WO2000011476A1 - Procedes d'evaluation de la fonction immunitaire - Google Patents

Procedes d'evaluation de la fonction immunitaire Download PDF

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Publication number
WO2000011476A1
WO2000011476A1 PCT/US1998/024917 US9824917W WO0011476A1 WO 2000011476 A1 WO2000011476 A1 WO 2000011476A1 US 9824917 W US9824917 W US 9824917W WO 0011476 A1 WO0011476 A1 WO 0011476A1
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Prior art keywords
cells
sample
immune cells
hiv
blasting
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PCT/US1998/024917
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English (en)
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Ronald B. Moss
Wieslawa K. Giermakowska
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The Immune Response Corporation
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Priority to AU15330/99A priority Critical patent/AU1533099A/en
Publication of WO2000011476A1 publication Critical patent/WO2000011476A1/fr

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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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV

Definitions

  • ⁇ -chemokines has been associated predominantly but not exclusively with CD8 and natural killer cell (NK) lymphocytes (Cocchi et al., Science 270:1811-1815 (1995); Moss et al., J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 14:343-350 (1997) and Oliva et al, J. Clin. Invest. 102(1):223-231 (1998)).
  • NK natural killer cell lymphocytes
  • lymphocyte proliferation assay involving culturing peripheral blood mononuclear cells (PBMC's) in the presence of HIV-1 antigens.
  • PBMC's peripheral blood mononuclear cells
  • the drawbacks of this assay include the required use of radioactive thymidine and specialized harvesting equipment, as well as the inability to distinguish and measure helper T cell-specific immune responses.
  • Newer methods using flow cytometry also have drawbacks in that they utilize dyes or measure intracellular cytokines which may not be indicative of cell proliferation.
  • flow cytometric analysis of T cells only provides information about the quantity, but not the function of T helper lymphocytes.
  • the present invention provides a novel method for evaluating specific and nonspecific immune responses measured from a sample of immune cells obtained from a subject.
  • the method employs flow cytometry to evaluate blasting immune cells in the sample as an indication of an immune response.
  • Preferred subjects are humans.
  • a sample of immune cells e.g., peripheral blood mononuclear cells or "PBMC”
  • PBMC peripheral blood mononuclear cells
  • Blasting (e.g., proliferating) cells are then evaluated by flow cytometry (e.g., fluorescence activated cell sorting or "FACS”) to determine the strength and nature of the immune response to the antigen.
  • flow cytometry e.g., fluorescence activated cell sorting or "FACS”
  • the novel method of the invention can be used to evaluate a change in immune function. This requires comparing the immune function measured from two or more samples of immune cells taken from a subject at different time points. Accordingly, in another embodiment, the method involves evaluating blasting cells by flow cytometry from two or more samples of immune cells obtained from a subject (e.g., following culturing the cells with an antigen), and comparing the results from the two or more samples to measure a change in immune function.
  • the two or more samples of immune cells can be obtained, for example, before and after administration to the subject of a vaccine, an immunosuppressant, an antiviral agent, an antitumor agent or other therapeutic to determine a change in immune function associated with these treatments.
  • the method of the invention can be used to quantify the number of blasting immune cells in a sample obtained from a subject using flow cytometry as an indication of the strength of an immune response.
  • the method can additionally or alternatively be used to identify the types of blasting cells in the sample, thereby providing information about the functional nature of the immune response. In one embodiment, this is done by labelling the immune cells (e.g., with fluorescent labelled antibodies) in the sample prior to evaluating the sample by flow cytometry so that the different blasting cell types associated with the immune response can be identified.
  • the method of the invention can be used to evaluate any specific or non-specific immune response.
  • the method can be used to determine changes in immune function after infection with a pathogen (e.g., virus or bacterium), after development of tumors or following transplantation of tissues or organs.
  • a pathogen e.g., virus or bacterium
  • the method can be used to determine changes in immune function after treatment of patients with vaccines, immunosuppressants, antiviral agents, antitumor agents or other therapeutic agents.
  • the method can be used to evaluate non-specific immune function by, for example, evaluating responses to mitogens (e.g., Con A, PHA etc.), CMV or tetanus.
  • the method can be used to evaluate or to predict the efficacy of a vaccine treatment administered to a subject.
  • the method of the invention is used to evaluate an HlV-specific immune response in a sample of immune cells obtained from a subject.
  • the method involves contacting the immune cells in the sample with an HIV antigen, and then analyzing the blasting immune cells in the sample by flow cytometry.
  • the subject is infected with HIV and, after obtaining a sample of immune cells from the subject, the cells are cultured with an HIV antigen such as gpl20 depleted HIV-1 or native p24.
  • the cells in the sample are then evaluated by flow cytometry to quantify the blasting cells (e.g., by gating on a region of blasting cells) in response to the stimulating antigen.
  • the cells are labelled with fluorescently labelled monoclonal antibodies specific for certain classes and types of lymphocytes prior to evaluation by flow cytometry (e.g., FACS) so that different types of blasting cells in the sample can be determined during evaluation.
  • flow cytometry e.g., FACS
  • the method can be performed on samples of immune cells obtained before and after the subject is treated with, for example, an HIV vaccine, to determine the efficacy of the vaccine (e.g., to determine an increase in immunoreactivity of the immune cells from the subject when cultured with an HIV antigen following immunization with the vaccine).
  • the invention provides a method of evaluating the efficacy of a vaccine by administering a vaccine to a subject, and then analyzing blasting immune cells in a sample of cells obtained from the subject, preferably after labelling the cells, by flow cytometry (e.g., FACS).
  • flow cytometry e.g., FACS
  • the cells are cultured with an antigen (e.g., the same or a similar antigen as was used to immunize) prior to evaluation to induce proliferation.
  • the results of the evaluation e.g., the quantity and/or types of blasting immune cells
  • Figure 1 is a graph depicting the lymphocyte proliferative response to the HIV-1 antigen, gpl20-depleted HIV-1, and native p24, at baseline and four weeks post treatment (*p ⁇ 0.01). A 5-fold, and 10-fold increase in lymphocyte proliferation was observed with gpl20-HIV-l antigen, and native p24, respectively, four weeks post treatment.
  • Figure 3 A is a graph depicting the distribution of blasting (proliferating*) cells by flow cytometry in response to HIV-1 antigen at baseline and four weeks post treatment with HIV-1 Immunogen (* ⁇ 0.01).
  • CD4 T helper cells
  • CD8 CD8 lymphocytes
  • NK natural killer cells
  • ⁇ monocytes
  • B B lymphocytes).
  • Figure 3B is a graph depicting the kinetics of CD4, CD8, and natural killer (NK) cells blasting in response to HIV-1 antigen after two treatments with HIV-1 Immunogen in a patient not undergoing antiviral drug therapy. Arrows denote treatment with the HIV-1 Immunogen.
  • Figure 4 is a flow cytometric dot blot of CD4 and CD8 cells blasting in PBMC culture before (A) and after (B) treatment with HIV-1 antigen in response to i) no antigen, ii) HIV-1 antigen, iii) p24 antigen.
  • Figure 5 is a graph showing memory (CD4) cells and na ⁇ ve (CD8) cells blasting four weeks after treatment with HIV-1 Immunogen with, and without antigen stimulation (*p ⁇ 0.01).
  • Figure 6 is a graph showing the effect of CD4, CD8, or CD56 (NK) depletion in
  • This invention provides a novel method for evaluating immune function using flow cytometry.
  • the method provides information concerning the strength of an immune response by measuring the quantity of immune cells which proliferate (e.g., blast) in response to an antigen stimulus, without the use of radioactive agents.
  • the method provides information concerning the nature of the immune response by identifying the types of cells which are proliferating and which are thus involved in the immune response.
  • the method is highly accurate and practical, and can be used in place of conventional methods for evaluating and predicting immune function
  • the term "evaluating" includes measuring a positive immune response (e.g., an increase in immune cell function or upregulation in response to an antigen), a negative immune response (e.g., a decrease in immune cell function or downregulation in response to an antigen), or no response (e.g., no change in immune response to an antigen or anergy).
  • the term “evaluating” also includes quantifying an immune response, by determining the number of cells as a percent of the total population that are modulated (e.g., upregulated or downregulated).
  • the term “evaluating” is also intended to include determining the types of cells that are modulated.
  • flow cytometery shall have its art recognized meaning which generally refers to a technique for characterizing biological particles, such as whole cells or cellular constituents, by flow cytometry (See e.g., Jaroszeski et ⁇ l, Method in Molecular Biology, (1998), Vol 91 : Flow Cytometry Protocols, Hummama Press; Longobanti Givan, (1992) Flow Cytometry, First Principles, Wiley Liss). All known forms of flow cytometery are intended to be included, particularly fluorescence activated cell sorting (FACS), in which fluorescent labelled molecules are evaluated by flow cytometry.
  • FACS fluorescence activated cell sorting
  • the term “blasting” refers to cells which are actively dividing and multiplying.
  • the term “blasting” also refers to cells which elicit a functional response to an antigen, or cells which are activated and stimulated to divide by an antigen.
  • the term “immune cells” refers to any cell that elicits an immune response, such as peripheral blood mononuclear cells (PBMCs), including CD4 cells, CD8 cells, natural killer cells (NK), monocytes, macrophages, granulocytes, B lymphocytes, T lymphocytes and leukocytes.
  • PBMCs peripheral blood mononuclear cells
  • NK natural killer cells
  • monocytes macrophages
  • macrophages granulocytes
  • B lymphocytes granulocytes
  • T lymphocytes T lymphocytes
  • leukocytes leukocytes
  • vaccine refers to any immunogen, including but not limited to viral, bacterial and tumor immunogens.
  • subject includes any mammal capable of having an immune response. Preferred subjects are humans.
  • the phrase “labelling immune cells” refers to the use of any label which is specific for a cell type, such that the cell type can be distinguished by FACS.
  • labels include but are not limiting to a fluorescent label, for example on an antibody specific for a cell type.
  • HlV-specific immune response is intended to include a positive or negative immune response following exposure of cells to an HIV antigen.
  • HIV antigens include whole (e.g., inactivated) virus or any immunogenic components of HIV, e.g., gpl20 and p24.
  • HIV antiviral agent is intended to include any known HIV antiviral agent, for example, AZT and protease inhibitors.
  • the method of the invention involves evaluating blasting immune cells by flow cytometry in a sample of immune cells obtained from a subject as an indication of an immune response.
  • the immune cells are cultured with an antigen prior to evaluation so that the strength of their response (e.g., stimulation) to the antigen is being measured.
  • the cells can also be labelled prior to evaluation, but generally following stimulation, so that the different classes (e.g., CD4 cells, CD8 cells, natural killer cells, macrophages, B cells etc.) and types (e.g., na ⁇ ' ve T helper cells verses memory cells) of immune cells which are blasting can be identified when the cells are valuated by flow cytometry (e.g., by FACS).
  • Flow cytometry analysis is then performed on samples of immune cells, both with and without prior antigen stimulation.
  • the quantity of blasting cells in the sample provides an indication of the strength of the immune response.
  • the types of blasting cells in the sample provides an indication of the nature of the immune response (e.g., the lymphocytes involved).
  • a comparison of the immune response between samples exposed and not exposed to antigen stimulus represents the "antigen-specific" immune response (e.g., the strength and nature of the immune response caused by exposure to the particular antigen).
  • a comparison of the immune response (specific or non-specific) between samples obtained from subjects before and after treatment with, for example, a vaccine or an immunosuppressant indicates the change in immune function caused by the treatment.
  • the invention provides a method of evaluating a change in an immune response by obtaining a first sample of immune cells from a subject, evaluating the blasting immune cells in the first sample by flow cytometry (e.g., by FACS), typically following exposure to an antigen.
  • the method further involves obtaining a second sample of immune cells from the subject, evaluating the blasting immune cells in the second sample by flow cytometry, typically following exposure to an antigen.
  • the method further involves comparing the evaluations for the first and second samples to evaluate any change in the immune response between the samples.
  • This method can be performed after administering to the subject a therapeutic agent, such as a vaccine, an immunosuppressant, an antiviral agent or an antitumor agent to determine a change in immune function attributable to the therapeutic agent.
  • a therapeutic agent such as a vaccine, an immunosuppressant, an antiviral agent or an antitumor agent to determine a change in immune function attributable to the therapeutic agent.
  • the immune cells are also labelled prior to evaluating the blasting cells in the samples by flow cytometry.
  • the invention provides a method of evaluating an HIV- specific immune response by contacting a sample of immune cells from a subject with an HIV antigen, analyzing the cells by FACS, and evaluating the blasting immune cells in the sample.
  • the sample of immune cells can be obtained from a subject infected with HIV, for example, who has been treated with an HIV vaccine.
  • the method includes comparing the HIV-specific immune response in the subject prior to administration of the HIV vaccine with the HIV-specific immune response in the subject following administration of the HIV vaccine.
  • another aspect of the invention is a method of evaluating the efficacy of a vaccine by administering a vaccine to a subject, analyzing a sample of immune cells obtained from the subject by flow cytometry to evaluate the blasting immune cells in the sample, and comparing the results with a sample of immune cells obtained from the subject prior to administering the vaccine, all as described above.
  • Samples of immune cells can be obtained from a subject using techniques well known in the art.
  • whole blood can be removed from a subject by standard methods and immune cells (e.g., peripheral blood mononuclear cells or "PBMCs") obtained, for example, by removing red blood cells.
  • immune cells e.g., peripheral blood mononuclear cells or "PBMCs"
  • PBMCs peripheral blood mononuclear cells
  • One technique for doing so uses density gradients that pellet red blood cells at the bottom of the centrifuge tube, leaving PMBCs at an interface from which they are collected.
  • Another technique uses red cell lysis techniques using ammonium chloride, which preferentially lyses red blood cells.
  • a preferred apparatus for performing flow cytometry in the method of the invention is a fluorescence activated cell sorter (FACS).
  • the FACS apparatus commonly includes a light source, usually a laser, and several detectors for the detection of cell particles or subpopulations of cells in a mixture using light scatter or light emission parameters.
  • the underlying mechanisms of FACS are well known in the art, and essentially involve scanning (e.g., counting, sorting by size or fluorescent label) single particles as they flow in a liquid medium past an excitation light source. Light is scattered and fluorescence is emitted as light from the excitation source strikes the moving particle.
  • Forward scatter FSC, light scattered in the forward direction, i.e. the same direction as the beam
  • SSC side angle scatter
  • Orange-red light generated from, for example, the fluorochrome R- phycoerythrin (PE) is detected in another band of wavelengths, designated the FL2 parameter.
  • red fluorescence generated by flourochromes such as PerCP (BEcton Dickinson, San Jose, CA), Quantum red (Sigma, St. Louis, MO) and CyChrome (Pharmingen, La Jolla, CA), is detected in a third band of wavelengths, designated the FL3.
  • Flow cytomeric analysis can involve the use of a single fluorochrome conjugated to an antibody.
  • the antibody is then used to determined the absence or presence of the antigen in the heterogenous sample.
  • Antibodies are conjugated to different fluorescent dyes by any conventional procedure, as, for example, the procedures described by Wofsy et al., "Modification and Use of Antibodies to Label Cell Surface Antigens," Selected methods in Cellular Immunology, B.B. Mishell and S.M. Siigi, ed., W.H. Freeman and Co. (1980). If two fluorescent dyes are used for the FACS analysis, the two dyes should have a detectable fluorescence emission difference.
  • Various pairs of dyes may be used, for example, combinations of fluorescein, R-phycoerythrin, rhodamine, Texas Red, PerCP, or phycobiliprotein groups.
  • the dye-conjugated labelled antibodies may be combined with a sample isolated from a subject, so that the antibodies bind to different cell types in the sample. If desired, an incubation step may be used to promote binding.
  • the sample may be analyzed with a flow cytometer for their light scatter or fluorescence properties, or both parameters may be analyzed simultaneously. Scatter signals may be detected in both the forward and wide angle directions and may be used to identify subpopulations of activated cells based on cell size, morphology and granularity.
  • Two color cell analysis may be performed as described by Parks, et al., Flow Cytometry and Fluorescence- Activated Cell Sorting. Handbook for Experimental Immunology, 4th ed. D.M. Weir, L.A. Touchberg, C.C. Blackwell, and L.A. Herzenberg, eds. Blackwell Scientific Publications, Edinburg (1984).
  • the complete sample of labeled cells may be passed, at a time, through a flow cytometer equipped with one or more lasers, such as argon and helium-neon 30 mW lasers, to provide excitation energy to excite the two dyes.
  • the flow cytometer may be equipped with a single light source that can be used to excite multiple fluorescent dyes.
  • the samples are evaluated by gating on particles with defined characteristics.
  • Treating refers to the selection of a population of particles from a sample, based on the characteristics of the particle. For example, characteristics of a particle can be defined based on the FSC, SSC and/or fluorescence intensity numbers. Therefore, particles with the required characteristics will pass through the gate and are selected for further analysis, while those that do not have the required characteristics will not be selected for further analysis.
  • immune cells are evaluated by gating on blasting, or actively dividing, cells.
  • the FSC, SSC and/or fluorescence characteristics for population of immune cells that is blasting is different from a population of immune cells that are not blasting.
  • a sample of immune cells having had no prior antigen contact is analyzed by flow cytometry, and blasting cells are gated.
  • a sample of immune cells which has had prior antigen contact and, therefore, which generally contains more blasting cells, is then analyzed and the same gate is used. This ensures analysis of the same region of blasting cells.
  • the immune cells are labelled prior to evaluating the sample by flow cytometry so that different blasting cell types can be identified.
  • the label can be any label that can identify different cell types by flow cytometry, for example a fluorescent label, as described above.
  • Antibodies can be conjugated to different fluorescent dyes by any conventional procedure, as, for example, the procedures described by Wofsy et al., "Modification and Use of Antibodies to Label Cell Surface Antigens," Selected methods in Cellular Immunology, B.B. Mishell and S.M. Siigi, ed., W.H. Freeman and Co. (1980).
  • Evaluating the blasting immune cells can involve quantifying the blasting immune cells. This can be performed by determining the number of cells as a percent of the total population blasting in a region prior to antigen contact, and comparing the change in the number of cells as a percent of the total population blasting in the same region after antigen contact. Standard techniques for gating and determining the percentage of cells on a gated region are known in the art (See e.g., Jaroszeski et al, Method in Molecular Biology, (1998), Vol 91 : Flow Cytometry Protocols, Hummama Press; Longobanti Givan, (1992) Flow Cytometry, First Principles, Wiley Liss).
  • Evaluating the blasting immune cells can also involve identifying the types of immune cells which are blasting.
  • the different types of immune blasting can be determined using fluorescent labelled antibodies specific for antigens on the different cell types.
  • fluorescent labelled antibodies specific for different cell types include, but is not limited to CD3 FITC/CD 16+CD56PE for NK cells; CD3 FITC/CD 19PE for B cells; CD45 FITC/CD 14PE for monocytes; CD3 FITC/CD4PerCP for T helper cells; CD3 FITC/CD8PE for CD8 cells; CD4PerCP/CD45 RO PE for T helper memory cells; CD8 FITC/CD45RO PE for CD8 memory cells; CD62 FITC/CD45RA PE/CD4PerCP for naive T helper cells; CD62 FITC/CD45RA PE/CD8PerCP for naive CD8 cells.
  • the method can be used to evaluate specific and non-specific immune responses.
  • the method can be used to determine changes in immune function after infection with a pathogen (e.g., virus or bacterium), development of tumors or transplantation of tissues or organs.
  • a pathogen e.g., virus or bacterium
  • the method can be used to determine changes in immune function after treatment of patients with vaccines, immunosuppressants, antiviral agents, antitumor agents or other therapeutics.
  • the method can be used to evaluate non-specific immune function by, for example, evaluating responses to mitogens (e.g., Con A, PHA etc.), CMV or tetanus.
  • mitogens e.g., Con A, PHA etc.
  • the method of the invention can be used to assess whether a subject afflicted with a disease is likely to respond to a therapeutic regimen.
  • the baseline (i.e., before treatment) level of immune function in a subject, evaluated by the method of the invention can be used as an indication of how the patient will respond to treatment.
  • the presence of blasting CD4 (particularly memory cells), CD8 and NK cells among peripheral blood cells isolated from HIV-1 infected patients prior to HIV vaccine treatment indicates that the patients are more likely to respond to the vaccine treatment (e.g., exhibit an increase in immune function in response to an HIV antigen stimulus following administration of the vaccine).
  • Example 1 Evaluation of an HIV-Specific Immune Response Following Vaccination by Lymphocyte Proliferation Assay and Chemokine Production
  • CD4 cells are the target for the HIV-1 virus, which infects these cells and reduces their numbers in patients afflicted with the disease. Accordingly, diagnosis of HIV-1 involves the enumeration of CD4 cells in the peripheral blood.
  • the baseline demographics for the study are listed in Table 1. The mean CD4 count at baseline was 607 cells/ ⁇ l. The mean plasma HIV-1 RNA copy number at baseline was 1319 copies/mL using the Hoffman La Roche Amplicor assay. Concomitant antiviral drug therapies are also listed in Table 1.
  • HIV-1 Immunogen received one intramuscular injection of the HIV-1 Immunogen at Day 1 and every 12 weeks which consisted of 10 units p24 (100 ⁇ g total protein inactivated) gpl20-depleted HIV-1 in Incomplete Freund's Adjuvant (IF A).
  • Table 1 Baseline demographics of eight HIV-1 seropositive subjects
  • Inactivated HIV-1 antigens used in the study were prepared as follows.
  • the gpl20-depleted HIV-1 antigen was purified by ultrafiltration and ion exchange chromatography using methods previously described by Prior et al. BioPharm. October:22-34 (1996) from the extracellular supernatant fluid of HZ321 Hut-78 cells (Choi et al. AIDS Res. Hum. Retroviruses 13(4):357-361 (1997)).
  • the outer envelope protein (gpl20) was depleted at the ultrafiltration stage of the purification process.
  • Antigen preparations were inactivated through a sequential application of beta- propiolactone (BPL) (LoGrippo Ann. N. Y. Acad. Sci. 83:578-594 (I960)) and 60 Co irradiation (Kitchen et al. Vox Sang 56:223-229 (1989)).
  • BPL beta- propiolactone
  • I960 60 Co irradiation
  • p24 Native p24 (referred to as p24) was lysed from purified gpl20-depleted, inactivated HIV-1 with 2% Triton X-100 and purified using Pharmacia Sepharose Fast Flow S resin. Chromatography was carried out at pH 5.0 and p24 was eluted using a linear salt gradient. Purity of the final product was estimated by both SDS-PAGE and reverse phase HPLC to be >99%.
  • C. Lymphocyte Proliferation Assay To compare the recognition of HIV-1 antigens in HIV infected patients before (at baseline) and after treatment with immunizing antigen, in vitro lymphocyte proliferation assays were performed. Fresh PBMCs isolated from HIV-1 seropositive subjects were purified and cultured with media alone or with inactivated HIV antigens including whole gpl20-depleted HIV-1 at a concentration of 5 ⁇ g/ml, or purified native p24 at a concentration of 5 ⁇ g/ml, prepared as described above.
  • PBMCs were seeded in a round bottom 96 well plate (Falcon) at 2 xlO 5 cells/well in complete RPMI [RPMI (Hy clone) containing 10% heat-inactivated (56°C x 30 minutes) human AB serum (Gemini) and 1% antibiotics (100 U/ml penicillin; lOO ⁇ g/ml streptomycin (Gibco) and L-glutamine 1% (Hyclone)]. All assays were performed in triplicate. After 6 days of incubation, cells were labeled with 1 ⁇ Ci of 3 H-thymidine in complete RPMI for 16-18 hours.
  • BPL beta-propiolactone
  • MIP-1 ⁇ HIV-1 antigen stimulated ⁇ - chemokine
  • ⁇ -chemokines can be made principally, but not exclusively, by CD 8 and natural killer cells (Cocchi et al. Science 270:1811-1815 (1995); Moss et al. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 14:343-350 (1997); and Oliva et al. J. Clin.
  • CD4 and CD8 cell populations were depleted from PBMCs using Dynabeads M-450 CD4 and Dynabeads M-450 CD8 (Dynal, Lake Success, New York). Cells were resuspended in PBS+2% human serum and two consecutive incubations for 30 minutes at 4°C with Dynabeads (Dynabeads to target cell ratio 5:1) and were processed to obtain a complete depletion. Indirect methods were used for CD56 (NK cells) depletion. Cells (in PBS+2% human serum) were incubated with CD56 biotin-conjugated mouse anti-human monoclonal antibody (Pharmingen, San Diego, California) for 30 minutes at room temperature.
  • results from cell depletion experiments demonstrate that depletion of CD4 lymphocytes from the PBMC most efficiently abrogates the HIV-1 specific proliferative response in two different patients, as shown in Figure 6.
  • the study also shows that the HIV-1 specific imune response which results in the proliferation of T helper cells, can be associated with the activation and proliferation of CD8 and natural killer cells (CD56). Therefore, CD4 plays a key role in effecting the proliferation of other cell types.
  • Example 3 Evaluation of an HIV-Specific Immune Response by a Novel Non- Radioactive Flow Cytometry Assay and Comparison With Lymphocyte Proliferation and Chemokine Production Assays
  • Samples obtained from HIV infected subjects at baseline and following HIV immunization as described in Example 1 were also analyzed by a novel, non-radioactive flow cytometry assay to evaluate the increase in immunoreactivity to HIV antigen following immunization.
  • This assay was used not only to measure an increase in the number of proliferating (blasting) cells in response to HIV antigen following immunization, without the use of radioactive compounds, but also to identify the types of cells which were proliferating, thereby providing further information about the nature of the immune response.
  • Fresh PBMCs from patients were cultured for 6 days with or without HIV-1 antigen in tissue culture tubes.
  • PBMCs were washed 2 times in PBS + 2% FBS and resuspended in the same solution.
  • the following monoclonal antibodies were added at a concentration of 1 : 10: CD3 FITC/CD 16+CD56PE for NK cells; CD3 FITC/CD 19PE for B cells; CD45 FITC/CD 14PE for monocytes; CD3 FITC/CD4PerCP for T helper cells; CD3 FITC/CD8PE for CD8 cells; CD4PerCP/CD45 RO PE for T helper memory cells; CD8 FITC/CD45RO PE for CD8 memory cells; CD62 FITC/CD45RA PE/CD4PerCP for naive T helper cells; CD62 FITC/CD45RA PE/CD8PerCP for naive CD8 cells.
  • Control antibodies were mouse gamma 1-FITC, mouse gamma 2-PE, and mouse gamma 1-PerCP.
  • the PerCP antibodies were obtained from Becton Dickinson, Mountain View, California. All other monoclonal antibodies were obtained from Pharmingen, Inc., San Diego, California. Cells were incubated with antibody at 4°C for 30 minutes in the dark, then washed twice and fixed in 1% paraformaldehyde for at least one hour and stored at 4°C.
  • the cells were analyzed by fluorescence activated cell sorter (FACSort, Becton Dickinson) within 24 hours. Analysis was conducted by gating on cells that were blasting in respone to antigen stimulation. Results were measured in terms of % total cells. Gating on blasting cells selects only those cells with an increase in size (e.g., which are proliferating), while excluding cells of a smaller size. The same selection gate was subsequently used to analyse cells in samples which had not been incubated with antigen so that the same region of cells was being compared. A comparison of the data in the same region from both stimulated and unstimulated samples accurately reflects an increase in cell proliferation in response to an antigen.
  • FACSort fluorescence activated cell sorter
  • FIG. 3 A shows the distribution of blasting cells by flow cytometry in response to HIV-1 Immunogen at baseline and four weeks post treatment with HIV-1 Immunogen (*p ⁇ 0.01).
  • T helper CD4 lymphocytes pO.Ol
  • CD8 lymphocytes pO.Ol
  • natural killer cells p ⁇ 0.01
  • proliferation of monocytes ⁇
  • B lymphocytes four weeks post treatment
  • the results of a study of the kinetics of HIV-1 antigen stimulated cell proliferation in a patient not on antiviral drug therapy by flow cytometry is shown in Figure 3B.
  • the HIV-1 antigen was administered on two separate occasions to the patient, the first administration was at day 0.
  • the number of CD4, CD8, and NK cells proliferating in response to HIV-1 increased by approximately 4-fold, 3-fold and 2-fold, respectively.
  • the number of CD4 and CD8 cells continued to increase steadily, to approximately 8-fold and 4-fold, respectively, 12 weeks post treatment.
  • the second adminstration of the HIV-1 antigen was at 12 weeks.
  • the number of CD4 and CD8 cells continued to increase four weeks after the second administration of the antigen.
  • the results of the study again shows that CD4, CD8, and NK cells are the predominant cell types involved in HIV-1 anergy reversal exhibited in patients receiving HIV-1 immunization.
  • FIG. 4 The dot blots shown in Figure 4 (Panel B) compare CD4 and CD8 cells blasting in PBMC culture after immunization with HIV-1 antigen in response to i) no antigen, ii) HIV-1 antigen, and iii) p24 antigen.
  • CD4, CD8, and NK cells were the main cell types identified as proliferating in response to HIV-1 antigen or p24 antigen, correlating well with the results obtained in both the lymphocyte proliferation assay and chemokine production assays described in Example 1 and the cell depletion assay described in Example 2.
  • the data shown below in Table 2 demonstrates just how strong the correlation was between the data obtained using the novel flow cytometric method and the corresponding lymphocyte stimulation index (LSI) (determined from the lymphocyte proliferation assay described in Example 1, which measures thymidine incorporation).
  • LSI lymphocyte stimulation index
  • the correlation values in Table 2 demonstrate a high correlation between the data obtained from flow cytometry and lymphocyte proliferation assay to meaure proliferation of cells.
  • Table 2 The correlation of CD4, CD8, and NK cells proliferating in response to
  • HIV-1 antigen or p24 antigen by the flow cytometric method and thymidine incorporation.
  • the novel flow cytometry method allows greater phenotypic analysis of proliferating cells.
  • lymphocytes e.g., CD4, CD8, NK, B Cells, Macrophages
  • subtypes within lymphocyte classes e.g., CD4 memory cells (associated with CD45RO antigen) and naive T helper cells (associated with CD45RA antigen)
  • CD4 memory cells associated with CD45RO antigen
  • naive T helper cells associated with CD45RA antigen
  • phenotypic analysis by flow cytometry of antigen stimulated cells in the present studies revealed that the predominant cell types that were proliferating four weeks after treatment with HIV-1 Immunogen were CD4 lymphocyte memory cells (pO.Ol) and CD8 lymphocyte memory cells (pO.Ol), as shown in Figure 5. This information can then be used to determine that, after treatment with an HIV-1 immunogen, cells proliferate and differentiate into predominantly CD4 and CD8 memory cells.
  • the novel flow cytometry method allows the characterization and comparison of specific and non-specific immune responses.
  • the method can be used to determine changes in immune function after infection with a pathogen (e.g., virus or bacterium), development of tumors or transplantation of tissues or organs.
  • a pathogen e.g., virus or bacterium
  • the method can be used to determine changes in immune function after treatment of patients with vaccines, immunosuppressants, antiviral agents, antitumor agents or other therapeutics.
  • the method can be used to evaluate nonspecific immune function by, for example, evaluating responses to mitogens (e.g., Con A, PHA etc.), CMV or tetanus.
  • mitogens e.g., Con A, PHA etc.

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Abstract

L'invention concerne un procédé mettant à profit la cytométrie de flux afin d'évaluer les cellules immunitaires blastiques contenues dans l'échantillon servant à indiquer la réponse immunitaire. Un échantillon de cellules immunitaires (p. ex. les cellules mononucléaires périphériques sanguines ou « PMBC ») est mis en contact avec un antigène pour induire une stimulation cellulaire précédant l'évaluation. Les cellules blastiques (p. ex. proliférantes) sont ensuite évaluées par la cytométrie de flux (p. ex. le tri cellulaire activé par fluorescence ou « FACS ») pour déterminer la résistance et la nature de la réponse immunitaire face à l'antigène. Le procédé peut servir à évaluer toute réponse immunitaire qu'elle soit spécifique ou non.
PCT/US1998/024917 1998-08-25 1998-11-25 Procedes d'evaluation de la fonction immunitaire WO2000011476A1 (fr)

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WO2005085864A1 (fr) * 2004-02-27 2005-09-15 The Government Of The United States Of America, Asrepresented By The Secretary Of The Department Of Health And Human Services Essais pharmacodynamiques à cytométrie de flux
WO2006004588A2 (fr) * 2004-05-05 2006-01-12 Sixty Eight, L.L.C. Complexes d'interaction immunitaire dynamique et procedes pour l'utilisation et la preparation de tels complexes
WO2007106939A1 (fr) * 2006-03-20 2007-09-27 St Vincent's Hospital Sydney Limited Procédé de détection de cellules t spécifiques d'antigènes ou activées par des mitogènes
WO2010026328A1 (fr) * 2008-09-05 2010-03-11 Horiba Abx Sas Procede et dispositif de classification, de visualisation et d'exploration de donnees biologiques
CN115774102A (zh) * 2021-09-08 2023-03-10 复旦大学 确定新冠疫苗介导的保护免疫力应答状态的方法

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EP1495134A2 (fr) * 2002-04-11 2005-01-12 Cyclex, Inc. Methode de controle de la reponse immunitaire et de prediction de resultats cliniques chez des receveurs d'organe
EP1495134A4 (fr) * 2002-04-11 2008-02-13 Cyclex Inc Methode de controle de la reponse immunitaire et de prediction de resultats cliniques chez des receveurs d'organe
EP2322928A1 (fr) * 2002-04-11 2011-05-18 Cyclex, Inc. Procédé de mésure de la réponse immune et de prédiction du résultat clinique des patients de greffe utilisant la mésure de l'ATP dans les lymphocytes
WO2005085864A1 (fr) * 2004-02-27 2005-09-15 The Government Of The United States Of America, Asrepresented By The Secretary Of The Department Of Health And Human Services Essais pharmacodynamiques à cytométrie de flux
US9389223B2 (en) 2004-02-27 2016-07-12 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Pharmacodynamic assays
AU2005218769B8 (en) * 2004-02-27 2012-04-12 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Pharmacodynamic assays using flow cytometry
AU2005218769B2 (en) * 2004-02-27 2011-12-22 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Pharmacodynamic assays using flow cytometry
WO2006004588A2 (fr) * 2004-05-05 2006-01-12 Sixty Eight, L.L.C. Complexes d'interaction immunitaire dynamique et procedes pour l'utilisation et la preparation de tels complexes
WO2006004588A3 (fr) * 2004-05-05 2006-05-26 Sixty Eight L L C Complexes d'interaction immunitaire dynamique et procedes pour l'utilisation et la preparation de tels complexes
US8030005B2 (en) 2006-03-20 2011-10-04 St. Vincent's Hospital Sydney Limited Method for detecting antigen specific or mitogen-activated T cells
WO2007106939A1 (fr) * 2006-03-20 2007-09-27 St Vincent's Hospital Sydney Limited Procédé de détection de cellules t spécifiques d'antigènes ou activées par des mitogènes
FR2935802A1 (fr) * 2008-09-05 2010-03-12 Horiba Abx Sas Procede et dispositif de classification, de visualisation et d'exploration de donnees biologiques
JP2012502266A (ja) * 2008-09-05 2012-01-26 ホリバ アベイクス エスアーエス 生体データの分類、可視化並びに探索の方法及び装置
WO2010026328A1 (fr) * 2008-09-05 2010-03-11 Horiba Abx Sas Procede et dispositif de classification, de visualisation et d'exploration de donnees biologiques
CN115774102A (zh) * 2021-09-08 2023-03-10 复旦大学 确定新冠疫苗介导的保护免疫力应答状态的方法

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