WO2009111042A2 - Hiv co-receptor tropism assays and compositions thereof - Google Patents

Abstract

The invention relates to methods of detecting HIV and methods of determining HIV co-receptor tropism.

Description

HIV-CO-RECEPTOR TROPISM ASSAYS AND COMPOSITIONS THEREOF

BACKGROUND

HIV uses a receptor-mediated pathway in the infection of host cells. HIV-I requires contact with two cell-surface receptors to gain entry into cells and initiate infection; CD4 is the primary receptor and CXCR4 and CCR5 serve as secondary coreceptors for HIV-I isolates that are tropic for T-cell lines or macrophages, respectively. Deng et al. (1996) Nature 381:661-6; Doranz et al. (1996) Cell 86:1149- 59; and Berger et al. (1998) Nature 391 :240. CXCR4 or CCR5, in conjunction with CD4, form a functional cellular receptor for entry of certain strains of HIV into cells. Whereas all infective HIV isolates interact with CD4 not all isolates interact with the same coreceptor, that is an HIV particle selectively interacts with either CXCR4, CCR5 or both (mixed tropic).

Coreceptor use plays a critical role in viral tropism, pathogenesis, and disease progression. HIV-I strains transmitted in vivo generally use CCR5 (CCR5 viruses). Fenyo et al. (1998) Nature 391 :240; Samson et al. (1996) Nature 382:722-5; Shankarappa et al. (1999) J. Virol. 73:10489-502; and Scarlatti et al. (1997) Nature Med. 3:1259-65. These viruses typically infect macrophages and primary CD4+ lymphocytes, and do not form syncytia in vitro. Bjδrndal et al. (1997) J. Virol. 71 :7478-87. These viruses are said to be macrophage tropic (M-tropic).

Years after chronic infection is established, strains using CXCR4 emerge in ~50% of infected individuals. Berger et al. (1998); Scarlatti et al. (1997); Koot et al. (1993); and Connor et al. (1997) J. Exp. Med. 185:621-8. In many cases, the CXCR4 and CCR5 strains coexist in the viral population. CXCR4 strains not only infect primary T-lymphocytes but also replicate in T-cell lines and induce syncytia. Bjδrndal et al. (1997). These viruses are said to be T-cell tropic (T-tropic). This difference in cell tropism correlates with disease progression. During HIV infection, strains isolated from individuals early in the course of their infection are usually M-tropic, while viruses isolated from approximately 50% of individuals with advanced immunodeficiency also include viruses that are T-tropic.

The finding that change from M-to T-tropic viruses over time in infected individuals correlates with disease progression suggested that the ability of the viral envelope to interact with CXCR4 represents an important feature in the pathogenesis of immunodeficiency and the development of full blown Acquired Immunodeficiency Syndrome (AIDS). The emergence of CXCR4 virus is predictive of rapid depletion of CD4+ cells and acceleration of HIV-I disease progression. Berger et al. (1998); Scarlatti et al. (1997); Koot et al. (1993); and Connor et al. (1997). In vitro results suggest that selective blockade of CXCR4 receptors may prevent the switch from the less pathogenic CCR5 strains to the more pathogenic CXCR4 strains. Este et al. (1999) J. Virol. 73:5577-85.

Accordingly, diagnostic methods for use in detecting HIV tropism and/or monitoring shifts in coreceptor use (e.g. shifts from CXCR4-specific HIV to CCR5- specific HIV and vice versa) would be beneficial for predicting disease progression over time or in response to treatment. In particular, diagnostic methods for measuring, monitoring, evaluating, detecting, etc. patient-derived HIV samples for coreceptor usage would be beneficial for evaluating HIV disease progression in the face of various anti-HFV treatment and therapies.

SUMMARY OF THE INVENTION

The present invention is directed to compositions, kits and methods for determining HIV co-receptor tropism. The invention offers a way for determining whether an individual infected with HIV has CCR5 tropic viruses, CXCR4 tropic viruses, or is infected with a mix of both CCR5 and CXCR4 tropic viruses. The assays of the invention utilize a phenotypic selection step followed by a nucleic acid amplification/detection step, thus allowing for rapid and sensitive identification of HIV tropism. The diagnostic assays of the invention can be used to monitor shifts in coreceptor usage, measure the efficacy of a particular treatment protocol, assess or predict the degree of HIV progression, or determine when to start or change antiretroviral treatment. In a first aspect, the invention is directed to a method for detecting HIV. First, a reaction mixture is formed by contacting a sample with a binding buffer containing a CD4 polypeptide so as to allow CD4 to bind HIV gpl20. Next, the reaction mixture is incubated with either a CCR5 or a CXCR4 polypeptide under conditions permissible for CCR5 or CXCR4 binding to HIV gp 120. The presence of HIV bound to either CCR5 or CXCR4 is then detected in a nucleic acid detection assay. The presence of a HIV nucleic acid in the CCR5 or CXCR4 reaction mixture indicates HIV is present in the sample. Furthermore, the presence of a HIV nucleic acid in the CCR5 reaction not only indicates that HIV is present, but also that the HIV is CCR5 topic, similarly the presence of a HIV nucleic acid in the CXCR4 reaction indicates that HIV is CXCR4 tropic.

The invention is also directed to additional assays for determining HIV co- receptor tropism. In this assay a reaction mixture includes a sample and a CD4 polypeptide. The reaction is incubated under conditions permissible for CD4 binding to HIV gpl20. Next, the reaction mixture is incubated with immobilized (e.g., solid support bound) CCR5 or CXCR4 under suitable binding conditions. After a sufficient incubation period the solid support is washed so as to remove any unbound sample. Generally, a sample is divided into two fractions and is tested in both a CCR5 binding assay and CXCR4 binding assay. The HIV that remains bound to either CCR5 or CXCR4 is then detected in a nucleic acid detection assay. The detection of a HIV nucleic acid in the CCR5 reaction indicates the presence of a CCR5 tropic HIV in the sample, while the detection of a HIV nucleic acid in the CXCR4 reaction indicates the presence of a CXCR4 tropic HIV in the sample.

The present invention also relates to a method for determining HIV co- receptor tropism by first acidifying a serum or plasma sample from an individual infected with HIV. The sample is then concentrated so as to form an HIV pellet. The pellet is solubilized in a buffer containing a CD4 polypeptide and incubated under suitable for CD4-gpl20 binding After a sufficient amount of time, the reaction mixture is incubated with immobilized (e.g., bound to a solid support) CCR5 or CXCR4, thereby permitting binding of the HIV to either CCR5 or CXCR4.

Generally, a sample will be divided into two fractions and is tested in both a CCR5 binding assay and CXCR4 binding assay. The solid support is washed so as to remove the unbound virus (e.g., CXCR4 tropic virus would not bind to a CCR5 coated plate), while the remaining coreceptor bound virus is treated so as to extract the HIV genome. The presence of the HIV is then determined by a nucleic acid detection assay that is specific for the HIV viral genome. The detection of a HIV nucleic acid in the CCR5 reaction indicates the presence of a CCR5 tropic HIV in the sample, while the detection of a HIV nucleic acid in the CXCR4 reaction indicates the presence of a CXCR4 tropic HIV in the sample.

The invention is also directed to kits for practicing the methods. For example, the invention relates to a kit for determining HIV co-receptor tropism, wherein the kit includes a CD4 polypeptide, a solid support, CCR5 and CXCR4 polypeptides, a lysis buffer, a primer and a probe that are complementary to the HIV genome. In another embodiment, the kit contains a CD4 polypeptide, a solid support, a CCR5 and/or CXCR4 polypeptide and a DNA polymerase. In yet another embodiment, the kit includes a soluble CD4 polypeptide, a CCR5 and/or CXCR4 polypeptide and a labeled probe.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the results of an assay in which CXCR4 tropism was determined. Figure 2 illustrates an amino acid sequence of CCR5. Figure 3 illustrates an amino acid sequence of CXCR4. Figure 4 illustrates the amino acid sequence of CD4.

DETAILED DESCRIPTION

The present invention is directed to compositions, kits and methods for determining HIV co-receptor tropism. The invention offers a way for determining whether an individual infected with HIV has CCR5 tropic viruses, CXCR4 tropic viruses or is infected with a combination of both CCR5 and CXCR4 tropic viruses. The assays of the invention utilize a combination of a phenotypic selection step followed by a nucleic acid amplification/detection step which together allow for the rapid and sensitive identification of HIV tropism.

The diagnostic assays of the invention can be used to monitor shifts in coreceptor usage, measure the efficacy of a particular treatment protocol, assess or predict the degree of HIV progression, or determine when to start or change antiretroviral treatment. Unlike many other tropism assays the present assay does not introduce potential bias by cloning the HIV-I env gene, it is very sensitive, rapid (less than 24 hours), does not rely on preexisting databases to correlate sequence specific information with related characteristics, directly measures tropism in a clinical sample without prior genetic manipulation, reduces the risk of contamination as real time RT-PCR is preferably performed in a closed tube system in the final step, is compatible with a large number of nucleic acid detection technologies, and it can be performed using plasma or serum which as been previously frozen.

I. Definitions

"HIV" is Human Immunodeficiency Virus, a virus that causes immunodeficiency by attacking CD4+ cells in the body. The term "HIV" is intended to include HIV-I and HIV-2. HIV-I includes but is not limited to extracellular virus particles and the forms of HIV-I associated with HIV-I infected cells. HIV-2 includes but is not limited to extracellular virus particles and the forms of HIV-2 associated with HIV-2 infected cells. HIV is also meant to include SFV and FIV. As used herein, the term "determining HIV co-receptor tropism" refers to distinguishing experimentally between the affinity of HIV for a particular cellular receptor. In one embodiment, the method entails determining whether a subject is infected with HIV that binds to and infects a cell by associating with either the CCR5 or CXCR4 co-receptor. For example, the infected individual may be infected with a CCR5 tropic HIV, a CXCR4 tropic HIV, a dual tropic HIV or a combination of the three.

As used herein, "CCR5" is a chemokine receptor which binds members of the C-C group of chemokines. CCR5 refers to naturally occurring or recombinant CCR5 proteins and to proteins having an amino acid sequence which is the same as that of a naturally occurring or recombinant CCR5 protein. Accordingly, as defined herein, the term includes mature receptor protein, polymorphic or allelic variants, and other isoforms of CCR5 (e.g., produced by alternative splicing or other cellular processes), and modified or unmodified forms of the foregoing (e. g., glycosylated, unglycosylated). CCR5 proteins can be isolated and/or recombinant proteins (including synthetically produced proteins). Naturally occurring or recombinant CCR5 proteins include wild type proteins such as mature CCR5, polymorphic or allelic variants and other isoforms which occur naturally in mammals (e.g., humans, non-human primates). Such proteins can be recovered or isolated from a source which naturally produces mammalian CCR5. Generally, the CCR5 polypeptide will have an amino acid sequence is substantially identical to that provided in Genbank Accession Number 1705896 (SEQ ID NO:1); i.e., at about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 1. In one embodiment, the CCR5 polypeptide is a CCR5 functional fragment.

As used herein, "CXCR4" is a chemokine receptor which binds members of the C-X-C group of chemokines. CXCR4 refers to naturally occurring or recombinant CXCR4 proteins and to proteins having an amino acid sequence which is the same as that of a naturally occurring or recombinant corresponding CXCR4 protein. Accordingly, as defined herein, the term includes mature receptor protein, polymorphic or allelic variants, and other isoforms of a CXCR4 (e.g., produced by alternative splicing or other cellular processes), and modified or unmodified forms of the foregoing (e. g., glycosylated, unglycosylated). CXCR4 proteins can be isolated and/or recombinant proteins (including synthetically produced proteins). Naturally occurring or recombinant CXCR4 proteins include wild type proteins such as mature CXCR4, polymorphic or allelic variants and other isoforms which occur naturally in mammals (e.g., humans, non-human primates). Such proteins can be recovered or isolated from a source which naturally produces mammalian CXCR4.

Generally, the CXCR4 polypeptide will have an amino acid sequence is substantially identical to that provided in Genbank Accession Number 400654 (SEQ ID NO:2) i.e., at about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:2. hi one embodiment, the CXCR4 polypeptide is a CXCR4 functional fragment.

A "functional fragment" of a CCR5 or CXCR4 polypeptide refers to an isolated and/or recombinant protein or polypeptide which has at least one function characteristic of a CCR5 or CXCR4 protein as described herein, such as a binding activity (e.g. binding to one or more ligands such as gpl20, MIP- lot, MIP- 1/3, RANTES), a signaling activity and/or ability to stimulate a cellular response. Suitable functional CCR5 and CXCR4 fragments include the extracellular portions of the molecules. Suitable CCR5 and CXCR4 polypeptides and fragments thereof are described herein and known in the art. As used herein "CD4 protein" refers to naturally occurring or recombinant CD4 proteins and to proteins having an amino acid sequence which is the same as that of a naturally occurring or recombinant corresponding CD4 protein. Accordingly, as defined herein, the term includes mature receptor protein, polymorphic or allelic variants, and other isoforms of a CD4 (e.g., produced by alternative splicing or other cellular processes), and modified or unmodified forms of the foregoing (e. g., glycosylated, unglycosylated). CD4 proteins can be isolated and/or recombinant proteins (including synthetically produced proteins). Naturally occurring or recombinant CD4 proteins include wild type proteins such as mature CD4, polymorphic or allelic variants and other isoforms which occur naturally in mammals (e.g., humans, non-human primates). Such proteins can be recovered or isolated from a source which naturally produces mammalian CD4, for example.

In general, the CD4 polypeptide will have an amino acid sequence which is substantially identical to that provided in Accession Number CAA60883 (SEQ ID NO:3); i.e., at about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:3. In one embodiment, the CD4 polypeptide is a functional fragment of CD4.

A "functional fragment" of a CD4 polypeptide refers to an isolated and/or recombinant protein or polypeptide which has at least one function characteristic of a CD4 protein as described herein, such as a binding activity (e.g. binding to gpl20 of HIV), a signaling activity and/or ability to stimulate a cellular response. In one embodiment, the functional CD4 fragment is a soluble CD4.

The term "detecting" as used herein refers to any method of verifying the presence of a given molecule (e.g., a CCR5 or CXCR4 tropic HIV), directly or indirectly. The techniques used to accomplish this may include, but are not limited to, RT, PCR, sequencing, molecular beacons, and hybridization. Examples of reagents which might be used for detection include, but are not limited to, radiolabeled probes, enzymatic labeled probes (horseradish peroxidase, alkaline phosphatase), affinity labeled probes (biotin, avidin, or streptavidin), the INVADER detection assay (Third Wave Technologies; Madison, WI) and TAQMAN detection assays (supplied by a variety of commercial sources including Roche) which are described in U.S. Patent Nos. 5,723,591; 5,925,517 and 5,804,375. Additional assays useful for practicing the invention are described in U.S. Patent No: 6,548,250. In addition nucleic acid detection/amplification assays which are specific for the detection of HIV nucleic acids and are particularly useful in practicing the invention include the COBAS AMPLICOR HIV-I MONITOR test, version 1.5 (Roche; Indianapolis, IN), REAL- TIME HIV-I assay (Abbott; Des Plaines, IL) and NUCLISENS HIV-I assay (BioMerieux; Marcy l'Etoile, France).

The term "sample" as used herein, may be from any source that may contain the molecule of interest, e.g., HIV. Such samples include, but are not limited to, virus grown in vitro and biological fluids, such as plasma, serum, saliva, whole blood, semen, cerebrospinal fluid, urine, sputum, nasal material, bronchial secretions, as well as tissue, all of which may be isolated from primates, e.g., humans, or rodents, e.g., mice, and rats. Preferably, the sample is plasma or serum.

The term "contacting" as used herein, refers generally to providing access of one component, reagent, polypeptide or sample to another. For example, contacting can involve mixing a solution comprising a CD4 polypeptide with a sample. The solution comprising one component, reagent, polypeptide or sample may also comprise another component or reagent, such as a detergent, which facilitates mixing, interaction, uptake, or other physical or chemical phenomenon advantageous to the contact between components, reagents, polypeptides and/or samples.

The term "solid support" refers any solid phase that can be used to immobilize e.g., a polypeptide, an antibody or a complex. Suitable solid supports will be well known in the art and include the walls of wells of a reaction tray, such as a microti ter plate, the walls of test tubes, polystyrene beads, paramagnetic or non-magnetic beads, dextran beads, agarose beads, acrylic beads, nitrocellulose membranes, nylon membranes, and microparticles such as latex particles. Typical materials for solid supports include, but are not limited to, polyvinyl chloride (PVC), polystytrene, cellulose, nylon, latex and derivatives thereof. Further, the solid support may be coated, derivatized or otherwise modified to promote adhesion of the desired molecules (e.g., CXCR5 polypeptide) and/or to deter non-specific binding or other undesired interactions. The choice of a specific "solid phase" is usually not critical and can be selected by one skilled in the art depending on the assay employed. Thus, latex particles, microparticles, paramagnetic or non-magnetic beads, membranes, plastic tubes, walls of micro titer wells, glass or silicon chips are all suitable sold supports. Conveniently, the solid support can be selected to accommodate various detection methods. For example, 96 or 384 well plates can be used for assays that will be automated, for example by robotic workstations, and/or those that will be detected using, for example, a plate reader.

As used herein, the terms "identity" or "percent identity", refers to the subunit sequence similarity between two polymeric molecules, e.g., two polynucleotides or two polypeptides. When a subunit position in both of the two molecules is occupied by the same monomelic subunit, e.g., if a position in each of two peptides is occupied by serine, then they are identical at that position. The identity between two sequences is a direct function of the number of matching or identical positions, e.g., if half (e.g., 5 positions in a polymer 10 subunits in length), of the positions in two peptide or compound sequences are identical, then the two sequences are 50% identical; if 90% of the positions, e.g., 9 of 10 are matched, the two sequences share 90% sequence identity. The identity between two sequences is a direct function of the number of matching or identical positions. Thus, if a portion of the reference sequence is deleted in a particular peptide that deleted section is not counted for purposes of calculating sequence identity. Identity is often measured using sequence analysis software e.g., BLASTN or BLASTP (available at the world wide web site ("www") of the National Center for Biotechnology Information (".ncbi") of the National Institutes of Health (".nih") of the U.S. government (".gov"), in the "Blast" directory ("/BLAST/"). The default parameters for comparing two sequences {e.g., "Blast"-ing two sequences against each other), by BLASTN (for nucleotide sequences) are reward for match = 1, penalty for mismatch = -2, open gap = 5, extension gap = 2. When using BLASTP for protein sequences, the default parameters are reward for match = 0, penalty for mismatch = 0, open gap = 11, and extension gap = 1. Additional, computer programs for determining identity are known in the art.

"Similarity" or "percent similarity" in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues, or conservative substitutions thereof, that are the same when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms, or by visual inspection. By way of example, a first polypeptide can be considered similar to a second polypeptide when the amino acid sequence of the first polypeptide is at least 40%, 50%, 60%, 70%, 75%, 80%, 90%, or even 95% or more identical, or conservatively substituted, to a region of the second polypeptide when compared to any sequence of an equal number of amino acids as the number contained in the first polypeptide as aligned by a computer similarity program known in the art and described herein.

Preferably, the polypeptide region of the first protein and the second protein includes one or more conserved amino acid residues.

II. Assays of the Invention

The present invention, at least in part, is directed to a method for determining HIV co-receptor tropism by obtaining a sample suspected of containing HIV. The sample is then concentrated so as to form an HIV pellet. The pellet is solubilized in a buffer containing a CD4 polypeptide and incubated under suitable for CD4-gpl20 binding After a sufficient amount of time, the reaction mixture is incubated with immobilized (e.g., bound to a solid support) CCR5 or CXCR4, thereby permitting binding of the HIV to either CCR5 or CXCR4. Generally, a sample will be divided into two fractions and is tested in both a CCR5 binding assay and CXCR4 binding assay. The solid support is washed so as to remove the unbound virus (e.g., CXCR4 tropic virus would not bind to a CCR5 coated plate), while the remaining coreceptor bound virus is treated so as to extract the HIV genome. The presence of the HIV is then determined by a nucleic acid detection assay that is specific for the HIV viral genome. The detection of a HIV nucleic acid in the CCR5 reaction indicates the presence of a CCR5 tropic HIV in the sample, while the detection of a HIV nucleic acid in the CXCR4 reaction indicates the presence of a CXCR4 tropic HIV in the sample. 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. One may determine the stage of disease by assessing co-receptor usage as the change from CCR5 topic to CXCR4 tropic correlates with disease progression. The shifts can be monitored over the course of an HIV infection. For example, an individual who is infected with HIV may have a sample taken and analyzed for coreceptor usage early during the course of infection and at multiple times thereafter. The coreceptor usage can be assayed and compared over each time point.

In another embodiment, the coreceptor assay includes quantifying the proportion of virus using each coreceptor. Quantitation can be performed in a number of ways, including assaying a viral primary isolate using the methods described herein and applying a statistical method. For example, one may semi-quantitatively determine the proportion of viruses using the CCR5 and/or CXCR4 coreceptor by assaying coreceptor usage by the methods described herein. The signal generated in each assay can be compared with a suitable control. Suitable controls include samples having known concentrations of CCR5 or CXCR4 tropic HIV. Comparing the signal generated from the sample with that generated from the control can then be performed so as to semi-quantitatively determine the amount of each virus in a sample.

The present invention further relates to a diagnostic method to monitor the suppression of CXCR4-or CCR5 -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.

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.

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 antiretro viral therapy.

In any of the methods of the invention discussed herein, the sample may be first acidified before performing a binding assay. It has been found that acidifying the sample enhances CD4-gpl20 binding. Thus, preferably the sample is acidified to a pH of about 5.75 to about 6.75. Suitable methods for acidifying the sample are known in the art and include the addition of glycine-HCL.

The present invention utilizes CD4 polypeptides. The CD4 polypeptides utilized in the invention can be obtained from a variety of sources. For example, they may be recombinant, natural, or synthetic polypeptides. Furthermore, the use of functional fragments, rather than the full length CD4, is also contemplated as long as the fragment retains the ability to bind gpl20. For example, one may use only the extracellular (e.g., soluble) portion of the molecule which is involved in the CD4- gpl20 interaction.

The CCR5 and CXCR4 polypeptides utilized in the invention can also be obtained from a variety of sources. For example, they may be recombinant, natural, or synthetic polypeptides. In some embodiments, one will utilize a membrane preparation from a cell that expresses either a CCR5 or a CXCR4 polypeptide. In addition to full-length CCR5 and CXCR4 polypeptides, it is also contemplated that functional fragments of these polypeptides will be particularly useful in practicing the invention. For example, the extracellular N-terminal portion of the CD4 polypeptide is particularly useful. The CCR5 and CXCR4 polypeptides may include modifications, e.g., biotinylation, etc.

In a preferred embodiment, the CCR5 and CXCR4 polypeptides are attached to a solid support. Suitable solid supports include micro titer plates (e.g., polystyrene, derivatized polystyrene), membranes, beads (e.g., glass, latex, silica, etc.), paramagnetic microspheres, and latex microspheres.

The methods of the invention include a nucleic acid detection step. Numerous nucleic acid detection assays known in the art and described herein, are suitable for use in the present invention. These include target based amplification methods such as RT-PCR, transcription mediated amplification, branched DNA detection, NASBA and rolling circle amplification; signal based amplification such as bDNA and immunoPCR. In some of these assays, e.g., RT-PCR, the HIV genome is detected with an intercalating dye or labeled oligonucleotide probe. Suitable labels include radioisotopes, biotin, fluorescent moieties, flurophores, chemical luminescent moieties and enzymatic moieties.

IH. Practicing the Methods of the Invention

A. CD4 Binding to HIV SO 120

Generally, HIV in a serum or plasma sample will first be concentrated by centrifugation. It has been found that acidifying the sample enhances virus recovery (See Example 2). Thus, preferably the sample is acidified to a pH of about 5.75 to about 6.75 prior to centrifugation. Suitable methods for acidifying the sample are known in the art and include the addition of glycine-HCL.

Once the sample is concentrated, the supernatant is aspirated and the pellet is resuspended in a suitable buffer (e.g., PBS and Tris-NaCl) containing an appropriate concentration of CD4 polypeptide. Suitable concentrations of CD4 for use in the binding reaction are described herein and can be readily determined by those having ordinary skill in the art. For example, a sCD4 polypeptide (Cat. No. 3002; Protein Sciences) can be added to the binding reaction at a 1:100-1 :1000 dilution. The virus and CD4 polypeptide are incubated for a period of time sufficient to allow the virus and CD4 to bind. Generally, the incubation proceeds from between about 10 minutes and about 60 minutes, but may require overnight. The temperature at which each of the incubation steps of the methods is performed is not critical, but is generally performed at between about 18° C and about 37° C.

B. 2D120-CD4 and CCR5 or CXCR4 Binding

Binding of CD4 to gpl20 results in a conformational change in gpl20 which allows it to bind the coreceptor (CCR5 or CXCR4). In one embodiment, the CD4 binding reaction and the coreceptor binding reaction are performed simultaneously under the same reaction conditions. In another embodiment, a first CD4 binding reaction is performed and then a second coreceptor binding reaction is performed. The coreceptor polypeptides (CCR5 and CXCR4) may be recombinant, natural, or synthetic polypeptides. In some embodiments, one will utilize a membrane preparation from a cell that expresses either a CCR5 or a CXCR4 polypeptide. In addition to full-length CCR5 and CXCR4 polypeptides, it is also contemplated that functional fragments of these polypeptides will be particularly useful in practicing the invention.

Generally the coreceptor molecule is bound to a solid support. For example, the coreceptor molecule may be biotinylated and a microtiter plate may be coated with streptavidin, thus allowing the coreceptor molecule to bind the microtiter plate. The CD4-gpl20 bound sample is then added to the coreceptor-coated plate and incubated for a suitable period of time, e.g., 1 hour, at a suitable temperature, e.g., 37C, so as to allow the coreceptor to bind the CD4-gpl20 complex. Preferably, the incubation proceeds from between about 5 minutes to about 60 minutes, but may require overnight. The temperature at which the incubation step is performed is not critical. Preferably, the temperature at which the incubations occur is between about 18° C and about 37° C. After a suitable incubation time the plate bound HIV is washed with a suitable buffer so as to remove any unbound virus (e.g., 4X in PBS).

C. Detection Assay

After both the CD4 and coreceptor binding reactions are complete, the coreceptor bound HIV can be treated with a lysis solution so as to extract the HIV genome. The HIV genome is further processed in a nucleic acid detection assay so as determine the presence and/or quantity of HIV bound by the coreceptor and thus determining the tropism of the virus.

The nucleic acid detection assay may be performed in the same reaction vessel as the coreceptor binding reaction or in a separate reaction vessel. Any nucleic detection assay can be adapted for use in the present invention. Preferably, the nucleic acid detection reaction first employs a reverse transcriptase reaction (RT) followed by a polymerase chain reaction (PCR) or RT-PCR.

As used herein, the term "RT-PCR" refers to the replication and amplification of RNA sequences. In this method, reverse transcription is coupled to PCR, e.g., as described in U.S. Patent No. 5,322,770, herein incorporated by reference in its entirety. In RT-PCR, the RNA template is converted to cDNA due to the reverse transcriptase activity of an enzyme, and then amplified using the polymerizing activity of the same or a different enzyme. Both thermostable and thermolabile reverse transcriptase and polymerase can be used.

RT-PCR reactions can be performed as a one-step (e.g., one-step RT-PCR) or a two-step (e.g., two-step RT-PCR) reaction. According to the invention, one-step RT-PCR type reactions may be accomplished in one tube thereby lowering the possibility of contamination. Such one-step reactions comprise (a) mixing a nucleic acid template (e.g., HIV RNA) with suitable amplification reagents and (b) incubating the mixture under conditions sufficient to permit amplification. Two-step RT-PCR reactions may be accomplished in two separate steps. Such a method comprises (a) mixing a nucleic acid template (e.g., HIV RNA) with suitable amplification reagents (b) incubating the mixture under conditions sufficient to permit cDNA synthesis, (c) mixing the reaction mixture in (b) with one or more DNA polymerases and (d) incubating the mixture of step (c) under conditions sufficient to permit amplification.

The nucleic acid detection assays utilize oligonucleotide primers and/or probes that are specific for the HIV genome and/or compliment thereof. The primers and/or probes are preferably specific for a conserved region of the HIV genome. For example, the highly conserved region of the HIV-I gag gene is particularly suited for targeting probes and primers (Kwok et al. 1993. PCR detection of human immunodeficiency virus type 1 proviral DNA sequences. Inn: Diagnostic Molecular Biology Principles and Applications. PCR. eds. Persing D.H., et al. ASM, Washington, DC). Other suitable probes and primers for use in detecting and amplifying the HIV genome are known in the art and included in many of the nucleic acid based HIV detection kits described herein. Additional, primer and probes that are specific for HIV-I and HIV-2 are described in U.S. Patent No. 6,881,537 and U.S. Patent No. 6,316,183, both of which are herein incorporated by reference in their entirety.

Suitable RT-PCR assays for use in the present invention are well known in the art and are commercially available. Reverse transcriptases and kits for performing a quantitative RT-PCR assay may be obtained commercially, for example, from Invitrogen, Inc. (Carlsbad, Calif.), Pharmacia (Piscataway, N.J.), Sigma (Saint Louis, Mo.) Stratagene (BRILLIANT II QRT-PCR AFFINITYSCRIPT master mix; La Jolla, CA), or Applied Biosystems (TAQMAN One-Step RT-PCR Master Mix kits; Forest City, CA).

Suitable PCR based detection assays for use in the present methods are also well known in the art and include , but not limited to, (a) direct detection of a released cleavage product on a gel; (b) indirect or direct detection of a signal generated during a nucleic acid cleavage reaction (TAQMAN reaction); (c) fluorescent change upon a probe binding a target (MOLECULAR BEACONS); or SYBR Green detection assay. Cleavage reactions utilizing an endonuclease activity include the INVADER detection assay (Third Wave Technologies; Madison, WI) which is described in U.S. Patent No. 6,348,314 and is herein incorporated by reference in its entirety. Cleavage reaction assays encompassed by the present methods also include MOLECULAR BEACON detection assays (supplied by a variety of commercial sources) and TAQMAN detection assays (supplied by a variety of commercial sources including Roche) which are described in U.S. Patent Nos. 5,723,591; 5,925,517 and 5,804,375, each of which is herein incorporated by reference in its entirety. Cleavage reactions useful in the present invention are also described in U.S. Patent No: 6,548,250 which is herein incorporated by reference.

Nucleic acid detection/amplification assays which are specific for the detection of HIV nucleic acids and are particularly useful in practicing the invention include the COBAS AMPLICOR HIV-I MONITOR test, version 1.5 (Roche; Indianapolis, IN), REAL-TIME HIV-I assay (Abbott; Des Plaines, IL) and NUCLISENS HIV-I assay (BioMerieux; Marcy l'Etoile, France).

In addition to PCR amplification, the methods of the invention may be practiced using any method for the detection of a HIV genome, including Strand Displacement Amplification (SDA), Rolling Circle Amplification (RCA), Transcription Mediated Amplification (TMA) or Ligase Chain Reaction (LCR), branched DNA detection, NASBA; signal based amplification such as bDNA and immunoPCR. D. Samples The present invention relates to assays for determining coreceptor usage in HIV samples. Generally the samples are obtained from a patient. Patient-derived virus includes, but is not limited to, primary viral isolates and cultured virus, e.g., biological clones. Patient-derived viruses can be obtained from clinical specimens 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-I 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).

In the methods of the present invention the sample, e.g., serum, plasma or tissue culture sample, may be acidified in order to enhance HIV recovery from the sample. Thus, preferably the sample is acidified to a pH of about 5.75 to about 6.75. Suitable methods for acidifying the sample are known in the art and include the addition of glycine-HCL.

For purposes of conducting the coreceptor assays CCR5- and CXCR4-specific positive control viruses can be employed, such as HIV JR-FL and LAV/HTLV-IIIB. Infection with CCR5- and CXCR4-specifϊc positive control viruses can be assayed parallel to infection with primary isolates. Uninoculated cells/animals can be negative controls.

E. Solid Support

In one embodiment, the invention is practiced in the presence of a solid support. As used herein, a "solid support" or "solid surface" refers to any structure that provides a support for the capture molecule (e.g., CD4, CCR5 or CXCR4). Suitable solid supports include polystyrene, derivatized polystyrene, a membrane, such as nitrocellulose, PVDF or nylon, a latex bead, a glass bead, a silica bead, paramagnetic or latex microsphere, dextran beads, agarose beads, acrylic beads, or microtiter well. As a further example, the solid support may be a modified microtiter plate, such as a Top Yield plate, which allows for covalent attachment of a capture molecule to the plate. When the solid support is a material such as a bead, paramagnetic microsphere or latex microsphere, the solid support may be contained in an open container, such as a multi-well tissue culture dish, or in a sealed container, such as a screw-top tube, both of which are commonly used in laboratories.

The solid support may be modified to facilitate binding of the capture molecule to the surface of the support, such as by coating the surface with poly L- lysine, or siliconized with amino aldehyde silane or epoxysilane. The skilled artisan will understand that the circumstances under which the methods of the current invention are performed will govern which solid supports are most preferred and whether a container is used.

Quantities of the capture molecule to be attached to the solid support may be determined empirically by checkerboard titration with different quantities of analyte that would be expected to mimic quantities in a test sample. Generally, the quantity of the analyte in the test sample is expected to be in the attogram to milligram range. An unknown concentration of the analyte in a test sample will be added at specified volumes, and this will influence the sensitivity of the test. If large volumes of the test sample (e.g., 200-400 uL) are used, modification of the test format may be needed to allow for the larger sample volumes. Generally, however, the concentration of the capture molecule will be about 1 to about 10 micrograms per mL.

The capture molecule can be attached to a solid support by routine methods that have been described for attachment of an analyte to plastic or other solid support systems (e.g., membranes or microspheres). Examples of such methods may be found in U.S. Pat. No. 4,045,384 and U.S. Pat. No. 4,046,723, both of which are incorporated herein by reference.

Attachment of the capture molecule to surfaces such as membranes, microspheres, or microtiter wells may be performed by direct addition in PBS, or other buffers of defined pH, followed by drying in a convection oven.

The capture molecule may be attached to the solid support by an attachment means, such as via adsorption, covalent linkage, avidin-biotin linkage, streptavidin- biotin linkage, heterobifunctional cross-linker, Protein A linkage or Protein G linkage. Each of the attachment means should permit the use of stringent washing conditions with minimal loss of the capture molecule from the surface of the solid support. As an example, the adsorption may be hydrophilic adsorption. As a further example, the heterobifunctional cross-linker may be maleic anhydride, 3-aminopropyl trimethoxysilane (APS), N- 5 azido, 2-nitrobenzoyaloxysuccinimide (ANB-NOS) or mercaptosilane.

The capture molecule may be attached to the solid support though a portion of the capture molecule, such as an amino acid residue, preferably a lysine or arginine residue, a thiol group or a carbohydrate residue.

The solid support may be derivatized with avidin or streptavidin, and the capture molecule may be modified to contain at least one biotin moiety, to aid in the attachment of the capture molecule to the solid support. Alternatively, the solid support may be derivatized with biotin, and the capture molecule may be modified to contain at least one avidin or at least one streptavidin moiety.

In one embodiment, what-germ agglutinin is bound to a solid support in order to capture CXCR4 or CCR5 containing cell membranes.

F. Washing

In embodiments which utilize a solid support between the additions of reagents in the methods of the present invention, the assay system is preferably subjected to washing to reduce the incidence of non-specific binding. Stringent wash conditions which do not cause dissociation of the CD4-gpl20 or coreceptor-gpl20 binding reaction can be employed. For example, heating, pH changes, or (and) the addition of formamide, detergents and salts can be used to increase the efficiency of the wash step. Too stringent conditions can lead to dissociation of the CD4 and/or coreceptors from gpl20. The stringent conditions should be experimentally optimized for each assay. However, in washing to reduce non-specific binding, if some of the CD4 and/or coreceptor interactions are lost, this can be compensated for by additional target replication realized by increasing the number of temperature recycle steps. While the number of wash cycles and soak times is empirically determined, in general either water or a low or high molarity salt solution with a detergent such as Tween 20, Triton X-IOO, or NP -40 may be used as the washing solution. 1-8 washes, each lasting 5 seconds to 10 minutes may be performed, after incubation of each of the reagents used in the methods. The detergent concentration is typically 0 to .1% with a salt concentration of 0 to 100OnM (e.g., NaCl). Preferably, washing takes place between each incubation step, e.g., after addition of the coreceptor molecules to the solid support, after addition of the test sample and after addition of the detector molecule. Exemplary washing conditions are described in the Examples.

G. Polypeptides of the Invention

Described herein are CD4, CXCR4 and CCR5 polypeptides which are used in practicing the invention. CD4 has a 370 amino acid extracellular region containing four immunoglobulin-like domains, a membrane spanning domain, and a charged intracellular region of 40 amino acid residues. Maddon, P. et al., Cell 42:93 (1985); Clark, S. et al., Proc. Natl. Acad. Sci. (USA) 84:1649 (1987). The CD4 polypeptides utilized in the invention can be obtained from a variety of sources. For example, they may be recombinant, natural, or synthetic polypeptides.

In general, the CD4 polypeptide will have an amino acid sequence is substantially identical to that provided in (SEQ ID NO:3); i.e., at about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 3. Furthermore, additional amino acid and nucleic acid sequences of CD4 are readily available to those of skill in the art.

The CD4 polypeptides of the invention include functional fragments, functional mutant proteins, and/or functional fusion proteins (e.g., produced via mutagenesis and/or recombinant techniques). Generally, fragments or portions of CD4 proteins include those having a deletion (i. e., one or more deletions) of an amino acid (i.e., one or more amino acids) relative to the mature CD4 protein (such as N-terminal, C-terminal or internal deletions). Fragments or portions in which only contiguous amino acids have been deleted or in which non-contiguous amino acids have been deleted relative to mature CD4 protein are also envisioned. Generally, mutants of CD4 proteins include natural or artificial variants of a CD4 protein differing by the addition, deletion and/or substitution of one or more contiguous or non-contiguous amino acid residues (e.g., receptor chimeras). The CD4 polypeptides and fragments of the invention exhibit binding to gpl20 of HIV. In one embodiment, the functional CD4 fragment is a soluble CD4.

A "soluble CD4" or "sCD4" is a variant of the CD4 protein which binds gpl20 and which is soluble in water-based pharmaceutical preparations (or pharmaceutically acceptable solvents or compositions which include components in addition to water) and in physiological fluids, including plasma, at a level which is sufficient to achieve a therapeutically effective concentration in circulation. sCD4 proteins include those in which part or the entire transmembrane domain of the primary structure of CD4 has been deleted, for example through truncation of the coding sequence; the cytoplasmic domain of the protein may likewise be deleted without the loss of the desired biological activity of HIV gpl20 binding. sCD4 molecules capable of binding gpl20 are well known in the art and described, for example, in EP Publication No. 385 909; Deen et al. (1988) Nature 331 :82-84; Capon, D., et al. (1988) Nature, 337:525-531; and Till, M., et al. (1988) Science

242:1166-1168; European Patent Application No. 0, 331, 356, each of which is herein incorporated by reference in its entirety. Suitable CD4 and sCD4 polypeptides are readily available form many commercial suppliers including R&D Systems, Inc. (Minneapolis, MN) and Protein Sciences (Meriden, CT).

The coreceptor polypeptides (CCR5 and CXCR4) utilized in the invention can also be obtained from a variety of sources. For example, they may be recombinant, natural, or synthetic polypeptides. In some embodiments, one will utilize a membrane preparation from a cell that expresses either CCR5 or CXCR4.

CCR5 is a chemokine receptor which binds members of the C-C group of chemokines. Generally, the CCR5 polypeptide will have an amino acid sequence is substantially identical to that provided in Genbank Accession Number 1705896 (SEQ ID NO:1); i.e., at about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:1. In one embodiment, the CCR5 polypeptide only includes the first 31 amino acids of CCR5, which corresponds to the extracellular portion of CCR5. Accordingly, the extracellular portion extends from the methionine at position number 1 to the arginine at position number 31 of SEQ ID NO:1. Additional CCR5 peptides and active fragments are known in the art, including those described in U.S. Publication No.2002/0068813, which is herein incorporated by reference in its entirety.

In addition to using the full-length CCR5 and CXCR4 polypeptides in the methods of the invention, it is also contemplated that functional fragments of these may also be used. CCR5 polypeptides include functional fragments, functional mutant proteins, and/or functional fusion proteins (e.g., produced via mutagenesis and/or recombinant techniques). Generally, fragments or portions of CCR5 proteins include those having a deletion (i. e., one or more deletions) of an amino acid (i.e., one or more amino acids) relative to the mature CCR5 protein (such as N-terminal, C- terminal or internal deletions). Fragments or portions in which only contiguous amino acids have been deleted or in which non-contiguous amino acids have been deleted relative to mature CCR5 protein are also envisioned. Generally, mutants of CCR5 proteins include natural or artificial variants of a CCR5 protein differing by the addition, deletion and/or substitution of one or more contiguous or non-contiguous amino acid residues (e.g., receptor chimeras). Such mutations can be in a conserved region or nonconserved region (compared to other CXC and/or CC chemokine receptors), extracellular, cytoplasmic, or transmembrane region, for example.

CXCR4 is a chemokine receptor which binds members of the C-X-C group of chemokines. Generally, the CXCR4 polypeptide will have an amino acid sequence is substantially identical to that provided in Genbank Accession Number 400654 (SEQ ID NO:2) i.e., at about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:2 or a functional fragment thereof.

CXCR4 polypeptides of the invention include functional fragments, functional mutant proteins, and/or functional fusion proteins (e.g., produced via mutagenesis and/or recombinant techniques). Generally, fragments or portions of CXCR4 proteins include those having a deletion (i. e., one or more deletions) of an amino acid (i.e., one or more amino acids) relative to the mature CXCR4 protein (such as N-terminal, C-terminal or internal deletions). Fragments or portions in which only contiguous amino acids have been deleted or in which non-contiguous amino acids have been deleted relative to mature CXCR4 protein are also envisioned. Generally, mutants of CXCR4 proteins include natural or artificial variants of a CXCR4 protein differing by the addition, deletion and/or substitution of one or more contiguous or non-contiguous amino acid residues (e.g., receptor chimeras). Such mutations can be in a conserved region or nonconserved region (compared to other CXC and/or CC chemokine receptors), extracellular, cytoplasmic, or transmembrane region, for example.

A CCR5 or CXCR4 protein as described herein will bind to a gp 120 molecule and generally will also exhibit binding activity to one or more ligands such as MIP- lα, MIP- IjS, RANTES, a signaling activity and/or ability to stimulate a cellular response. Suitable functional CCR5 and CXCR4 fragments include the extracellular portions of the molecules. Suitable CCR5 and CXCR4 polypeptides and fragments thereof are readily available form many commercial suppliers including Millipore, Inc. and American Peptide Company, Inc. (CCR5 , Product No. 3002)

In order to express the polypeptides of the invention, DNA molecules obtained by any of the methods described herein or those that are known in the art, can be inserted into appropriate expression vectors by techniques well known in the art. For example, a double stranded cDNA can be cloned into a suitable vector by homopolymeric tailing or by restriction enzyme linking involving the use of synthetic DNA linkers or by blunt-ended ligation. DNA ligases are usually used to ligate the DNA molecules and undesirable joining can be avoided by treatment with alkaline phosphatase. Therefore, the invention comprises vectors (e.g., recombinant plasmids and bacteriophages) that include nucleic acid molecules (e.g., genes or recombinant nucleic acid molecules comprising genes) as described herein. The term "recombinant vector" includes a vector (e.g., plasmid, phage, phasmid, virus, cosmid, fosmid, or other purified nucleic acid vector) that has been altered, modified or engineered such that it contains greater, fewer or different nucleic acid sequences than those included in the native or natural nucleic acid molecule from which the recombinant vector was derived.

For eukaryotic hosts, different transcriptional and translational regulatory sequences may be employed, depending on the nature of the host. They may be derived from viral sources, such as adenovirus, bovine papilloma virus, Simian virus or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Examples include, but are not limited to, the TK promoter of the Herpes virus, the SV40 early promoter, the yeast gal 4 gene promoter, etc. Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the genes can be modulated.

In some embodiments, one or more DNA molecules comprising a nucleotide sequence encoding one or more polypeptide chains of a hybrid protein are operably linked to one or more regulatory sequences, which are capable of integrating the desired DNA molecule into a host cell. Cells which have been stably transformed by the introduced DNA can be selected, for example, by introducing one or more markers which allow for selection of host cells which contain the expression vector. A selectable marker gene can either be linked directly to a nucleic acid sequence to be expressed, or be introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of proteins described herein. It would be apparent to one of ordinary skill in the art which additional elements to use, if necessary. Factors of importance in selecting a particular plasmid or viral vector include, but are not limited to, the ease with which recipient cells that contain the vector are recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species. Once the vector(s) is constructed to include a DNA sequence for expression, it may be introduced into an appropriate host cell by one or more of a variety of suitable methods that are known in the art, including but not limited to, for example, transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, etc. Host cells may either be prokaryotic or eukaryotic. Examples of eukaryotic host cells include, for example, mammalian cells, such as human, monkey, mouse, and Chinese hamster ovary (CHO) cells. Such cells facilitate post-translational modifications of proteins, including, for example, correct folding or glycosylation. Additionally, yeast cells can also be used to express hybrid proteins of the invention. Like most mammalian cells, yeast cells also enable post-translational modifications of proteins, including, for example, glycosylation. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number plasmids that can be utilized for production of proteins in yeast. Yeast transcription and translation machinery can recognize leader sequences on cloned mammalian gene products, thereby enabling the secretion of peptides bearing leader sequences (i.e., pre-pep tides). A particularly preferred method of high- yield production of the hybrid proteins of the invention is through the use of dihydro folate reductase (DHFR) amplification in DHFR-deficient CHO cells, by the use of successively increasing levels of methotrexate as described in US 4,889,803. The polypeptide obtained may be in a glycosylated form.

After the introduction of one or more vector(s), host cells are usually grown in a selective medium, which selects for the growth of vector-containing cells. Purification of the recombinant proteins can be carried out by any of the methods known in the art or described herein, for example, any conventional procedures involving extraction, precipitation, chromatography and electrophoresis. A further purification procedure that may be used for purifying proteins is affinity chromatography using monoclonal antibodies which bind a target protein. Generally, crude preparations containing a recombinant protein are passed through a column on which a suitable monoclonal antibody is immobilized. The protein usually binds to the column via the specific antibody while the impurities pass through. After washing the column, the protein is eluted from the gel by changing pH or ionic strength, for example.

H. Primers and Probes for Use in Practicing the Invention

The terms "polynucleotide", "oligonucleotide" and "nucleic acid (molecule)" are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes single-, double- stranded and triple helical molecules. Polynucleotides may be isolated from genes, or chemically synthesized by methods known in the art.

The invention provides for oligonucleotide primers and probes useful for detecting, measuring or amplifying a nucleic acid (e.g., HIV genome). The term "primer" may refer to more than one primer and refers to an oligonucleotide, whether occurring naturally, as in a purified restriction digest, or produced synthetically, which is capable of acting as a point of initiation of synthesis along a complementary strand when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is catalyzed. Such conditions include the presence of four different deoxyribonucleoside triphosphates and a polymerization-inducing agent such as DNA polymerase or reverse transcriptase, in a suitable buffer ("buffer" includes substituents which are cofactors, or which affect pH, ionic strength, etc.), and at a suitable temperature. The primer is preferably single-stranded for maximum efficiency in amplification. In some embodiments, the primer may have a 3' flap which is cleaved by a 3' nuclease.

Oligonucleotide primers useful according to the invention are single-stranded DNA or RNA molecules that are hybridizable to a template nucleic acid sequence and prime enzymatic synthesis of a second nucleic acid strand. The primer is complementary to a portion of a target molecule. It is contemplated that oligonucleotide primers according to the invention are prepared by synthetic methods, either chemical or enzymatic. Alternatively, such a molecule or a fragment thereof is naturally-occurring, and is isolated from its natural source or purchased from a commercial supplier. Oligonucleotide primers and probes are generally 5 to 100 nucleotides in length, ideally from 17 to 40 nucleotides, although primers and probes of different length are of use. Primers for amplification are preferably about 17-25 nucleotides. Primers useful according to the invention are also designed to have a particular melting temperature (Tm) by the method of melting temperature estimation. Commercial programs, including Oligo, Primer Design and programs available on the internet, including Primer3 and Oligo Calculator can be used to calculate a Tm of a nucleic acid sequence useful according to the invention. Preferred, Tm's of a primer will depend on the particular embodiment of the invention that is being practiced. For example, in one embodiment the primer will dissociate from a target at a temperature of 41⁰C or more. While in other embodiments it is preferable to have a Tm between about 45 and 65⁰C and more preferably between about 50 and 6O⁰C. The oligonucleotides of the invention include polynucleotide templates (modified or non- modified) and primers. The polynucleotide templates can be prepared with lengths ranging in length from at least 10 bases in length, typically at least 20 bases in length, for example, at least 30, 40, 50, 60, 70, 80, 90 or 100 bases in length. While the oligonucleotide can be large nucleic acid fragments, it is generally limited to nucleic acids of 500 bases or less. Generally, the primers and/or probes for use in detecting and amplifying the HIV genome are generally complementary to a conserved region of the HIV genome. For example, the highly conserved region of the HIV-I gag gene is particularly suited for targeting probes and primers (Kwok et al. 1993. PCT detection of human immunodeficiency virus type 1 proviral DNA sequences. Inn: Diagnostic Molecular Biology Principles and Applications. PCR. eds. Persing D.H., et al. ASM, Washington, DC). Additional, primer and probes specific for HIV-I and HIV-2 are described in U.S. Patent No. 6,881,537 and U.S. Patent No. 6,316,183, both of which are herein incorporated by reference in their entirety. Additional probes and primers for use in detecting and amplifying the HIV genome are known in the art and included in many of the nucleic acid based HIV detection kits described herein.

I. Kits

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. The invention relates to a kit for determining HIV co-receptor tropism, wherein the kit includes a CD4 polypeptide, a solid support, a CCR5 and CXCR4 polypeptide, a lysis buffer and a primer and a probe that are complementary to the HIV genome. In another embodiment, the kit contains a CD4 polypeptide, a solid support, a CCR5 and/or CXCR4 polypeptide and a DNA polymerase. In yet another embodiment, the kit includes a soluble CD4 polypeptide, a CCR5 and/or CXCR4 polypeptide and a labeled probe.

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.

EXAMPLES

Example 1. HIV Tropism Assay

The following assay can be performed to determine whether a sample contains HIV which is CCR5 tropic, CXCR4 tropic or whether the sample contains a combination of CCR5 and CXCR4 tropic viruses. Briefly, a human serum sample is obtained from an individual infected with HIV. 50ul of 1.0 M Glycine buffer (pH2.75) is added to 500ul of the serum sample, mixed and pelleted at 24,000 RPM (60,000 G forces). The supernatant is carefully removed and the pellet is reususpended in 200ul of PBS or Tris-NaCl buffer containing sCD4 polypeptide (Cat. No. 3002 Protein Sciences; suitable dilutions from 1:100 to 1 :1000 of the stock solution).

Meanwhile, a coreceptor coated plate is prepared by adding purified CCR5 or CXCR4 polypeptides (CCR5, Product No. 3002, from American Peptide Company, Inc.; lmg/ml stock) to each well of a Pierce streptavidin coated plate at a dilution of 1 : 100 in PBS. The plate-solution is allowed to incubate at room temperature for 2 hours. The plates are then washed 4X with 300ul of PBS and blocked.

Alternatively, the plate may be prepared by adding CCR5 or CXCR4 polypeptide membrane preparations (CXCR4, Product No. HTS004M, from Millipore). In this embodiment, the wells are first incubated with wheat germ agglutinin (Vector Laboratories) at lOug/ml in PBS overnight at 4C. Following incubation the wells are washed and the polypeptide membrane preparation is added at a suitable dilution (e.g., at a dilution of 1 : 100 in PBS of CXCR4 membrane preparation). The plate-solution is allowed to incubate at room temperature for 2 hours. The plates are then washed 4X with 300ul of PBS.

The entire volume of the each microcentrifuge tube containing the solubilized pellet is added to the peptide-coated Streptavidin wells and incubated for lhr at 37C. Each well is aspirated and washed 4X with PBS and the bound HIV is processed for detection. Various nucleic acid methods described herein and known in the art can be used for assay the presence and/or amount of HIV in a sample. For example, the COBRAS AMPLICOR HIV- 1 MONITOR Test, version 1.5 (Roche Diagnostics) is used to quantitate the amount of HIV present in the CCR5 or CXCR4 tropism assay according to manufacturer's instructions.

This test utilizes five major processes: specimen preparation (lysis); reverse transcription of target RNA to generate cDNA; PCR amplification of cDNA utilizing HIV-I specific complimentary primers; hybridization of the amplified products to oligonucleotide probes specific to the targets; and detection of the probe-bound amplified products by colorimetric determination. Thus, identification of the presence or the amount of HIV in the particular coreceptor assay will determine the tropism of the virus contained in the sample

Example 2. CXCR4 HIV-I Tropism Assay CXCR4 HIV-I tropism assays were performed on two patients; one infected with a

CCR5 tropic virus (Patent A) and one infected with a CXCR4 tropic virus (Patient B).

Briefly, serum samples were obtained from both patients and split into two fractions;

(1) a non-treated serum and (2) an acidified serum sample that was treated with 50 ul of 1.0

M Glycine buffer (pH2.75). The HIV was then pelleted by centrifugation so as to produce an HIV pellet (24,000 RPM-60,000 G forces) and the supernatant was removed. The pellet was resuspended in 200ul of PBS having sCD4 polypeptide (Cat. No. 3002 Protein Sciences; suitable dilutions from 1:100 to 1 :1000 of the stock solution).

A coreceptor coated plate was prepared with a CXCR4 polypeptide membrane preparation (CXCR4, Product No. HTS004M, from Millipore). In this embodiment, the wells are first incubated with wheat germ agglutinin (Vector Laboratories) at lOug/ml in PBS overnight at 4C. Following incubation the wells are washed and the polypeptide membrane preparation is added at a suitable dilution (e.g., at a dilution of 1 : 100 in PBS of CXCR4 membrane preparation). The plate-solution is allowed to incubate at room temperature for 2 hours. The plates are then washed 4X with 300ul of PBS.

The entire volume of the each microcentrifuge tube containing the solubilized pellet was then added to the peptide-coated Streptavidin wells and incubated for lhr at 37C. Each well was aspirated and washed 4X with PBS. The bound HIV was then processed for detection of the HIV genome. The sample was processed according to the COBRAS AMPLICOR HIV-I MONITOR Test, version 1.5 (Roche Diagnostics) in order to assay the relative amounts of HIV present in the CXCR4 tropism assay. The results are illustrated in Figure 1 which illustrates no CXCR4 tropic HIV in either of the Patient A processed samples (having only CCR5 tropic viruses), but a significant amount of CXCR4 tropic HIV in both the Patient B samples. Furthermore, it is evident from the data that acidifying the serum sample prior to centrifugation increases the HIV yield obtained from the samples.

All patents, patent applications, and published references cited herein are hereby incorporated by reference in their entirety. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims

Claims

CLAIMSWhat is claimed is:

1. A method for detecting HIV, said method comprising: forming a reaction mixture by contacting a sample with CD4 under conditions permissible for CD4 binding to HIV; contacting the reaction mixture with either CCR5 or CXCR4 under conditions permissible for CCR5 or CXCR4 binding to HIV; and detecting the presence of HIV bound to either CCR5 or CXCR4 in a nucleic acid detection assay, wherein the presence of a HIV nucleic acid indicates HIV is present in the sample.

2. A method for determining HIV co-receptor tropism, said method comprising: forming reaction mixture by contacting a sample with CD4 under conditions permissible for binding of HIV to CD4; contacting the reaction mixture with a solid support having CCR5 or CXCR4 under conditions permissible for binding of HIV to CCR5 or CXCR4; removing unbound HIV; detecting the presence of HIV bound to either CCR5 or CXCR4 in a HIV nucleic acid detection assay, wherein the presence of CCR5 bound HIV indicates the HIV is CCR5 tropic and the presence of CXCR4 bound HIV indicates the HIV is CXCR4 tropic.

3. A method for determining HIV co-receptor tropism, said method comprising: acidifying a serum or plasma sample from an individual infected with HIV; concentrating the serum or plasma sample so as to form a pellet having HIV; forming a reaction mixture by solublizing the pellet and contacting the HIV with CD4 thereby permitting binding of HIV to CD4; contacting the reaction mixture with a solid support having CCR5 or CXCR4 thereby permitting binding of HIV to CCR5 or CXCR4; removing unbound HIV; extracting the RNA of HIV which is bound to CCR5 or CXCR4; and detecting the presence of HIV bound to either CCR5 or CXCR4 in a HIV nucleic acid detection assay, wherein the presence of CCR5 bound HFV indicates the HIV is CCR5 tropic and the presence of CXCR4 bound HIV indicates the HIV is CXCR4 tropic.

4. The method of any one of the proceeding claims, wherein the sample is acidified with glycine-HCL.

5. The method of any one of the proceeding claims, wherein the sample is acidified to a pH of about 5.75 to about 6.75

6. The method of any one of the proceeding claims, wherein CD4 is soluble CD4.

7. The method of any one of the proceeding claims, wherein CD4 is a recombinant polypeptide.

8. The method of any one of the proceeding claims, wherein CD4 is a synthetic polypeptide.

9. The method of any one of the proceeding claims, wherein CD4 is a functional fragment.

10. The method of any one of the proceeding claims, wherein CCR5 and/or CXCR4 is a recombinant polypeptide.

11. The method of any one of the proceeding claims, wherein CCR5 or CXCR4 is a synthetic polypeptide.

12. The method of any one of the proceeding claims, wherein CCR5 and/or CXCR4 is biotinylated.

13. The method of any one of the proceeding claims, wherein CCR5 is a functional fragment.

14. The method of any one of the proceeding claims, wherein the CCR5 fragment is an N-terminal fragment.

15. The method of any one of the proceeding claims, wherein CXCR4 is a functional fragment.

16. The method of any one of the proceeding claims, wherein CCR5 or CXCR4 is a membrane preparation from a cell that expresses either CCR5 or CXCR4.

17. The method of any one of the proceeding claims, wherein CCR5 and/or CXCR4 is biotinylated.

18. The method of any one of the proceeding claims, wherein the nucleic acid detection assay is RT-PCR.

19. The method of any one of the proceeding claims, wherein the CCR5 or CXCR4 is bound to a solid support.

20. The method of claim 19, wherein the solid support is a microtiter plate.

21. The method of any one of the proceeding claims, wherein the nucleic acid detection assay is selected from the group consisting of RT-PCR, transcription mediated amplification, branched DNA detection and NASBA.

22. The method of claim 21, wherein the nucleic acid detection step includes detecting a signal from a labeled probe or intercalating dye.

23. The method of any one of the proceeding claims, wherein the nucleic acid detection step includes detecting a signal from a label selected from the group consisting of a radioisotope, biotin, fluorescent moiety, flurophore, chemical luminescent moiety and an enzymatic moiety.

24. The method of any one of the proceeding claims, wherein the solid support is selected from the group consisting of polystyrene, derivatized polystyrene, a membrane, a latex bead, a glass bead, a silica bead, dextran bead, agarose bead, acrylic bead, a paramagnetic microsphere, a latex microsphere and a microtiter well.

25. The method of any one of the proceeding claims, wherein the method is used to assess or predict the degree of HIV progression, to determine when to start or change antiretroviral treatment or to monitor the efficacy of an anti-retro viral treatment.

26. The method of claim 1, wherein the sample is plasma or serum.

27. A kit for determining HIV co-receptor tropism, said kit comprising: a CD4 polypeptide; a solid support; a CCR5 polypeptide; a CXCR4 polypeptide; a lysis buffer; and a primer and probe that is complementary to a portion of the HIV genome and instructions for use.

28. A kit for determining HIV co-receptor tropism, said kit comprising: a CD4 polypeptide; a solid support; a CCR5 and/or a CXCR4 polypeptide; and a DNA polymerase; and instruction for use

29. A kit for detecting HIV, said kit comprising: a soluble CD4 polypeptides; a CCR5 and/or a CXCR4 polypeptide; and a labeled probe and instructions for use.

30. The kit of any one of claims 27-28, wherein CD4 is soluble CD4.

31. The kit of any one of claims 27-29, wherein CD4 is recombinant CD4.

32. The kit of any one of claims 27-29, wherein CD4 is synthetic CD4.

33. The kit of any one of claims 27-29, wherein CD4 is a functional fragment.

34. The kit of any one of claims 27-29, wherein CCR5 or CXCR4 is recombinant.

35. The kit of any one of claims 27-29, wherein CCR5 or CXCR4 is a synthetic polypeptide.

36. The kit of any one of claims 27-29, wherein CCR5 or CXCR4 is biotinylated.

37. The kit of any one of claims 27-29, wherein CCR5 is a functional fragment.

38. The kit of any one of claims 27-29, wherein the CCR5 functional fragment is an N-terminal fragment of CCR5.

39. The kit of any one of claims 27-29, wherein CXCR4 is a functional fragment.

40. The kit of any one of claims 27-29, wherein CCR5 or CXCR4 is a membrane preparation.

41. The kit of any one of claims 27-29, wherein CCR5 or CXCR4 is biotinylated.

42. The kit of any one of claims 27-29, wherein the CCR5 or CXCR4 is bound to a solid support.

43. The kit of claim 42, wherein the solid support is a microtiter plate.

44. The kit of any one of claims 27-29, further comprising a labeled probe or intercalating dye.

45. The kit of claim 44, wherein the labeled probe has a detectable moiety, a radioisotope, biotin a fluorescent moiety a flurophore, a chemical luminescent moiety or an enzymatic moiety.

46. The kit of claim 44, wherein the solid support is selected from the group consisting of polystyrene, derivatized polystyrene, a membrane, a latex bead, a glass bead, a silica bead, dextran bead, agarose bead, acrylic bead, a paramagnetic microsphere, a latex microsphere and a microtiter well.

47. The method of any one of claims 1-26, wherein the HIV is HIV-I.

48. The kit of any one of claims 27-46, wherein the HIV is HIV-I.