

The present invention further relates to a diagnostic method to monitor the suppression of CXCR4-specific strains in patients infected with HIV undergoing antiretroviral therapy comprising transforming cells with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line expressing an HIV envelope-compatible coreceptor, and assaying for fusion before and/or after initiating antiretroviral therapy.

The present invention further relates to a diagnostic method to monitor the suppression of CXCR4-specific strains in patients infected with HIV undergoing antiretroviral therapy comprising transforming cells containing an HIV Tat-activatable reporter gene construct with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line containing a constitutively active tat gene and an HIV envelope-compatible coreceptor, and assaying for fusion by detection of reporter gene expression before and/or after initiating antiretroviral therapy.

The present invention further relates to a diagnostic method to monitor the suppression of CXCR4-specific strains in patients infected with HIV undergoing antiretroviral therapy comprising obtaining patient-derived virus and assaying the virus for coreceptor use before and/or after initiating antiretroviral therapy.

The present invention further relates to a diagnostic method to monitor the suppression of CXCR4-specific strains in patients infected with HIV undergoing antiretroviral therapy comprising determining CXCR4 coreceptor use, CCR5 coreceptor use, and a ratio of HIV using the CXCR4 coreceptor compared to HIV using the CCR5 coreceptor before and/or after initiating antiretroviral therapy.

The present invention further relates to a diagnostic method to monitor the suppression of CXCR4-specific strains in patients infected with HIV undergoing antiretroviral therapy comprising obtaining primary viral isolates and deriving biological clones therefrom, assaying the clones for coreceptor use, and applying a method for quantitating the proportion of virus in a primary isolate that uses each coreceptor before and/or after initiating antiretroviral therapy.

The present invention further relates to a diagnostic method to monitor the suppression of CXCR4-specific strains in patients infected with HIV undergoing antiretroviral therapy comprising determining the sequence of HIV envelope gene before and/or after initiating antiretroviral therapy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a diagnostic method to determine whether CXCR4 or CCR5 strains are present in a patient infected with HIV.

The present invention further relates to a diagnostic method to determine whether CXCR4 or CCR5 strains are present in a patient comprising transforming cells with an HIV envelope gene cloned from an infected patient, selectively fusing the cells with an indicator cell line expressing an HIV envelope-compatible coreceptor, and assaying for fusion. Cell surface envelope protein variants will selectively interact with either CCR5 or CXCR4. Fusion only occurs when an envelope protein interacts with a compatible coreceptor present on the surface of indicator cells. Cells expressing a particular envelope gene will fuse with either CCR5 or CXCR4 indicator cells, depending on the patient's envelope gene specificity. Therefore, whether fusion occurs with either CCR5 or CXCR4 indicator cells will indicate coreceptor usage.

A patient or subject can be any animal, preferably a mammal, and even more preferably a human, infected with HIV. The infectious AIDS virus can be, but is not limited to, HIV-1 and HIV-2.

An indicator cell line is a cell line comprising the CD4 receptor and a coreceptor or functional fragments thereof, suceptible to infection with HIV. Preferably the coreceptor is CXCR4 or CCR5. An indicator cell line can be, for example, HOS-CD4.CCR5 and HOS-CD4.CXCR4 (Deng et al. (1996); and Equils et al. (2000) J. Inf. Dis. 182:751-757), available from the AIDS Research and Reference Reagent Program Catalog. Other cell lines available from this catalog that are suitable for use as indicator cell lines include U373-MAGI (Fred Hutchinson Research Cancer Center); 3T3.T4, GHOST and U87.CD4 (New York University Medical Center). An indicator cell line can be any known in the art, such as described in Glushakova et al. (1999); and Dreyer et al. (1999). Although engineered primarily to screen HIV strains for drug sensitivity, the indicator cell lines described in WO 99/67429 are also suitable for use as indicator cell lines.

An indicator cell line can be constructed by methods known in the art. Nucleic acids encoding HIV receptors or coreceptors, such as CD4, CXCR4 and/or CCR5, or functional fragments thereof capable of effecting receptor binding, can be cloned into recombinant vectors and introduced into cells of choice in vitro. HIV receptors and coreceptors or fragments thereof can be expressed by the recombinant vectors in cells of choice. Methods for generation and use of recombinant vectors in vitro are well known in the art. See Sambrook, Fritsch and Maniatis, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989 (e.g., procedures for isolating DNA, constructing recombinant vectors, transfecting and transforming cells and producing heterologous peptides).

The term "or (a) fragment(s) thereof" as employed in the present invention and in context with polypeptides of the invention, comprises specific peptides, amino acid stretches of the polypeptides as disclosed herein. It is preferred that said "fragment(s) thereof" is/are functional fragment(s). The term "functional fragment" denotes a part of the above identified polypeptide of the invention which fulfils, at least in part, physiologically and/or structurally related activities of the polypeptide of the invention. It is also envisaged that the fragments, like the full-length polypeptides, can distinguish between HIV strains in effecting binding. The polypeptides of the present invention can be recombinant polypeptides expressed in eukaryotic cells, like mammalian cells.

Generally, recombinant DNA technology has enabled the expression of foreign (heterologous) proteins in cell lines of choice. In this process, a vector containing genetic material directing a cell to produce a protein encoded by a portion of a heterologous DNA sequence is introduced into the host, and the transformed host cells can be fermented, cultured or otherwise subjected to conditions which facilitate the expression of the heterologous DNA, leading to the formation of large quantities of the desired protein. Plasmids are extensively used as vectors to clone DNA molecules. Most plasmid vectors are made by taking DNA from a variety of replicons (plasmids, bacteriophage chromosomes and bacterial chromosomes) and joining the DNA together (using restriction enzymes and DNA ligase) to form a plasmid that has an origin of replication, a selection marker (usually an antibiotic-resistance gene) and a promoter for expressing genes of interest in the required host cell. A vector can be, for example, as in U.S. Pat. Nos. 5,990,091 and 6,004,777, and as in PCT/US00/04203.

Furthermore, the recombinant vector can, in addition to the nucleic acid sequences of the invention (e.g. those encoding HIV receptors or coreceptors or functional fragments thereof), comprise expression control elements, allowing proper expression of the coding regions in suitable hosts. Such control elements are known in the art and can include a promoter, a splice cassette, translation initiation codon, translation and insertion site for introducing an insert into the vector. Preferably, the nucleic acid molecule is operatively linked to expression control sequences allowing expression in eukaryotic or prokaryotic cells.

Control elements ensuring expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned herein above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements can include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in for example mammalian cells comprise the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous sarcoma virus), human elongation factor 1.alpha.-promoter, aPM-I promoter (Schaffer et al. (1999) Biochem. Biophys. Res. Commun. 260:416-425), or inducible promoter(s), like, metallothionein or tetracyclin, or enhancers, like CMV enhancer or SV40-enhancer. For the expression in prokaryotic cells, a multitude of promoters including, for example, the tac-lac-promoter or the trp promoter, has been described. Besides elements that are responsible for the initiation of transcription, such regulatory elements can also comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL), Casper, Casper-HS43, pUAST, or prokaryotic expression vectors, such as lambda gt11.

Furthermore, depending on the expression system leader sequences capable of directing the polypeptide to a cellular compartment can be added to the coding sequence of the nucleic acid molecules of the invention and are well known in the art. The leader sequence(s) is assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a protein thereof, into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization of expressed recombinant products. Once the vector has been incorporated into the appropriate cell line, the cells are maintained under conditions suitable for high level expression of the nucleotide sequences.

A cell can be transfected or transformed with a recombinant vector or plasmid. Methods of transformation and transfection are well known in the art. The transformed cells can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth. The resulting transformed or transfected cell lines are genetically modified with a nucleic acid molecule according to the invention or with a vector comprising such a nucleic acid molecule. The term "genetically modified" means that the cell comprises in addition to its natural genome a nucleic acid molecule or vector according to the invention which was introduced into the cell or host or into one of its predecessors/parents. The nucleic acid molecule or vector can be present in the genetically modified cell either as an independent molecule outside the genome, preferably as a molecule that is capable of replication, or it can be stably integrated into the genome of the cell.

The present invention can utilize any suitable prokaryotic or eukaryotic cell to construct an indicator cell line. Suitable prokaryotic cells are those generally used for cloning like Escherichia coli or Bacillus subtilis. Eukaryotic cells comprise, for example, fungal or animal cells, and are preferable for constructing an indicator cell line. Animal cells are preferably used for conducting the specificity assay. Suitable animal cells are, for instance, insect cells, vertebrate cells, preferably mammalian cells, such as e.g. GHOST, CHO, Hela, NIH3T3, MOLT-4, Jurkat, K562, HepG2, 3T3-L1 (and derivatives thereof), HIB-1B (Villena et al. (1998) Biochem. J. 331:121-127), HEK 293, PAZ6 (Strobel et al. (1999) Diabetologia 42:527-533). Further suitable cell lines known in the art are obtainable from cell line depositories, like the American Type Culture Collection (ATCC) and the AIDS Research and Reference Reagent Program Catalog. For example, HL3T1 and HeLa CD4+ (NIH) and HeLa T4+ (Columbia University) cell lines are available through the catalog and are easily adaptible for use in constructing an indicator cell line. Derivation of primary cells from an animal, preferably a mammal, and even more preferable a human, can also be undertaken for the purposes of establishing a suitable cell line.

Preferably, the cell transformed with the receptor/coreceptor encoding vector of the invention is a blood cell, such as a macrophage or T cell, or an immortalized cell line derived therefrom.

Cloning strategies for isolating envelope genes of interest are well known in the art. See Sambrook, Fritsch and Maniatis, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989. Advantageously, envelope gene sequences can be obtained from a variety of patient tissues, including blood and mucosal tissues. High fidelity cloning of the samples above can be achieved by routine performance of multiple long RT-PCR reactions on limiting dilutions of RNA, followed by multiple PCR's on cDNAs obtained from each RT reaction. Performance of multiple PCR's on each cDNA preparation increases the likelihood of amplifying a different HIV-1 RNA species. These measures also decrease the chance of recombination.

Clinical specimans comprising tissues and/or fluids from HIV-infected patients can be utilized for cloning envelope genes of interest. Advantageously, patient-derived virus can be obtained from sites in addition to peripheral blood, particularly those sites from which cultured virus cannot be obtained. For example, while circulating macrophages and CD4.sup.+ T cells are the dominant reservoir of HIV-1, viral populations distinct from those in the peripheral blood exist in many tissue reservoirs, including the genital mucosa and lymphoid tissue.

Determination of cell fusion can be carried out using a variety of assays. The assay for cell fusion can be carried out, for example, with the use of an inducible reporter gene construct. Preferably, the inducible reporter gene construct is activated upon fusion with a cell containing a suitable transcriptional activator and/or transcription factor. A Tat-inducible reporter gene construct can be utilized, comprising a reporter gene linked to an HIV-1 LTR promoter. The reporter gene construct can encode a wide variety of colormetric, enzymatic and/or fluorescent reporter genes, such as the green fluorescent protein, placental alkaline phosphatase, firefly luciferase, .beta.-galactosidase (encoded by the lacZ gene) and chloramphenicol acetyltransferase (encoded by the CAT gene). The assay for cell fusion can also be carried out using labeled antibodies, which specifically detect HIV envelope proteins, and CXCR4 or CCR5 coreceptors on or within a fused cell. This method can be combined with cell sorting techniques, to separate populations of fused cells.

The present invention further relates to a diagnostic method to determine whether CXCR4 or CCR5 strains are present in a patient comprising transforming cells containing an HIV Tat-activatable reporter gene construct with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line containing a constitutively active tat gene and an HIV envelope-compatible coreceptor, and assaying for fusion by detection of reporter gene expression. Upon fusion of cells mediated by the envelope variant-coreceptor interaction, Tat inducible reporter gene expression will be activated.

The present invention further relates to a diagnostic method to determine whether CXCR4 or CCR5 strains are present comprising obtaining patient-derived virus and assaying the virus for coreceptor use (i.e. coreceptor assay).

Patient-derived virus includes, but is not limited to, primary viral isolates, biological clones, and molecular clones. Patient-derived viruses can be obtained from clinical specimans comprising any fluid or tissue obtained from an HIV infected individual, such as peripheral blood.

Patient-derived virus can be obtained by methods known in the art. For instance, peripheral blood of HIV-infected individuals can be separated into plasma and cell components by methods known in the art. Fang et al. (1995) Proc. Natl. Acad. Sci. USA 92:12110-4. Primary viral isolates of HIV-1 can be obtained by co-culture with normal donor peripheral blood mononuclear cells (PBMCs). Fang et al. (1995). Titration of viral isolates in PBMCs can be carried out, for example, by using the methods previously described by Fang et al (1995).

Biological clones can be derived from primary isolates by methods known in the art, such as short-term limited dilution cloning. Connor et al. (1997). Quantitation of HIV-1 RNA in plasma can be carried out, for example, by using NucliSens (Organon Teknika Corp., Durham, N.C.). Quantitation methods can set a lower limit, preferably at .ltoreq.80 copies/ml.

Biological clones are applicable for determining the proportion of virus using each receptor. It is desirable to quantitate the proportion of virus using each coreceptor when rigorously comparing coreceptor use over time.

Assaying for coreceptor use can comprise inoculating indicator cell lines with primary isolates and/or biological clones (i.e. coreceptor assay) followed by determining whether infection occurred (i.e. specificity assay).

For purposes of conducting the coreceptor assay, the indicator cells can be seeded, for example, onto 12-well plates and inoculated, preferably after 12-36 hours, such as 24 hours, with a standard quantity of titered virus, preferably 10.sup.2 TCID.sub.50 of first passage primary viral isolates or biological clones.

For purposes of conducting the coreceptor assay CCR5- and CXCR4-specific positive control viruses can be employed, such as HIV JR-FL and LAV/HTLV-IIIB. Infection with CCR5- and CXCR4-specific positive control viruses can be carried out in parallel to infection with primary isolates and/or biological clones. Uninoculated cells can be negative controls.

Prior to conducting the coreceptor assay, indicator cell lines can be tested by inoculation with duplicate primary and control isolates to eliminate the possibility of any artifacts resulting from infection via low levels of endogenous coreceptor expression.

Following the coreceptor assay, a specificity assay is conducted to determine whether infection occurred. A suitable method for determining infection of the indicator cell line can be measurement of a complex formation. The measurement of a complex formation is well known in the art and comprises, inter alia, heterogeneous and homogeneous assays. Homogeneous assays comprise assays wherein the binding partners remain in solution. Heterogeneous assays comprise assays like, inter alia, immuno assays, for example, ELISAs, RIAs, IRMAs, FIAs, CLIAs or ECLs. Such assays are, inter alia, disclosed in U.S. Pat. No. 5,854,003 or in U.S. Pat. No. 5,639,858. Specificity assays like ELISA are preferred. Any specificity or detection step of the present invention can be assisted by computer technology or other means of automation, including flow cytometry.

A suitable method for determining whether infection of the indicator cell line occurred is contacting an epitope of HIV and identifying whether binding occurs, without binding to a control. In particular, the specificity assay of the invention can be carried out by employing antibodies directed against the HIV p24 antigen, as described by Kusunoki et al. (1999) Nucleosides Nucleo. 18:1705, or by using commercial ELISA assay kits, available, for example, from NEN Life Science Products, Boston.

Therefore, the coreceptor assay of the invention can be easily performed using the disclosure herein and methods known in the art such as described herein.

The present invention yet further relates to a diagnostic method to monitor shifts in coreceptor use associated with changes in HIV disease progression.

The present invention further relates to a diagnostic method to monitor shifts in coreceptor use associated with changes in HIV disease progression comprising transforming cells with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line expressing an HIV envelope-compatible coreceptor, and assaying for fusion.

The present invention further relates to a diagnostic method to monitor shifts in coreceptor use associated with changes in HIV disease progression comprising transforming cells containing an HIV Tat-activatable reporter gene construct with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line containing a constitutively active tat gene and an HIV envelope-compatible coreceptor, and assaying for fusion by detection of reporter gene expression.

The present invention further relates to a diagnostic method to monitor shifts in coreceptor use associated with changes in HIV disease progression comprising determining CXCR4 coreceptor use, CCR5 coreceptor use, and a ratio of HIV using the CXCR4 coreceptor compared to HIV using the CCR5 coreceptor.

The present invention further relates to a diagnostic method to monitor shifts in coreceptor use associated with changes in HIV disease progression comprising obtaining patient-derived virus from a clinical speciman and deriving biological clones therefrom, assaying the clones for coreceptor use, and applying a method of quantitating the proportion of virus in the clinical specimen that uses each coreceptor.

In a preferred embodiment, the coreceptor assay includes quantifying the proportion of virus using each coreceptor. Quantitation can be performed in a number of ways, including determining coreceptor specificity of multiple biologic clones, preferably at least 5; determining the viral sequence of portions of the HIV envelope gene, particularly the V3 region which predicts coreceptor use; or assaying a viral primary isolate using a semiquantitative method and applying a statistical method, for example, as described herein.

Determination of the proportion of CCR5 or CXCR4 virus can be carried out by determining coreceptor specificity of multiple biologic clones. A system devised for determining coreceptor specificity through the use of an indicator cell line is provided herein above. Biologic clones derived from the patients' primary viral isolates can be assayed for coreceptor use by employing an indicator cell line, such as the HOS-CD4+ cell line.

Determination of the proportion of CCR5 or CXCR4 virus can be carried out by determining the viral sequence of portions of the HIV envelope gene, particularly the V3 region which predicts coreceptor use. The envelope protein can be gp120, gp 160 or a portion thereof. Envelope sequences are predictive of coreceptor use on the basis of the overall charge of the V3 loop and the presence of basic or acidic residues at positions 275 and 287 of the env gene. Bhattacharya et al. (1996) AIDS Res. Hum. Retrovir. 12:83-90; and Hung et al. (1999) J. Virol. 73:8216-26. An example of a system devised for determining the viral sequence of portions of the HIV envelope gene is provided herein. Reverse transcriptase polymerase chain reaction can be used to amplify the V3 region of the env gene from plasma or other body fluid. Amplified products can be sub-cloned, verified by restriction digestion, and sequenced.

It is furthermore envisaged, that the diagnostic method involves the use of micro-chips comprising nucleic acid molecules encoding a envelope protein, or a fragment thereof, preferably a V3 region fragment, on "gene chips"; or an envelope protein, or a fragment thereof, preferably a V3 region fragment, on "protein-chips" (See U.S. Pat. Nos. 6,066,454; 6,045,996; 6,043,080; 6,040,193; 6,040,138; 6,033,860; 6,033,850; 6,025,601; 6,022,963; 6,013,440; 5,968,740; 5,925,525; 5,922,591; 5,919,523; 5,889,165; 5,885,837; 5,874,219; 5,858,659; 5,856,174; 5,856,101; 5,843,655; 5,837,832; 5,834,758; 5,831,070; 5,770,722; 5,770,456; 5,753,788; 5,744,305; 5,733,729; 5,710,000; 5,631,734; 5,599,695; 5,593,839; 5,578,832; and 5,556,752). Diagnostic gene chips can comprise a collection of polypeptides that specifically detect a envelope protein, or fragments thereof, preferably V3 region fragments; or nucleic acid molecules that specifically detect a nucleic acid molecule encoding a envelope protein, or fragments thereof, preferably V3 region fragments; all of which can be used for the purposes of determining coreceptor use. The envelope protein can be gp160, gp 120, or a portion thereof.

Determination of the proportion of CCR5 or CXCR4 strains from patient-derived virus can be carried out using a quantitative statistical method. An example of a system devised for quantitation of the proportion of CCR5 and CXCR4 strains in a clinical specimen is provided herein. In this system, lambda (.lamda.) is a continuous, nonlinear variable between one and zero derived from the results of coreceptor use by biologically and molecularly cloned virus; it describes the mixed proportion of viruses using CCR5 and CXCR4. A .lamda. value near one describes a population of viruses that almost all use CCR5; a value near zero describes a population that almost all use CXCR4.

More in particular, this system comprises a variable, .lamda., that is constructed as the proportion of strains using CCR5. Lambda=1 represents an isolate in which all strains prefer the CCR5 coreceptor but .lamda.=0 indicates that all prefer CXCR4. Lambda values can be assessed by utilizing qualitative assay data derived from patient-derived virus and sequences of the V3 portion of the env gene in patient-derived virus. Lambda values can be constructed by relating data derived from the same patient sample by using three different analyses: biologic cloning, V3 sequencing, and semiquantitative assays of primary isolates. To construct .lamda. values, the proportion of biologic and, if available, molecular clones using CCR5 at each time point is calculated, then the proportion is linked to the semiquantitative coreceptor use score (- to 3+) of primary isolates obtained simultaneously. The data are transformed to approximate a Poisson distribution. Poisson regression analysis can then be performed to determine the factors associated with changes in .lamda. values.

The Wilcoxon Rank Sum Test can be used to make comparisons between the magnitude of log viral level, CD4+ counts, and Lambda (.lamda.) values. Data for factors relating to changes in .lamda. values can be analyzed by multivariate Poisson regression. Variables can include log HIV-1 RNA levels, changes in viral levels, CD4+ cell counts, changes in CD4+ cell counts, and indicator variables for levels of antiretroviral therapy.

Application of the diagnostic methods to detect and/or monitor shifts in coreceptor use is useful for predicting disease prognosis over time.

The present invention yet further relates to a diagnostic method to determine whether CXCR4-specific strains are present in patients infected with HIV undergoing antiretroviral therapy.

The present invention further relates to a diagnostic method to determine whether CXCR4 or CCR5 strains are present in patients infected with HIV undergoing antiretroviral therapy comprising transforming cells with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line expressing an HIV envelope-compatible coreceptor, and assaying for fusion before and/or after initiating antiretroviral therapy.

The present invention further relates to a diagnostic method to determine whether CXCR4 or CCR5 strains are present in patients infected with HIV undergoing antiretroviral therapy comprising transforming cells containing an HIV Tat-activatable reporter gene construct with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line containing a constitutively active tat gene and an HIV envelope-compatible coreceptor, and assaying for fusion by detection of reporter gene expression before and/or after initiating antiretroviral therapy.

The present invention further relates to a diagnostic method to determine whether CXCR4-specific strains are present in HIV-infected patients receiving antiretroviral therapy comprising obtaining patient-derived virus and assaying for coreceptor use before and/or after initiating antiretroviral therapy.

The present invention yet further relates to a diagnostic method to monitor the suppression of CXCR4-specific strains in HIV-infected patients receiving antiretroviral therapy.

The present invention further relates to a diagnostic method to monitor the suppression of CXCR4-specific strains in HIV-infected patients receiving antiretroviral therapy comprising transforming cells with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line expressing an HIV envelope-compatible coreceptor, and assaying for fusion before and/or after initiating antiretroviral therapy.

The present invention further relates to a diagnostic method to monitor the suppression of CXCR4-specific strains in HIV-infected patients receiving antiretroviral therapy comprising transforming cells containing an HIV Tat-activatable reporter gene construct with an HIV envelope gene variant cloned from an infected patient, selectively fusing the cells with an indicator cell line containing a constitutively active tat gene and an HIV envelope-compatible coreceptor, and assaying for fusion by detection of reporter gene expression before and/or after initiating antiretroviral therapy.

The present invention further relates to a diagnostic method to monitor the suppression of CXCR4-specific strains in HIV-infected patients receiving antiretroviral therapy comprising determining CXCR4 coreceptor use, CCR5 coreceptor use, and a ratio of HIV using the CXCR4 coreceptor compared to HIV using the CCR5 coreceptor before and/or after initiating antiretroviral therapy.

The present invention further relates to a diagnostic method comprising obtaining patient-derived virus from a clinical specimen and deriving biological clones therefrom, assaying the clones for coreceptor use, and applying a method for quantitating the proportion of virus in the clinical specimen that uses each coreceptor before and/or after initiating antiretroviral therapy.

The present invention yet further relates to a diagnostic method to monitor the suppression of CXCR4-specific strains in HIV-infected patients receiving antiretroviral therapy comprising determining the sequence of HIV envelope gene before and/or after initiating antiretroviral therapy.

Application of the diagnostic method further provides a way to monitor the effectiveness of antiretroviral therapy. Aspects of antiretroviral therapy that can be monitored, for example, are development of drug resistance and/or sensitivity. The diagnostic methods of the invention can be applied before initiating antiretroviral therapy to determine a suitable antiretroviral treatment regimen. The diagnostic methods of the claimed invention can also be applied after initiating antiretroviral therapy to monitor efficacy of a viral treatment regimen and where efficacy of the treatment is directly related to decrease of CXCR4 coreceptor use. The diagnostic methods of the invention can also be used to determine whether a putative antiretroviral therapy or treatment is efficacious in decreasing CXCR4 coreceptor use.

Antiretroviral therapy can include, but is not limited to, HAART, protease inhibitors, fusion inhibitors, integrase inhibitors, co-receptor specific agents, 3TC, AZT, nevirapine, non-nucleoside analogue reverse transcriptase inhibitors and nucleoside analogue reverse transcriptase inhibitors. HAART can be three or more antiretroviral drugs in combination, including at least one protease inhibitor, or at least a reverse transcriptase inhibitor and a protease inhibitor; or at least two reverse transcriptase inhibitors with at least one protease inhibitor.

Typical reverse transcriptase inhibitors include nucleoside analogs, e.g., AZT (Zidovudine), ddi (didanosine), ddc (zalcitabine), D4T (stavudine), 3TC (lamivudine), Ziagen (abacavir), combivir (mix of AZT and 3TC), and non-nucleoside analogs, e.g., viramune (nevirapine), rescriptor (delavirdine), sustiva (efavirenz). Protease inhibitors include invirase (saquinavir), norvir (ritonavir), crixivan (indinavir), viracept (nelfinavir), agenerase (amprenivir), kaletra (lopinavir and ritonavir) and fortovase (saquinavir in a soft gelatin form). Thus, HAART can also be "triple cocktail" therapy--a three drug regimen to combat HIV wherein one of the three drugs is usually a protease inhibitor (and the other two are usually reverse transcritase inhibitors).

One skilled in the art (e.g. a physician, preferably specializing in the treatment of infectious disease) would use appropriate judgment and discretion in determining how often to apply the diagnostic methods to a test subject (e.g. a patient). Frequency of application can vary, depending on, for example, the age, sex, type of antiretroviral therapy administered to, or stage of disease progression in, a test subject.

One skilled in the art further understands the results of the diagnostic method to provide additional information about the stage of disease progression or therapeutic efficacy, depending on the amount of CXCR4 specific strain specificity of a test subject.

Application of the diagnostic methods to detect and/or monitor shifts in coreceptor use is useful for assessing the effectiveness of antiretroviral therapy.

The present invention further relates to a composition that is a diagnostic composition which can be, for example in the form of a kit.

The diagnostic composition can comprise the components as defined herein above wherein said components are bound to/attached to and/or linked to a solid support. It is furthermore envisaged, that the diagnostic composition comprises nucleic acid sequences encoding a envelope protein, or a fragment thereof, preferably a V3 region fragment; or indicator cell lines of this invention; all of which can be contained on micro-chips identifiable with a suitable means for detection.

Solid supports are well known in the art and comprise, inter alia, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction trays, plastic tubes etc. Suitable methods for fixing/immobilizing cells, nucleic acid sequences, or polypeptides of the invention are well known and include, but are not limited to ionic, hydrophobic, covalent interactions and the like.

The diagnostic composition of the present invention can be advantageously used as a kit, inter alia, for carrying out the method of the invention and could be employed in a variety of applications, e.g., as diagnostic kits, as research tools. Additionally, the kit of the invention can contain means for detection suitable for scientific, medical and/or diagnostic purposes. The manufacture of the kits follows preferably standard procedures that are known to the person skilled in the art. Kits can advantageously include instructions for use and/or admixture of ingredients.

In the present invention, it is additionally understood that HIV is a lentivirus, and the skilled artisian can readily understand that from the teachings herein, and the knowledge in the art, within the ambit of the invention are herein embodiments wherein the virus is a lentivirus other than HIV, including SIV and FIV, as in U.S. Pat. Nos. 5,863,542 and 5,766,598, and wherein the coreceptors are analogous (e.g. homologous) to CCR5 and CXCR4. As used herein, acquired immunodeficiency virus is interchangeable with HIV and encompasses other such viruses such as SIV and FIV. One skilled in the art can follow the teachings in the art to identify analogous coreceptors.

Claim 1 of 46 Claims

1. A diagnostic method of determining viral tropism comprising: (a) obtaining a population of patient-derived acquired immunodeficiency virus; (b) determining CXCR4 coreceptor use; (c) determining CCR5 coreceptor use; and (d) determining the ratio of acquired immunodeficiency virus using the CXCR4 coreceptor compared to virus using the CCR5 coreceptor.

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