WO2003097088A2

WO2003097088A2 - Methods to detect, isolate, target and manipulate hiv-infected t cells

Info

Publication number: WO2003097088A2
Application number: PCT/US2003/015371
Authority: WO
Inventors: Alex Franzusoff
Original assignee: The Regents Of The University Of Colorado
Priority date: 2002-05-15
Filing date: 2003-05-15
Publication date: 2003-11-27
Also published as: WO2003097088A3; AU2003243243A1

Abstract

Disclosed is a method for detecting or isolating human immunodeficiency virus-infected T cells in a patient by detecting or isolating CD4+, CTLA4+ T cells from a patient sample. The method is generally applicable to a diagnostic assay for the detection and/or isolation of HIV-infected T cells in a patient sample; for the isolation of HIV virions from an individual patient T cells; for monitoring the impact of various anti-viral therapies on control of HIV infection in a patient, and for testing the efficacy of various HIV therapeutic drugs and protocols. Methods for reducing HIV virion assembly and proliferation in a patient are also described.

Description

METHODS TO DETECT, ISOLATE, TARGET AND MANIPULATE HIV-INFECTED T CELLS

Government Support This invention was made in part with government support under NIH Grant No.

AI34747, awarded by the National Institutes of Health. The government may have certain rights to this invention.

Field of the Invention The present invention generally relates to methods for detecting or isolating human immunodeficiency virus (HIN)-infectedT cells in patients, for diagnosing HIV-infection and monitoring therapy for HIV using the isolated cells, for identifying agents that inhibit HIV virion assembly and proliferation, to therapeutic agents that can be identified using such methods, and to the treatment of HIN-infected patients using such agents.

Background of the Invention HIV-1 virions that bud from the cell surface contain most, but not all, of the viral components encoded by the viral RΝA genome. Besides the two copies of the viral RΝA genome, the budded HIV- 1 virion contains structural (Gag), catalytic (Pol) and the accessory viral proteins Vpr and Νef, all encased within a membrane bilayer studded with trimers of the viral envelope glycoproteins gpl20 and gp41. The viral envelope is derived from the infected cell's plasma membrane, a process that inadvertently, as well as specifically, contributes cellular membrane proteins to the budded virions. These components constitute the immature virion that buds from the plasma membrane of infected cells. The biosynthesis of the various HIV-1 components is initiated in stages during the viral replicative cycle, which proceeds efficiently in activated, but not quiescent T cells. These stages are defined by HIV- 1 RΝA splicing and exit of the viral-specific messages from the nucleus. The extent to which HIV-1 RΝA is spliced dictates which HIN-encoded components will be produced. The first, or early, stage of viral protein production depends on the translation of doubly spliced messages whose expression yields the HIV-1 Tat, Rev and Νef proteins. Following a build-up of Tat and Rev proteins in the cell, the second, or intermediate, stage of HIV-1 production can begin. The Rev protein interrupts HIV-1 RΝA splicing and serves as an export factor to promote HIV- 1 RΝA exit from the nucleus. During the intermediate stage of viral replication, singly spliced messages are delivered to the cytoplasm for the translation of HIV-1 Env, Vpu, Vpr and Vif proteins. With further buildup of Rev protein in the cell, the final (or late) stage of viral replication can begin, since unspliced HIV transcripts can exit the nucleus. The unspliced messages direct the synthesis of the Gag and Gag-Pol proteins, as well provide the full-length HIV RNA genome to be incorporated into virions. The time between initiation of early stage viral production until the budding of assembled HIV-1 virions is separated by as much as 24 hours. Env protein produced during the intermediate stage of viral replication is present in the cell for several hours before initiation of the late stage of viral replication, when the RNA genome and the Gag and Gag-Pol proteins become available for virion production. Coordinating the timing of synthesis with the cellular localization of the different HIV-1 components during viral replication may be important to the regulation of HIV-1 virion assembly in T cells.

The organization of HIV-1 components within the virions is important for viral infectivity. Virion maturation is completed after budding from the cell and is accompanied by morphogenesis of the virion interior. A cone-shaped protein shell forms around the viral genome and the complement of structural, catalytic and accessory viral proteins. Viral protease (PR) activity is crucial for virion maturation, since the structural (p55 gag) and catalytic (p66 pol) protein precursors are cleaved within the immature virion after budding. The structural proteins of the mature HIV- 1 virion derived by cleavage of the HIV- 1 p55 Gag precursor protein include the matrix (pi 7), capsid (p24), nucleocapsid (p7), pi, p2 and p6 proteins. The catalytic proteins derived by cleavage of the Pol precursor protein include reverse transcriptase (RT), integrase (IN) and the viral PR protease. Proper folding within the virion, which is necessary for generating infectious particles, is dependent on how viral components encounter each other. Studies dissecting how virions are assembled have focused heavily on the role of Gag proteins. The literature describing Gag assembly in vitro and in a variety of cell types is extensive. HIV-1 Gag proteins can assemble by themselves to create viral-like particles (VLPs) that can bud from the cell surface. Many site-directed mutations have been generated to examine the contributions to VLP assembly of various domains in the Gag p55 precursor protein. These studies showed that the p 17 matrix (MA) subunit at the N- terminus of the p55 Gag precursor protein is important to virion assembly. The MA subunits constitute the protein lining underneath the lipid envelope of mature virions. Gag proteins are myristoylated at the N-terminus of the MA subunit. Myristoylation appears to stabilize Gag binding to lipid membrane bilayers. Interestingly, myristoylation is not essential for Gag binding to membranes in cells, as shown by analysis of myristoylation-defective Gag mutants. However, wild-type HIN-1 Gag becomes myristoylated, binds to membranes and multimerizes into NLPs that can bud from the plasma membrane - all in the absence of other HIV-1 components.

Additional interactions besides Gag multimerization are required for the proper assembly of HIV-1 virions. For instance, Vif appears to interact with Gag and Gag-Pol proteins for incorporating the RΝA genome during virion assembly. Despite a role for Vif in RΝA incorporation during virion assembly, Vif is not consistently incorporated into virions released from infected cells. The requirement for several cellular proteins to promote HIV-1 assembly has been noted, such as TsglOl, HP68, ubiquitin ligase and chaperone proteins. Therefore, HlV-encoded proteins (in addition to Gag), the viral RΝA and several cellular components are required for normal assembly of infectious HIN-1 virions.

Key to HIN-1 infectivity is the recruitment of the Env glycoproteins during virion assembly. The cytoplasmic tail of Env gp41 is essential for Env incorporation in budded virions. Mutations in the sequence of the Env gp41 cytosolic tail have been characterized that interfere with Env recruitment into the virion. HIV-1 Env mislocalization due to these gp41 tail mutations permits the pseudotyping of HIV virions, meaning that the membrane envelope proteins from other viruses, such as VS V-G, MuLV or Mo-MLV, can be substituted for HIV-1 Env in the budded virions. However, in order to pseudotype other viruses with HIN-1 Env, the HIV-1 Gag proteins must be provided. Therefore, the interaction between HIV- 1 Gag and Env is vital for assembling these two components together into infectious virions.

While it is clear that Gag binds to the gp41 cytoplasmic tail in budded virions, two different domains of the Gag precursor protein (pi 7 MA and p6), as well as the Nif protein, have been implicated for recruiting Env glycoproteins during virion assembly. At the plasma membrane, Env is found co-localized with Gag proteins in lipid rafts prior to virion budding but not randomly distributed around the cell surface. According to the previously held view of HIN virion assembly, Env proteins are delivered to the plasma membrane directly after synthesis by the constitutive secretory pathway. Env trafficking to the cell surface is independent of Gag delivery to the plasma membrane. In that model, lateral diffusion in the plasma membrane would be necessary for the Env and Gag proteins to encounter each other. Several problems arise from that model. Gag proteins, bound to the plasma membrane via its myristoyl groups, are restricted in their lateral diffusion due to their localization in lipid rafts. Furthermore, since Env protein synthesis precedes Gag protein production by several hours, then according to the previously held view, Env proteins would be present on the cell surface for several hours before the late stage of HIV-1 replication begins. However, the presence of Env proteins at the cell surface for protracted periods before Gag proteins and full length RNA become available to initiate virion assembly is an unfavorable scenario for HIV proliferation. For instance, circulating anti-gpl20 antibodies would have extended opportunities to bind Env on the surface of infected cells. The binding of anti-Env antibodies would inactivate Env function for subsequent infection of target cells. Furthermore, antibody crosslinking of Env on the surface of T cells has been suggested as a mechanism for triggering apoptosis of HlV-infected cells. Therefore, protracted surface exposure of Env could lead to T cell death before virions are released.

Other negative impacts from prolonged Env residence at the cell surface include the probability for Env binding to CD4, its normal receptor. CD4 binding to Env during infection triggers conformational changes in Env that would inactivate gpl20 function for infectivity if this event took place during virion assembly. Furthermore, the levels of CD4 protein could potentially reach stoichiometric levels with Env in budded virions, which is not observed. However, HIV- 1 has evolved mechanisms to reduce interference by CD4 binding during virion assembly. HIV-1 encodes two proteins, Nef and Npu, whose function is to reduce the surface density of CD4 on HIV-1 infected cells. HIN-1 Νef is responsible for down-regulating CD4 receptors already on the cell surface and HIN-1 Npu is suggested to retain nascent CD4 molecules in the ER, thus further reducing CD4 presentation on the cell surface.

Defining the mechanisms for assembling infectious virus in T cells is key to understanding the regulation of HIV-1 proliferation and can lead to methods for identifying agents useful for disruption of HIN virion assembly and proliferation for the treatment of patients. The biology of CD4⁺ T cells, compared to other cell types, greatly influences the process of HIV- 1 virion assembly. Hence, it is essential to use human CD4⁺ T cells to investigate the mechanisms of HIV-1 virion assembly.

Summary of the Invention One embodiment of the present invention relates to a method to detect or isolate human immunodeficiency virus (HJN)-infected T cells in a patient. The method includes the step of detecting or isolating CD4⁺ T cells that express cytotoxic T lymphocyte antigen 4 (CTLA4) in a T cell-containing biological sample from a patient who is infected with HIN, wherein T cells that express CD4 and CTLA4 are predicted to be infected with HIV. A biological sample can include, but is not limited to, a blood sample or a sample of peripheral blood mononuclear cells. In one aspect, the step of detecting or isolating comprises a method selected from flow cytometry, magnetic bead isolation, immunoaffinity chromatography, immunoassay, and radioimmunoassay. In another aspect, the step of detecting or isolating comprises fluorescent activated cell sorting. In another aspect, the step of detecting or isolating comprises sorting CD4⁺, CTLA4⁺ T cells using flow cytometry. In yet another aspect, the step of detecting or isolating comprises using flow cytometry to isolate CD4⁺ T cells from the sample, followed by detecting or isolating CD4⁺ cells that express CTLA4.

In yet another aspect of this embodiment of the invention, the step of detecting or isolating comprises contacting CD4⁺ T cells in the sample with an agent that selectively binds to CTLA4. Such an agent includes, but is not limited to, an anti-CTLA4 antibody, a protein that selectively binds to CTLA4, and a soluble B7 receptor. In one aspect, this step of detecting or isolating comprises detecting or isolating a labeling reagent that is attached to the agent or to an antibody that selectively binds to the agent. In another embodiment, the step of detecting or isolating comprises contacting CTLA4⁺ T cells in the sample with an agent that selectively binds to CD4. Such an agent includes, but is not limited to, an anti- CD4 antibody, a protein that selectively binds to CD4, a soluble majorhistocompatibility complex (MHC) class II protein, and a soluble HIV-gpl20 molecule. In one aspect, this step of detecting or isolating comprises detecting or isolating a labeling reagent that is attached to the agent or to an antibody that selectively binds to the agent. In another aspect of this embodiment of the invention the method further includes a step of detecting or isolating CD4⁺, CTLA4⁺ T cells that also express CD45RO and HLA- DR, wherein T cells that express CD4, CTLA4, CD45RO, and HLA-DR are predicted to be infected with HIV. In another aspect, the method further includes a step of detecting or isolating CD4⁺, CTLA4⁺ T cells that do not express or have low expression of a molecule selected from the group consisting of CCR7 and CD62L, wherein T cells that express CD4 and CTLA4 and that do not express or have low expression of CCR7 or CD62L are predicted to be infected with HIV. In yet another aspect, the method further includes a step of detecting or isolating CD4⁺, CTLA4⁺ T cells that also express CD45RO and HLA-DR and that do not express or have low expression of a molecule selected from the group consisting of CCR7 and CD62L, wherein T cells that express CD4, CTLA4, CD45RO, and HLA-DR, and that do not express or have low expression of CCR7 or CD62L, are predicted to be infected with HIN.

In one aspect, the method of the invention further comprises a step of detecting or isolating human immunodeficiency virions from the CD4⁺, CTLA4⁺ T cells. In yet another embodiment, the method further includes destroying CD4⁺, CTLA4⁺ T cells in the sample and returning the treated sample to the patient.

Yet another embodiment of the present invention relates to a method to monitor the efficacy of a treatment for human immunodeficiency virus (HIN)-infection in a patient. The method includes the steps of: (a) detecting or isolating CD4⁺, CTLA4⁺ T cells in a T cell- containing biological sample from a patient who is infected with and undergoing treatment for HIV; (b) detecting or isolating human immunodeficiency virions produced by the CD4⁺, CTLA4⁺ T cells in the sample; and (c) comparing the quantity or infectivity of human immunodeficiency virions from (b) to human immunodeficiency virions detected or isolated from CD4⁺, CTLA4⁺ T cells in a prior sample from the patient. In this method, a reduction in the number or infectivity of the human immunodeficiency virions in (b) as compared to the quantity or infectivity of human immunodeficiency virions in the prior sample indicates that the treatment is having a beneficial effect. The sample can include, but is not limited to, a blood sample or a sample of peripheral blood mononuclear cells.

In one aspect of this embodiment of the invention, step (c) comprises comparing the quantity of human immunodeficiency virions in (b) to the quantity of human immunodeficiency virions in the prior sample. In another aspect, step (c) comprises comparing the infectivity of human immunodeficiency virions in (b) to the infectivity of human immunodeficiency virions in the prior sample. In another aspect, the method includes measuring active virion assembly, wherein a reduction in virion assembly of the virions in (b) as compared to the virions in the prior sample indicates that the treatment is having a beneficial effect. In yet another aspect, the method includes measuring active virion proliferation, wherein a reduction in active virion proliferation of the virions in (b) as compared to the virions in the prior sample indicates that the treatment is having a beneficial effect. In yet another embodiment, the method further includes a step of testing virions isolated in (b) for their ability to infect a test culture of non-HIN infected T cells in vitro, as compared to the virions isolated from a prior sample, wherein a reduction in infectivity of the virions isolated in (b) indicates a positive effect of the treatment. The prior sample can include a sample collected from the patient prior to an administration of the treatment for HIN-infection, or after an administration of the treatment for HIN- infection. In one aspect, the method further includes detecting genetic mutations in the virions isolated in (b).

Another embodiment of the present invention relates to a method to monitor the efficacy of treatment for human immunodeficiency virus (HJN)-infection in a patient. The method includes the steps of: (a) detecting or isolating CD4⁺, CTLA4⁺ T cells in a T cell- containing biological sample from a patient who is infected with and undergoing treatment for HIN; and (b) comparing the number of CD4⁺, CTLA4⁺ T cells in (a) to the number of CD4⁺, CTLA4⁺ T cells in a prior biological sample from the patient, wherein detection of a reduction in the number of CD4⁺, CTLA4⁺ T cells in the sample as compared to in the prior sample indicates that the treatment for HIN is reducing the number of H-N-infected cells in the patient. In one aspect, step (b) of comparing further comprises comparing the number of CD4⁺, CTLA4⁺ T cells in the sample to a number of CD4⁺, CTLA4⁺ T cells in a normal control sample, wherein detection of a change in the number of CD4⁺, CTLA4⁺ T cells toward the number of CD4⁺, CTLA4⁺ T cells in the normal control sample indicates that the treatment for HIV is reducing the number of HlV-infected cells in the patient. The sample can include, but is not limited to, a blood sample or a sample of peripheral blood mononuclear cells.

Yet another embodiment of the present invention relates to a method to diagnose human immunodeficiency virus infection in a patient. The method includes the steps of: (a) detecting CD4⁺, CTLA4⁺ T cells in a T cell-containing biological sample from a patient who is suspected of being infected with HIV; (b) isolating the CD4⁺, CTLA4⁺ T cells from (a); and (c) detecting HIV in the cells from (b). In one aspect, step (c) of detecting comprises detecting human immunodeficiency virion infection and viral assembly and proliferation in the T cells isolated in (b). In another aspect, step (c) of detecting comprises detecting human immunodeficiency virions in the T cells isolated in (b) by in situ RNA hybridization.

Another embodiment of the present invention relates to a method to identify a regulatory compound that inhibits human immunodeficiency virus (HIV) virion assembly and proliferation. The method includes the steps of: (a) contacting a putative regulatory compound with a cell that expresses CTLA4 and that is infected with HIN; and (b) identifying regulatory compounds that disrupt the regulation by Env of the intracellular localization of the CTLA4 secretory granule within the cell. In one aspect, the regulatory compound is contacted with the cell intracellularly. In one embodiment, the cell is a CD4⁺ T cell.

In one aspect, the step (b) comprises identifying regulatory compounds that disrupt the inhibition by Env of transport of the CTLA4 secretory granule to the plasma membrane. For example, step (b) can include detecting cell surface CTLA4 expression, wherein an increase in CTLA4 cell surface expression after contact with the putative regulatory compound as compared to prior to or in the absence of contact with the compound indicates that the compound disrupts the inhibition by Env of transport of the CTLA4 secretory granule to the plasma membrane.

In another aspect, the step (b) comprises identifying regulatory compounds that prevent the transport of CTLA4 secretory granules to the plasma membrane.

In yet another aspect, step (b) comprises identifying regulatory compounds that induce transport to and expression of Env protein at the cell surface prior to the expression of other HIN proteins required for HIV virion assembly. For example, step (b) can include detecting cell surface expression of Env and the expression of at least one other HIV protein prior to and after contact with the putative regulatory agent, wherein detection of an increase in Env cell surface expression after contact with the compound as compared to prior to contact with the compound and as compared to expression of the other HIV protein indicates that the compound induces transport to and expression of Env protein at the cell surface prior to the expression of other HIV proteins required for HIV virion assembly. Such an other HIN protein can include, but is not limited to, Gag.

In another aspect, step (b) comprises identifying regulatory compounds that result in the release of an increased number of non-infectious virions from the cell after contact with the compound as compared to prior to or in the absence of contact with the agent.

In yet another aspect, step (b) comprises identifying regulatory compounds that disrupt the association of a cellular protein with the cytoplasmic tail sequence of Env in CTLA4 secretory granules, wherein the association sequesters CTLA4 secretory granules within the cell in the absence of the regulatory compound. For example, such a cellular protein can include, but is not limited to, a protein involved in the targeting and fusion or trafficking of intracellular granules to fuse with the plasma membrane.

Yet another embodiment of the invention relates to a method to identify a regulatory compound that inhibits human immunodeficiency virus (HIN) virion assembly and proliferation, the method including the steps of: (a) isolating CD4⁺ T cells that express cytotoxic T lymphocyte antigen 4 (CTLA4) in a T cell-containing biological sample from a patient who is infected with HIV, wherein T cells that express CD4 and CTLA4 are predicted to be infected with HIN; (b) contacting a putative regulatory compound for inhibition of HIV infection with the cells of (a); and (c) detecting regulatory compounds that reduce HIV virion assembly and proliferation in the cells of (a) after contact with the compound as compared to prior to or in the absence of contact with the compound. In one aspect, the step of detecting comprises measuring the quantity of human immunodeficiency virions released from the cells of (a) after contact with the compound, wherein a decrease in the quantity of human immunodeficiency virions released from the cells indicates that the compound is a regulatory compound for inhibition of HIV. In another aspect, the step of detecting comprises measuring the infectivity of human immunodeficiency virions released from the cells of (a) after contact with the compound, wherein a decrease in the infectivity of human immunodeficiency virions released from the cells indicates that the compound is a regulatory compound for inhibition of HIN. In another aspect, the step of detecting comprises measuring active virion assembly in the cells of (a), wherein a reduction in active virion assembly after contact with the compound indicates that the compound is a regulatory compound for inhibition of HIV. In yet another aspect, the step of detecting comprises measuring the number of T cells of (a) that are infected with actively proliferating HIV, wherein a decrease in the number of T cells that are infected with actively proliferating HIV after contact with the compound indicates that the compound is a regulatory compound for inhibition of HIV. In another aspect, the step of detecting comprises measuring death of the T cells, wherein an increase in the death of the T cells after contact with the compound indicates that the compound is a regulatory compound for inhibition of HIV.

Yet another embodiment of the invention relates to a method to inhibit human immunodeficiency virus (HIV) virion assembly and proliferation, comprising contacting a population of cells containing HIV-infected CD4⁺ T cells with a regulatory compound that disrupts the regulation by Env of the intracellular localization of the CTLA4 secretory granule within the cell. In one aspect, the regulatory compound is a modified Gag protein that causes the premature delivery of Env and CTLA4 containing granules to the surface. For example, the modified Gag protein can include, but is not limited to, an isolated Gag-Tat fusion protein. In one aspect, the modified Gag protein is a portion of Gag protein sufficient to bind to Env and fused to a Tat protein. In another aspect, the regulatory compound is administered to an HIV-infected patient in vivo.

Yet another embodiment of the invention relates to a method to target an HIV- infected T cell for destruction, comprising contacting a population of cells containing HIV- infected CD4⁺ T cells with an agent that activates CTLA4 expressed by a CD4⁺ T cell in a manner effective to induce apoptosis of the cell. In one aspect, the agent includes, but is not limited to, an anti-CTLA4 antibody, an agent that selectively binds to and activates CTLA4, or a soluble B7 receptor.

Another embodiment of the invention relates to another method to target an HIV- infected T cell for destruction, comprising contacting a population of cells containing HIV- infected CD4⁺ T cells with a complex comprising: (1) a first agent that targets the complex to cells expressing CTLA4; and (2) a second agent selected from the group consisting of: a toxin, an anti-viral agent, an agent that induces apoptosis in the cell, and an agent that antagonizes the activity of Env, wherein the first and second agents are complexed together. The first agent can include, but is not limited to, an anti-CTLA4 antibody, an agent that selectively binds to CTLA4, and a soluble B7 receptor. Another embodiment of the present invention relates to a method to identify a regulatory compound that inhibits human immunodeficiency virus (HIV) virion assembly and proliferation, comprising contacting an immobilized Env protein or cytoplasmic portion thereof with a Gag/cellular docking protein in the presence and absence of a putative regulatory compound, wherein an inhibition of the interaction between the Env protein or cytoplasmic portion thereof and the Gag or cellular docking protein in the presence of the putative regulatory compound as compared to in the absence of the putative regulatory compound, indicates that the putative regulatory compound inhibits HIV virion assembly.

Detailed Description of the Invention

This invention generally relates to the present inventor's elucidation of a specific role for the T cell costimulatory molecule, cytotoxic T lymphocyte antigen 4 (CTLA4), in the susceptibility of cells to HIV infection, including elucidating the discrete roles of CTLA4 and CD28 in determining the outcome of HIV infectivity. The present invention also relates to the discovery by the present inventor of a mechanism by which Env regulates secretory granules containing CTLA4 in order to delay transport of Env to the cell surface until a time when virion assembly is favorable and initiated. More specifically, the present inventor has shown that delivery of Env to the cell surface of HIV-infected cells is accompanied by CTLA4 surface expression, as would be observed if both molecules reside in the same intracellular granule. The present inventor's studies (described in detail in the Examples section) showed that Env transits from the ER to the Golgi, and then transits directly to the CTLA4-containing granules, without first trafficking to the cell surface. Without being bound by theory, the present inventor believes that within the CTLA4 secretory granules, Env associates with one or more cellular proteins and sequesters the secretory granules within the cell until a time when it is beneficial for virion assembly to transit to the cell surface. These results indicate that a novel regulatory event in control of Env trafficking to the cell surface and in HIV viral assembly may provide an additional regulatory step in HIV virion assembly.

More specifically, the intracellular trafficking of HIN-1 Env in CD4+ cells is crucial to the assembly and budding of infectious virus. Although HIV buds from the surface of infected cells, the present inventor shows herein that Env is not constitutively delivered to the cell surface as has been previously assumed. Instead, Env traffics from the Golgi to intracellular granules of the regulated secretory pathway of human T cells. These intracellular granules contain the immunomodulatory protein, CTLA4, which is normally recruited to the cell surface following T cell activation (Linsley et al., (1996) Immunity 4:535-43; Alegre et al., (1996)J Immunol. 157:4762-70). Delivery of Env to the cell surface of HIV-infected cells is accompanied by CTLA4 surface expression, as would be expected if both molecules reside in the same intracellular granule. The results from the inventor's studies reveal that a novel regulatory event in control of Env trafficking to the cell surface and in HIV viral assembly can provide an additional regulatory step in HIV virion assembly. The observations that Env does not constitutively reside at the cell surface of HIN- infected T cells has been previously described (Willey et al., (1988) Proc. Natl. Acad. Sci. U S A 85:9580-4). However, the present invention provides the elucidation of the intracellular compartments where Env is found during HIV-1 infection. This added definition is possible because most studies of intracellular Env trafficking have been performed in cell types, such as fibroblasts, that are not normal targets for HIN infection (Earl et al., (1991)J. F/ro/.65:2047-55; SanJose etal., (1997) Virology 239:303-14; Otteken et al., (1996) J. Virol. 70:3407-15). The organization of the secretory pathway branches in T cells and macrophages contributes to the distinctive itinerary of Env trafficking in those cells. Hence, defining the intermediates in the trafficking pattern of Env in HIN-infected T cells is crucial to understanding the regulation of the assembly and budding of nascent HTV virions.

Rather than being delivered directly from the Golgi to the cell surface, the Env protein is diverted into granules of the regulated branch of the secretory pathway (see Examples). This result supports observations that gpl60 cleavage is not dependent on the furin protease, which functions in the constitutive branch of the secretory pathway (Ohnishi et al., (1994) J Virol. 68:4075-4079; Molloy et al., (1999) Trends Cell Biol. 9:28-35). Rather, Env is cleaved by the PC6 protease which inhabits the regulated secretory pathway (Hu et al., submitted to Proc. Natl. Acad. Sci. USA). Therefore, Env is sorted into the regulated pathway, where its delivery to the cell surface would depends on specific signals, which influences the timing of viral assembly and budding. The results of this study indicate that Env transits from the ER to the Golgi, and then transits directly to the CTLA4-containing granules compartment, without first trafficking to the cell surface. This conclusion is based on the failure to uptake gpl20 antibody from the cell surface (see Examples), and the absence of Env in membrane fractions containing endosomes and plasma membrane (see Examples). In this respect, Env trafficking to the CTLA4-containing granules resembles that of Fas ligand, which is transported directly to thesegranules withouttransitingtothe cell surface (Blottetal.,. (2001)J CellSci. 114:2405- 2416). The trafficking itineraries of Env and Fas ligand are therefore distinct from that of CTLA4, which is endocytosed from the cell surface to the intracellular granules (Iida et al., (2000) J. Immunol. 165:5062-8; Linsley et al., (1996), supra; Alegre et al., (1996), supra; Chuang et al., (1997) J. Immunol. 159:144-51; Blott et al.,. (2001), supra; Barrat et al., (1999) Proc. Natl. Acad. Sci. USA 96:8645-50). Sorting Env from the Golgi to the CTLA4- containing granules seems to be an inherent property of the protein since identical results were observed whether Env was expressed by recombinant vaccinia virus or by HIV-1. Hence, more than one pathway exists for the delivery of molecules to the intracellular CTLA4-containing granules. Without being bound by theory, the present inventor believes that there are several reasons for concluding that HIV, by choosing to proliferate in T cells, evolved multiple mechanisms to support the production of infectious virions. HIV-1 replication is dependent on maintaining T cells in an activated state. HIN must interfere with the strategies that T cells normally use to attenuate the signaling for T cell activation, such as control of CTLA4 surface presentation. Furthermore, the lifetime ofactivated T cells is carefully regulated. T cells are more apt than other cell types to undergo apoptosis rather than prolong the survival of activated T cells which may be dangerous to the host if damaged. Therefore it is reasonable to conclude that HIN evolved a mechanism for retaining Env intracellularly until all the components for assembling virions were produced. The mechanism elucidated by the present inventor redirects the emphasis of the previously held view that HIN virion assembly is entirely conducted at the cell surface. Instead, the present inventor's observations that Env is retained intracellularly in regulated secretory granules provides the key to a paradigm where Gag (possibly together with other HIN components) binds to Env on the intracellular CTLA4-containing granule. Gag-Env binding is followed by the delivery of CTLA4, and the secretory granules harboring the Gag-Env complexes, to the cell surface. Now virion assembly can proceed before antibodies or CD4 can interfere with the process, and before the T cell can commit apoptosis. Therefore, the pre-packaging of HIN components, initiated intracellularly before delivery to the cell surface, serves to orchestrate efficient virion assembly, at the proper time in the viral replicative cycle.

The present inventor's research provides evidence as to what events during HIN-1 infection trigger Env delivery to the cell surface. Cell surface expression of CTLA4 increases during HIV protein production (see Examples) suggesting that CTLA4-containing granules are signaled to translocate to the plasma membrane at some late stage of HIV virus assembly. Because of their shared residence in granules, CTLA4 and Env are recruited concomitantly to the cell surface (see Examples). The trigger that drives CTLA4-containing granules to translocate to the cell surface is not provided solely by the Env protein, since increased CTLA4 expression at the cell surface is not observed when Env is the only HIV protein expressed by recombinant vaccinia virus. Nevertheless, T cell activation seems to be sufficient to induce cell surface expression of CTLA4 (Alegre et al., (1996), supra; Steiner et al., (1999) Clin. Expt. Immunol. 115:451-7) and gpl 20. Furthermore, when Env is finally delivered to the cell surface, its distribution is clustered in focused regions on the plasma membrane (see Examples), consistent with previously reported observations on the intracellular localization of the matrix protein (MA) of HIV-1 (Fais et al., (1995) Aids 9:329- 35). Since viral assembly depends on the recruitment of HIN-1 Gag protein, the binding of Gag protein to Env on the cytosolic surface of these intracellular CTLA4-containing granules is predicted to initiate the translocation of the granules to the cell surface (Ono et al., (2000) J. Virol. 74:2855-2866).

Without being bound by theory, the present inventor believes that intracellular Env storage in the CTLA4regulated secretory granules provides two benefits in support of HIV proliferation. First, a prolonged residence of Env at the cell surface would prematurely alert the immune system before productive virions would be assembled and released for proliferation. HIV-1 has apparently developed multiple mechanisms for escaping detection by the immune system. One example is the Νef-mediated down-regulation of MHC-1 proteins to prevent viral antigen presentation and cytotoxic T cell mediated destruction of HIV-infected cells (Scheppler et al., (1989) J Immunol. 143:2858-66; Collins & Baltimore (1999) Immunol. Rev. 168:65-74; Piguet et al., (2000) Nat. Cell Biol. 2:163-7). It would be similarly advantageous to reduce the probability of antibody-mediated detection of HIV- infected cells by limiting exposure time of Env at the cell surface until productive viral progeny can be rapidly assembled.

The second predicted benefit for Env retention in the regulated granules relates to the role of CTLA4 in attenuating activation signals of the infected T cells (Lee et al., (1998) Science 282:2263-6; Oosterwegel et al., (1999) Curr. Opin. Immunol. 11 :294-300). Since HIV proliferation is dependent on T cell activation, the cellular machinery co-opted to produce HIV virions may depend on maintaining cells in an activated state. Therefore, it may be important to prevent CTLA4 from attenuating T cell activation during HIV protein production. The timing of CTLA4 translocation to the cell surface maybe delayed by events related directly or indirectly to the presence of Env in the intracellular granules. However, since other regulatory molecules of the immune system may be present in the CTLA4- containing granules, further studies must be conducted to determine which molecules are involved in controlling HIN proliferation, and/or evasion of immune surveillance. The results provided by this study therefore suggest new avenues for research into the potential regulation of Env trafficking to the cell surface and of HIV virion assembly therapeutic disruption of HIV proliferation.

In resting T cells, CTLA4 binds to B7 proteins on antigen presenting cells (APCs), (which can be mimicked by crosslinking surface CTLA4 with anti-CTLA4 antibodies) and causes T cells to undergo cell-cycle arrest. On activated T cells, CTLA4 binding to B7 or crosslinking with anti-CTLA4 antibodies signals the T cells to undergo apoptosis. In the context of HIV replication, T cells must remain activated to support viral protein production. Therefore, premature CTLA4 surface presentation might trigger termination of the activation signals, and may cause HIV-infected T cells to commit suicide before virion production would be completed. Therefore, it is reasonable to conclude that HIV-1 has evolved mechanisms to prevent the premature delivery of CTLA4-containing regulated granules to the surface of infected cells. The present invention discloses methods which take advantage of these mechanisms for diagnostic and therapeutic purposes, as well as for compound screening.

CTLA4 (CD152) is an important T cell regulatory protein that acts as a negative regulator of the immune response when expressed on the cell surface. Cell surface presentation of CTLA4 is tightly controlled in T cells, as might be expected for a signal that is used to terminate immune activation. The CTLA4-containing granules are directed to fuse with the plasma membrane about 24-36 hours after T cells become activated to terminate the activation signals.

Antigen-specific stimulation of T cells requires two signals for activating the immune response. The binding of the T cell receptor (TCR) to its cognate MHC protein on the surface of antigen-presenting cells (APCs) represents one half of the signal for T cell activation. The second signal for T cell activation depends on the binding of CD28 proteins on the surface of T cells to B7 receptors on APCs. Therefore, both signals arising from the binding of TCR and of CD28 are required to trigger T cell activation. The disruption of one of the two activation signals attenuates the immune response.

The delivery of CTLA4 to the surface of activated T cells displaces CD28 binding to the B7 receptors on APCs. The substitution of CTLA4 for CD28 thus interferes with the dual signals required to maintain T cell activation. As a result, CTLA4 presentation on the cell surface attenuates the activated response. Three clinical studies have been published showing that CTLA4 expression is enhanced on CD4⁺ T cells during HIV infection (Steiner et al., 1999, Clin. Exp. Immunol. 115:451-457; Leng et al., 2002, AIDS 16:519-529; Leng et al., 2001, J. Acquired Immune Deficiency Syndrome 27:389-397). Finding no change in the expression of CTLA4 in CD8⁺ T cells in HIV-infected patients, however, these authors suggest that expression of CTLA4 on CD4⁺ T cells may be a general feature of the human immune system and not affected by disease states. Alternatively, the authors note that an increase in CTLA4 expression and a decrease in CD28 expression by T cells may represent two means of achieving a decrease in T cell responsiveness. The authors question whether blockade of CTLA4 might aid in the reversal of immune dysfunction in HIV-infected patients. However, this discussion was speculative, as these authors did not describe a particular mechanism or reason for the increased expression of CTLA4. Moreover, blockade of CTLA4 after it reaches the cell surface would be too late to avoid virion assembly, as indicated by the present invention.

Without being bound by theory, the present inventor believes that using isolation techniques such as flow sorting for CD4 and CTLA4, HIN-1 infected T cells can be enriched greater than 50-fold from a biological sample, such as peripheral blood mononuclear cells. It is known that there is a direct correlation between the number of HIV-infected T cells in a patient and the viral load in the patient. The detection and isolation method of the present invention is the first method known to the inventor that can identify individually infected T cells from a patient by using a cellular marker rather than viral markers. In addition, this method can be used to further isolate the virus itself from individually infected T cells. This is significant, because viral markers mutate at a great rate during infection, such that no single viral detection system will be as effective for detecting viral infection in different patients, or for isolating virus from an individual patient. This method is useful, therefore, as a diagnostic assay for the detection and/or isolation of HIV-infected T cells in a patient sample, and particularly, T cells that are actively replicating new virus; for the isolation of HIN virions from an individual patient T cells; and/or for monitoring the impact of various anti-viral therapies, including immune-based therapies, on control of HIV infection in a patient. Further, the method can be used to test the efficacy of various anti-viral drugs, immune-based anti-viral therapies, vaccines, antibodies, etc., on the T cells of an individual patient in vitro, since HIV-infected T cells can be readily detected and isolated using the method. Additionally, this method can be used for any research or other therapeutic or diagnostic application, wherein it is desirable to detect and/or isolate HIV-infected cells, regardless of whether the virus is mutating or has mutated. Other methods derived from the discovery of the present invention (e.g., methods to identify therapeutic agents) will be discussed below. Accordingly, one embodiment of the present invention relates to a method to detect or to isolate human immunodeficiency virus (H-V)-infected T cells in a patient or sample containing HIN-infected cells. The method includes the steps of detecting or isolating CD4⁺ T cells that express cytotoxic T lymphocyte antigen 4 (CTLA4) in a T cell-containing biological sample from a patient who is infected with HIV. T cells that express CD4 and CTLA4 are predicted to be infected with HIV. Essentially, this method includes any steps and the use of any reagents by which one can detect and/or isolate individual T cells within a population of cells which are infected with HIV, using the cellular markers (i.e., CD4 and CTLA4). In one embodiment, the method can further include a step of detecting and/or isolating virus from an individually infected T cell that is detected and isolated using the method described above. The method of the present invention involves the use of a biological sample containing H-N-infected T cells. The biological sample can be a cell sample, a tissue sample and/or a bodily fluid sample collected from a patient that contains T cells. According to the present invention, a cell sample is a specimen of cells, typically in suspension or separated from connective tissue which may have connected the cells within a tissue in vivo, which have been collected from an organ, tissue or fluid by any suitable method which results in the collection of a suitable number of cells for evaluation by at least one of the methods of the present invention. A cell sample can include a previously isolated and/or cultured cell sample, such as an isolated culture of T cells or a culture of cells that has been enriched for a given cell type, such as T cells. Methods for enriching for T cells in a cellular sample are well known in the art. Preferred cell samples include, but are not limited to, isolated peripheral blood mononuclear cells and enriched cultures of T cells. A tissue sample, although similar to a cell sample, is defined herein as a section of an organ or tissue of the body which typically includes several cell types and/or cytoskeletal structure which holds the cells together. One of skill in the art will appreciate that the term "tissue sample" may be used, in some instances, interchangeably with a "cell sample", although it is preferably used to designate a more complex structure than a cell sample. A tissue sample can be obtained by a biopsy, for example, including by cutting, slicing, or a punch. A bodily fluid sample is a fluid excreted or secreted by a tissue or organ to be evaluated or contained within a bodily vessel. A bodily fluid is obtained by any method suitable for the particular bodily fluid to be sampled. A preferred bodily fluid to sample includes blood.

According to the present invention, the step of detecting cells that are CD4⁺ and CTLA4⁺ (i.e., that express CD4 and CTLA4) can be performed using any known method for detecting these cellular markers. For example, suitable techniques for the detection of CD4 or CTLA4 expression include, but are not limited to, measurement of transcription of the RNA encoding the protein and measurement of the protein, including measurement of the expression of the protein on the surface of a cell.

For RNA expression, methods of detection include but are not limited to: extraction of cellular mRNA and northern blotting using labeled probes that hybridize to transcripts encoding all or part of a nucleic acid sequence encoding the target protein; in situ RNA hybridization; amplification of mRNA expressed from a gene encoding the target protein using sequence-specific primers and reverse transcriptase-polymerase chain reaction (RT- PCR), followed by quantitative detection of the product by any of a variety of means; extraction of total RNA from the cells, which is then labeled and used to probe cDNAs or oligonucleotides encoding all or a portion of the target protein, arrayed on any of a variety of surfaces. The term "quantifying" or "quantitating" when used in the context of quantifying transcription levels can refer to absolute or to relative quantification. Absolute quantification may be accomplished by inclusion of known concentration(s) of one or more target nucleic acids and referencing the hybridization intensity of unknowns with the known target nucleic acids (e.g. through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of hybridization signals between two or more genes, or between two or more treatments to quantify the changes in hybridization intensity and, by implication, transcription level.

Methods to measure protein expression levels include, but are not limited to: western blotting, immunocytochemistry, flow cytometry or other immunologic-based assays; assays based on a property of the protein including but not limited to ligand binding or interaction with other protein partners. Binding assays are also well known in the art. For example, a BIAcore machine can be used to determine the binding constant of a complex between two proteins. The dissociation constant for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip (O'Shannessyetal. Anal. Biochem.212:457-468 (1993); Schuster etal., Nature 365:343-347 (1993)). Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme linked immunoabsorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UN absorption, circular dichrosim, or nuclear magnetic resonance (ΝMR).

Methods of isolating cells that express CD4 or CTLA4 include, but are not limited to, flow cytometry (including fluorescent activated cell sorting), magnetic bead isolation, and immunoaffinity chromatography. Cells expressing CD4 and CTLA4 can be isolated in sequential steps, such as by first isolating cells expressing either one of CD4 or CTLA4 and then isolating from that population cells that express the other molecule. One method of detecting and/or isolating cells according to the present invention includes contacting cells in the sample with a compound (agent, reagent, molecule) that selectively binds to either CD4 or CTLA4 under conditions whereby CD4 or CTLA4 expressed by the T cells, respectively, will bind to the compound. CD4⁺ and CTLA4⁺ T cells can be identified using reagents or methods in the art suitable for selecting and/or detecting CD4 or CTLA4 expression (e.g., cell sorting, antibody labeling and/or isolation). Compounds that selectively bind to CTLA4 can be any reagent that can selectively bind to CTLA4 such that cells expressing CTLA4 can be positively identified (detected) and in some embodiments, isolated. Such reagents include, but are not limited to, antibodies, antigen binding fragments, or any other protein, peptide or small molecule binding partner, that selectively binds to CTLA4, including soluble natural ligands of CTLA4 (e.g., a soluble B7 receptor). Compounds that selectively bind to CD4 can be any reagent that can selectively bind to CD4 such that cells expressing CD4 can be positively identified (detected) and in some embodiments, isolated. Such reagents include, but are not limited to, antibodies, antigen binding fragments, or any other protein, peptide or small molecule binding partner, that selectively binds to CD4, including soluble natural ligands of CD4 (e.g., soluble maj orhistocompatibility complex (MHC) class II protein, or a soluble HIN-gp 120 molecule). Other means of detecting CD4 or CTLA4 expression will be apparent to those of skill in the art and the present method is not limited solely to protein detection methods. The conditions under which the cells are contacted with a reagent for identifying CD4 or CTLA4, such as by mixing or combining, are any suitable conditions in which the compound will bind to the CD4 or CTLA4 molecule if it is expressed by a T cell.

According to the present invention, the phrase "selectively binds to" refers to the ability of an antibody, antigen binding fragment or binding partner (e.g., protein, soluble ligand, etc.) used in the present invention to preferentially bind to specified proteins (e.g., to CD4 or CTLA4). More specifically, the phrase "selectively binds" refers to the specific binding of one molecule to another (e.g., an antibody, fragment thereof, or binding partner to CD4 or CTLA4), wherein the level of binding, as measured by any standard assay (e.g., an immunoassay), is statistically significantly higher than the background control for the assay. For example, when performing an immunoassay, controls typically include a reaction well/tube that contain antibody or antigen binding fragment alone (i.e., in the absence of antigen), wherein an amount of reactivity (e.g., non-specific binding to the well) by the antibody or antigen binding fragment thereof in the absence of the antigen bound by the antibody is considered to be background. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA), immunoblot assays, etc. Cells that express CD4 or CTLA4 can be identified and/or isolated by detecting the reagent/compound that is bound to CD4 or CTLA4, such as by using a labeled reagent (or compound that can be detected in combination with any suitable method (flow cytometry, immunoassay, etc.) for detection of the reagent, indicating the expression of CD4 or CTLA4. In one embodiment, an antibody that selectively binds to the CD4- or CTLA4-binding agent can be used to identify the reaction. Such an antibody can be labeled as described below. Detectable labels suitable for use in the present invention include any reagent or composition detectable by spectroscopic, photochemical, biochemical, immunochemical, magnetic, electrical, optical or chemical means. Useful labels in the present invention include, but are not limited to, biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads.TM.), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels. Means of detecting such labels and/or using such labels to isolate cell populations are well known to those of skill in the art. Therefore, the compound that binds to CD4 or CTLA4 can be used to isolate the CD4- or CTLA4-expressing T cells (e.g., by cell sorting, including FACS, affinity isolation techniques, etc.). Reagents useful for detection and/or isolation of CD4 or CTLA4 expressing cells are well known in the art and are publicly available.

In one embodiment of the invention, the step of detecting or isolating is performed using a method that includes, but is not limited to: flow cytometry, magnetic bead isolation, immunoaffinity chromatography, immunoassay, and radioimmunoassay.

In another embodiment of the invention, the method includes an additional step of detecting one or more markers that associate CTLA4 expression with HIV infection. While HIN-1 is capable of infecting naive as well as memory CD4⁺ T cells, HIV-l replicates preferentially in activated CD45RO⁺ CD62L¹⁰ CCR7 HLA-DR⁺ T_EM cells rather than in quiescent naive or memory T cell populations (Lawn et al., (2001) Clin Microbiol Rev 14:753-77; Harari et al., (2002) Blood 100: 1381-7; Chun et al., (1997) J Virol 71 :4436-44; Ostrowski et al., (1999) J Virol 73:6430-5; Douek et al. (2002) Nature 417:95-8). Recent evidence has shown that HIV-infected memory T cells account for 0.1-0.2% of all CD4⁺ T cells in blood, regardless of whether the infected individuals received anti-retroviral therapy (Douek et al. (2002), supra). The reasons that HIN proliferates more effectively in memory CD45RO⁺ T cells are that the reverse transcription of HIN RΝA to DΝA needed for integration into the genome is compromised in CD45RA⁺ naive T cells. Furthermore, T cell activation drives HJN-LTR promoter-dependent expression of the viral genes. Therefore, the viral life cycle is more likely to proceed in activated memory T cell populations than in quiescent naive cells. This preference is exacerbated by the pro-inflammatory signaling that stems from the opportunistic infections that occur in HIV-infected individuals.

The activation state of the immune system impacts the pathogenesis of HIV-1 infection and AIDS disease progression. The pro-inflammatory environment in HIV-infected individuals spurs the immune system into a state of heightened, or "chronic" immune activation (Lawn et al., (2001), supra; Harari et al., (2002), supra; Leng et al., (2001) J Acquir Immune DeficSyndr 21 -.389-91; Leng etal., (2002) Aids 16:519-29). HIV-1 -infected individuals exhibit elevated blood levels of the soluble factors that represent surrogate markers of immune activation, including tumor necrosis factor (TΝF)-α, TNF-α receptor II, soluble IL-2 receptor (sIL-2R), neopterin (reflecting IFN-γ activation of macrophages), CD23 (reflecting IL-4 activity) and β2-microglobulin (Fauci, A. S. (1993) Science 262:1011-8; Fahey, J. L. (1998) ClinDiagn Lab Immunol 5: 597-603). Paradoxically, the heightened state of immune activation resulting from chronic HIV infection, while essential for mounting an effective host immune response against pathogens, supports HIV replication in infected individuals. As a result, the ratio of CCR7^" T_EM to CCR7⁺ T_CM cells is increased in HIN- infected individuals, as might be expected in response to chronic immune activation.

Of significance for this invention is that chronic immune activation in HIV-infected individuals is believed to result in abortively activated T cells that are "anergic" and unable to contribute effective immune responses against HIV proliferation (Lawn et al., (2001), supra; Harari et al., (2002), supra; Leng et al., (2001), supra; Leng et al., (2002), supra; Fauci, A. S. (1993), supra; Plaeger et al., (1999) Clin Immunol 90:238-46; Fahey, J. L. (1998), supra). Disease progression to AIDS, which stems from depletion of functional CD4⁺ T cells, is attributed to multiple causes, including: i) specific virus-induced cell death; ii) widespread activation-induced loss of the memory (CD45RO⁺) T cell pool; and iii) impaired renewal of the naive (CD45RA⁺) T cell pool (Lawn et al., (2001), supra). Therefore, while a significant percentage of CD4⁺ CCR7^" T_EM cells appear to be activated during HIV infection, only a subset of these cells are productively activated through T cell receptor signaling in response to antigen exposure, while the remaining cell population is unproductively activated as a consequence of the pro-inflammatory environment (Lawn et al., (2001), supra; Douek et al. (2002), supra; Leng et al., (2001), supra; Fauci, A. S. (1993), supra; Fahey, J. L. (1998), supra; Betts et al., (2001) J Virol 75: 11983-91). For example, in one aspect of the invention, the includes a step of detecting or isolating CD4⁺, CTLA4⁺ T cells that do not express CCR7 and/or have low expression of CD62L, wherein T cells that express CD4, CTLA4, and CD45RO and HLA-DR are predicted to be infected with HIV.

Once one has used the novel discovery by the present inventors to detect and isolate

HIN-infected cells from a patient sample (i.e., identified by expression of CD4 and CTLA4), the cells can be used in a variety of different valuable protocols. For example, one can destroy the CD4⁺, CTLA4⁺ T cells ex vivo and then return the remaining cells in the sample to the patient (e.g., using extracorporeal phoresis systems). One can additionally use the isolated T cells in diagnostic assays, patient monitoring, drug efficacy testing, drug screening methods, characterization of virions from the individual patient (e.g., by genetic testing or infectivity testing), or any other methods of using isolated HIN-infected T cells. An advantage of the invention described herein is that one can now isolate individually infected T cells before they produce virions in vivo. By allowing the cells to then proceed with HIV assembly and release after sorting for the CD4⁺, CTLA4⁺ cells (and since it is not necessary to permeabilize the cells to determine which cells are HIV-infected, thus destroying their ability to function post-sorting), the researcher or clinician will be able to monitor how many virions are released from a known number of T cells and compare this data to untreated T cells (preferably from the same patient, before administration of drugs, or at different times after drug administration). This is, for example, an effective way to monitor the pharmacokinetics of drug activity in the body. Furthermore, the virions that are released from the treated and untreated cells may be quantified and tested for their relative ability to infect a test culture of non-HIV infected T cells in an infectivity assay. This will reveal to what extent the vitality of virions produced by treated cells is impacted by treatment, or by mutations in response to the treatment. For instance, the current drugs AZT, ddl, ddC, etc., put selective pressure on HIN-infected cells to make mutations in the reverse transcriptase in order to 'bypass' the drugs. T cells infected with these mutant genomes may yield virions whose infectivity is affected by the drug treatment, so that the same number of budded virions may exhibit a shallow or steep inhibition of vitality. This is only one of the benefits of being able to examine directly the virions released from T cells, as opposed to examining virions that are circulating. The latter process may under- or over-represent the profile of virions in a patient's blood. For instance, the virions circulating in the blood may have poorer ability to infect cells, compared to siblings from the same cell that immediately bind and infect new cells before they can be isolated from the blood. Hence, by sampling virions in the blood, one may get a gross misrepresentation of the impact of a drug on virion vitality, whereas the present invention will be invaluable for providing accurate information about the virions in a given patient. Therefore, in one embodiment of the invention, the method includes a step of detecting or isolating human immunodeficiency virus (HIN) virions from the T cells that are detected or isolated as described above. Detection of HIN virions produced by T cells can be performed using a variety of techniques known in the art, and include detection of the HIN genome integrated into the host genome and more preferably, detection of HIV RΝA (to detect cells that are actively replicating the virus), as well as detection of HIV proteins (e.g., either intracellular or after viral assembly and release from the cell). Methods of detecting RΝA and protein expression are well known in the art and have been described in general above. In one aspect, HIV is detected by in situ RΝA hybridization. In another aspect, HTV is detected by the binding of an antibody against one or more HIV proteins (e.g., gpl20). Reagents useful in detecting viral DΝA, RΝA and proteins can also be used to isolate the virions from the cell or cell culture. Methods for isolating HIV virions include, but are not limited to, centrifugation, use of soluble CD4-Ig fusion protein to bind to virions (Landau et al., (1988) Nature 334: 159-162), or use of soluble CD4 in a capture ELISA.

Another embodiment of the present invention relates to a method to monitor the efficacy of a treatment for human immunodeficiency virus (HIN)-infection in a patient. The method includes the steps of: (a) detecting or isolating CD4⁺, CTLA4⁺ T cells in a T cell- containing biological sample from a patient who is infected with and undergoing treatment for HIV; (b) detecting or isolating human immunodeficiency virions produced by the CD4⁺, CTLA4⁺ T cells in the sample; and (c) comparing the quantity or infectivity of human immunodeficiency virions from (b) to human immunodeficiency virions detected or isolated from CD4⁺, CTLA4⁺ T cells in a prior sample from the patient, wherein a reduction in the number or infectivity of the human immunodeficiency virions in (b) as compared to the quantity or infectivity of human immunodeficiency virions in the prior sample indicates that the treatment is having a beneficial effect. The prior sample can be taken at any one or more suitable time points, including before a first administration of a given therapeutic protocol, drug or regimen, and at various time points before and after the first and subsequent administrations of the therapeutic protocol. The method can be used to quickly and effectively assess the HIV-infected T cells from a single patient at any given time before, during and/or after therapy for HIV or an associated condition. The prior sample can also include a sample of non-HIN-infected T cells from the patient, which provides another type of control (e.g., an autologous "background" control). A prior sample can also include a T cell control from a non-HIV-infected patient (e.g., a different patient), or from a population of normal (non-infected) controls, or a control value can be predetermined from normal control data collected over time. The comparison of a test sample to another sample, and particularly an autologous sample, is a valuable and straightforward means of directly evaluating the efficacy of a given drug or therapeutic regimen for HIN treatment (or for determine any effect of a treatment for an associated or other condition on HIV-infection) in a specific patient throughout the treatment process. The clinician can use information derived from these assays to adjust, change, maintain or even cease the therapy for the individual patient. In this embodiment of the invention, CD4⁺, CTLA4⁺ T cells are detected or isolated as described above. In addition, human immunodeficiency virions produced by the CD4⁺, CTLA4⁺ T cells in the sample are detected or isolated as described above. In the step of comparing, the method can include the step of comparing the quantity of human immunodeficiency virions in the test sample to the quantity of human immunodeficiency virions in the prior sample or determining the infectivity of human immunodeficiency virions in the test sample to the infectivity of human immunodeficiency virions in the prior sample. For example, one can measure active virion assembly by the test cells, wherein a reduction in assembly of the virions in the test sample as compared to the virions in the prior sample indicates that the treatment is having a beneficial effect. In another aspect, one can measure active virion proliferation (e.g., viral budding and release from the cells), wherein a reduction in active virion proliferation of the virions in the test sample as compared to the virions in the prior sample indicates that the treatment is having a beneficial effect. One can also perform a genetic analysis of virions isolated by the present method to look for mutations in the virions, such as might occur in response to therapy with a given drug. In this embodiment, cells are typically isolated from the patient before viral assembly and release has occurred in vivo. The cells can then be cultured in vitro and monitored for viral assembly and release using any of the analyses described herein. In vitro methods for culturing HIV- infected T cells are well known in the art.

Virion quantities are typically measured using any suitable method for quantifying the amount of virions released from a cell, and include quantification by p24 Gag protein ELISAs, or by copies of the HIV RNA genome using RT-PCR. Virion infectivity can be measured, for example, by testing virions isolated from the test sample for their ability to infect a standardized test culture of non-HIN infected human T cells in vitro, known as TCID₅₀ (T cell infectivity dose), as compared to the virions isolated from a prior sample, wherein a reduction in infectivity of the virions isolated in the test sample indicates a positive effect of the treatment.

Another embodiment of the invention is a method to monitor the efficacy of treatment for human immunodeficiency virus (HIN)-infection in a patient, which includes the steps of: (a) detecting or isolating CD4⁺, CTLA4⁺ T cells in a T cell-containing biological sample from a patient who is infected with and undergoing treatment for HIV; and (b) comparing the number of CD4⁺, CTLA4⁺ T cells in (a) to the number of CD4⁺, CTLA4⁺ T cells in a prior biological sample from the patient, wherein detection of a reduction in the number of CD4⁺, CTLA4⁺ T cells in the sample as compared to in the prior sample indicates that the treatment for HIN is reducing the number of HIN-infected cells in the patient. In this embodiment, step (a) is performed as described previously herein. The detected or isolated HIV-infected cells are then evaluated simply by comparing the number of HIV-infected cells isolated from the patient sample as compared to the prior sample. Alternatively, or in addition, the number of HIN-infected cells in the test sample can be compared to a normal control sample, such as a predetermined average number of CD4⁺, CTLA4⁺ T cells in a normal control sample (e.g., from a non-HIN-infected patient), wherein detection of a change in the number of CD4⁺, CTLA4⁺ T cells toward the number of CD4⁺, CTLA4⁺ T cells in the normal control sample indicates that the treatment for HIN is reducing the number of HIV-infected cells in the patient toward that of a non-infected patient (e.g., the background number).

Yet another embodiment of the invention relates to a method to diagnose human immunodeficiency virus infection in a patient, including the steps of: (a) detecting CD4⁺, CTLA4⁺ T cells in a T cell-containing biological sample from a patient who is suspected of being infected with HIV; (b) isolating the CD4⁺, CTLA4⁺ T cells from (a); and (c) detecting HIV in the cells from (b). In this method, steps (a) and (b) are performed as previously described herein. The step of detecting preferably uses a method that detects not only the presence of virus in the T cell, but also active production of virus by the cell (e.g., viral production, assembly, proliferation), such as by detecting viral RΝA or viral proteins in the cell. For example, the method of detecting can be performed by in situ RΝA hybridization, although other suitable methods will be known in the art and some have been described above.

The terms "diagnosis" or "diagnosing" refer to the identification of a disease or condition (e.g., HIN-infection) on the basis of its signs and symptoms or a diagnostic marker (e.g., expression of CD4 and CTLA4). As used herein, a "positive diagnosis" indicates that the disease or condition, or a potential for developing the disease or condition, has been identified. In contrast, a "negative diagnosis" indicates that the disease or condition, or a potential for developing the disease or condition, has not been identified.

Another embodiment of the present invention relates to a method to identify a regulatory compound that inhibits human immunodeficiency virus (HIN) virion assembly and proliferation. This method generally includes the steps of: (a) contacting a putative regulatory compound with a cell that expresses cytotoxic T lymphocyte antigen 4 (CTLA4); and (b) identifying regulatory compounds that target the mechanism by which HIV-Env makes use of CTLA4 secretory granules to be delivered to the cell surface at the appropriate time in HIV virion replication and assembly (e.g., by identifying regulatory compounds that disrupt the regulation by Env of the intracellular localization of the CTLA4 secretory granule within the cell). As discussed above, the present inventor has provided evidence that Env sequesters the CTLA4 secretory granules within the cell until a time when Gag can associate with Env, thereby releasing the inhibition and allowing the complex to transit to the cell surface for assembly into infectious virions. By disrupting this regulation of transit imposed by Env, the Env/CTLA4-containing granules can be induced to transit to the surface prematurely (e.g., before Env associates with Gag), thereby disrupting virion assembly and/or exposing Env to the extracellular milieu, where host defenses can be mobilized against the viral protein and infected T cell. Preferably, compounds are identified which disrupt the regulation by Env of the intracellular localization of the CTLA4 secretory granule within the cell. In one embodiment, the compound is contacted with the cell intracellularly.

In one aspect, compounds are identified which disrupt the inhibition by Env of transport of the CTLA4 secretory granule to the plasma membrane. For example, the step of detecting can include detecting cell surface CTLA4 expression after contact with the putative regulatory compound, wherein an increase in CTLA4 cell surface expression after contact with the putative regulatory compound as compared to prior to or in the absence of contact with the compound indicates that the compound disrupts the inhibition by Env of transport of the CTLA4 secretory granule to the plasma membrane. Reagents for detection of CTLA4 are well known in the art and have been previously described herein (e.g., antibodies, soluble natural ligands, other binding proteins). In another embodiment, compounds are identified which induce transport to and expression of Env protein at the cell surface prior to the association of Env with other HIV proteins and/or prior to expression of other HIV proteins required for assembly. For example, one can detect cell surface expression of Env prior to and after contact with the putative regulatory agent, wherein detection of an increase in Env cell surface expression after contact with the compound as compared to prior to contact with the compound and, in one aspect, as compared to cellular expression (e.g., intracellular or surface expression) of the other HIV protein indicates that the compound induces transport to and expression of Env protein at the cell surface prior to the expression of other HIV proteins required for HIN virion assembly. An example of an HIV protein other than Env that is required for HIV virion assembly at the cell surface is Gag. Detection of increased Env at the cell surface after contact with the putative regulatory agent in the absence of a commensurate increase in Gag in the cell (e.g., Gag binds to the intracellular face of the plasma membrane) would indicate that the putative regulatory agent has induced premature transport of Env to the cell surface (e.g., before other HIN proteins, such as Gag, have been synthesized for virion assembly). HIV proteins other than Gag that are involved in virion assembly and that could also be detected in this method include, but are not limited to, Pol, Vpr, Νef, or copies of the full- length RΝA genome for virion incorporation. The sequences of multiple isolates of these proteins are well known in the art (e.g., Pol: GenBank Accession No. AAN74523/gi25807936; Vpr: GenBank Accession No. AAN74530/gi25807943; Nef: GenBank Accession No. AAM10902/gi30410842, each of which is incorporated herein by reference).

In another aspect, regulatory compounds are identified that prevent the transport of CTLA4 secretory granules to the plasma membrane. For example, one can compare the expression of CTLA4 at the cell surface after contact with the putative regulatory compound as compared to in the absence of contact with the putative regulatory compound, wherein a decrease in CTLA4 expression after contact with the compound indicates that the compound is inhibiting transport of CTLA4 granules to the plasma membrane.

In another aspect, regulatory compounds are identified that disrupt the association of one or more cellular protein with Env, wherein the association normally enables the sequestration of CTLA4 secretory granules within the cell in the absence of said regulatory compound. For example, the cellular protein could include a protein involved in the targeting and fusion or trafficking of intracellular granules to fuse with the plasma membrane, such as, but not limited to, syntaxin 6, syntaxin 16, rab4 or rabl 1. In one aspect, compounds are identified that disrupt the activity of Env by binding to the cytoplasmic tail of Env and particularly, to a region of Env that is approximately 60 to 80 amino acids from the C-terminus of the cytoplasmic tail of Env. Essentially, virtually any compound that targets and disrupts this mechanism of HIN virion assembly is encompassed by the present invention. The sequences (nucleotide and amino acid) for multiple isolates of Env (and the other HIV proteins) are well known in the art. For example, GenBank Accession No. AAN74523 (gi25807937), the sequence of which is incorporated by reference in its entirety, discloses an amino acid sequence for an isolate of Env. Numerous other sequences for Env proteins are also publicly available. The cytoplasmic domain of Env, to which the cellular proteins and Env bind, are encoded after the transmembrane domain of Env gp41. The Env gp41 cytoplasmic domain is generally referred to as amino acids 700-850 of the Env gpl60 precursor protein from the rflB isolate. While these numbers are not identical for each viral isolate, the cytoplasmic domain is encoded to the carboxyterminus of the conserved transmembrane domain in each of HIN-1 and HTV-2.

In another aspect, regulatory compounds are identified that result in the release of an increased number of non-infectious virions from the cell after contact with the compound as compared to prior to or in the absence of contact with the agent. The infectivity of virions isolated from a cell can be evaluated as previously described herein (e.g., by infection of a test cell culture).

It is to be understood that the present invention also contemplates identifying putative regulatory compounds via any of the above-described mechanisms using cell lysates and/or immobilized proteins or any other suitable non-cell based assays. For example, one can provide components such as Env protein or portions thereof, other HIN proteins, CTLA4 secretory granules and/or cellular docking proteins and identify regulatory compounds using high throughput analyses of large libraries of compounds. Νon-cell based assays are discussed below.

As used herein, the term "putative" refers to compounds having an unknown or previously unappreciated regulatory activity in a particular process. As such, the term "identify" is intended to include all compounds, the usefulness of which as a regulatory compound to inhibit the mechanism by which HIV-Env associates with CTLA4 secretory granules and is delivered to the cell surface is determined by a method of the present invention. According to the present invention, putative regulatory compounds to test include, for example, compounds that are products of molecular diversity strategies (a combination of related strategies allowing the rapid construction of large, chemically diverse molecule libraries), rational drug design, natural products and compounds having partially defined regulatory properties. Such a compound can be a protein-based compound, a carbohydrate- based compound, a lipid-based compound, a nucleic acid-based compound, a natural organic compound, or a synthetically derived organic compound. Such a compound can be obtained, for example, from libraries of natural or synthetic compounds, in particular from chemical or combinatorial libraries (i.e., libraries of compounds that differ in sequence or size but that have the same building blocks; see for example, U.S. Patent Nos. 5,010,175 and 5,266,684 of Rutter and Santi, or Maulik et al., 1997, Molecular Biotechnology: Therapeutic Applications and Strategies, Wiley-Liss, Inc., which are incorporated herein by reference in their entirety) or by rational drug design (see Maulik et al., ibid.). The method to identify compounds can include both cell-based assays and non-cell- based assays, with non-cell based assays being preferred for the screening of large compound libraries which take advantage of the novel discovery presented herein. If the assay is a cell- based assay, suitable cells for use with the present invention include any cell that endogenously expresses CTLA4 as disclosed herein (such as a T cell), or which has been transfected with and expresses recombinant CTLA4. Preferably, the cell can be infected with HIV and uses mechanisms of viral assembly comparable to a T cell. Preferably, the method includes contacting a cell that can express CTLA4, and preferably a T cell that can express CTLA4, and more preferably a CD4⁺ T cell that can express CTLA4, with a putative regulatory compound. In another embodiment the putative regulatory compound is contacted with an artificial membrane comprising CTLA4 or other components of the system which is to be disrupted (e.g., HIV-Env, secretory granule membranes and associated proteins, CTLA4).

The step of contacting can be performed by any suitable method, depending on how the cellular components (e.g., CTLA4, granules, Env, etc.) and/or putative regulatory compound are provided. For example, HIV-infected cells expressing CTLA4 can be grown in liquid culture medium or grown on solid medium in which the liquid medium or the solid medium contains the compound to be tested. In addition, the liquid or solid medium contains components necessary for cell growth, such as assimilable carbon, nitrogen and micro- nutrients. A cell-based assay is conducted under conditions which are effective to screen for regulatory compounds useful in the method of the present invention. Effective conditions include, but are not limited to, appropriate media, temperature, pH and oxygen conditions that permit cell growth. Cell lysates can be combined with other cell lysates and/or the compound to be tested in any suitable medium.

Cells of the present invention can be cultured in a variety of containers including, but not limited to, tissue culture flasks, test tubes, microtiter dishes, and petri plates. Culturing is carried out at a temperature, pH and carbon dioxide content appropriate for the cell. Such culturing conditions are also within the skill in the art. Acceptable protocols to contact a cell with a putative regulatory compound in an effective manner include the number of cells per container contacted, the concentration of putative regulatory compound(s) administered to a cell, the incubation time of the putative regulatory compound with the cell, and the concentration of compound administered to a cell. Determination of such protocols can be accomplished by those skilled in the art based on variables such as the size of the container, the volume of liquid in the container, the type of cell being tested and the chemical composition of the putative regulatory compound (i.e., size, charge etc.) being tested.

In one embodiment, the conditions under which a cell is contacted with a putative regulatory compound, such as by mixing, are conditions in which the cell behaves in a manner indicative of a T cell or an HIV-infected T cell if essentially no regulatory compound is present. For example, such conditions include normal culture conditions in the absence of a putative regulatory compound. The present methods involve contacting cells with the compound being tested for a sufficient time to allow for the compound to be evaluated. The period of contact with the compound being tested can be varied depending on the result being measured, and can be determined by one of skill in the art. In one embodiment, the compound is delivered to the intracellular compartment of the cell using methods known to those of skill in the art. For example, for binding assays, a shorter time of contact with the compound being tested is typically suitable, than when a cellular process, such as expression of a cellular marker, transport, inhibition of an activity, etc., is assessed. As used herein, the term "contact period" refers to the time period during which the cells or molecules are in contact with the compound being tested. The term "incubation period" refers to the entire time during which, for example, cells are allowed to grow prior to evaluation, and can be inclusive of the contact period. Thus, the incubation period includes all of the contact period and may include a further time period during which the compound being tested is not present but during which growth is continuing (in the case of a cell based assay) prior to scoring. It will be recognized that shorter incubation times are preferable because compounds can be more rapidly screened. A preferred incubation time is between about 1 minute to about 48 hours. The step of detecting or identifying is designed to indicate whether the putative regulatory compound disrupts the normal mechanism by which HIN-Env associates with CTLA4 secretory granules and is delivered to the cell surface, which includes, but is not limited to, inhibition of the association of Env with cellular proteins effective to sequester CTLA4 secretory granules within the cell until a time when virion assembly is driven forward, induction of transport to and expression of Env protein at the cell surface prior to the association of Env with other HIV proteins and/or prior to expression of other HIV proteins required for assembly, prevention of the transport of CTLA4 granules to the cell surface, etc. Methods of identifying compounds meeting a given criterion can include any suitable detection methods, including, but not limited to, detection or measurement of CTLA4 and/or Env expression in the presence and absence of the putative regulatory compound, detection or measurement of association of Env with cellular proteins (e.g., cellular proteins involved in the normal targeting and fusion or trafficking of intracellular granules to fuse with the plasma membrane), detection or measurement of the cellular localization of CTLA4/Env-containing secretory granules, detection or measurement of binding of a compound to Env, etc. Techniques for performing such methods are known in the art, and include a variety of binding assays, western blotting, immunocytochemistry, flow cytometry, other immunological based assays, phosphorylation assays, kinase assays, immunofluorescence microscopy, RNA assays, immunoprecipitation, evaluation of cell morphology, in situ hybridization, and other biological assays. Binding assays include BIAcore machine assays, immunoassays such as enzyme linked immunoabsorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichrosim, or nuclear magnetic resonance (NMR).

In vitro cell-based and non-cell based assays may be designed to screen for compounds that regulate HlY-infection of T cells via disruption of the association of Env with CTLA4-containing granules at either the transcriptional or translational level.

In one embodiment, large scale screening of compound libraries or computer-based drug design methods are used to identify regulators of the interaction between Env and the CTLA4 secretory granules and/or related proteins. For example, proteins such as the Env protein, cellular proteins that associate with Env, and/or cell lysates containing such proteins, can be immobilized to a solid substrate such as a test tube, microtiter well or a column, by means well known to those in the art, such substrates including, but not limited to: artificial membranes, organic supports, biopolymer supports and inorganic supports. The protein can be immobilized on the solid support by a variety of methods including adsorption, cross- linking (including covalent bonding), and entrapment. Adsorption can be through van del Waal's forces, hydrogen bonding, ionic bonding, or hydrophobic binding. Exemplary solid supports for adsorption immobilization include polymeric adsorbents and ion-exchange resins. Solid supports can be in any suitable form, including in a bead form, plate form, or well form. The putative regulatory compound can be contacted with the immobilized protein by any suitable method, such as by flowing a liquid containing the compound over the immobilized protein. In one embodiment, a suitable non-cell based assay includes a competition assay wherein binding of Gag/cellular docking protein to immobilized cytoplasmic Env domain is evaluated in the presence and absence of a putative regulatory compound. Techniques for evaluating binding and competition are well known in the art and have been referenced previously herein.

In another embodiment of the invention, a method to identify a regulatory compound that inhibits human immunodeficiency virus (HIV) virion assembly and proliferation includes the steps of: (a) isolating CD4⁺ T cells that express cytotoxic T lymphocyte antigen 4 (CTLA4) in a T cell-containing biological sample from a patient who is infected with HIV, wherein T cells that express CD4 and CTLA4 are predicted to be infected with HIV; (b) contacting a putative regulatory compound for inhibition of HIN infection with the cells of (a); and (c) detecting regulatory compounds that reduce HIV virion assembly and proliferation in the cells of (a) after contact with the compound as compared to prior to or in the absence of contact with the compound. In this method, steps (a), (b) and (c) have been described in various aspects of other embodiment of the invention above. The step of detecting can include, for example, measuring the quantity of human immunodeficiency virions released from the cells of (a) after contact with the compound, wherein a decrease in the quantity of human immunodeficiency virions released from the cells indicates that the compound is a regulatory compound for inhibition of HIN. In another aspect, the step of detecting comprises measuring the infectivity of human immunodeficiency virions released from the cells of (a) after contact with the compound, wherein a decrease in the infectivity of human immunodeficiency virions released from the cells indicates that the compound is a regulatory compound for inhibition of HIV. For example, the step of detecting can include measuring active virion assembly in the cells of (a), wherein a reduction in active virion assembly after contact with the compound indicates that the compound is a regulatory compound for inhibition of HIN. The step of detecting can also include measuring the number of T cells of (a) that are infected with actively proliferating HIN, wherein a decrease in the number of T cells that are infected with actively proliferating HIN after contact with the compound indicates that the compound is a regulatory compound for inhibition of HIV. In one aspect, the step of detecting comprises measuring death of the T cells, wherein an increase in the death of the T cells after contact with the compound indicates that the compound is a regulatory compound for inhibition of HIV. Compounds identified by any of the above-identified methods can be used in therapeutic methods of treatment of HIV-infected patients. For example, by prematurely triggering the presentation of Env on the surface of a cell along with CTLA4, well before new viruses would be able to form, it is possible to "trick" HIV-infected T cells into revealing themselves early, thereby allowing for the destruction of the infected cells by the immune system and other host defenses or by interactions which have negative consequences for the infected cell which may ordinarily be inhibited or ineffective during HIV infection. In addition, premature delivery of Env to the cell surface may prevent the assembly of an infectious virus. Further, one can take advantage of the expression of CTLA4 at the cell surface of infected cells, and particularly, early expression of CTLA4 (based on disruption of the Env control of expression) by triggering apoptosis in the cell through CTLA4 (e.g., by anti-CTLA4 or other inducers of apoptosis in the cell). Additionally, CTLA4 can be used as a target for the delivery of various agents to HIV-infected cells, such as toxins, anti -viral agents, agents which induce apoptosis of the cell, and/or agents with inhibit Env activity. For example, complexes and chimeric compounds can be developed which take advantage of the association of CTLA4 expression with HIV-infected cells and active virion assembly.

Compounds of the present method can be used in therapeutic methods to inhibit human immunodeficiency virus (HTV) virion assembly and proliferation, comprising contacting a population of cells containing HIV-infected CD4⁺ T cells with the regulatory compound. The population of cells can be contacted in vivo or ex vivo (described below). Accordingly, one embodiment of the invention also relates to a therapeutic method that recognizes the mechanism discovered by the present inventor. This method inhibits human immunodeficiency virus (HIN) virion assembly and proliferation, and includes the step of contacting a population of cells containing HIV-infected CD4⁺ T cells with a regulatory compound that disrupts the regulation by Env of the intracellular localization of the CTLA4 secretory granule within the cell. In one aspect, the regulatory compound is a modified Gag protein that causes the premature delivery of Env and CTLA4 containing granules to the surface. For example, the modified Gag protein can be an isolated Gag-Tat fusion protein, where Gag is either a full-length, native Gag protein or simply a portion of Gag protein sufficient to bind to Env and fused to a Tat protein. The amino acid and nucleic acid sequences for multiple isolates of Gag and Tat are well known in the art (e.g., see GenBank Accession Νos. AAΝ74522/gi25807935 (Gag) or AAN74528/gi25807941 (Tat), incorporated by reference in their entirety). In one aspect, the regulatory compound (e.g., Gag-Tat) is administered to an HIV-infected patient in vivo, or alternatively, to T cells within samples of PBMCs taken from patients. The addition of the Tat-Gag fusion protein will prematurely trigger CTLA4 surface delivery in those cells that were expressing the Env protein. In this manner, the cells producing virus would be revealed. In one aspect, the full- length Gag or at least the portion of Gag that binds to Env cyto tail will be fused to the cationic peptide of HIV- 1 Tat (residues 47-57) that confers its ability to deliver heterologous cargo into mammalian cells. The construct also contains an hemeagglutinin (HA) or hexahistidine epitope to facilitate purification of the fusion protein from bacterial cells. The plasmid encoding the Tat peptide and epitope with multiple cloning site for inserting Gag has been described (Becker-Hapak et al., (2001) Methods pp247-256).

Another embodiment of the invention includes a method to target an HIV-infected T cell for destruction, comprising contacting a population of cells containing HIV-infected CD4⁺ T cells with a compound, complex, or composition that selectively binds to CTLA4 expressed by a CD4⁺ T cell and induces the cell expressing CTLA4 to undergo apoptosis or otherwise be destroyed. In this embodiment, CTLA4 can serve as a target molecule for the delivery of toxic, anti-viral, apoptosis-inducing compounds, or anti-Env compounds to an HIV-infected cell, or alternatively, CTLA4 can itself be triggered to induce apoptosis in the cell. In this latter aspect, the CTLA4 can be contacted with a triggering compound, including, but not limited to, a anti-CTLA4 antibody, a compound (e.g., a protein) that selectively binds to and activates CTLA4, or a soluble B7 receptor, including multimeric soluble B7 receptor. HIV-infected cells are abortively activated in a manner that is very different from normal, uninfected T cells. Infected cells may be triggered to undergo apoptosis by contacting the cell with anti-CTLA4 antibodies (with or without leukophoresis) or by targeting other compounds (e.g., PMA/ionomycin) to these cells to drive them into premature apoptosis. By crosslinking CTLA4 on the surface of cells, with antibody, multimeric soluble B7, or some other compound that mimics this activity, these cells would be induced to undergo apoptosis, if they were in the process of producing virions. Such methods can inhibit HlV-infection in an individual and provide a therapeutic benefit to the patient. In one aspect, a population of cells containing HIV-infected CD4⁺ T cells is contacted with a complex comprising: (1) a first agent that targets the complex to cells expressing CTLA4; and (2) a second agent selected from: a toxin, an anti-viral agent, an agent that induces apoptosis in the cell, and an agent that antagonizes the activity of Env, wherein the first and second agents are complexed together. Agents that target the complex to cells expressing CTLA4 are as described above and include, but are not limited to, anti-CTLA4 antibody, a compound (e.g., a protein) that selectively binds to and activates CTLA4, or a soluble B7 receptor.

In general, a composition to be administered to an individual that contains a regulatory reagent useful for inhibiting human immunodeficiency virus (HIV) virion assembly and proliferation or for targeting destruction of HIV infected T cells includes a pharmaceutically acceptable carrier, which includes pharmaceutically acceptable excipients and/or delivery vehicles, for delivering the agent(s) to a patient. According to the present invention, a "pharmaceutically acceptable carrier" includes pharmaceutically acceptable excipients and/or pharmaceutically acceptable delivery vehicles, which are suitable for use in administration of the composition to a suitable in vitro, ex vivo or in vivo site. A suitable in vitro, in vivo or ex vivo site is the site of delivery of the composition in the patient. Preferred pharmaceutically acceptable carriers are capable of maintaining the compound useful in the present invention in a form that, upon arrival of the compound at the cell target in a culture or in patient, the compound is capable of interacting with its target (e.g., an HIN- infected T cell). Suitable excipients of the present invention include excipients or formularies that transport or help transport, but do not specifically target a composition to a cell (also referred to herein as non-targeting carriers). Examples of pharmaceutically acceptable excipients include, but are not limited to water, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution, other aqueous physiologically balanced solutions, oils, esters and glycols. Aqueous carriers can contain suitable auxiliary substances required to approximate the physiological conditions of the recipient, for example, by enhancing chemical stability and isotonicity.

Suitable auxiliary substances include, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, calcium chloride, and other substances used to produce phosphate buffer, Tris buffer, and bicarbonate buffer. Auxiliary substances can also include preservatives, such as thimerosal, m- or o-cresol, formalin and benzol alcohol. Compositions of the present invention can be sterilized by conventional methods and/or lyophilized.

One type of pharmaceutically acceptable carrier includes a controlled release formulation that is capable of slowly releasing a composition of the present invention into a patient or culture. As used herein, a controlled release formulation comprises a compound useful in the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Other carriers of the present invention include liquids that, upon administration to a patient, form a solid or a gel in situ. Preferred carriers are also biodegradable (i.e., bioerodible).

A pharmaceutically acceptable carrier which is capable of targeting can be referred to as a "delivery vehicle" or more particularly, a "targeting delivery vehicle." Delivery vehicles of the present invention are capable of delivering a composition of the present invention to a target site in a patient. A "target site" refers to a site in a patient to which one desires to deliver a composition. A cell or tissue can be targeted, for example, by including in the vehicle a targeting moiety, such as a ligand capable of selectively (i.e., specifically) binding another molecule at a particular site (e.g., a molecule on the surface of the target cell such as CTLA4). Examples of such ligands include antibodies, antigens, receptors and receptor ligands. Alternatively, particular modes of administration (e.g., direct injection) and/or types of delivery vehicles (e.g., liposomes) can be used to deliver a composition preferentially to a particular site. Examples of delivery vehicles include, but are not limited to, artificial and natural lipid-containing delivery vehicles, viral vectors, and ribozymes. Natural lipid-containing delivery vehicles include cells and cellular membranes. Artificial lipid-containing delivery vehicles include liposomes and micelles.

One preferred delivery vehicle of the present invention is a liposome. A liposome is capable of remaining stable in an animal for a sufficient amount of time to deliver a therapeutic nucleic acid molecule or protein to a preferred site in the animal. A liposome, according to the present invention, comprises a lipid composition that is capable of delivering a nucleic acid molecule or protein to a particular, or selected, site in a patient. A liposome according to the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver a nucleic acid molecule or protein into a cell. Suitable liposomes for use with the present invention include any liposome. Preferred liposomes of the present invention include those liposomes commonly used in, for example, gene or protein delivery methods known to those of skill in the art. More preferred liposomes comprise liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol.

Another preferred delivery vehicle comprises a viral vector. A viral vector includes an isolated nucleic acid molecule encoding a protein useful in the present invention, in which the nucleic acid molecules are packaged in a viral coat that allows entrance of DNA into a cell. A number of viral vectors can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, lentiviruses, adeno-associated viruses and retroviruses.

In accordance with the present invention, acceptable protocols to administer an agent including the route of administration and the effective amount of an agent to be administered to an animal can be determined and executed by those skilled in the art. Effective dose parameters can be determined by experimentation using in vitro cell cultures, in vivo animal models, and eventually, clinical trials if the patient is human. Effective dose parameters can be determined using methods standard in the art for the treatment of HIV-infected patients. Such methods include, for example, determination of survival rates, side effects (i.e., toxicity) and progression or regression of disease. As used herein, the phrase "protected from a disease" refers to reducing the symptoms of the disease; reducing the occurrence of the disease, and/or reducing the severity of the disease. Protecting a patient can refer to the ability of a therapeutic composition of the present invention, when administered to a patient, to prevent a disease from occurring and/or to cure or to treat the disease by alleviating disease symptoms, signs or causes. As such, to protect a patient from a disease includes both preventing disease occurrence (prophylactic treatment) and treating a patient that has a disease or that is experiencing initial symptoms or later stage symptoms of a disease (therapeutic treatment). In particular, protecting a patient from a disease can be accomplished by reducing HIN-infection, reducing HIN virion assembly and proliferation, and/or reducing HIN-virion production and/or infectivity in a cell. The term, "disease" refers to any deviation from the normal health of a patient and includes a state when disease symptoms are present, as well as conditions in which a deviation (e.g., infection) has occurred, but symptoms (e.g., symptoms of acquired immunodeficiency syndrome) are not yet manifested. In accordance with the present invention, a suitable single dose size is a dose that results in regulation of HlV-infection, HIV virion assembly and proliferation, and/or HIN- virion production and/or infectivity in a cell, when administered one or more times over a suitable time period. Doses can vary depending upon the disease being treated. One of skill in the art can readily determine appropriate single dose sizes for a given patient based on the size of a patient and the route of administration. One of skill in the art can monitor the effectiveness of the treatment using methods as described previously herein.

As discussed above, a therapeutic composition of the present invention is administered to a patient in a manner effective to deliver the composition to a cell, a tissue, and/or systemically to the patient, whereby the desired result is achieved as a result of the administration of the composition. Suitable administration protocols include any in vivo or ex vivo administration protocol. The preferred routes of administration will be apparent to those of skill in the art, depending on whether the composition is nucleic acid based, protein based, or cell based. For proteins or nucleic acid molecules, preferred methods of in vivo administration include, but are not limited to, intravenous administration, intraperitoneal administration, intramuscular administration, intranodal administration, infracoronary administration, intraarterial administration (e.g., into a carotid artery), subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, inhalation (e.g., aerosol), intracranial, intraspinal, intraocular, intranasal, oral, bronchial, rectal, topical, vaginal, urethral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue. Routes useful for deliver to mucosal tissues include, bronchial, intradermal, intramuscular, intranasal, other inhalatory, rectal, subcutaneous, topical, transdermal, vaginal and urethral routes. Combinations of routes of delivery can be used and in some instances, may enhance the therapeutic effects of the composition.

Ex vivo administration refers to performing part of the regulatory step outside of the patient, such as administering a composition (nucleic acid or protein) of the present invention to a population of cells removed from a patient under conditions such that the composition contacts and/or enters the cell, and returning the cells to the patient. Ex vivo methods are particularly suitable when the target cell type can easily be removed from and returned to the patient. Many of the above-described routes of administration, including intravenous, intraperitoneal, intradermal, and intramuscular administrations can be performed using methods standard in the art. Aerosol (inhalation) delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein by reference in its entirety). Oral delivery can be performed by complexing a therapeutic composition of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal.

Examples of such carriers, include plastic capsules or tablets, such as those known in the art.

One method of local administration is by direct injection. Direct injection techniques are particularly useful for administering a composition to a cell or tissue that is accessible by surgery, and particularly, on or near the surface of the body. Administration of a composition locally within the area of a target cell refers to injecting the composition centimeters and preferably, millimeters from the target cell or tissue.

Various methods of administration and delivery vehicles disclosed herein have been shown to be effective for delivery of a nucleic acid molecule to a target cell, whereby the nucleic acid molecule transfected the cell and was expressed. In many studies, successful delivery and expression of a heterologous gene was achieved in preferred cell types and/or using preferred delivery vehicles and routes of administration of the present invention. All of the publications discussed below and elsewhere herein with regard to gene delivery and delivery vehicles are incorporated herein by reference in their entirety.

For example, using liposome delivery, U.S. Patent No. 5,705,151, issued January 6, 1998, to Dow et al. demonstrated the successful in vivo intravenous delivery of a nucleic acid molecule encoding a superantigen and a nucleic acid molecule encoding a cytokine in a cationic liposome delivery vehicle, whereby the encoded proteins were expressed in tissues of the animal, and particularly in pulmonary tissues. In addition, Liu et al., Nature Biotechnology 15:167, 1997, demonstrated that intravenous delivery of cholesterol- containing cationic liposomes containing genes preferentially targets pulmonary tissues and effectively mediates transfer and expression of the genes in vivo. Several publications by Dzau and collaborators demonstrate the successful in vivo delivery and expression of a gene into cells of the heart, including cardiac myocytes and fibroblasts and vascular smooth muscle cells using both naked DNA and Hemagglutinating virus of Japan-liposome delivery, administered by both incubation within the pericardium and infusion into a coronary artery (infracoronary delivery) (See, for example, Aoki et al., 1997, J. Mol. Cell, Cardiol. 29:949- 959; Kaneda et al., 1997, Ann NY. Acad. Sci. 811 :299-308; and von der Leyen et al., 1995, Proc Natl Acad Sci USA 92:1137-1141).

Delivery of numerous nucleic acid sequences has been accomplished by administration of viral vectors encoding the nucleic acid sequences. Using such vectors, successful delivery and expression has been achieved using ex vivo delivery (See, of many examples, retroviral vector; Blaeseetal., 1995, Science 270:475-480; Bordignonetal., 1995, Science 270:470-475), nasal administration (CFTR-adenovirus-associated vector), infracoronary administration (adenoviral vector and Hemagglutinating virus of Japan, see above), intravenous administration (adeno-associated viral vector; Koeberl et al., 1997, Proc Natl Acad Sci USA 94: 1426-1431). A publication by Maurice et al. (1999, J. Clin. Invest. 104:21-29) demonstrated that an adenoviral vector encoding a β2-adrenergic receptor, administered by infracoronary delivery, resulted in diffuse multichamber myocardial expression of the gene in vivo, and subsequent significant increases in hemodynamic function and other improved physiological parameters. Levine et al. describe in vitro, ex vivo and in vivo delivery and expression of a gene to human adipocytes and rabbit adipocytes using an adenoviral vector and direct injection of the constructs into adipose tissue (Levine et al., 1998, J. N-.tr. Sci. Vitaminol. 44:569-572).

In the area of neuronal gene delivery, multiple successful in vivo gene transfers have been reported. Millecamps et al. reported the targeting of adenoviral vectors to neurons using neuron restrictive enhancer elements placed upstream of the promoter for the transgene (phosphoglycerate promoter). Such vectors were administered to mice and rats intramuscularly and intracerebrally, respectively, resulting in successful neuronal-specific transfection and expression of the transgene in vivo (Millecamps et al., 1999, Nat. Biotechnol. 17:865-869). As discussed above, Bennett et al. reported the use of adeno- associated viral vector to deliver and express a gene by subretinal injection in the neural retina in vivo for greater than 1 year (Bennett, 1999, ibid.).

Gene delivery to synovial lining cells and articular joints has had similar successes. Oligino and colleagues report the use of a herpes simplex viral vector which is deficient for the immediate early genes, ICP4, 22 and 27, to deliver and express two different receptors in synovial lining cells in vivo (Oligino et al., 1999, Gene Ther. 6:1713-1720). The herpes vectors were administered by intraarticular injection. Kuboki et al. used adenoviral vector- mediated gene transfer and intraarticular injection to successfully and specifically express a gene in the temporomandibular joints of guinea pigs in vivo (Kuboki et al., 1999, Arch. Oral. Biol. 44:701-709). Apparailly and colleagues systemically administered adenoviral vectors encoding IL-10 to mice and demonstrated successful expression of the gene product and profound therapeutic effects in the treatment of experimentally induced arthritis (Apparailly et al., 1998, J. Immunol. 160:5213-5220). In another study, murine leukemia virus-based retro viral vector was used to deliver (by intraarticular injection) and express a human growth hormone gene both ex vivo and in vivo (Ghivizzani et al., 1997, Gene Ther.4:977-982). This study showed that expression by in vivo gene transfer was at least equivalent to that of the ex vivo gene transfer. As discussed above, Sawchuk et al. has reported successful in vivo adenoviral vector delivery of a gene by intraarticular injection, and prolonged expression of the gene in the synovium by pretreatment of the joint with anti-T cell receptor monoclonal antibody (Sawchuk et al., 1996, ibid. Finally, it is noted that ex vivo gene transfer of human interleukin-1 receptor antagonist using a retrovirus has produced high level intraarticular expression and therapeutic efficacy in treatment of arthritis, and is now entering FDA approved human gene therapy trials (Evans and Robbins, 1996, Curr. Opin. Rheumatol. 8:230-234). Therefore, the state of the art in gene therapy has led the FDA to consider human gene therapy an appropriate strategy for the treatment of at least arthritis. Taken together, all of the above studies in gene therapy indicate that delivery and expression of a recombinant nucleic acid molecule according to the present invention is feasible.

Another method of delivery of recombinant molecules is in a non-targeting carrier (e.g., as "naked" DNA molecules, such as is taught, for example in Wolff et al., 1990, Science 247, 1465-1468). Such recombinant nucleic acid molecules are typically injected by direct or intramuscular administration. Recombinant nucleic acid molecules to be administered by naked DNA administration include an isolated nucleic acid molecule of the present invention, and preferably includes a recombinant molecule of the present invention that preferably is replication, or otherwise amplification, competent. A naked nucleic acid reagent of the present invention can comprise one or more nucleic acid molecules of the present invention including a dicistronic recombinant molecule. Naked nucleic acid delivery can include intramuscular, subcutaneous, intradermal, transdermal, intranasal and oral routes of administration, with direct injection into the target tissue being most preferred. A preferred single dose of a naked nucleic acid vaccine ranges from about 1 nanogram (ng) to about 100 μg, depending on the route of administration and/or method of delivery, as can be determined by those skilled in the art. Suitable delivery methods include, for example, by injection, as drops, aerosolized and/or topically. In one embodiment, pure DNA constructs cover the surface of gold particles (1 to 3 μm in diameter) and are propelled into skin cells or muscle with a "gene gun."

In the method of the present invention, compositions can be administered to and methods performed using cells from any member of the Vertebrate class, Mammalia, that can be infected with human immunodeficiency virus or a virus related thereto. The method of the present invention is preferably used in humans, although all animal models of HIV infection, including HIN-infected scid-hu mice, can be used.

The entire disclosure of U.S. Provisional Patent Application Serial No. 60/380,987, filed May 15, 2002, and entitled "Methods to Detect, Isolate, Target and Manipulate HIV- Infected T Cells", is incorporated herein by reference. The following examples are provided for the purpose of illustration and are not intended to limit the scope of the present invention.

Examples The following Materials and Methods were used to perform the experiments described in Examples 1-3. Cell Lines

The human T cell line H9 was obtained from the American Type Culture Collection (ATCC, Rockville, MD). Reagents were obtained from Sigma (St. Louis, MO), unless otherwise indicated. Fresh blood from healthy adult donors was used to isolate CD4+ cells from PBMCs using CD4 MicroBeads (Miltenyi Biotec Inc., Auburn, CA), as described by the manufacturer. H9 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) (Gemini Bio-Products, Woodland, CA) and 10 μg/ml gentamicin (Gibco-BRL, Rockville, MD), and 41.4 μg/ml 2-mercaptoethanol. 10 IU/ml of recombinant human IL-2 (Boehringer-Marmheim, Indianapolis, IN) and 3 μg/ml of phytohemagglutinin (PHA) were added to the human CD4+ cells 3 days prior to HIV-1 infection. This treatment was also utilized to increase the levels of expression of the endogenous CTLA4 (Steiner et al., (1999), supra). Generation of H9 Cells Stably Expressing CTLA4 Full-length mouse CTLA4 gene fused directly to GFP at the C-terminus under the control of the human ubiquitin C promoter was engineered in plasmid pUp. H9 cells (8 x 10⁶) in serum free DMEM were mixed with 30 μg DNA in a 4mm gap cuvette and electroporated in a BTX electroporator (Genetronics Inc., San Diego, CA) that was set for 500 V capacitance/resistance mode, 1050 μF capacitance, 720 ohms resistance, and 260 V charging voltage. Cells were incubated 48 h in DMEM after which Geneticin (Gibco-BRL) was added to 800 μg/ml. H9 cells stably expressing CTLA4-GFP were obtained from three rounds of fluorescent activated cell sorting (FACS). Viruses and Antibodies

The D47 (gpl20) and D61 (gp41) hybridomas (Earl et al., (1994) J. Virol. 68:3015- 26; Earl et al., (1997) J. Virol. 71:2674-84), as well as the recombinant vaccinia viruses vPE16 (Earl et al., (1990)J Virol. 64:2448-51), expressing the gp 160 precursor were kindly provided by Dr. Patricia Earl (NIH). Ascites for these antibodies were produced in Balb/c mice as described (36). The 902 monoclonal antibody against gp 120 was obtained from the NTH AIDS Research and Reference Program. HIV-1 strain DIB was purchased from Advanced Biotechnologies Inc. (Columbia, MD). A monoclonal antibody against GFP (Clontech Laboratories Inc., Palo Alto, CA), a polyclonal antibody against MG160 (from K Howell, UCHSC), a monoclonal antibody against human CTLA4 (J. Bluestone, UIC, Chicago, IL.), polyclonal antibodies against human CD4 receptor, Fgr kinase, calreticulin, and the extracellular domain of mouse CTLA4 (Santa Cruz Biotechnology Inc., CA) were used in these studies. PE-conjugated anti-mouse CTLA4 antibody was purchased from BD Biosciences-Pharmingen (San Diego, CA). Fluorescent-conjugated secondary antibodies were obtained from Jackson Laboratories (West Grove, PA). Virus infections

For infection with the recombinant vaccinia virus vPE16, H9 cells were infected at a MOI of 30 PFU/cell for 3h (37°C , 5% CO₂), after which cells were washed and incubated with fresh medium. At 14h post- infection, cells were processed for immunological analysis. In experiments involving HIV-1, CD4⁺ cells isolated from PBMCs, as well as H9 cells were infected at a MOI of 0.5 TCID₅₀/cell for 3h. Cells were then extensively washed with fresh medium and incubated for additional 72h (37°C, 5%> CO₂). Cells were pelleted, washed and processed for further experiments. Imunocytochemistry, Cell Imaging Analysis, and Flow Cytometry

For immunofluorescence, 5 x 10⁴ HIV-infected T cells were bound to washed, poly- D-lysine-coated cover slips (3 mg/ml), fixed for 30 min with 3% paraformaldehyde/3% sucrose (Fisher Scientific, New Jersey, NJ) , and quenched 10 min in 50 mM NH₄C1 (Fisher). Cells were permeabilized 4 min with 0.1% Triton X-100 in PBS, and blocked 5 min with 0.2%) gelatin in PBS. Cells were incubated with antibodies then observed using a Zeiss Axiovert M100 epifluorescence microscope (Karl Zeiss Inc., Thornwood, NY). Visual data were acquired using a Cooke Corporation SensiCam CCD camera and were digitally deconvolved using a nearest neighbors algorithm with SlideBook software (Intelligent Imaging Inc., Denver, CO). In addition, colocalization analysis was performed using the masking and statistics capabilities of SlideBook. Initially, stacks of images were acquired in 0.2 μm steps throughout the cell volume. Stacks were deconvolved using a constrained iterative algorithm.

For the antibody uptake experiments, 3 x 10⁴ cells were incubated 45 min on ice with

1 μg D47 or mouse CTLA4 (extracellular domain) antibody. After washing 5 times with PBS, cells were resuspended in DMEM medium and incubated for 45 min at 37 °C , 5% CO₂, to allow antibody internalization. After fixation, cells were incubated with the corresponding secondary antibodies conjugated to biotin, followed by streptavidin-Cy3.

For flow cytometry analysis of CTLA4 surface expression, HIV-1 infected H9- CTLA4-GFP cells were washed three times in ice-cold PBS and incubated with saturating concentration of the PE-conjugated CTLA4 antibody for 2h on ice. Cell were then washed five times with ice-cold PBS and analyzed in Becton Dickinson FACSan. Metabolic Labeling, Immunoprecipitations, and Western Blots

For labeling of Env expressed by recombinant vaccinia viruses, at 14h post-infection, 3 x 10⁶ cells were washed and then starved 30 min in methionine-free DMEM with 5% dialyzed FBS. Cells were pulsed 40 min in the presence of 100 μCi of ³⁵S-methionine (ICN Biomedicals, Irvine, CA) and chased for different time points. To immunoprecipitate Env, gradient fractions of lysed cells were incubated 10 min in lysis buffer (PBS containing 1% TX-100, 0.4%) deoxycholic acid, lOOmM PMSF, and lx Complete™ protease inhibitor cocktail (Boehringer-Mannheim), after which the lysate was clarified by centrifugation at 14000g for 10 min at 4°C. Typically, 1 μl of ascites fluid or 1 μg of polyclonal antibody was used per immunoprecipitation. Incubations for at least 3h at 4°C were followed by addition of protein- A Sepharose for at least 2 hr. Complexes were washed 4 times with lysis buffer and eluted from beads by SDS-loading buffer. Proteins were separated by SDS-PAGE (8%) and visualized by autoradiography. For Western blot analyses, infected cells were washed with PBS and the pellet treated with lOOμl of lysis buffer. The lysate was clarified by centrifugation (14,000g) and SDS- loading buffer was added to lOOμg of the clarified supernatant. Total proteins from the gradient fractions were precipitated by methanol/chloroform (4: 1) extraction, and the pellet washed with methanol. Proteins were then subjected to SDS-PAGE (8%) and transferred (16h) onto Immobilon membranes (Millipore, Bedford, MA), which were incubated with the primary antibody, followed by peroxidase conjugated secondary antibody, and revealed by ECL (NEN Life Science Products Inc., Boston, MA). Density Gradient Fractionation

Separation of granules from other membranes was performed as described previously (Waites et al., (2001) J Cell Biol. 152:1159-68), with a few modifications. After infection of H9 cells with vaccinia virus vPE16 for 14h, cells were labeled as indicated above, rinsed with PBS, harvested in 0.25 M sucrose, 5 mM Tris-HCl pH 7.3, ImM EGTA, 2mM PMSF, and IX protease inhibitor cocktail. Cells were lysed by multiple passage through a 22.5 gauge needle on an insulin syringe. Upon 60-80% cell lysis (as judged by phase contrast microscopy), the homogenates were sedimented twice at lOOOg for 10 min to pellet nuclei and unbroken cells. The resulting post-nuclear supernatant (PNS) was layered onto linear 0.3-1.6M sucrose gradient and sedimented to equilibrium at 30,000 rpm (154,325g, RCFmax) for 16h in a SW41 rotor at 4°C. Fractions were collected from the top. Membrane fractionation were analyzed by immunoblots of the fractions using antibodies for calreticulin (ER marker protein), MG160 (Golgi marker protein), CD4 (cell surface/endosome marker protein), and CTLA4-GFP (regulated granule marker protein). Immunoprecipitation of proteins from the sucrose fractions was achieved by adding 10X lysis buffer prior to the addition of antibodies and protein A-sepharose.

Example 1

This example demonstrates the co-localization of CTLA4 and Env in the same intracellular secretory granules.

Indirect immunofluorescence was performed to detect the intracellular compartments where Env was targeted in the T cell secretory pathway. Briefly, HIN-1 infected CD4⁺ cells were double stained for immunofluorescence with an anti-gp41 antibody and calreticulin (ER marker protein), or anti-gp41 and anti-Fgr kinase (Golgi marker protein). Co-localization of the two markers in the cell was revealed by the staining. In one experiment, staining with an anti-gp 120 antibody showed surface staining on cells. The same field was also analyzed by bright field microscopy (BF) to reveal the contours of the cell. In another experiment, double staining for immunofluorescence with anti-gp 120 antibody and anti-CTLA4 antibody was performed. In yet another experiment, HIN-1 infected H9-CTLA4-GFP cells labeled with anti-gp 120 antibody was evaluated. The frequency of these patterns was assessed by examining approximately 90 cells for the staining of each compartment.

A panel of representative images for Env localization in HIN-1 infected CD4⁺ cells was isolated from PBMCs (data not shown). The expected appearance of Env in ER and Golgi organelles was observed as previously reported (Willey et al., (1988), supra; Earl et al., (1994), supra; San Jose et al., (1997), supra). In the present study, -40% cells showed a pattern of Env localization in the ER and ~25%> showed labeling associated with the Golgi (data not shown). About 25%> of Env-expressing cells revealed cell surface labeling. A significant fraction of HIV-1 infected cells (10%>) showed an intracellular punctate pattern of Env localization. Double-labeling with the granular marker CTLA4 identified the punctate staining as intracellular regulated secretory granules. The granule localization of Env was also observed with antibodies to gp41, as well as with either human and mouse T cells infected with Env-expressing vaccinia virus vPE 16 (data not shown). The localization of Env in the regulated secretory granules was confirmed with HIN-infected human H9 T cells that stably express a CTLA4-GFP fusion protein. The co-localization of CTLA4-GFP and Env in the same intracellular granules was verified by three dimensional analysis of multiple focal planes using digital deconvolution fluorescence microscopy. The results from immunofluorescence microscopy suggested that Env normally traffics to CTLA4-containing regulated secretory granules as part of its intracellular trafficking itinerary following HIV-l infection of human T cells.

Example 2

The following example shows that HIV- 1 Env traffics directly from Golgi to CTLA4- containing granules. The steady-state localization of CTLA4 in the intracellular granules is achieved by rapid endocytosis from the cell surface (Chuang et al., (1997), supra). Antibody uptake experiments were performed to determine whether Env was similarly directed to the intracellular granules by endocytosis from the cell surface. Briefly, an antibody against the CTLA4 lumenal domain was incubated with H9-CTLA4-GFP cells in culture prior to fixation, permeabilization and staining with a Cy3 -conjugated second antibody. In one experiment, 3d after infection by HIV-1 (MOI: 0.5), H9-CTLA4-GFP cells were incubated on ice with anti-gp 120 antibody. Cells were then washed and gp 120 antibody internalization was allowed at 37 °C prior to fixation, permeabilization, and staining with a secondary Cy3- conjugated antibody.

As previously reported (Chuang et al., (1997), supra), the present study confirmed that antibodies against the extracellular domain of CTLA4 were internalized to the same intracellular granules as those harboring CTLA4 at steady-state in the H9-CTLA4-GFP cell line (data not shown). In contrast, no endocytic uptake of antibodies to the extracellular domain of Env was detected in HTV- infected H9 cells, even though these antibodies label Env protein at the surface of some cells (data not shown). These experiments suggested that, unlike CTLA4, Env localization to the intracellular granules does not utilize the pathway of endocytosis from the cell surface.

In studying Env trafficking, the exit of Env from the ER is slow, most likely due to complications in protein folding, trimerization and due to the potential for binding CD4 proteins in the ER of human T cells (Earl et al., (1991), supra; Raja et al., (1993) J. Gen. Virol. 74( Pt 10):2085-97). A significant fraction of nascently synthesized Env proteins are degraded by ER quality control mechanisms. Because Env protein "trickles" out of the ER, post-ER trafficking of Env can be effectively monitored. To dissect the intracellular trafficking itinerary of Env, pulse-chase radiolabeled H9 T cells were analyzed by membrane fractionation in sucrose density gradients. This approach resolved the key organelles of the secretory pathway involved in Env trafficking, i.e., ER, Golgi, plasma membrane, and the CTLA4-containing granules (data not shown). Briefly, lysates of radiolabeled H9-CTLA4- GFP cells infected with vaccinia virus vPE16 (encoding HIV-1 Env) were resolved by density sucrose gradient centrifugation. Experiments representing a 30 min radiolabel pulse followed by 0, 2, 4 or 16h chase periods were performed. Each fraction from the gradients was analyzed for the presence of radiolabeled gpl20 Env by immunoprecipitation. In addition, the distribution of gp41 in the gradients at 4h chase was evaluated. The resolution of marker proteins of specific organelles on the gradient was analyzed by immunoblots. The resolved fractions were blotted for the marker proteins calreticulin (ER), MG160 (Golgi), CTLA4 (regulated secretory granules) and CD4 (plasma membrane and endosomes). For each fraction, the amount of the marker was expressed as a percentage of total immunoreactivity across the gradient. At different chase times, the organelles from lysates of H9-CTLA4-GFP cells infected with vaccinia virus vPE 16 were resolved by density gradient fractionation. At the start of the chase experiment (following the 30 min pulse), there was no detectable cleavage of H1N- gpl60, and most of Env was found in dense fractions 10-12 (data not shown), corresponding to ER membranes. A small proportion of HIN-gpl60 was also found in lighter gradient fractions correlating with the Golgi marker protein (data not shown). Following a 2h chase period, the proportion of HIN-gpl60 in the Golgi fractions increased, while the fraction of Env in the ER diminished. The Env cleavage products were evident in fractions 5-7, corresponding to Golgi compartments. The appearance of gpl20 shed into the medium starting at the 2h chase time point is consistent with partial Env distribution in post-Golgi compartments.

A shift in intracellular Env localization began to be apparent by 4h chase. The peak of HIN-gpl60 and the mature gpl20-gp41 products were recovered in fractions 6-10, which overlaps with the distribution of CTLA4-containing granules. At even longer chase-times, i.e., 16h chase, most of the envelope glycoproteins remain in fractions 6-10. Interestingly, Env was not detected in more buoyant membrane fractions containing plasma membrane marker proteins, even though a fraction of the total gpl20 pool was shed into the media. Immunoprecipitation analysis of gpl20 from the media started as early as 2h post-chase, yet was greatly elevated by 4- 16 h. The release of gp 120 into the medium would imply that gp41 would be present to a significant degree at the cell surface. Nevertheless, Env fractionation experiments performed with a gp41 antibody failed to detect Env in cell surface fractions, yet they show significant overlap with gpl20-containing membranes. This result suggests that the granule compartment constitutes the major post-Golgi fraction of Env protein in the cell. At longer chase-times, gpl20 continued to be shed even though most of the intracellular Env pool was localized to CTLA4-containing granules. Taken together, these results showed that Env transited the secretory pathway from ER to Golgi (as expected), yet was directly delivered to and retained in CTLA4-containing granules rather than trafficking to the cell surface. Shedding of gpl20 into the medium seems to represent the outcome of Env trafficking in a minor population of cells, while trafficking to and residence in the regulated secretory granules is the principal itinerary of Env prior to virion assembly. Example 3

The following example demonstrates cell surface recruitment of the CTLA4 granule during

HIN infection.

HIV-1 infection of H9-CTLA4-GFP cells triggered a visible alteration in the immunofluorescent pattern for CTLA4 and for Env. In this experiment, the impact of HIV infection on expression of CTLA4 at the cell surface of H9-CTLA4-GFP cells was evaluated by examining GFP fluorescence and double staining by immunofluorescence of CTLA4 and Env. In place of the typical punctate staining of the granules, the majority of cells incubated with HIV now revealed CTLA4 and Env localization at the cell surface (data not shown). However, infection of D10 IL-2 mouse T cells stably expressing CTLA4-GFP protein with the recombinant vaccinia virus vPE 16, followed by fixing, permeabilization and staining for immunofluorescence with antibody 902 to HIV- 1 gp 120 for comparison with the localization of CTLA4 GFP fluorescence, showed no change in surface CTLA4 expression in cells that solely expressed Env by vaccinia virus. Flow cytometry of CTLA4 protein at the cell surface of mock-infected or HIN-1 infected H9-CTLA4-GFP T cells was performed. Cells were stained with antibody to PE- conjugated mouse anti-CTLA4 antibody prior to fixation and flow cytometry. In addition, flow cytometry of gpl20 at the cell surface of untreated or PHA + IL-2 treated H9-CTLA4 T cells infected with recombinant vaccinia virus vPE 16 encoding HIN- 1 Env was performed. Cells were stained with antibody to gp 120 (D47) and PE-conjugated anti-mouse IgG antibody prior to fixation and flow cytometry. Results showed that the fluorescent intensity of CTLA4 and the number of cells expressing CTLA4 at the cell surface significantly increased following HIN infection. The concomitant expression of CTLA4 and gpl20 at the cell surface strongly suggested that the CTLA4-containing granules were recruited to the cell surface following HIV-1 infection.

While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims.

Claims

What is claimed:

1. A method to detect or isolate human immunodeficiency virus (HIN)-infected T cells in a patient, comprising detecting or isolating CD4⁺ T cells that express cytotoxic T lymphocyte antigen 4 (CTLA4) in a T cell-containing biological sample from a patient who is infected with HIN, wherein T cells that express CD4 and CTLA4 are predicted to be infected with HIV.

2. The method of Claim 1 , wherein said step of detecting or isolating comprises a method selected from the group consisting of flow cytometry, magnetic bead isolation, immunoaffinity chromatography, immunoassay, and radioimmunoassay.

3. The method of Claim 1 , wherein said step of detecting or isolating comprises fluorescent activated cell sorting.

4. The method of Claim 1 , wherein said step of detecting or isolating comprises sorting CD4⁺, CTLA4⁺ T cells using flow cytometry.

5. The method of Claim 1 , wherein said step of detecting or isolating comprises using flow cytometry to isolate CD4⁺ T cells from said sample, followed by detecting or isolating CD4⁺ cells that express CTLA4.

6. The method of Claim 1 , wherein said step of detecting or isolating comprises contacting CD4⁺ T cells in said sample with an agent that selectively binds to CTLA4.

7. The method of Claim 6, wherein said agent is selected from the group consisting of an anti-CTLA4 antibody, a protein that selectively binds to CTLA4, and a soluble B7 receptor.

8. The method of Claim 6, wherein said step of detecting or isolating comprises detecting or isolating a labeling reagent that is attached to said agent or to an antibody that selectively binds to said agent.

9. The method of Claim 1 , wherein said step of detecting or isolating comprises contacting CTLA4⁺ T cells in said sample with an agent that selectively binds to CD4.

10. The method of Claim 9, wherein said agent is selected from the group consisting of an anti-CD4 antibody, a protein that selectively binds to CD4, a soluble majorhistocompatibility complex (MHC) class π protein, and a soluble HIV-gp 120 molecule.

11. The method of Claim 9, wherein said step of detecting or isolating comprises detecting or isolating a labeling reagent that is attached to said agent or to an antibody that selectively binds to said agent.

12. The method of Claim 1, further comprising detecting or isolating CD4⁺, CTLA4⁺ T cells that also express CD45RO and HLA-DR, wherein T cells that express CD4,

CTLA4, CD45RO, and HLA-DR are predicted to be infected with HIN.

13. The method of Claim 1, further comprising detecting or isolating CD4⁺, CTLA4⁺ T cells that do not express or have low expression of a molecule selected from the group consisting of CCR7 and CD62L, wherein T cells that express CD4 and CTLA4 and that do not express or have low expression of CCR7 or CD62L are predicted to be infected with HΓV.

14. The method of Claim 1, further comprising detecting or isolating CD4⁺, CTLA4⁺ T cells that also express CD45RO and HLA-DR and that do not express or have low expression of a molecule selected from the group consisting of CCR7 and CD62L, wherein T cells that express CD4, CTLA4, CD45RO, and HLA-DR, and that do not express or have low expression of CCR7 or CD62L, are predicted to be infected with HIV.

15. The method of Claim 1, wherein said biological sample is a blood sample.

16. The method of Claim 1, wherein said biological sample is a sample of peripheral blood mononuclear cells.

17. The method of Claim 1, further comprising a step of detecting or isolating human immunodeficiency virions from said CD4⁺, CTLA4⁺ T cells.

18. The method of Claim 1 , further comprising destroying CD4⁺, CTLA4⁺ T cells in said sample and returning said treated sample to said patient.

19. A method to monitor the efficacy of a treatment for human immunodeficiency virus (HIN)-infection in a patient, comprising: a. detecting or isolating CD4⁺, CTLA4⁺ T cells in a T cell-containing biological sample from a patient who is infected with and undergoing treatment for HIN; b. detecting or isolating human immunodeficiency virions produced by said CD4⁺, CTLA4⁺ T cells in said sample; and c. comparing the quantity or infectivity of human immunodeficiency virions from (b) to human immunodeficiency virions detected or isolated from CD4⁺, CTLA4⁺ T cells in a prior sample from said patient, wherein a reduction in the number or infectivity of said human immunodeficiency virions in (b) as compared to the quantity or infectivity of human immunodeficiency virions in the prior sample indicates that said treatment is having a beneficial effect.

20. The method of Claim 19, wherein said step (c) comprises comparing the quantity of human immunodeficiency virions in (b) to the quantity of human immunodeficiency virions in the prior sample.

21. The method of Claim 19, wherein said step (c) comprises comparing the infectivity of human immunodeficiency virions in (b) to the infectivity of human immunodeficiency virions in the prior sample.

22. The method of Claim 19, comprising measuring active virion assembly, wherein a reduction in virion assembly of said virions in (b) as compared to said virions in the prior sample indicates that the treatment is having a beneficial effect.

23. The method of Claim 19, comprising measuring active virion proliferation, wherein a reduction in active virion proliferation of said virions in (b) as compared to said virions in the prior sample indicates that the treatment is having a beneficial effect.

24. The method of Claim 19, further comprising testing virions isolated in (b) for their ability to infect a test culture of non-H-V infected T cells in vitro, as compared to said virions isolated from a prior sample, wherein a reduction in infectivity of said virions isolated in (b) indicates a positive effect of said treatment.

25. The method of Claim 19, wherein said prior sample was collected from said patient prior to an administration of said treatment for HIN-infection.

26. The method of Claim 19, wherein said prior sample was collected from said patient after an administration of said treatment for HIV-infection.

27. The method of Claim 19, further comprising detecting genetic mutations in said virions isolated in (b).

28. The method of Claim 19, wherein said biological sample is a blood sample.

29. The method of Claim 19, wherein said biological sample is a sample of peripheral blood mononuclear cells.

30. A method to monitor the efficacy of treatment for human immunodeficiency virus (HIN)-infection in a patient, comprising: a. detecting or isolating CD4⁺, CTLA4⁺ T cells in a T cell-containing biological sample from a patient who is infected with and undergoing treatment for HIV; and b. comparing the number of CD4⁺, CTLA4⁺ T cells in (a) to the number of CD4⁺, CTLA4⁺ T cells in a prior biological sample from the patient, wherein detection of a reduction in the number of CD4⁺, CTLA4⁺ T cells in said sample as compared to in the prior sample indicates that the treatment for HIN is reducing the number of HIV-infected cells in said patient.

31. The method of Claim 30, wherein said step (b) of comparing further comprises comparing the number of CD4⁺, CTLA4⁺ T cells in said sample to a number of CD4⁺, CTLA4⁺ T cells in a normal control sample, wherein detection of a change in the number of CD4⁺, CTLA4⁺ T cells toward the number of CD4⁺, CTLA4⁺ T cells in the normal control sample indicates that the treatment for HIN is reducing the number of HIV-infected cells in said patient.

32. The method of Claim 30, wherein said biological sample is a blood sample.

33. The method of Claim 30, wherein said biological sample is a sample of peripheral blood mononuclear cells.

34. A method to diagnose human immunodeficiency virus infection in a patient, comprising: a. detecting CD4⁺, CTLA4⁺ T cells in a T cell-containing biological sample from a patient who is suspected of being infected with HIN; b. isolating said CD4⁺, CTLA4⁺ T cells from (a); and c. detecting HIV in said cells from (b).

35. The method of Claim 34, wherein said step (c) of detecting comprises detecting human immunodeficiency virion infection and viral assembly and proliferation in said T cells isolated in (b).

36. The method of Claim 35, wherein said step (c) of detecting comprises detecting human immunodeficiency virions in said T cells isolated in (b) by in situ RΝA hybridization.

37. A method to identify a regulatory compound that inhibits human immunodeficiency virus (HIN) virion assembly and proliferation, comprising: a. contacting a putative regulatory compound with a cell or cell lysate that expresses CTLA4 and that is infected with HIV; b. identifying regulatory compounds that disrupt the regulation by Env of the intracellular localization of the CTLA4 secretory granule within the cell.

38. The method of Claim 37, wherein said regulatory compound is contacted with said cell intracellularly.

39. The method of Claim 37, wherein said step (b) comprises identifying regulatory compounds that disrupt the inhibition by Env of transport of the CTLA4 secretory granule to the plasma membrane.

40. The method of Claim 39, wherein said step (b) comprises detecting cell surface CTLA4 expression, wherein an increase in CTLA4 cell surface expression after contact with said putative regulatory compound as compared to prior to or in the absence of contact with said compound indicates that said compound disrupts the inhibition by Env of transport of the CTLA4 secretory granule to the plasma membrane.

41. The method of Claim 37, wherein said step (b) comprises identifying regulatory compounds that prevent the transport of CTLA4 secretory granules to the plasma membrane.

42. The method of Claim 37, wherein said step (b) comprises identifying regulatory compounds that induce transport to and expression of Env protein at the cell surface prior to the expression of other HIN proteins required for HIN virion assembly.

43. The method of Claim 42, wherein said step (b) comprises detecting cell surface expression of Env and the expression of at least one other HIN protein prior to and after contact with said putative regulatory agent, wherein detection of an increase in Env cell surface expression after contact with said compound as compared to prior to contact with said compound and as compared to expression of said other HIV protein indicates that said compound induces transport to and expression of Env protein at the cell surface prior to the expression of other HIN proteins required for HIV virion assembly.

44. The method of Claim 43, wherein said other HIN protein is Gag.

45. The method of Claim 37, wherein said step (b) comprises identifying regulatory compounds that result in the release of an increased number of non-infectious virions from said cell after contact with said compound as compared to prior to or in the absence of contact with said agent.

46. The method of Claim 37, wherein said step (b) comprises identifying regulatory compounds that disrupt the association of a cellular protein with the cytoplasmic tail sequence of Env in CTLA4 secretory granules, wherein said association sequesters CTLA4 secretory granules within the cell in the absence of said regulatory compound.

47. The method of Claim 46, wherein said cellular protein is a protein involved in the targeting and fusion or trafficking of intracellular granules to fuse with the plasma membrane.

48. The method of Claim 37, wherein said cell is a CD4⁺ T cell.

49. A method to identify a regulatory compound that inhibits human immunodeficiency virus (HIV) virion assembly and proliferation, comprising: a. isolating CD4⁺ T cells that express cytotoxic T lymphocyte antigen 4

(CTLA4) in a T cell-containing biological sample from a patient who is infected with HI , wherein T cells that express CD4 and CTLA4 are predicted to be infected with HIY; b. contacting a putative regulatory compound for inhibition of HIN infection with said cells of (a); and c. detecting regulatory compounds that reduce HIN virion assembly and proliferation in said cells of (a) after contact with said compound as compared to prior to or in the absence of contact with said compound.

50. The method of Claim 49, wherein said step of detecting comprises measuring the quantity of human immunodeficiency virions released from said cells of (a) after contact with said compound, wherein a decrease in the quantity of human immunodeficiency virions released from said cells indicates that said compound is a regulatory compound for inhibition of HIV.

51. The method of Claim 49, wherein said step of detecting comprises measuring the infectivity of human immunodeficiency virions released from said cells of (a) after contact with said compound, wherein a decrease in the infectivity of human immunodeficiency virions released from said cells indicates that said compound is a regulatory compound for inhibition of HIN.

52. The method of Claim 49, wherein said step of detecting comprises measuring active virion assembly in said cells of (a), wherein a reduction in active virion assembly after contact with said compound indicates that said compound is a regulatory compound for inhibition of HIV.

53. The method of Claim 49, wherein said step of detecting comprises measuring the number of T cells of (a) that are infected with actively proliferating HIY, wherein a decrease in the number of T cells that are infected with actively proliferating HIV after contact with said compound indicates that said compound is a regulatory compound for inhibition of HIN.

54. The method of Claim 49, wherein said step of detecting comprises measuring death of said T cells, wherein an increase in the death of said T cells after contact with said compound indicates that said compound is a regulatory compound for inhibition of HIN.

55. A method to inhibit human immunodeficiency virus (HIN) virion assembly and proliferation, comprising contacting a population of cells containing HIV-infected CD4⁺ T cells with a regulatory compound that disrupts the regulation by Env of the intracellular localization of the CTLA4 secretory granule within the cell.

56. The method of Claim 55, wherein said regulatory compound is a modified Gag protein that causes the premature delivery of Env and CTLA4 containing granules to the surface.

57. The method of Claim 56, wherein said modified Gag protein is an isolated Gag-Tat fusion protein.

58. The method of Claim 55, wherein said modified Gag protein is a portion of Gag protein sufficient to bind to Env and fused to a Tat protein.

59. The method of Claim 55, wherein said regulatory compound is administered to an HIN-infected patient in vivo.

60. A method to target an HIN-infected T cell for destruction, comprising contacting a population of cells containing HIN-infected CD4⁺ T cells with an agent that activates CTLA4 expressed by a CD4⁺ T cell in a manner effective to induce apoptosis of said cell.

61. The method of Claim 60, wherein said agent is selected from the group consisting of an anti-CTLA4 antibody, an agent that selectively binds to and activates CTLA4, and a soluble B7 receptor.

62. A method to target an HIV-infected T cell for destruction, comprising contacting a population of cells containing HIN-infected CD4⁺ T cells with a complex comprising: (1) a first agent that targets said complex to cells expressing CTLA4; and (2) a second agent selected from the group consisting of: a toxin, an anti-viral agent, an agent that induces apoptosis in said cell, and an agent that antagonizes the activity of Env, wherein said first and second agents are complexed together.

63. The method of Claim 62, wherein said first agent is selected from the group consisting of: an anti-CTLA4 antibody, an agent that selectively binds to CTLA4, and a soluble B7 receptor.

64. A method to identify a regulatory compound that inhibits human immunodeficiency virus (HIV) virion assembly and proliferation, comprising contacting an immobilized Env protein or cytoplasmic portion thereof with a Gag/cellular docking protein in the presence and absence of a putative regulatory compound, wherein an inhibition of the interaction between said Env protein or cytoplasmic portion thereof and said Gag or cellular docking protein in the presence of said putative regulatory compound as compared to in the absence of said putative regulatory compound, indicates that said putative regulatory compound inhibits HIN virion assembly.