WO2005114187A2 - Procédés et compositions de diagnostic du sida et d’autres maladies et conditions impliquant l’activation du système immunitaire - Google Patents

Procédés et compositions de diagnostic du sida et d’autres maladies et conditions impliquant l’activation du système immunitaire Download PDF

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WO2005114187A2
WO2005114187A2 PCT/US2005/013554 US2005013554W WO2005114187A2 WO 2005114187 A2 WO2005114187 A2 WO 2005114187A2 US 2005013554 W US2005013554 W US 2005013554W WO 2005114187 A2 WO2005114187 A2 WO 2005114187A2
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trail
hiv
cells
disease
immunoassay
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WO2005114187A3 (fr
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Gene Shearer
Jean-Philippe Herbeuval
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US Department of Health and Human Services
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153 or CD154

Definitions

  • This invention relates to methods and compositions suitable for monitoring the progression of AIDS, and other diseases whose progression involves immune system activation.
  • the invention relates to the use of the TNF-Related Apoptosis-Inducing Ligand (TRAIL) and TRAIL Compounds to monitor the progression of AIDS, and such other diseases.
  • TRAIL TNF-Related Apoptosis-Inducing Ligand
  • a number of diseases and conditions are characterized by a progression that involves immune system activation. These diseases include Acquired Immunodeficiency Syndrome (AIDS), allergic asthma, Alzheimer's disease, autoimmune disorders, Crohn's disease, Grave's disease, leukemia, atherosclerosis, lupus, multiple sclerosis, Parkinson's disease, transplant rejection, etc. (Ringheim, G.E. et al. (2004) "NEURODEGENERATIVE DISEASE AND THE NEUROIMMUNE AXIS (ALZHEIMER'S AND PARKINSON'S DISEASE, AND VIRAL INFECTIONS),” J. Neuroimmunol.
  • HIV Human Immunodeficiency Virus
  • CD4 + and CD8 + T cells are activated by HIV- 1 infection, and none of the current models adequately accounts for the preferential loss of CD4 + T cells (Groux, H. et al.
  • AICD initiated by a variety of stimuli has been proposed as a potential mechanism for "bystander” cell killing.
  • these stimuli noninfectious viruses, which represent more than 95% of HIV- 1 particles in plasma of patients, were shown to induce apoptosis of uninfected CD4 + and CD8 + T cells (Dimitrov, D. S. et al. (1993) "QUANTITATION OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 INFECTION KINETICS,” J Virol 67, 2182-90; Piatak, M., Jr. et al.
  • a surrogate marker should, ideally, identify patients at highest risk of disease progression, provide information on how an individual was infected, help clinicians accurately determine the stage of the HIV infection, predict development of opportunistic infections associated with the infection, and monitor the efficacy of antiviral treatments.
  • surrogate markers should be easily quantifiable, reliable, chemically available, and affordable.
  • surrogate markers are measurable traits that correlate with disease progression from symptomless HIV infection to full blown clinical AIDS (Tsoukas, CM. et al. (1994) "MARKERS PREDICTING PROGRESSION OF HUMAN IMMUNODEFICIENCY VIRUS-RELATED DISEASE,” Clin Microbiol Rev 7( 1 ): 14-28).
  • Prognostic or surrogate markers of HIV- 1 disease progression include viral load, CD4 + T cell number, soluble immune system activation markers and T helper cell functional profiles (Ho, D. D. (1996) "VIRAL COUNTS COUNT IN HIV INFECTION,” Science 272:1 124-1 125; Mellors, J.W. et al. (1996) "PROGNOSIS IN HIV-1 INFECTION PREDICTED BY THE QUANTITY OF VIRUS IN PLASMA,” Science 272 1 167-1 170). The number of HIV-1 particles in infected patient blood has been reported to be the best available surrogate marker of HIV-1 disease progression (Ho, D. D.
  • the total amount of HIV virus present in a patient at a given time i.e., the "viral load” is a particularly widely employed HIV surrogate marker (Mellors, J. et al. (1996) "PROGNOSIS IN HIV-1 INFECTION PREDICTED BY THE QUANTITY OF VIRUS IN PLASMA," Science 272: 1 167-1 170; Baker, R. (1996) "HIV VIRAL LOAD
  • PCR amplification is highly susceptible to cross-contamination; a drawback that has limited the clinical use of PCR in monitoring HIV infection (Schnittman, S.M.
  • HIV surrogate markers have been proposed.
  • cellular markers like HLA-DR + , IL-2R + , and T cells; as well as soluble markers like beta-2 microglobulin ( ⁇ -M), neopterin, soluble Interleukin receptor (sIL-2R), soluble CD4 (sCD4), and soluble CD8 (sCD8) have been proposed (Tsoukas, CM. et al. ( 1994) "MARKERS PREDICTING PROGRESSION OF HUMAN IMMUNODEFICIENCY VIRUS-RELATED DISEASE, " Clin Microbiol Rev. 7(1 ): 14-28).
  • a study performed to test the ability of cellular markers i.e., the number of CD4 T cells, the number of CD8 + T cells and the ratio of CD4 + T cells to CD8 + T cells
  • serologic markers i.e., serum levels of neopterin, beta 2-microglobulin, soluble interleukin-2 receptors, IgA, and HIV p24 antigen
  • serum levels of neopterin, beta 2-microglobulin, soluble interleukin-2 receptors, IgA, and HIV p24 antigen serum levels of neopterin, beta 2-microglobulin, soluble interleukin-2 receptors, IgA, and HIV p24 antigen
  • a marker must have a biologically relevant relationship to disease progression, the relationship cannot effectively be utilized in evaluating a treatment regimen unless the marker also responds quickly to effective treatment.
  • the level of plasma HIV RNA and the absolute numbers of peripheral CD4 + T cells have become de facto reference markers, additional parameters are still needed in clinical practice in order to refine antiretroviral treatments and to discriminate adequately between apparently equivalent stages of disease (see, De Milito, A. et al. (2003) "SURROGATE MARKERS AS A GUIDE TO EVALUATE RESPONSE TO ANTIRETROVIRAL THERAPY, " Curr. Med. Chem.10 (5):349-365).
  • prognostic markers of immune system activation that can be used to assess disease progression and which might be suitable for the monitoring and prognosis of antiretroviral therapies are desired (De Milito, A. et al. (2003) "SURROGATE MARKERS As A GUIDE TO EVALUATE RESPONSE TO ANTIRETROVIRAL THERAPY,” Curr. Med. Chem.10 (5):349-365; Fahey, J.L. et al.
  • TNF-Related Apoptosis-Inducing Ligand TNF-Related Apoptosis-Inducing Ligand (TRAIL) TNF-Related Apoptosis-Inducing Ligand (TRAIL) is a member of the TNF superfamily. It induces apoptosis in human tumor cell lines and in virus-infected cells, but does not induce apoptosis in normal cells (Wiley, S. R. et al. (1995) "IDENTIFICATION AND CHARACTERIZATION OF A NEW MEMBER OF THE TNF FAMILY THAT INDUCES APOPTOSIS," Immunity 3, 673-682; Griffith, T. S., et al.
  • TRAIL has been implicated in apoptosis of multiple cell types in HIV- infected patients, including CD4 + and CD8 + T cells (Lum, J. J. et al. (2001) "INDUCTION OF CELL DEATH IN HUMAN IMMUNODEFICIENCY VIRUS-INFECTED MACROPHAGES AND RESTING MEMORY CD4 T CELLS BY TRAIL/APO21 ,” J Virol 75, 11128-1 1 136; Katsikis, P. D. et al.
  • TRAIL TRAIL
  • mTRAIL membrane-bound
  • sTRAIL soluble TRAIL
  • type I interferon interferon-alpha and beta: IFN- ⁇ and IFN- ⁇
  • TRAIL can be secreted by leukocytes, including T lymphocytes, natural killer cells (Smyth, M.J. et al.
  • HIV- 1 infection activates circulating monocytes by inducing IFN- (Zagury, D. et al. (1998) "Interferon alpha and Tat involvement in the immunosuppression of uninfected T cells and C- C chemokine decline in AIDS,” Proc Natl Acad Sci U S A.
  • Soluble TRAIL (“sTRAIL”) is upregulated in HIV- 1 -infected patients (Liabakk, N.B. et al (2002) “DEVELOPMENT, CHARACTERIZATION AND USE OF MONOCLONAL ANTIBODIES AGAINST sTRAIL: MEASUREMENT OF STRAIL BY ELISA," J Immunol Methods. 259(1 -2): 1 19-28) and in vitro studies have linked TRAIL to the depletion of T cells from HIV-1 -infected patients (Lum, J. J. et al.
  • TRAIL-R1 death receptor 4 DR4
  • TRAIL-R2 DR5
  • DR5 gene is regulated by the transcription factor p53 which can be upregulated by type I interferons (Wu, G. S. et al.
  • the amino acid sequence of TRAIL is known, as is the N-terminal cytoplasmic domain (residues TRAIL residues 38-281 ), which comprises sTRAIL.
  • sTRAIL production is presently thought to involve a shedding mechanism that involves cleavage by proteases (see, Liabakk, N.B. et al.
  • This invention relates to methods and markers for monitoring the progression of AIDS, and other diseases whose progression involves immune system activation.
  • the invention relates to the use of the TNF-Related Apoptosis-Inducing Ligand (TRAIL) and TRAIL Compounds to monitor the progression of HIV infection, and to diagnose AIDS and other immune system diseases.
  • TRAIL TNF-Related Apoptosis-Inducing Ligand
  • this is accomplished by assessing the presence or concentration of TRAIL and especially mTRAIL, sTRAIL, the TRAIL DR5 receptor molecule, and biological molecules that activate TRAIL or its receptor or that are expressed by cells that express activated TRAIL or TRAIL DR5 receptor molecules in an individual's blood or other bodily fluids.
  • the invention further contemplates assessing the presence or concentration of molecules that are not TRAIL Compounds (as that term is defined herein) in an individual ' s blood or other bodily fluids in concert with an assessment of the presence or concentration of one or more TRAIL Compound(s) in order to determine the presence or severity of a disease involving immune system activation.
  • additional compounds include CD69 and HLA-DR.
  • the present invention thus provides a new surrogate marker that has mechanistic implications for HIV-1 pathogenesis, and which directly correlates with viral load but not necessarily inversely with CD4 + T cell count.
  • Soluble TRAIL produced by monocytes can interact with TRAIL death receptors that can be induced by CD 4 + T cells, and contribute to apoptosis-induced loss of CD4 + T cells in progression to AIDS.
  • the present invention thus permits, by measuring levels of the TNF-Related Apoptosis-Inducing Ligand, one to distinguish among HIV-1 infected patients with high viral load, HIV-1 infected patients with low viral loads and uninfected healthy individuals.
  • the invention provides an advantage over other surrogate marker (e.g., viral load measurement and CD4 + T cell count) in monitoring the progression of retroviral infection in that TRAIL is a death molecule involved in CD4 + T cell depletion in HIV/AIDS.
  • TRAIL is a marker of immune function that directly correlates with HIV viral load.
  • the invention provides a method for determining whether a mammal suffers from a disease or condition of involving immune system activation, wherein the method comprises assaying for the presence or concentration of a TRAIL Compound in a biological fluid of the mammal.
  • the invention particularly concerns the embodiment of such method wherein the mammal is selected from the group consisting of a human, simian, feline, bovine, equine, canine, ovine or porcine mammal.
  • the method comprises an immunoassay that determines the presence or concentration of the TRAIL Compound
  • the immunoassay comprising the steps of: (a) contacting a sample of the biological fluid with an antibody specific for the TRAIL Compound, the contacting being under conditions sufficient to permit the TRAIL Compound if present in the sample to bind to the antibody and form a TRAIL Compound - antibody complex; (b) contacting the formed TRAIL Compound - antibody complex with a molecule capable of specific binding to the complex, the contacting being under conditions sufficient to permit the molecule to bind to the complex and form an extended complex; and (c) determining the presence or concentration of the TRAIL Compound in the biological fluid by determining the presence or concentration of the formed extended complex in the sample.
  • the TRAIL Compound is selected from the group consisting of sTRAIL, mTRAIL, the TRAIL DR5 receptor molecule, a biological molecule that activates TRAIL, and a biological molecule that activates the TRAIL DR5 receptor (especially, wherein the biological molecule that activates the TRAIL DR5 receptor is p53.
  • the invention particularly concerns the embodiments of such methods wherein the method reveals the presence of the TRAIL Compound, or wherein the method reveals the concentration of the TRAIL Compound.
  • the invention particularly concerns the embodiments of such methods wherein the disease or condition involving immune system activation is selected from the group consisting of HIV infection, AIDS, cancer, atherosclerosis, Alzheimer's disease, inflammation, autoimmune disorder, allergic asthma, Crohn ' s disease, Grave ' s disease, lupus, multiple sclerosis, Parkinson ' s disease, allograft transplant rejection, and graft vs host disease.
  • the disease or condition involving immune system activation is cancer, and particularly colon cancer, lung cancer, breast cancer, pancreatic cancer, leukemia, myeloma, skin cancer, brain cancer, cervical cancer, or testicular cancer.
  • the invention additionally concerns an immunoassay that determines the presence or concentration of a TRAIL Compound in a biological fluid of a mammal, wherein the immunoassay comprises the steps of: (a) contacting a sample of the biological fluid with an antibody specific for the TRAIL Compound, the contacting being under conditions sufficient to permit the TRAIL Compound if present in the sample to bind to the antibody and form a TRAIL Compound - antibody complex; (b) contacting the formed TRAIL Compound - antibody complex with a molecule capable of specific binding to the complex, the contacting being under conditions sufficient to permit the molecule to bind to the complex and form an extended complex; and (c) determining the presence or concentration of the TRAIL Compound in the biological fluid by determining the presence or concentration of the formed extended complex in the sample.
  • the invention additionally concerns the embodiment of such immunoassay wherein the TRAIL Compound is selected from the group consisting of sTRAIL, mTRAIL, the TRAIL DR5 receptor molecule, a biological molecule that activates TRAIL, and a biological molecule that activates the TRAIL DR5 receptor (especially, wherein the biological molecule that activates the TRAIL DR5 receptor is p53
  • the invention additionally concerns the embodiment of such immunoassay wherein the mammal is selected from the group consisting of a human, simian, feline, bovine, equine, canine, ovine or porcine mammal.
  • the invention additionally concerns the embodiments of such immunoassays wherein the immunoassay is an immunochromatographic immunoassay, wherein: in the step (a), the biological sample is placed in contact with a first porous carrier, the first porous carrier containing a non-immobilized, labeled antibody specific for the TRAIL Compound; in the step (b), the formed TRAIL Compound- antibody complex is placed in contact with a second porous carrier, the second porous carrier being in communication with the first porous carrier, and containing an immobilized molecule capable of specific binding to the complex; and in the step (c), the presence or concentration of the TRAIL Compound in the biological fluid is determined by detecting the presence of the labeled antibody specific for the TRAIL Compound in the second porous carrier.
  • the immunoassay is an immunochromatographic immunoassay, wherein: in the step (a), the biological sample is placed in contact with a first porous carrier, the first porous carrier containing a non-immobilized, labeled antibody specific
  • the invention additionally concerns the embodiments of such immunoassays wherein the immunoassay reveals the presence of the TRAIL Compound or wherein the immunoassay reveals the concentration of the TRAIL Compound.
  • the invention additionally concerns the embodiments of such immunoassays wherein the immobilized molecule capable of specific binding to the complex is an rsTRAIL molecule.
  • the invention additionally concerns the embodiments of such immunoassays wherein the TRAIL Compound is sTRAIL, and the immobilized molecule capable of specific binding to the complex is an anti-sTRAIL antibody.
  • the invention additionally concerns the embodiments of such immunoassays wherein the immunoassay determines whether a mammal suffers from a disease or condition involving immune system activation selected from the group consisting of HIV infection, AIDS, cancer, atherosclerosis, Alzheimer's disease, inflammation, autoimmune disorder, allergic asthma, Crohn's disease,
  • the invention further concerns the embodiments of such methods wherein the disease or condition involving immune system activation is cancer, and particularly colon cancer, lung cancer, breast cancer, pancreatic cancer, leukemia, myeloma, skin cancer, brain cancer, cervical cancer, or testicular cancer.f
  • the invention additionally concerns a kit for measuring the presence or concentration of a TRAIL Compound in a biological fluid of a mammal, wherein the kit comprises a hollow casing comprising a multilayer filter system, and first and second porous carriers, wherein the second porous carrier is in communication with the first porous carrier, and the first porous carrier is in communication with the multilayer filter system, a portion of which is accessible from the casing; wherein: the first porous carrier contains a non-immobilized, labeled antibody that specifically binds the TRAIL Compound; and the second porous carrier contains an immobilized, unlabeled molecule that binds to a TRAIL Compound - antibody complex.
  • the invention additionally concerns the embodiments of such kits wherein the kit permits the presence of the TRAIL Compound or the concentration of the TRAIL Compound to be determined.
  • the invention additionally concerns the embodiments of such kits wherein the immobilized molecule capable of specific binding to the complex is an rsTRAIL molecule.
  • the invention additionally concerns the embodiments of such kits wherein the TRAIL Compound is selected from the group consisting of sTRAIL, mTRAIL, the TRAIL DR5 receptor molecule, a biological molecule that activates TRAIL, and a biological molecule that activates the TRAIL DR5 receptor (especially, wherein the biological molecule that activates the TRAIL DR5 receptor is p53) , and the immobilized molecule capable of specific binding to the complex is an anti-sTRAIL antibody.
  • the TRAIL Compound is selected from the group consisting of sTRAIL, mTRAIL, the TRAIL DR5 receptor molecule, a biological molecule that activates TRAIL, and a biological molecule that activates the TRAIL DR5 receptor (especially, wherein the biological molecule that activates the TRAIL DR5 receptor is p53) , and the immobilized molecule capable of specific binding to the complex is an anti-sTRAIL antibody.
  • the invention additionally concerns the embodiments of such kits wherein the kit is suitable for determining whether a mammal suffers from a disease or condition of involving immune system activation selected from the group consisting of HIV infection, AIDS, cancer, atherosclerosis, Alzheimer's disease, inflammation, autoimmune disorder, allergic asthma, Crohn's disease, Grave's disease, lupus, multiple sclerosis, Parkinson's disease, allograft transplant rejection, and graft vs host disease.
  • the invention further concerns the cmbodi ents of such kits wherein the disease or condition involving immune system activation is cancer, and particularly colon cancer, lung cancer, breast cancer, pancreatic cancer, leukemia, myeloma, skin cancer, brain cancer, cervical cancer, or testicular cancer.
  • one aspect of the present invention concerns the recognition that anti-type I interferon antibodies can be used as an anti-HIV-1 therapeutic agent.
  • Figure 1 shows increased plasma levels of TRAIL in AIDS patients.
  • Panel A shows plasma samples from 107 HIV-1 -infected patients and 53 uninfected controls which are tested for their soluble TRAIL content by ELISA.
  • Two groups of HIV-1 -infected patients were defined depending on their viral load.
  • the mean values of plasma TRAIL were 852 ⁇ 52 pg/ml for 55 control donors, 1339 ⁇ 79 pg/ml for 49 HIV- 1 -infected patients with undetectable viral load ( ⁇ 50 RNA copies/ml of blood) and 2242 ⁇ 131 pg/ml for 58 HIV-1 -infected patients with higher viral load (>50 RNA copies/ml of blood).
  • FIG. 1 Panel B shows correlation between TRAIL levels observed in patients and TRAIL levels predicted by the invention.
  • Figure 2 shows effects of AT-2 HIV-1 particles on TRAIL secretion.
  • Panel A shows PBMC from HIV-1 uninfected or HIV-1 infected individuals cultured for 3 days in the presence or absence of AT-2 HIV-1 MN OI HIV- 1 Ada - Levels of TRAIL are detected using ELISA.
  • Panel B shows CD4 + , CD8 + T cells, DC, macrophages and monocytes from infected and noninfected individuals cultured for 3 days in presence of AT-2 HIV-1 N Mean values (+/- standard errors) are shown for six independent experiments for each condition tested.
  • Panel C shows monocytes from HIV-1 uninfected donors cultured for 1 day in the presence of microvesicles (controls) or AT-2 HIV- 1 M Cells are analyzed for mTRAIL by FACS.
  • Panel D shows monocytes from HIV-1 uninfected donors cultured for 1 day in the presence of microvesicles or AT-2 HIV- 1 MN - TRAIL mRNA level quantified by real time PCR. Mean values with standard errors are shown for four independent experiments.
  • Panel E shows monocytes from HIV-1 uninfected donors cultured in the presence of different concentration of AT-2 HIV- I MN and sTRAIL quantified by ELISA. Mean values with standard errors are shown for six independent experiments.
  • Figure 3 shows effect of IFN type 1 antibodies on TRAIL production by monocytes from HIV-1 uninfected donors.
  • Panel A shows monocytes cultured for 3 days in the presence of AT-2 HIV-1 MN with blocking antibodies against IFN- ⁇ , IFN- ⁇ or both.
  • the level of sTRAIL is quantified by ELISA.
  • Panel B shows monocytes cultured for 1 day in the presence of AT-2 HIV-1 MN , with or without blocking antibodies against IFN- ⁇ and IFN- ⁇ .
  • the level of TRAIL mRNA is quantified by real time PCR analysis.
  • Panel C shows monocytes cultured for 3 days in the presence of recombinant IFN- ⁇ or IFN- ⁇ .
  • the level of sTRAIL is quantified by ELISA. Mean values with standard errors presented in Figure 3, Panels A, B, and C are representative of four independent experiments for each condition are tested.
  • Panel D shows IFN- ⁇ levels by monocytes cultured for 1 (open bars) or 3 (closed bars) days in presence of microvesicles (control), AT-2 HIV-1 MN or AT-2 HIV-1
  • Panel E shows STAT1 and STAT2 expression.
  • Monocytes were cultured 24h in the presence of AT-2 HIV-1 MN, AT-2 HIV-1 A d d or recombinant IFN- ⁇ / ⁇ (10 ng/mL) and production of STAT1 and STAT2 was analyzed by western- blot. ⁇ -actin was used as a loading control.
  • Panel F shows the results of a blocking assay: monocytes were cultured for 3 days with AT-2 HIV-1 MN and in presence of: (1) isotype control antibody; (2) the CD4 binding inhibitor soluble CD4 (2 ⁇ g/ml); (3) the CXCR4 inhibitor AMD-3100 (2 ⁇ g/ml); (4) the fusion inhibitor T20 (2 ⁇ g/ml). Monocytes cultured without AT-2 HIV-1 MN were used as control. TRAIL level was quantified by ELISA. Mean values with standard errors are shown for 3 independent experiments for each condition tested.
  • Figure 4 shows effect of infectious HIV-1 on TRAIL production by monocytes.
  • Panel A shows monocytes cultured for 3 days in the presence of AT-2 HIV-1 MN , HIV- 1 M N or infectious HIV-1 LAV.
  • sTRAIL level is determined by ELISA. Mean values with standard errors are shown for six independent experiments for each condition tested.
  • Panel B shows monocytes from HIV-1 - donors cultured 3 days in the presence of live HIV- 1 N with blocking antibodies against IFN , IFN ⁇ or both.
  • the level of sTRAIL is quantified by ELISA. Mean values with standard errors are shown for 6 independent experiments for each condition tested.
  • Panel C shows monocytes from HIV-1 " donors cultured for 1 day in the presence of microvesicles (controls) or live HIV-1 MN .
  • Cells are analyzed for mTRAIL by FACS.
  • Monocytes exposed to HIV-1 MN overexpressed mTRAIL compared to controls (p ⁇ .0001 ). Data are representative of 6 independent experiments performed.
  • Figure 5 shows effect of infectious HIV-1 on sTRAIL production in tonsil culture.
  • Ex vivo human lymphoid tissue were culture for 12 days in presence or absence of HIV-1 LA V- STRAIL was measured at day 3, 6, 9 and 12 by ELISA. Culture medium was changed at 3-day intervals.
  • Panel B Plasma TRAIL levels and flow cytometric analysis of Annexin V + DR5 + CD4 + T cells from 16 uninfected control donors and 22 HI V-l -infected patients, p values were calculated using a two-tailed Student's t test.
  • Figure 7 shows annexin V and STAT expression in T cells:
  • Figure 7, Panel A Flow cytometric analysis of positively-selected CD4 + and CD8 + T cells from healthy, HIV-1 -uninfected blood donors cultured for 1 (left) and 6 (right) days alone or in the presence of microvesicles (controls) or with: H1V-1 MN ; HIV- l ⁇ da ; influenza A virus (Flu); HIV-1 MN plus Flu; or HIV-l Ada plus Flu. Infectious HIV-1 was used in the example illustrated. Very similar results were obtained when AT-2 HIV-1 was used.
  • Figure 7, Panel D Effect of culturing CD4 + T cells with Flu, AT-2 HIV-1 MN or AT-2 HIV-1 MN plus Flu on IFN- ⁇ production
  • Figure 7, Panel E phosphorylated STAT1 activation
  • Figure 7, Panel F Western blot analysis of STAT1 and STAT2 protein after exposure of CD4 + T cells to AT-2 HIV-1 MN , Flu, AT-2 HIV-1 MN plus Flu, and effect of anti-IFN- ⁇ / ⁇ antibodies on STAT expression.
  • Data of Figure 7, Panel A are mean values with standard errors of 6 independent experiments for each condition tested.
  • Data of Figure 7, Panels B, C, D, E and F are representative of 4 independent experiments.
  • Figure 8 shows an analysis of TRAIL expression and production:
  • Panel A Flow cytometric analysis of CD4 + and CD8 + T cells cultured with Flu, infectious HIV-I MN, or HIV-I MN plus Flu.
  • Panel B TRAIL gene expression after culture of CD4 + T cells with AT-2 HIV-1 MN plus Flu , and effect of anti-IFN- ⁇ / ⁇ antibodies.
  • Panel C Effect of anti-IFN- ⁇ / ⁇ antibodies on mTRAIL expression induced by culture with AT-2 HIV-1 MN plus Flu.
  • Panel D Effect of culturing CD4 + T cells with IFN- ⁇ , IFN- ⁇ or IFN- ⁇ / ⁇ on the percentage of CD4 + T cells expressing mTRAIL.
  • Panel E Western blot analysis of TRAIL production after culture of CD4 + T cells with AT-2 HIV-1 MN , Flu, AT-2 HIV-1 MN plus Flu, AT-2 HIV-1 MN plus Flu plus anti-IFN- ⁇ / ⁇ antibodies, or rlFN- ⁇ / ⁇ .
  • Panel F Effect of anti-IFN- ⁇ / ⁇ antibodies, monoclonal RIK-2 anti-TRAIL antibody, anti-DR5 antibody, and soluble CD4-IgG (sCD4) on the percentage of Annexin V + cells resulting from culture of CD4 + T cells with AT-2 HIV-I MN plus Flu
  • Panels B and F are mean values with standard errors of 6 independent experiments for each condition tested.
  • Panels A, C, D and E are representative of 4 independent experiments.
  • Figure 9 shows DR5 expression and p53 transcription and synthesis:
  • Panel A Flow cytometric analysis of DR5 expression on CD4 + and CD8 + T cells after culture with Flu, infectious HIV-1 MN , or Flu plus infectious HIV-I MN (Identical results were obtained using AT-2 HIV-1 MN ).
  • Panel B Two-color flow cytometric analysis of Annexin V/DR5 double positive CD4 + T cells cultured alone (control) or with AT-2 HIV-1 MN plus Flu.
  • Panel C Panel C: DR5 gene expression in CD4 + T cells cultured with AT-2 HIV-1 MN plus Flu, and inhibition by anti-IFN- ⁇ / ⁇ antibodies.
  • Panel D Flow cytometric analysis of: AT-2 HIV-1 MN plus Flu-induced DR5 expression by CD4 + T cells and inhibition by anti-IFN- ⁇ / ⁇ antibodies (upper) and lack of effect of recombinant IFN- ⁇ / ⁇ plus HIV-1 MN on DR5 expression (middle) and apoptosis (lower) of CD4 + T cells.
  • Panel E Western blot analysis of DR5 protein production by CD4 + T cells cultured with AT-2 HIV-1 MN) Flu, AT-2 HIV-1 MN plus Flu or rlFN- ⁇ / ⁇ , and inhibition of DR5 by anti-IFN- ⁇ / ⁇ antibodies.
  • Panel F Effect of culture of CD4 + T cells with AT-2 HIV-1 MN plus Flu on p53 gene expression and inhibition by anti-IFN- ⁇ / ⁇ antibodies.
  • Panel G Effect of culture of CD4 + T cells with AT-2 HIV-1 MN , Flu, or AT-2 HIV-1 MN plus Flu on p53 protein production.
  • Panel H Western blot analysis of p53 production by CD4 + T cells cultured with AT-2 H1V-1 MN , Flu, AT-2 HIV-1 MN plus Flu or rIFN- ⁇ / ⁇ , and inhibition by anti-IFN- ⁇ / ⁇ antibodies.
  • Panels C, F and G are mean values with standard errors of 6 independent experiments for each condition tested.
  • Panels A, B, D, E and H are representative of 4 independent experiments.
  • Figure 10 illustrates a "Two-Hit" model of type 1 interferon-dependent TRAIL ⁇ DR5-mediated selective apoptosis of CD4 + T cells in AIDS progression.
  • CD4 + T cells Exposure of CD4 + T cells to both hits is required to activate STAT2 (also increased STATl) and p53 that results in DR5 expression. Each of these virus- induced events is blocked by antibodies against IFN- ⁇ / ⁇ . None of the above virus- induced changes were observed in CD8 + T cells. Because rIFN- ⁇ / ⁇ did not replace the second virus for inducing p53, DR5 and apoptosis, "?” indicates a signal or event of the two-hit model (that triggers p53 and DR5).
  • pTRAIL plasma TRAIL
  • mTRAIL membrane TRAIL.
  • the present invention relates to methods and compositions suitable for monitoring the progression of AIDS, and other diseases whose progression involves immune system activation.
  • TRAIL TNF-Related Apoptosis-Inducing Ligand
  • CD4+ T cells that results from HIV-1 infection remains unsolved despite more than 20 years of investigation of AIDS pathogenesis.
  • TNF-related apoptosis-inducing ligand TRAIL
  • TNF superfamily a member of the TNF superfamily
  • TRAIL TNF-related apoptosis-inducing ligand
  • TRAIL induces apoptosis in human tumor cell lines (Griffith, T.S. et al. (1998) "INTRACELLULAR REGULATION OF TRAIL-INDUCED APOPTOSIS IN HUMAN MELANOMA CELLS,” J Immunol. 161 :2833-2840) and in infected cells (Jeremias, I. et al. (1998)
  • TRAIL Compound is intended to include not only the naturally occurring or recombinant fragments of TRAIL (such as mTRAIL and sTRAIL, ), but also the TRAIL DR5 receptor molecule, and biological molecules that activate TRAIL (such as IFN ⁇ / ⁇ ) or its receptor or that are expressed by cells that express activated TRAIL or TRAIL DR5 receptor molecules or fragments of such molecules.
  • TRAIL TRAIL
  • TRAIL is a membrane-associated protein composed of multiple domains: an N-terminal cytoplasmic domain, an internal transmembrane domain, and a C-terminal extracellular domain (amino acid residues 38-281 of SEQ ID NO:l); the C- terminal extracellular domain being responsible for inducing apoptosis (Wiley, S. R. et al. (1995) "IDENTIFICATION AND CHARACTERIZATION OF A NEW MEMBER OF
  • TRAIL The amino acid sequence of TRAIL (SEQ ID NO:l) is presented below, with the N-terminal cytoplasmic domain (residues 38-281 ) underlined:
  • TRAIL is encoded by residues 88-930 of SEQ ID NO:2 (the start and stop codons of TRAIL are underlined).
  • invention contemplates methods and compositions for monitoring the presence of biologically active forms of TRAIL, and in particular, for monitoring biologically active forms of TRAIL that are membrane associated or naturally secreted from cells.
  • biologically active forms of TRAIL include membrane bound TRAIL ("mTRAIL”) and soluble (i.e., non-membrane bound) TRAIL ("sTRAIL").
  • mTRAIL membrane bound TRAIL
  • sTRAIL soluble (i.e., non-membrane bound) TRAIL
  • TRAIL Compound is intended to refer to naturally occurring or recombinant fragments of TRAIL.
  • sTRAIL is the release form of membrane TRAIL, and corresponds to the extracellular domain of TRAIL (amino acid residues 38-281 of SEQ ID NO:l); it is encoded by residues 199-930 of SEQ ID NO:2).
  • the production of sTRAIL may involve a shedding mechanism that involves cleavage by proteases (see, Liabakk, N.B. et al. (2002) “DEVELOPMENT, CHARACTERIZATION AND USE OF MONOCLONAL ANTIBODIES AGAINST sTRAIL: MEASUREMENT OF sTRAIL BY ELISA,” J Immunol Methods.
  • sTRAIL has been used as a surrogate marker for assessing hepatitis B viral infection (Han, L.H. et al. (2002) “DETECTION OF SOLUBLE TRAIL IN HBV INFECTED PATIENTS AND ITS CLINICAL IMPLICATIONS,” World J Gastroenterol. 8(6): 1077-1080), and multiple sclerosis (Wandinger, K.P. et al.
  • rsTRAIL may be produced by expressing a TRAIL fragment comprising the C- terminal extracellular domain.
  • a TRAIL fragment comprising the C- terminal extracellular domain.
  • Wiley, S. R. et al. expressed a recombinant sTRAIL comprising amino acid residues 95-281 of SEQ ID NO:l (U.S Patent No. 5,763,223; Wiley, S. R. et al. (1995) "IDENTIFICATION AND CHARACTERIZATION OF A NEW MEMBER OF THE TNF FAMILY THAT INDUCES
  • rsTRAIL molecules comprising the C- terminal extracellular domain can be employed (for example, rsTRAIL molecules comprising amino acid residues 101-281, or 114-281, of SEQ ID NO:l) to induce apoptosis. See also, Wang, L.H. et al. (2002) “DENSITY CULTIVATION FOR PREPARATION OF RECOMBPNANT SOLUBLE HUMAN TRAIL. Acad. J. Sec. Mil. Med. Univ. 23(2):132- 135; Wang, X.J. et al.
  • sTRAIL has been expressed in Escherichia coli (Xia, X.X. et al. (2004) "Purification and Characterization of Recombinant sTRAIL Expressed in Escherichia coli," Acta Biochimica Biophysica Sinica 36(2): 1 18- 122).
  • sTRAIL ELISA detection kits are available commercially (Diaclone). Antibodies to sTRAIL and to TRAIL can be readily produced, or obtained commercially (Diaclone; Alexis Co ⁇ oration; Trinoca Biochem).
  • TRAIL DR5 Receptor The TRAIL DR5 receptor and polynucleotides that encode the TRAIL DR5 receptor, or fragments of such molecules, are TRAIL Compounds of the present invention.
  • the amino acid sequence of the TRAIL DR5 receptor is provided below as SEQ ID NO:ll.
  • a nucleic acid sequence that encodes the TRAIL DR5 receptor is provided below as SEQ ID NO:12 (see, GenBank Accession Nos. AAB71412 and AF020501, herein incorporated by reference).
  • the start and stop codons for the TRAIL DR5 receptor are underlined.
  • TRAIL DR5 may be a potential anti-cancer therapy (Yagita, H. et al. (2004) “TRAIL AND ITS RECEPTORS As TARGETS FOR
  • the p53 protein and polynucleotides that encode the p53 protein, or fragments of such molecules, are TRAIL Compounds of the present invention.
  • the p53 protein is encoded by the p53 gene, which is a very well described cancer gene (see, e.g., Vogelstein, B. et al. (2004) "CANCER GENES AND THE PATHWAYS THEY CONTROI,” Nat Med. 10(8):789-799; Okada, H. et al (2004) “PATHWAYS OF APOPTOTIC AND NON-APOPTOTIC DEATH IN TUMOUR CELLS,” Nat Rev Cancer. 4(8):592-603).
  • the amino acid sequence of the human p53 protein is provided below as SEQ ID NO:13.
  • SEQ ID NO: 14 A nucleotide sequence that encodes this protein is provided below as SEQ ID NO: 14 (see, GenBank Accession Nos. K03199 and AAA59989, herein inco ⁇ orated by reference). The start and stop codons for p53 are underlined.
  • CD69 is an early activation marker of peripheral lymphoctyte cells (Fogli, M. et al. (2004) "SIGNIFICANT NK CELL ACTIVATION ASSOCIATED WITH DECREASED CYTOLYTIC FUNCTION IN PERIPHERAL BLOOD OF HIV- 1 -INFECTED PAl lENTS,”Eur J Immunol. 34(8):2313-2321). CD69 is discussed in U.S. Patent 6,593,124.
  • Assays for CD69, and polynucleotides that encode CD69, or fragments of such molecules, may be used in concert with assays of TRAIL Compounds in order to monitor the progression of AIDS, and other diseases whose progression involves immune system activation.
  • the amino acid sequence of CD69 is provided by SEQ ID NO:15.
  • the sequence of a polynucleotide that encodes CD69 is provided by SEQ ID NO: 16 (see, GenBank Accession Nos. NM 001781 and NP 001772, herein inco ⁇ orated by reference).
  • the start and stop codons for CD69 arc underlined.
  • CD69 expression may be used in the diagnosis of HIV infection (Nielsen, S.D. et al. (1998) "EXPRESSION OF THE ACTIVATION ANTIGEN CD69 PREDICTS FUNCTIONALITY OF IN VITRO EXPANDED PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) FROM HEALTHY DONORS AND HIV-INFECTED PATIENTS," Clin Exp Immunol. 1 14( 1 ):66-72; Prince, H.E. et al.
  • the HLA-DR antigen is part of the major histocompatibility complex (MHC) that identifies self from non-self and is pivotal in immune recognition of foreign tissue and infective agents and presents a variety of cellular proteins necessary for immunity
  • MHC major histocompatibility complex
  • Assays for HLA-DR may be used in concert with assays of TRAIL Compounds in order to monitor the progression of AIDS, and other diseases whose progression involves immune system activation.
  • the amino acid sequence of HLA-DR alpha is provided by SEQ ID NO: 17.
  • the sequence of a polynucleotide that encodes HLA-DR alpha is provided by SEQ ID NO:18 (see, GenBank Accession Nos. NM 0191 1 1 and NP_061984, herein inco ⁇ orated by reference).
  • the start and stop codons for HLA-DR are underlined.
  • SEQ ID NO:17 MAISGVPVLG FFIIAVLMSA QES AIKEEH VIIQAEFYLN PDQSGEFMFD 10 20 30 40 50
  • HLA-DR may be used to predict HIV-progression (Gascon, R.L. et al. (2002) "INCREASED HLA-DR EXPRESSION ON PERIPHERAL BLOOD MONOCYTES IN SUBSETS OF SUBJECTS WITH PRIMARY HIV INFECTION IS ASSOCIATED WITH ELEVATED CD4 T-CELL APOPTOSIS AND CD4 T-CELL DEPLETION,” J Acquir Immune Defic Syndr. 30(2): 146-153; Choi, B.S. et al.
  • Interferon-alpha and beta IFN- ⁇ and IFN- ⁇ regulate membrane-bound (mTRAIL) and soluble TRAIL (sTRAIL)
  • mTRAIL membrane-bound
  • sTRAIL soluble TRAIL
  • SEQ ID NO:19 amino acid sequence of interferon alpha
  • SEQ ID NO:20 amino acid sequence of a polynucleotide that encodes interferon alpha
  • the gene encoding IFN ⁇ is located at positions 1806-2829 of SEQ ID NO:20. Position numbering is from AL390882. The start and stop codons for interferon alpha are underlined.
  • interferon beta The amino acid sequence of interferon beta is provided by SEQ ID NO:21.
  • sequence of a polynucleotide that encodes interferon beta is provided by SEQ ID NO:22 (see, GenBank Accession No. AL390882, herein inco ⁇ orated by reference).
  • the gene encoding IFN ⁇ is located at positions 90503-91361 of SEQ ID NO:22. Position numbering is from AL390882.
  • the start and stop codons for interferon beta are underlined.
  • the present invention provides a method for determining whether a mammal (especially a human, simian, feline, bovine, equine, canine, ovine or porcine mammal) suffers from a disease or condition of involving immune system activation, wherein the method comprises assaying for the presence or concentration of one or more TRAIL Compound(s) in a biological fluid of said mammal.
  • examples of such diseases and conditions whose progression involves "immune system activation” include HIV (FIV, SIV, etc.) infection, AIDS, certain cancers (e.g., colon cancer, lung cancer, breast cancer, pancreatic cancer, leukemia, myeloma, skin cancer, brain cancer, cervical cancer, testicular cancer, etc.), atherosclerosis, Alzheimer ' s disease, inflammatory diseases and autoimmune disorders (especially, allergic asthma, Crohn's disease, Grave ' s disease, lupus, multiple sclerosis, Parkinson ' s disease, allograft transplant rejection, graft vs. host disease, etc.
  • HIV HIV
  • the methods and compositions of the present invention may be used to diagnose whether such a mammal is healthy, or suffers from such diseases and conditions.
  • the methods and compositions of the present invention may be used to monitor the progression, stage, or severity of a disease or condition in humans and other mammals known to suffer from such a disease or condition.
  • the present invention particularly provides a method for diagnosing AIDS and other diseases and conditions involving immune system activation.
  • the present invention permits one to distinguish among HIV-1 -infected patients with high viral loads, patients with low viral loads and uninfected healthy control blood donors versus by measuring plasma levels of one or more TRAIL Compounds, and to relate the quantity of viral particles in an individual to the level(s) of TRAIL Compounds expressed or sectreted by cells (e.g., monocytes) and to recognize that the quantity of HIV-1 -mediated TRAIL Compound production by such cells is regulated by type I interferon.
  • cells e.g., monocytes
  • TRAIL Compound expression production is a marker of immune system activation (e.g., TRAIL is produced after HIV-1 infection)
  • the TRAIL Compounds can be used as markers for evaluating the progression of all diseases and conditions involving immune system activation.
  • the invention measures the presence or concentration of a TRAIL Compound in a fluid of a patient.
  • the invention measures the presence or concentration of sTRAIL or mTRAIL in a fluid of a patient.
  • Suitable fluid can comprise blood, serum, plasma, urine, saliva, semen, cerebrospinal fluid, lymph, etc.
  • sTRAIL a TRAIL Compound
  • mTRAIL a fluid of a patient.
  • Suitable fluid can comprise blood, serum, plasma, urine, saliva, semen, cerebrospinal fluid, lymph, etc.
  • a fluid will be blood, serum, or plasma.
  • the determination of the presence or concentration of sTRAIL may be accomplished using any molecule capable of specific binding with sTRAIL (e.g., a receptor or ligand of sTRAIL).
  • the invention will employ an immunoassay to detennine the concentration or presence of sTRAIL in such a fluid.
  • antibody is intended to encompass both naturally produced immunoglobulins (e.g., IgG, etc.), as well as fragments of such molecules (e.g., Fab, F(ab) 2 , etc.) that are capable of specifically binding an antigen, as well as recombinant antigen- binding molecules (e.g., humanized antibodies, single chain antibodies, etc.).
  • the invention will employ a PE-conjugated mouse IgGl anti-human TRAIL monoclonal antibody.
  • any of a wide variety of assay formats may be used in accordance with the methods of the present invention. Such formats may be heterogeneous or homogeneous, sequential or simultaneous, competitive or noncompetitive.
  • U.S. Patent Nos. 5,563,036; 5,627,080; 5,633,141 ; 5,679,525; 5,691 ,147; 5,698,41 1 ; 5,747,352; 5,811 ,526; 5,851,778; and 5,976,822 illustrate several different assay formats and applications.
  • Such assays can be formatted to be quantitative, so as to measure the concentration or amount of the selected TRAIL Compound, or they may be fonnatted to be qualitative, so as to measure the presence or absence of the selected TRAIL Compound.
  • Enzymatic-binding assay formats e.g., Enzyme- Linked Immunosorbent Assay (ELISA)
  • ELISA Enzymatic-binding assay formats
  • Heterogeneous immunoassay techniques typically involve the use of a solid phase material to which the reaction product becomes bound, but may be adapted to involve the binding of non-immobilized antigens and antibodies (i.e., a solution- phase immunoassay).
  • the reaction product is separated from excess sample, assay reagents, and other substances by removing the solid phase from the reaction mixture (e.g., by washing).
  • One type of solid phase immunoassay that may be used in accordance with the present invention is a sandwich immunoassay.
  • the more TRAIL Compound analyte e.g., sTRAIL, mTRAIL, etc.
  • This type of assay format is generally preferred, especially for the visualization of low analyte concentrations, because the appearance of label on the solid phase is more readily detected.
  • a "first" antibody bound to a solid support i.e., immobilized
  • a biological sample being tested for the presence of sTRAIL or mTRAIL is provided.
  • a "second” antibody which has been detectably labeled and which is capable of specifically binding with the TRAIL Compound is provided.
  • the "first” antibody” will be an anti-TRAIL antibody; alternatively, it may be an anti-"first” antibody.
  • the presence of a TRAIL Compound in the sample being tested results in the immobilization of the detectably labeled "second” antibody. The extent of such immobilization is proportional to the amount of TRAIL Compound present in the sample.
  • the "first" antibody may be incubated with the biological sample in an unbound state and then subsequently bound to the solid support (i.e., immobilizable).
  • the supports are then preferably extensively treated (e.g., by washing, etc.) to substantially remove unbound detectably labeled antibody that may be present.
  • Sandwich assay formats are described by Schuurs et al. U.S. Patent Nos. 3,791 ,932 and 4,016,043, and by Pankratz, et al, U.S. Patent No. 5,876,935.
  • the "second” antibody may be a natural immunoglobulin isolated from nonhuman primates (e.g., anti-human IgG murine antibody, anti-human IgG goat antibody, etc.), or can be produced recombinantly or synthetically. It may be an intact immunoglobulin, or an immunoglobulin fragment (e.g., FAb, F(Ab) 2 , etc.). As desired, other binding molecules (capable of binding to the TRAIL Compound employed) may be employed in concert with or in lieu of such second antibodies. To eliminate the bound-free separation step and reduce the time and equipment needed for a chemical binding assay, a homogeneous assay format may alternatively be employed.
  • one component of the binding pair may still be immobilized; however, the presence of the second component of the binding pair is detected without a bound-free separation.
  • homogeneous optical methods are the EMIT method of Syva, Inc. (Sunnyvale, CA), which operates through detection of fluorescence quenching; the laser nephelometry latex particle agglutination method of Behringwerke (Marburg, Germany), which operates by detecting changes in light scatter; the LPIA latex particle agglutination method of Mitsubishi Chemical Industries (Tokyo, Japan); the TDX fluorescence depolarization method of Abbott Laboratories (Abbott Park, IL); and the fluorescence energy transfer method of Cis Bio International (Paris, France).
  • the binding assay of the present invention may be configured as a competitive assay.
  • a competitive assay the more sTRAIL or mTRAIL present in the test sample, the lower the amount of label present on the solid phase.
  • a competitive assay can be conducted by providing a defined amount of a labeled selected TRAIL Compound (such as sTRAIL, mTRAIL or rsTRAIL) and determining whether the fluid being tested contains a TRAIL Compound that would compete with the labeled antibody for binding to the support.
  • a competitive assay the amount of captured label is inversely proportional to the amount of analyte present in the test sample.
  • Smith U.S. Patent No. 4,401 ,764 describes a competitive assay format using a mixed binding complex that can bind analyte or labeled analyte but in which the analyte and labeled analyte cannot simultaneously bind the complex.
  • Clagett (U.S. Patent No. 4,746,631) describes an immunoassay method using a reaction chamber in which an analyte/ligand/marker conjugate is displaced from the reaction surface in the presence of test sample analyte and in which the displaced analyte/ligand/marker conjugate is immobilized at a second reaction site.
  • the conjugate includes biotin, bovine serum albumin, and synthetic peptides as the ligand component of the conjugate, and enzymes, chemilumincscent materials, enzyme inhibitors, and radionucleotidcs as the marker component of the conjugate.
  • 4,661 ,444 describes a competitive immunoassay using a conjugate of an anti-idiotype antibody and a second antibody, specific for a detectable label, in which the detectable response is inversely related to the presence of analyte in the sample.
  • Allen European Patent Appln. No. 177,191 describes a binding assay involving a conjugate of a ligand analog and a second reagent, such as fluorescein, in which the conjugate competes with the analyte (ligand) in binding to a labeled binding partner specific for the ligand, and in which the resultant labeled conjugate is then separated from the reaction mixture by means of solid phase carrying a binding partner for the second reagent.
  • This binding assay format combines the use of a competitive binding technique and a reverse sandwich assay configuration; i.e., the binding of conjugate to the labeled binding member prior to separating conjugate from the mixture by the binding of the conjugate to the solid phase.
  • the assay result is determined as in a conventional competitive assay in which the amount of label bound to the solid phase is inversely proportional to the amount of analyte in the test sample.
  • Chieregatt et al. (GB Patent No. 2,084,317) describe a similar assay format using an indirectly labeled binding partner specific for the analyte.
  • Mochida et al. U.S. Patent No.
  • At least one component of the assay reagents e.g., a TRAIL Compound, an antibody, etc.
  • a detectable moieties e.g., a fluorescent moiety, a radioactive moiety, an enzyme, a magnetic or paramagnetic particle or bead, a chemiluminescent moiety, etc.
  • Radioisotopic-binding assay formats e.g., a radioimmunoassay, etc.
  • a radioisotopc as such label
  • the signal is detectable by the evolution of light in the presence of a fluorescent or fluorogenic moiety
  • Enzymatic-binding assay fonnats e.g., an ELISA, etc.
  • an enzyme as a label
  • the signal is detectable by the evolution of color or light in the presence of a chromogenic or fluorogenic moiety.
  • labels such as paramagnetic labels, materials used as colored particles, latex particles, colloidal metals such as selenium and gold, and dye particles (see U.S. Patent Nos. 4,313,734; 4,373,932, and 5,501 ,985) may also be employed.
  • enzymes especially alkaline phosphatase, ⁇ -galactosidase, horse radish peroxidase, or se
  • the presence of enzymatic labels may be detected through the use of chromogenic substrates (including those that evolve or adsorb fluorescent, UV, visible light, etc.) in response to catalysis by the enzyme label.
  • Detection of label can be accomplished using multiple detectors, multipass filters, gratings, or spectrally distinct fluors (see e.g., U.S. Patent No. 5,759,781 ), etc. It is particularly preferred to employ peroxidase as an enzyme label, especially in concert with the chromogenic substrate 3, 3', 5, 5'-tetramethylbenzidine (TMB).
  • peroxidase as an enzyme label, especially in concert with the chromogenic substrate 3, 3', 5, 5'-tetramethylbenzidine (TMB).
  • TMB 5'-tetramethylbenzidine
  • any of a wide variety of solid supports may be employed in the immunoassays of the present invention.
  • Suitable materials for the solid support are synthetics such as polystyrene, polyvinyl chloride, polyamide, or other synthetic polymers, natural polymers such as cellulose, as well as derivatized natural polymers such as cellulose acetate or nitrocellulose, and glass, especially glass fibers.
  • the support can take the form of spheres, rods, tubes, and microassay or microtiter plates. Sheet-like structures such as paper strips, small plates, and membranes are likewise suitable.
  • the surface of the carriers can be permeable and impermeable for aqueous solutions.
  • the substrate is coated on to a solid matrix.
  • any standard immunoassay solid matrix such as a microtitre plate, beads and the like can be used.
  • a TRAIL Compound e.g., sTRAIL
  • any biological sample that may be rendered fluidic (e.g., tissue or biopsy extracts, cellular extracts, extracts of feces, sputum, etc.) may likewise be employed in the assays of the present invention.
  • Materials for use in the assays of the invention are ideally suited for the preparation of a kit.
  • kit may comprise a carrier means being compartmentalized to receive in close confinement; one or more containers means vials, tubes and the like; each of the containers means comprising one of the separate elements to be used in the method.
  • one of the containers means may comprise as a first element a TRAIL Compound (especially, sTRAIL, mTRAIL or rsTRAIL) bound to a solid support.
  • a second container may comprise as a second element a detectably labeled antibody, preferably in lyophilized form, or in solution, that specifically binds to the employed TRAIL Compound.
  • a user adds to a container a premeasured amount of a sample suspected of containing a measurable yet unknown amount of a TRAIL Compound, and a premeasured amount of support- bound TRAIL Compound and the detectably labeled antibody.
  • a TRAIL Compound e.g., sTRAIL, etc.
  • it will compete for antibody binding to the immobilized TRAIL Compound.
  • an immune complex is formed and is separated from the supernatant fluid, and the immune complex or the supernatant fluid are detected, as by radioactive counting, addition of an enzyme substrate, and color development, or by inclusion of a chemical label (e.g., colloidal gold, latex beads, etc.).
  • a chemical label e.g., colloidal gold, latex beads, etc.
  • one of the containers means may comprise as a first element an anti-TRAIL Compound antibody bound to a solid support.
  • a second container may comprise as a second element a detectably labeled antibody, preferably in lyophilized form, or in solution, that specifically binds to a TRAIL Compound.
  • a user adds to a container a premeasured amount of a sample suspected of containing a measurable yet unknown amount of the TRAIL Compound (e.g., sTRAIL), and a premeasured amount of support-bound antibody and the detectably labeled antibody. To the extent that the TRAIL Compound is present in the sample being tested, it will bind to the immobilized antibody, and thus become itself immobilized.
  • an immune complex is formed and is separated from the supernatant fluid, and the immune complex or the supernatant fluid are detected, as by radioactive counting, addition of an enzyme substrate, and color development, or by inclusion of a chemical label (e.g., colloidal gold, latex beads, etc.).
  • a chemical label e.g., colloidal gold, latex beads, etc.
  • the kit may also contain one or more containers, each of which comprises a (different) predetermined amount of a TRAIL Compound (e.g., sTRAIL) or anti-TRAIL Compound antibody.
  • a TRAIL Compound e.g., sTRAIL
  • anti-TRAIL Compound antibody e.g., anti-TRAIL
  • the present invention particularly relates to the use of immuno- chromatographic assay formats to detect a TRAIL Compound (especially sTRAIL), and particularly in the diagnosis or monitoring of HIV infection and AIDS progression.
  • a TRAIL Compound especially sTRAIL
  • two contacting, but spatially distinct, porous carriers are employed.
  • the first such carrier may preferably contain a non-immobilized, labeled anti-sTRAIL antibody and the second such carrier will preferably contain an immobilized, but unlabeled antibody.
  • the unlabeled antibody may be an anti-sTRAIL antibody (especially one which binds to a different epitope from that recognized by the labeled antibody) or an antibody that specifically binds to the species of unlabeled antibody employed (e.g., where labeled murine anti-sTRAIL IgG antibodies are employed, the unlabeled antibody may be an anti-murine IgG antibody, etc.).
  • Other TRAIL Compounds may be employed in lieu of sTRAIL.
  • the device will comprise a hollow casing constructed of, for example, a plastic material, etc., in which the first carrier will communicate indirectly with the interior of the casing via a multilayer filter system that is accessible from the device (e.g., by protruding therefrom or by being incompletely covered by the device), such that a serum, plasma, or whole blood test sample can be applied directly to the filter system and will permeate therefrom into the first porous carrier.
  • the permeation of fluid containing the TRAIL Compound will cause the non-immobilized labeled antibody of the first carrier to become bound to the migrating TRAIL Compound, and will then permeate into the second carrier. Because the second carrier contains immobilized antibody that binds the migrating complex, immune complexes entering the second carrier will be trapped therein.
  • Detection of labeled antibody in the carrier containing the immobilized unlabeled antibody thus indicates that a TRAIL Compound is present in the sample being evaluated.
  • the assay can be made quantitative by measuring the quantity of labeled molecules that become bound within the second carrier.
  • Example 1 TNF-related Apoptosis-inducing Ligand (TRAIL) in HIV-1-infected Patients and its in vitro Production by Antigen-presenting Cells Since sTRAIL has been detected in HIV-1 -infected patients, the levels of plasma sTRAIL from HIV-1 -infected patients to those of uninfected control donors were compared in order to determine whether sTRAIL levels in AIDS patients were associated with viral load. Exposure of peripheral blood T cells from HIV-1 uninfected donors to infectious HIV-1 or to HIV-1 that had been rendered noninfectious by chemical treatment (Adrithiol-2) (AT-2 HIV-1 particles) results in the expression of membrane TRAIL on CD4 + but not CD8 + T cells.
  • Adrithiol-2 Adrithiol-2 HIV-1 particles
  • HIV-I MN CXCR4-tropic
  • HIV-l Ada CCR5-tropic
  • IFN- ⁇ and IFN- ⁇ type I interferons
  • One patient (I) was not on antiretroviral therapy (ART) prior to the commencement of the study, and was then started on Tenofivir, D4T, and Kaletra. The patient went off ART and did not start another regimen prior to the end of the study.
  • a second patient (II) was naive prior to starting the study and was enrolled into a study that included Trizivir, Combivir, and Sustiva. Therapy continued through week 40.
  • a third patient (III) was naive prior to starting the study and was enrolled in a study that included Trizivir, Combivir, and Sustiva. The patient was switched to DDI, Combivir, and Sustiva, which continued through week 40.
  • a fourth patient (IV) was on ART prior to the commencement of the study and was switched to Kaletra and Sustiva at the start of TRAIL study.
  • HIV- I N X4-tropic
  • HIV-l A da R5-tropic
  • Viral supernatants are inactivated with 1 mM AT-2 for 18 h at 4°C before purification.
  • Virus preparations are produced by sucrose gradient banding in a continuous-flow centrifuge (Rossio, J. L. et al.
  • Microvesicles used as a control reagent, are isolated from supernatants of uninfected cell cultures in a manner identical to that used for virus preparation from infected cells (Bess, J.W. et al. (1997) "MICROVESICLES ARE A SOURCE OF CONTAMINATING CELLULAR PROTEINS FOUND IN PURIFIED HIV-1 PREPARATIONS," Virology 230: 134-144).
  • PBMC peripheral blood obtained from healthy, HIV-1 -seronegative donors by the Department of Blood Transfusion, NIH, Bethesda.
  • Cells are cultured in RPMI medium (Invitrogen, Gaithersburg, MD) with 10% fetal bovine serum (Sigma, St. Louis, MO).
  • CD4 + and CD8 + cells were purified from PBMC by positive selection with anti-CD4 + or anti-CD8 + beads (Miltenyi Biotech, Auburn, CA).
  • CD4 + and CD8 + cells are purified from PBMC by positive selection with anti-CD4 or anti-CD8 beads (Miltenyi Biotech, Auburn, CA). Macrophages and dendritic cells are generated using monocytes as described by Chomarat, P. (2000) ("IL-6 SWITCHES THE DIFFERENTIATION OF MONOCYTES FROM DENDRITIC CELLS To MACROPHAGES," Nat Immunol. 1 :510-514). Cells were incubated with AT-2 HIV- 1 at a final concentration of 50 ng/mL p24 equivalent. Infectious HIV-IMN and HIV-1 LAV were used at the same concentration. For all the experiments, T cells isolated by both negative and positive selection are used interchangeably, without noticeable differences in the experimental outcome.
  • Tissues were then sectioned into 2 mm 3 blocks and placed on top of medium-hydrated collagen sponge gel (Gelfoam, Pharmacia & Upjohn) in complete medium at the air-liquid interface in 6 well plate (Costar, Cambridge, MA) with 9 blocks per sponge in each well in 4 ml of meidum. 24 h later, the culture supernatant was replaced with fresh medium containing 1% Pen/Strep (Gibco BRL), and each tissue block was infected with 1 ng p24 gag of an HIV-1
  • LAV.04 viral stock (3 ⁇ l, 120 TCID 50 ). Culture medium was sampled and changed every 3 days.
  • Incubations of PBMC and isolated T cell subsets with viruses are cultured at 1 x 10 6 cells/ml in RPMI 10% FCS in 12-well plates.
  • the inactivated virus preparations are added at the initiation of culture to a final concentration of 50 ng/mL p24 equivalent.
  • Infectious H1V- 1 MN and HIV-1 LAV are used at the same concentration.
  • Plasma quantification of HIV-1 RNA Plasma HIV-1 RNA is measured by quantitative RT-PCR and all data are expressed in copies/ml (Amplicor Monitor; Roche Diagnostic Systems, Brancburg, NY, USA; detection limit 50 copies/ml).
  • Membrane TRAIL detection Monocytes are cultured for 24 h in the absence or presence of AT-2 HIV-I MN , AT-2 HIV-l Ada and infectious HIV- I MN viruses. The cells are washed twice in PBS. Membrane TRAIL expression is determined by incubating cells for 20 minutes at room temperature with PE-conjugated mouse IgGl anti-human TRAIL monoclonal antibody, (clone B-S23 Diaclone, Besancon France) or with relevant control isotype-matched antibodies (at 5 ⁇ g/mL each); in PBS containing 2% mouse serum (SIGMA, St. Louis, MO).
  • PE-conjugated mouse IgGl anti-human TRAIL monoclonal antibody (clone B-S23 Diaclone, Besancon France) or with relevant control isotype-matched antibodies (at 5 ⁇ g/mL each); in PBS containing 2% mouse serum (SIGMA, St. Louis, MO).
  • Cells are acquired on FACSCalibur flow cytometer (Becton Dickinson), using CellQuest software (Becton Dickinson Immunocytometry System). Samples are gated on viable cells by forward and side light scatters, and at least 50,000 live cell events are acquired for each sample. Acquired data is analyzed using Cellquest software (Becton Dickinson).
  • Soluble TRAIL measurement levels of soluble TRAIL are measured in serum of HIV + or HIV " individuals, in PBMC and monocytes supernatants with the commercial TRAIL ELISA kit (Diaclone, Besancon, France), according to the manufacturer's instructions.
  • Blocking assay To block TRAIL production, monocytes are cultured 3 days with AT-2 HIV-1 MN/Ada in the presence of of CD4 binding inhibitor soluble CD4 (2 ⁇ g/ml), or the fusion inhibitor T20 (2 ⁇ g/ml), or the CXCR4 inhibitor AMD-310 (2 ⁇ g/ml) (AIDS Research and Reagents Program, NIA1D, Bethesda, MD), or in presence of both sheep polyclonal anti-human IFN- ⁇ (2000 IFN- ⁇ neutralizing U/ml ) and sheep polyclonal anti-human IFN- ⁇ antibodies (500 IFN- ⁇ neutralizing U/ml) (BioSource International, Camarillo, CA). Recombinant IFN- ⁇ and IFN- ⁇ (Peprotech Inc, Rocky Hill, NJ) are used at 200 U/ml. TRAIL production is quantified by ELISA.
  • RNA extraction and reverse transcription Total RNA is extracted from monocytes with the acid guanidium thiocyanate-phenol-chloroform method (Chomczynski, P. et al. (1987) "SF GLE-STEP METHOD OF RNA ISOLATION BY ACID GUANIDINIUM THIOCYANAE-PHENOL-CHLOROFORM EXTRACTION," Anal Biochem 162:156-159), modified for TRIzol (Invitrogen, Carlsbad, CA, USA). The amount and the purity of the extracted RNAs are determined by spectrophotometric analysis at 260nm and 280nm.
  • RNA is reverse transcribed into first-strand cDNA in a 20 ⁇ l final volume containing 1 ⁇ M of random hexanucleotide primers, 1 ⁇ M of oligo dT and 200 U of Molony murine leukemia virus reverse transriptase. (Promega, Madison, WI, USA).
  • Real Time PCR cDNA quantification for TRAIL and GAPDH is obtained with a real time PCR technique.
  • Real time PCR is conducted with the ABI Prism 7900HT (Applied Biosystems, Foster City, CA, USA). All reactions are performed using a SYBR green PCR mix (QuantiTect SYBR Green PCR, Qiagen, Valencia, CA, USA), according to the following thermal profile: denaturation at 95°C for 15 sec, annealing at 60°C (61 °C for TRAIL) for 15 sec, extension at 72°C for 15 sec (data collection is perfonned during the extension step).
  • the primer sequences are designed using the Primer Express Software v2.0 provided with the ABI Prism 7900HT
  • GAPDH Forward (SEQ ID NO:9) CCACCCATGG CAAATTCC Reverse (SEQ ID NO.10) TGGGATTTCC ATTGATGACA AG All reactions are performed in triplicate (denaturation at 95°C for 15 sec, annealing at 60°C (61 °C for TRAIL) for 15 sec, extension at 72°C for 15 sec).
  • Data analysis is performed with the SDS2.1 software, provided with the ABI Prism 7900HT.
  • the threshold level is determined by the software according to the optimization of the standard curve. Standards are obtained by amplification of a control sample in a PCR reaction, using the same primers, reagents and conditions optimized for the real time analysis. Arbitrary quantity values are assigned to the resulting standard and 4-fold serial dilutions are made to obtain an 8-point standard curve. Results are presented as ratios between the target gene and the GAPDH mRNA.
  • Monocytes were cultured with AT-2 HIV-I MN , AT-2 HIV- 1 ADA , recombinant IFN- ⁇ (10 ng/ml) or IFN- ⁇ (10 ng/ml), anti-IFN- ⁇ and anti-IFN- ⁇ (R & D Systems, Minneapolis MN). After 24 h, the cells were pelleted, washed in PBS and lysed (1% NP-40, 200mM NaCl, 50mM Tris pH 7.5, supplemented with protease inhibitor cocktail [Roche, Indianapolis, IN]).
  • Total protein was quantified by BCA (Pierce, Rockford, IL) and 10 ⁇ g of protein was mixed 1 :1 with SDS Protein Gel Buffer (Quality Biologies, Gaithersburg, MD). Samples were run on a 10% or 15% SDS-PAGE gel (Bio-Rad, Hercules, CA). After transfer, the blot was blocked in 3% milk/TBST then incubated with anti-STATl or anti-STAT2 (Upstate Biotechnology, Inc., Lake Placid, NY) antibodies followed by anti-rabbit HRP secondary antibody (Jackson, West Grove, PA). ECL was performed and bands were visualized on Hyperfilm (Amersham, Piscataway, NJ). B-actin was visualized as a loading control using a mouse monoclonal antibody (Sigma).
  • Panel A the mean values of plasma sTRAIL are 852 ⁇ pg/ml for 53 control donors, 1339 ⁇ 79 pg/ml for 49 HIV-1 -infected patients with undetectable viral load ( ⁇ 50 mRNA copies/ml of blood) and 2240 ⁇ 131 pg/ml for 58 HIV-1 -infected patients with detectable viral load (>50 mRNA copies/ml of blood).
  • Panel C provide a longitudinal study of four HIV-1- infected patients who began HAART therapy and were subsequently followed for 40 weeks with measurement of plasma viral load and sTRAIL.
  • Patient 1 exhibited an initial parallel drop in viral load and sTRAIL, followed by a concomitant rebound in both of these parameters. This rebound in viral load may reflect development of HIV-1 drug resistance.
  • Patient II showed a continuous parallel drop in viral load and sTRAIL.
  • Patient III showed three-of-four points in which changes in viral load and sTRAIL levels were similar.
  • Patient IV who had received ART prior to enrollment into this study, exhibited a parallel flat and low profile for both viral load and sTRAIL throughout the 40 weeks. This patient's initial low viral may have been due to successful ART prior to enrollment, which continued during this ART protocol.
  • Example 5 Noninfectious HIV-1 Induces TRAIL Production By Monocytes
  • PBMC from healthy HIV- uninfected donors and AIDS patients are cultured in the presence of microvesicles (negative control), AT-2 HIV-1 MN (CXCR4-tropic) or AT-2 HIV-1 Ada (CCR5- tropic) for 3-to-6 days.
  • sTRAIL levels in the culture supernatants are determined by ELISA.
  • CD8+ T cells nor by macrophages.
  • CD4+ T cells express mTRAIL but not sTRAIL after exposure to AT-2 HIV-1.
  • Example 6 Noninfectious HIV-1 Induced TRAIL Production By The Type I IFN Pathway
  • Anti-IFN- ⁇ Ab or anti-IFN- ⁇ Ab inhibited the sTRAIL production of AT-2 HIV-1 -exposed monocytes by 25 ⁇ 1 1 % and 40 ⁇ 3 % inhibition of TRAIL production, respectively.
  • AT-2 HIV-1 induction of TRAIL is IFN- ⁇ / ⁇ -dependent, it was verified that monocytes from uninfected donors cultured with AT-2 HIV- 1 MN or AT-2 HIV-1 Ada produced IFN- ⁇ ( Figure 3, Panel D). Moreover, it is demonstrated that AT-2 HIV-1 MN and AT-2 HIV-l Ada , as well as recombinant IFN- ⁇ / ⁇ , increased monocyte expression of STATl and STAT2 ( Figure 3, Panel E), the principal signaling molecules for type I IFN (Qureshi, S.A. et al.
  • Tests were conducted as to whether noninfectious HIV-1 -induced production of sTRAIL would be inhibited by: 1) soluble CD4, that blocks the interaction between viral gpl20 and CD4 expressed on CD4 + cells; 2) AMD-310, that blocks CXCR4 coreceptor binding; 3) T20, that prevents fusion of the virus with the CD4 cell membrane.
  • the results demonstrate that only soluble CD4 inhibited HIV-I MN from inducing sTRAIL production.
  • Example 7 Infectious HIV-1 Induces IFN Type I-Dependent TRAIL Production By Monocytes
  • PBMC are cultured for 3 days in the presence of infectious HIV-1 N at the same concentration used for noninfectious HIV-1.
  • Infectious HIV-1 LAV is also tested for TRAIL production.
  • Panel A HIV-1 MN -stimulated monocytes produced 1400 ⁇ 190 pg/ml of sTRAIL. This level of sTRAIL is not significantly different from the level detected after AT-2 HIV-I MN exposure (p ⁇ .5). HIV-1 LAV produces only a slightly lower level of sTRAIL.
  • Example 8 Infection With HIV-1 Induces TRAIL Production In Tonsil Tissue Culture
  • human tonsils were infected with infectious HIV-1 Lav , the HIV-1 strain used previously in this model (Grivel, J.C et al. (1999) "CCR5- AND CXCR4- TROPIC HIV-1 ARE EQUALLY CYTOPATHIC FOR THEIR T-CELL TARGETS IN HUMAN LYMPHOID TISSUE,” Nat Med. 1999;5:344-346), and the TRAIL production was measured at different times ranging from l -to-12 days.
  • TRAIL TNF-Related Apoptosis-Inducing Ligand
  • TRAIL production is associated with increased transcription of the TRAIL gene in monocytes exposed to HIV-1 particles, and is mediated by the type I IFN pathway. Induction of sTRAIL by HIV-1 is likely to be dependent on CD4-gpl20 interaction since it is blocked by soluble CD4. Finally, the ex vivo tonsil model demonstrates that TRAIL can be produced in primary lymphoid tissue after HIV-1 infection. Soluble TRAIL was reported in HIV-1 -infected patients (Liabakk, N.B. et al. (2002) “Development, Characterization And Use Of Monoclonal Antibodies against Strail: Measurement Of Strail By ELISA,” J Immunol Methods.
  • TRAIL Tumor Necrosis Factor-related Apoptosis-inducing Ligand
  • sTRAIL is higher in patients with elevated viral load, than in patients with low or undetectable viral loads.
  • plasma from all HIV-1 patients tested showed higher TRAIL content than plasma from healthy donors.
  • Our 40-week longitudinal study of patients on ART showed striking parallel changes between viral load and TRAIL levels.
  • ART decreased viral load, we observed a concomitant decrease in plasma TRAIL, which may be one of the reasons for the efficacy of antiviral therapy.
  • HIV-1 virions detected in HIV-1 -infected patients' plasma do not possess culturable infectivity (Piatak, M, Jr. et al. (1993) "HIGH LEVELS OF HIV-1 IN PLASMA DURING ALL STAGES OF INFECTION DETERMINED BY COMPETITIVE PCR,” Science. 259: 1749-1754), which raises the possibility that noninfectious virus particles contribute to HIV-1 pathogenesis.
  • the above Examples demonstrate that the major cellular source of TRAIL is monocytes, which produce TRAIL in response to both infectious and chemically-inactivated X4 (MN) and R5 (Ada) HIV-1.
  • PBMC and DC from HIV- 1 -infected donors produced sTRAIL without further stimulation, suggesting that previous contact with HIV-1 was sufficient to induce sTRAIL secretion.
  • sTRAIL production disappeared if the patients' PBMC were not stimulated by HIV-1 particles, suggesting that continuous cell-virus interaction is required to maintain sTRAIL production.
  • Type 1 interferons are produced as a result of viral infection and can exert anti-viral effects (Sato, K. et al. (2001 ) "ANTIVIRAL RESPONSE BY NATURAL KILLER CELLS THROUGH TRAIL GENE INDUCTION BY IFN-ALPHA/BETA," Eur J Immunol. 31 :3138-3146).
  • Type I interferons also induce TRAIL expression through the interferon-stimulated response element (ISRE), which binds IFN- inducible genes (Kayagaki, N. et al.
  • ISRE interferon-stimulated response element
  • HIV-1 induced TRAIL production was also tested in an ex vivo human tonsil model that resembles human lymphoid organ where critical events of HIV disease occur. Increased TRAIL secretion was seen in tonsil organ cultures from three different donors after HIV-1 infection. TRAIL production was maintained over an extended time period; higher TRAIL production was observed in infected rather than in uninfected tonsil cultures after 10 days. Detection of TRAIL in these HIV-1 -infected cultures is consistent with increased apoptosis of CD4 + T cells after HIV-1 infection of tonsil cultures (Grivel, J.C et al.
  • CD4 + T cells express mTRAIL but do not produce sTRAIL upon exposure to HIV-1 , the sTRAIL detected in plasma of HIV-1 - infected patients is unlikely to have been secreted by CD4 + T cells, and may have been produced by monocytes and/or dendritic cells after exposure to HIV-1. Furthermore, since B cells, present in large number in tonsil, did not produce
  • TRAIL TNF-related apoptosis-inducing ligand
  • Example 10 A Model for Type I Interferon-dependent CD4+ T Cell Apoptosis in AIDS: Two Hits to Death The pathogenic mechanisms responsible for the extensive depletion of
  • CD4 + T cells characteristic of AIDS have not previously been well understood. As indicated above, although HIV-1 -infected T cells die, the frequency of infected cells appears to be too low to account for the depletion of CD4 + T cells (Ha ⁇ er, M.E. et al. (1986) "DETECTION OF LYMPHOCYTES EXPRESSING HUMAN T- LYMPHOTROPIC VIRUS TYPE III IN LYMPH NODES AND PERIPHERAL BLOOD FROM INFECTED INDIVIDUALS BY IN SITU HYBRIDIZATION," Proc Natl Acad Sci U S A 83:772-776; Grivel, J.C et al.
  • Type I interferons have anti-viral activity, including against HIV-
  • TNF-related apoptosis-inducing ligand TRAIL
  • TNF superfamily a member of the TNF superfamily
  • TRAIL a member of the TNF superfamily
  • TRAIL has five receptors, two (DR4 and DR5) that induce cell death, and three others that lack the death domain (Pan, G.
  • the TRAIL gene is regulated by type I interferons (Gong, B. et al (2000) “GENOMIC ORGANIZATION AND TRANSCRIPTIONAL REGULATION OF HUMAN APO2/TRAIL GENE,” Biochem Biophys Res Commun 278:747-752), and the DR5 gene is transcriptionally regulated by p53 (Wu, G.S. et al. ( 1997) "KILLER DR5 Is A DNA DAMAGE-lNDUCIBLE P53-REGULATED DEATH RECEPTOR GENE,” Nat Genet 17:141 - 143).
  • TRAIL may be involved in CD4" T cell depletion, since TRAIL, produced by monocytes exposed to the HIV-1 Tat protein, killed uninfected CD4 + T cells (Yang, Y. et al. (2003) "MONOCYTES TREATED WITH HUMAN IMMUNODEFICIENCY VIRUS TAT KILL UNINFECTED CD4( + ) CELLS BY A TUMOR NECROSIS FACTOR-RELATED APOPTOSIS-INDUCED LIGAND-MEDIATED MECHANISM,” J Virol 77:6700-6708). Moreover, soluble TRAIL was found in HIV-1 -infected patients (Liabakk, N.B. et al.
  • TRAIL has been implicated, in vitro, in the apoptosis of HIV-1 -infected cells (Lum, J.J. et al.
  • results provided in this Example demonstrate that HIV-1 -infected patients exhibited: 1) elevated levels of TRAIL in their plasma, 2) increased pcrcentage of CD4 + T cells that expressed DR5 and the apoptotic marker Annexin V. Because a higher percentage of apoptotic CD4 + T cells was found than is known to be infected (see above), the possibility that uninfected CD4 + T cells can undergo TRAIL-mediated apoptotic death as the result of a noninfectious interaction with HIV-1 was investigated.
  • a two-hit model is presented in which infectious or noninfectious HIV-1 (first hit) can activate type 1 interferons and TRAIL, and in synergy with a second infection or event (second hit), result in p53 and DR5-mediated apoptosis of CD4 + T cells.
  • HIV-1 positive patients Venous blood was collected from 48 HIV-1 - infected patients. Preparation of AT-2-inactivated virions. HIV-I MN (X4-tropic) and HIV-
  • CD4 + and CD8 + cells were purified from PBMC by either positive selection with anti-CD4 or anti-CD8 beads or by negative selection using cocktail of antibodies that bind all but CD4 + or CD8 + T cells (Miltenyi Biotech, Auburn, CA). For all the experiments, T cells isolated by both negative and positive selection were used interchangeably, without noticeable differences in the experimental outcome. The purity of CD4 + and CD8 + cells after purification was >98% and >99%, respectively.
  • CD4 + and CD8 + T cells from HIV-1-seronegative donors were cultured for 24 h with or without influenza A and/or AT-2 HIV-1 viruses. Supernatants were collected and tested for IFN- ⁇ by ELISA (R & D Systems, Minneapolis, MN). Apoptosis study. Cells were centrifuged, washed in Annexin buffer (1.4 M
  • Cells were acquired on FACSCalibur flow cytometer (Becton Dickinson), using CellQuest software (Becton Dickinson Immunocytometry System). Samples were gated on viable cells by forward and side light scatters, and at least 50,000 live cell events were acquired for each sample. Acquired data were analyzed using Cellquest software (Becton Dickinson).
  • RNA extraction and reverse transcription Total RNA was extracted from CD4 + T cells with after 12 h of culture with HIV-IMN and influenza viruses, using the acid guanidium thiocyanate-phenol-chloroform method (Chomczynski, P. et al (1987) "SINGLE-STEP METHOD OF RNA ISOLATION BY ACID GUANIDINIUM THIOCYANATE-PHENOL-CHLOROFORM EXTRACTION," Anal Biochem 162: 156-159), modified for TRIzol (Invitrogen, Carlsbad, CA, USA).
  • cDNA quantification for TRAIL, DR-5, p53 and GAPDH was obtained with a real time PCR technique. Real time PCR was conducted as described above.
  • Primers for TRAIL and GAPDH are as described above.
  • Primers for DR-5 are: Forward (SEQ ID NO:5) 5'-GGGCCACAGGGACACCTT-3'; Reverse (SEQ ID NO:6) 5'-GCATCTCGCCCGGTTTT-3'.
  • Primers for p53 are: Forward (SEQ ID NO:7) 5'-CATGAGCGCTGCTCAGATAG-3'; Reverse (SEQ ID NO:8) 5'-ACACGCAAATTTCCTTCCAC-3'. All reactions were performed in triplicate.
  • the threshold level was determined by the software according to the optimization of the standard curve.
  • Standards were obtained by amplification of a control sample in a PCR reaction, using the same primers, reagents and conditions optimized for the real time analysis.
  • Arbitrary quantity values were assigned to the resulting standard and 4-fold serial dilution were made to obtain a 8-point standard curve. Results are presented as ratios between the target gene and the GAPDH mRNA.
  • Mouse IgG isotype control antibody (BD Bioscience) was used at 5 ⁇ g/ml. Apoptosis was assessed by the Annexin V method described above. Results shown are for 5 ⁇ g/mL (anti-TRAIL) and 1 ⁇ g/mL (anti-DR5).
  • CD4 T cells were culture for 24 h with or without influenza A and AT-2 HIV-1 viruses. Cells were lysed and nuclear p 53 was quantified by ELISA (Active Motif, Carlsbad, CA, USA) according to the according to the manufacturer's instructions. Western Blot. PBMCs from uninfected donors were separated from whole blood by Ficoll centrifugation and CD4 + T cells were isolated by magnetic beads (Miltenyi, Auburn , CA).
  • the cells were pelleted, washed in PBS and lysed (1 % NP-40, 200mM NaCl, 50mM Tris pH 7.5, supplemented with protease inhibitor cocktail [Roche, Indianapolis, IN]).
  • Total protein in the lysate was quantified using BCA (Pierce, Rockford, IL) and 10 ⁇ g of protein was mixed 1 :1 with SDS Protein Gel Buffer (Quality Biologies, Gaithersburg, MD). The samples were run on a 10% or 15% SDS-PAGE gel (BioRad, Hercules, CA).
  • the blot was blocked in 5% milk TBST then incubated with either anti-STATl , anti-STAT2 (Upstate Biotechnology, Inc., Lake Placid, NY), anti-TRAIL or anti-DR5
  • TRAIL and DR5 in HIV- 1 -infected patients and uninfected controls.
  • the levels of DR5 mRNA level in PBMC were compared from 16 HIV- 1 -infected patients and 9 HIV-1 -uninfected controls.
  • An additional 22 patients and 16 controls were tested for plasma TRAIL levels, and for the percentage of CD4 + T cells that were positive for Annexin V and DR5.
  • Annexin V and STAT expression in virus-exposed T cells were cultured from HIV-1-seronegative donors with infectious or noninfectious HIV-1.
  • infectious HIV-1 MN (X4) and HlV-l ⁇ da (R5), as well as their noninfectious viral counterparts that had been inactivated by aldrithiol 2 (AT-2 HIV-1) (Arthur, L.O. et al.
  • CD8 + T cells were positively-selected from PBMC and cultured for one-to-six days with these HIV-1 preparations, or with microvesicles (control), and the percentage of Annexin V-positive T cells was detennined, using multicolor flow cytometry. Infectious HIV-1 MN or HlV-l ⁇ dn did not induce significant apoptosis of either CD4 + or CD8 + T cells after one day. However, it was found that 32% and 31 % of CD4 + T cells were Annexin V positive when cultured 6 days with HIV-1 MN or HIV-1 Ada , respectively ( Figure 7, Panel A). This apoptosis was approximately two fold above T cells cultured with microvesicles (16%).
  • Figure 7, Panel C also illustrates how similar the flow cytometric Annexin V curves are between infectious (upper) and noninfectious HIV-1 (lower) plus Flu.
  • STATl and STAT2 are signaling molecules for type I interferons (Qureshi, S.A. et al. (1995) "TYROSINE-PHOSPHORYLATED STATI AND STAT2 PLUS A 48-KDA PROTEIN ALL CONTACT DNA IN FORMING INTERFERON-STIMUL ⁇ TED- GENE FACTOR 3," Proc Natl Acad Sci U S A 92:3829-3833; Leung, S. et al.
  • TRAIL expression was measured on T cells cultured with HIV-1 and Flu virus alone or in combination.
  • Membrane TRAIL was detected on CD4 + ( Figure 8, Panel A) but not CD8 + T cells cultured with Flu or HIV-1 MN alone, or with HIV-1 MN plus Flu.
  • Noninfectious AT-2 HIV-1 also induced TRAIL expression on CD4 + T cells (data not shown). Exposure of CD4 + T cells to HIV-1 MN plus Flu induced TRAIL mRNA expression that was blocked by IFN- ⁇ / ⁇ antibodies ( Figure 8, Panel B). Anti-IFN- ⁇ / ⁇ antibodies also inhibited virus-induced expression of mTRAIL, suggesting that type I interferons are paramount for the induction of TRAIL in virus exposed-T cells ( Figure 8, Panel C). IFN- ⁇ and/or IFN- ⁇ both induced mTRAIL expression on CD4 + T cells when added exogenously (D). Western blot analysis verified that TRAIL was produced upon culture of CD4 + T cells with AT- 2 HIV-1 MN , Flu, AT-2 HIV-1 MN plus Flu or IFN- ⁇ / ⁇ ( Figure 8, Panel E).
  • DR5 expression and p53 transcription and synthesis in virus-exposed T cells TRAIL-mediated apoptosis requires the expression of cellular TRAIL death receptors. Therefore, DR5 expression was measured on CD4 + and CD8 + T cells after culture with HIV-1 MN , Flu or HIV- 1 MN plus Flu. In contrast to TRAIL, which was induced by either HIV-1 MN or Flu alone, DR5 was selectively expressed only on CD4 + T cells cultured with both H1V- 1 MN and Flu ( Figure 9, Panel A). DR4 was not detected on CD4 + T cells, and CD8 + T cells did not express DR molecules.
  • DR5 expression is the limiting component of TRAIL-mediated death in HIV-1 -exposed cells.
  • DR5 expression is regulated by the tumor suppressor p53 (24, 40). Therefore, we tested whether p53 gene expression and synthesis would be induced by Flu, AT-2 HIV-I MN or AT-2 HIV- I N plus Flu, and whether p53 induction would be inhibited by lFN- ⁇ / ⁇ antibodies. Both p53 gene expression (Fig. 4F) and p53 production (Fig. 4G,H) required CD4 + T cell stimulation with AT-2 HIV-I MN plus Flu, and were blocked by anti-IFN- ⁇ / ⁇ . Thus, similar to the other components of this cascade to death, p53 activation and synthesis, which are necessary for DR5 expression (Wu, G.S. et al.
  • Puma model may be relevant here in that it revealed HIV-1 -induced apoptosis of CD4 expressing cells in the absence of infection via a p53-dependent mechanism.
  • This mechanism involves the synergistic effects of either infectious or noninfectious HIV-1 and a second event (viral infection), both of which are essential for activating rate-limiting expression of DR5 death receptor molecule by a type 1 interferon-dependent mechanism.
  • influenza A virus was employed as the second virus (because it co-localizes with HIV-1 in primary lymphoid tissue (Doherty, P.C. (1995) "ANATOMICAL ENVIRONMENT AS A DETERMINANT IN VIRAL IMMUNITY,” J Immunol 155: 1023-1027)
  • the second event was not limited to influenza; measles virus, for example, also synergized with AT-2 HIV-1 to induce p53, DR5 and apoptosis of CD4 + T cells.
  • influenza and measles virus infections have not been associated with AIDS progression, both of these viruses fulfill the requirement of activating p53 and DR5 in presence of HIV-1.
  • they provide an example of one way to selectively induce rapid and extensive CD4 T cell death upon exposure to HIV-1.
  • p53 expression required two viruses to induce DR5
  • other infection could provide the second event to induce DR5 and accelerate T cell depletion.
  • other non-viral events would induce p53 and DR5 resulting in apoptosis.
  • These other events might include agents that would induce DNA damage, since p53 is induced by DNA damage (Wu, G.S. et al.
  • CD4 + T cells cultured with AT-2 HIV-1 plus recombinant IFN- ⁇ / ⁇ did not induce apoptosis suggests that type I interferons (induced by HIV- 1 ) are required but not sufficient for inducing CD4 + T cell death.
  • the central event for CD4 + T cell death in our findings was activation of p53 by the combined effect of HIV- 1 and a second virus leading to the selective expression of DR5 on CD4 + T cells.
  • TRAIL/DR5-mediated death suggested by such model occurred within six days. Part of this rapid CD4 + depletion could be due to the fact that T cell repopulation did not occur in the above-reported in vitro studies, in contrast to the in vivo setting in which CD4 + T cell repopulation was reported (Wei, X. et al. (1995) "VIRAL DYNAMICS IN HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 INFECTION," Nature 373:1 17-122; Ho, D.D. et al. (1995) "RAPID TURNOVER OF PLASMA VIRIONS AND CD4 LYMPHOCYTES IN HIV-1 INFECTION,” Nature 373:123-126).
  • the present invention demonstrates that cultures of T cells enriched for CD4 + by 98-99% produced IFN- ⁇ when cultured with HIV-1 and/or Flu.
  • type I IFN production could be due to CD4 + monocytes.
  • IFN- ⁇ was reported to inhibit HIV-1 replication in vitro (Yamamoto, J.K. et al. (1986) "HUMAN ALPHA- AND BETA-INTERFERON BUT NOT GAMMA- SUPPRESS THE IN VITRO REPLICATION OF LAV, HTLV-III, AND ARV-2," J Interferon Res 6: 143- 152), and has been used for therapy in AIDS patients with Kaposis sarcoma (Frissen, P.H. et al.
  • the present findings raise the possibility that type I interferons can be harmful by setting the stage for the death of uninfected CD4 + T cells in patients whose CD4 + T cells have been activated to express TRAIL and DR5.
  • one aspect of the present invention concerns the recognition that anti-type I interferon antibodies can be used as an anti-HIV-1 therapeutic agent. See also reports that immune suppression is induced by IFN- ⁇ and can be blocked by anti-lFN-a antibodies (Zagury, D. et al.
  • FIG. 10 The above-described two-hit model, based on the above-described data is illustrated in Figure 10.
  • the model illustrates the requirement for STATl and STAT2 activation, as well as the induction of type 1 interferons, TRAIL, p53 and DR5 in CD4 + T cells.
  • the top panel of Figure 10 shows that only one hit is needed to induce TRAIL, but not p53 or DR5.
  • the bottom panel of Figure 10 indicates that two hits are required to induce the entire cascade of molecular events that are necessary for TRAIL-mediated death of CD4 + T cells.
  • mTRAIL membrane TRAIL
  • pTRAlL plasma TRAIL
  • the present invention provide a mechanistic explanation for CD4 + T cell death that is independent of productive HIV-1 infection, but which may contribute to CD4 + T cell depletion in HIV- 1 -infected patients.

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Abstract

Cette invention concerne des procédés et compositions appropriés pour contrôler l’évolution du SIDA, et d’autres maladies dont l’évolution implique l’activation du système immunitaire. En particulier, l’invention concerne l’utilisation du ligand induisant l’apoptose médiée par TNF (TRAIL) et les composés TRAIL pour contrôler l’évolution du SIDA, et d’autres maladies du système immunitaires.
PCT/US2005/013554 2004-04-23 2005-04-21 Procédés et compositions de diagnostic du sida et d’autres maladies et conditions impliquant l’activation du système immunitaire Ceased WO2005114187A2 (fr)

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US8173128B2 (en) 2002-11-27 2012-05-08 Irm Llc Methods and compositions for inducing apoptosis in cancer cells with an anti-DR5 antibody
US9120855B2 (en) 2010-02-10 2015-09-01 Novartis Ag Biologic compounds directed against death receptor 5
JP2022531058A (ja) * 2019-03-06 2022-07-06 ディアデム エス．アール．エル． アルツハイマー病の診断と予後判定におけるマーカーとしてのｐ５３ペプチド
US20230094142A1 (en) * 2020-01-29 2023-03-30 Memed Diagnostics Ltd. Methods of diagnosing and classifying viral infections

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CA2301202A1 (fr) * 1997-08-15 1999-02-25 Idun Pharmaceuticals, Inc. Recepteurs de trail, acides nucleiques codant ces recepteurs et procedes d'utilisation
JP2002543151A (ja) * 1999-05-04 2002-12-17 ヒューマン ジノーム サイエンシーズ， インコーポレイテッド 死ドメイン含有レセプター５
EP1223961A4 (fr) * 1999-09-30 2005-10-05 Univ Pennsylvania "trail" en tant qu'inhibiteur de l'inflammation auto-immune et de la progression du cycle cellulaire

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Publication number Priority date Publication date Assignee Title
US8173128B2 (en) 2002-11-27 2012-05-08 Irm Llc Methods and compositions for inducing apoptosis in cancer cells with an anti-DR5 antibody
EP2102370A4 (fr) * 2007-01-11 2010-01-13 Univ Duke Procede de surveillance d'une infection par le vih
US9120855B2 (en) 2010-02-10 2015-09-01 Novartis Ag Biologic compounds directed against death receptor 5
JP2022531058A (ja) * 2019-03-06 2022-07-06 ディアデム エス．アール．エル． アルツハイマー病の診断と予後判定におけるマーカーとしてのｐ５３ペプチド
JP7539399B2 (ja) 2019-03-06 2024-08-23 ディアデム エス．アール．エル． アルツハイマー病の診断と予後判定におけるマーカーとしてのｐ５３ペプチド
US20230094142A1 (en) * 2020-01-29 2023-03-30 Memed Diagnostics Ltd. Methods of diagnosing and classifying viral infections

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