GB2366294A

GB2366294A - Assay for evaluating cell responses to infection

Info

Publication number: GB2366294A
Application number: GB0021461A
Authority: GB
Inventors: Alberto Beretta; Josephine Braun
Original assignee: FOND JACQUELINE BEYTOUT
Current assignee: FOND JACQUELINE BEYTOUT
Priority date: 2000-09-01
Filing date: 2000-09-01
Publication date: 2002-03-06
Also published as: WO2002018624A3; GB0021461D0; WO2002018624A2; AU2001292011A1

Abstract

A rapid, quantitative assay for detection of an immune response factor to a preselected micro-organism (especially a virus such as HIV), produced by an <I>in vitro</I> cell culture exposed to said microorganism, comprises detection of the suppression of replication of said microorganism. The cell line used in the assay may produce a reporter analyte, such as an enzyme (particularly b -galactosidase) or a nucleic acid, indicative of such suppression and the immune response factor detected may be a cytokine, chemokine, antibody or, particularly, a CD8+ cell antiviral factor (CAF).

Description

<Desc/Clms Page number 1> ASSAY METHOD This invention relates to an assay method. More particularly, the present invention relates to an in vitro assay for evaluating cell responses to infection in different clinical conditions.

In the description which follows, the present invention will be described with particular reference to its preferred use with the detection of CD8 cell anti-viral factor (CAF) from CD8+ T cells in cell culture which are derived from individuals infected with the Human Immunodeficiency Virus (HIV), although the method has equal utility in the detection of other anti-viral factors or cytokines or antibodies especially those which are capable of neutralising HIV produced by other cells in in vitro culture.

It is known that CD8+ T cells derived from healthy human immunodeficiency virus (HIV)-infected individuals can suppress HIV replication in cultured CD4+ T cells without any apparent killing of infected cells [Walker CM, Moody DJ, Stites DP, Levy JA: CD8+ lymphocytes can control HIV infection in vitro by suppressing virus replication. Science 1986, 234: 1563-1566] (reviewed in [Levy JA, Mackewicz CE, Barker E: Controlling HIV pathogenesis: the role of the noncytotoxic anti-HIV response of CD8+ T cells. Immunol Today 1996, 17: 217-224]). This antiviral response (CD8 cell Antiviral Factor: CAF) is usually detected as a reduction of HIV p24 antigen and reverse transcriptase levels in culture supernatants of CD8+ T cells mixed with infected CD4+ T cells [Mackewicz C, Levy JA: CD8+ cell anti-HIV activity: nonlytic suppression of virus replication. AIDS Res Hum Retroviruses 1992, 8: 1039- 1050], it is mediated by a soluble factor detectable in trans-well cultures [Brinchmann JE, Gaudernack G, Vartdal F: CD8+ T cells inhibit HIV replication in naturally infected CD4+ T cells. Evidence for a soluble inhibitor. J Immunol 1990, 144: 2961-2966] and does not require HLA class I compatibility between the effector CD8+ cells and the target cells [Mackewicz C, Levy JA:

CD8+ cell anti-HIV activity: nonlytic suppression of virus replication. AIDS Res Hum Retroviruses 1992, 8: 1039-1050]. Unlike C-C chemokines, which are also produced by CD8+ T cells and suppress selectively HIV viruses which use CCR5 as a coreceptor (R5 viruses, [Berger EA, Doms RW, Fenyo EM, et al.: A new classification for HIVA [letter]. Nature 1998, 391: 240]), CAF suppresses all types of HIV-1, HIV-2 and simian immunodeficiency viruses (SIV) independently of their coreceptor usage [Walker CM, Thomson-Honnebier GA, Hsueh FC, Erickson AL, Pan LZ, Levy JA: CD8+ T cells from HIV-1-infected individuals inhibit acute infection by human and primate immunodeficiency viruses. Cell Immunol 1991, 137: 420-428]. It has also been demonstrated that the C-X-C chemokine stromal cell-derived factor (SDF-1 a), which inhibits selectively HIV viruses which use CXCR4 as a coreceptor (X4 viruses, [Berger EA, Doms RW, Fenyo EM, et al.: A new classification for HIVA [letter]. Nature 1998, 391: 240]) is not responsible for the CD8+ T cell suppression of this type of viruses [Lacey SF, McDanal CB, Horuk R, Greenberg ML: The CXC chemokine stromal cell-derived factor 1 is not responsible for CD8+ T cell suppression of syncytia-inducing strains of HIV-1. Proc Natl Acad Sci U S A 1997, 94: 9842-9847].

In addition to P-chemokines and CAF, CD8+ cells produce different cytokines which also can suppress HIV in vitro [Poli G: Laureate ESCI award for excellence in clinical science 1999. Cytokines and the human immunodeficiency virus: from bench to bedside. European Society for Clinical Investigation. Eur J Clin Invest 1999, 29: 723-732]. However, experiments with cytokine-neutralizing antibodies have clearly established that CAF is distinct from cytokines like IFNa and (3, TNFa, TGF(3, IL4, IL6, IL8, IL10 and IL13 [Brinchmann JE, Gaudernack G, Vartdal F: In vitro replication of HIV- 1 in naturally infected CD4+ T cells is inhibited by rIFN alpha 2 and by a soluble factor secreted by activated CD8+ T cells, but not by rIFN beta, rIFN gamma, or recombinant tumor necrosis factor-alpha. J Acquir Immune Defic

Syndr 1991, 4: 480-488], [Mackewicz CE, Ortega H, Levy JA: Effect of cytokines on HIV replication in CD4+ lymphocytes: lack of identity with the CD8+ cell antiviral factor. Cell Immunol 1994, 153: 329-343], [Moriuchi H, Moriuchi M, Combadiere C, Murphy PM, Fauci AS: CD8+ T-cell-derived soluble factor(s), but not beta-chemokines RANTES, MIP-1 alpha, and MIP-1 beta, suppress HIVA replication in monocytelmacrophages. Proc Nat/ Acad Sci U S A 1996, 93: 15341-15345]. Although a preliminary biochemical characterization of CAF has been reported [Levy JA, Mackewicz CE, Barker E: Controlling HIV pathogenesis: the role of the noncytotoxic anti-HIV response of CD8+ T cells. Immunol Today 1996, 17: 217-224], [Klotman MA, Mosoian A, Teixeria A: HIV-1 inhibitory activity of CD8+ cell supernatants is distinct from that of RANTES, MIP-1a and MIP-1 P. Marnes la Coquette, Paris, France, Elsevier, 1997.[Abstract p.46], its molecular nature has so far remained elusive.

Inhibition of HIV replication by CD8+ T cells has been associated to higher CD4 counts and better clinical status [Gomez AM, Smaill FM, Rosenthal KL: Inhibition of HIV replication by CD8+ T cells correlates with CD4 counts and clinical stage of disease. Clin Exp Immunol 1994, 97: 68-75], [Blackbourn DJ, Mackewicz CE, Barker E, et al.: Suppression of HIV replication by lymphoid tissue CD8+ cells correlates with the clinical state of HIV-infected individuals [see comments]. Proc Natl Acad Sci U S A 1996, 93: 13125- 13130], [Barker E, Bossart KN, Locher CP, Patterson BK, Levy JA: CD8+ cells from asymptomatic human immunodeficiency virus-infected individuals suppress superinfection of their peripheral blood mononuclear cells. J Gen Virol 1996, 77: 2953-2962] and it is detectable in the blood of patients with primary HIV infection a long time before neutralizing antibodies are detectable [Mackewicz CE, Yang LC, Lifson JD, Levy JA: Non-cytolytic CD8 T-cell anti- HIV responses in primary HIVA infection. Lancet 1994, 344: 1671-1673]. Similarly, in children born to seropositive mothers, CAF activity appears before

cytotoxic-T-lymphocytes (CTLs) appear and is associated with lower viral load and favourable early survival [Pollack H, Zhan MX, Safrit JT, et al.: CD8+ T-cell- mediated suppression of HIV replication in the first year of life: association with lower viral load and favorable early survival. Aids 1997, 11: F9-13], [Levy JA, Hsueh F, Blackbourn DJ, Wara D, Weintrub PS: CD8 cell noncytotoxic antiviral activity in human immunodeficiency virus-infected and -uninfected children. J Infect Dis 1998, 177: 470-472]. Strong CD8+ T cell noncytotoxic activity was also associated with a lack of infection in HIV exposed individuals bearing wild-type CCR5 genes [Stranford SA, Skurnick J, Louria D, et al.: Lack of infection in HIV-exposed individuals is associated with a strong CD8(+) cell noncytotoxic anti-HIV response. Proc Natl Acad Sci U S A 1999, 96: 1030-1035] and to the partial resistance to infection by an R5X4 primary virus in an exposed -uninfected individual homozygous for the CCR5 A32 deletion [Xiao L, Weiss SH, Qari SH, et al.: Partial resistance to infection by R5X4 primary HIV type 1 isolates in an exposed-uninfected individual homozygous for CCR5 32-base pair deletion. AIDS Res Hum Retroviruses 1999, 15: 1201-1208]. Further evidence for the association of CAF activity with resistance to HIV infection comes from a recent study which demonstrates that women who have been immunized with mononuclear cells derived from the partner are resistant to HIV infection and display a significant upregulation of the production of (3-chemokines and CAF [Wang Y, Tao L, Mitchell E, et al.: Allo- immunization elicits CD8+ T cell-derived chemokines, HIV suppressor factors and resistance to HIV infection in women. Nat Med 1999, 5: 1004- 1009]. Other studies reported the simultaneous upregulation of both CAF and Pchemokines [Scala E, D'Offizi G, Rosso R, et al.: C-C chemokines, IL-16, and soluble antiviral factor activity are increased in cloned T cells from subjects with long-term nonprogressive HIV infection. J Immunol 1997,158: 4485-4492], [Lehner T, Wang Y, Tao L, Bergmeier LA, Mitchell E, Doyle C: CD8-

suppressor factor and beta-chemokine function as a complementary mechanism to cognate immunity. Immunol Lett 1999, 66: 171-176].

Although CAF was originally described as being produced selectively by CD8+ T cells of HIV infected individuals, it has also been detected in cultures of CD8+ T cells from non-infected, non-exposed individuals [Rosok B, Voltersvik P, Larsson BM, Albert J, Brinchmann JE, Asjo B: CD8+ T cells from HIV type 1- seronegative individuals suppress virus replication in acutely infected cells. AIDS Res Hum Retroviruses 1997, 13: 79-85], [Kootstra NA, Miedema F, Schuitemaker H: Analysis of CD8+ T lymphocyte-mediated nonlytic suppression of autologous and heterologous primary human immunodeficiency virus type 1 isolates. AIDS Res Hum Retroviruses 1997, 13: 685-693] as well as in cultures of CD4+ T cells from an exposed-uninfected individual homozygous for the CCR5 032 deletion [Xiao L, Weiss SH, Qari SH, et al.: Partial resistance to infection by RSX4 primary HIV type 1 isolates in an exposed-uninfected individual homozygous for CCR5 32-base pair deletion. AIDS Res Hum Retroviruses 1999, 15: 1201-1208] and from uninfected control or HIV infected individuals [(Leith, J. G., Copeland, K. F., McKay, P. J., Bienzle, D., Richards, C. D., Rosenthal, K. L.: T cell-derived suppressive activity: evidence of autocrine noncytolytic control of HIV type 1 transcription and replication. AIDS Res Hum Retroviruses 1999,15(17): 1553-1561)].

At present, CAF activity is usually measured in one of two different ways: naturally or exogenously infected CD4+ T cells are cultured in the presence of autologous or heterologous CD8+ T cells at different CD8:CD4 cell ratios. Alternatively, supernatants from activated CD8+ T cells can be added to infected CD4+ T cells. In both cases CAF activity is measured as the reduction of viral replication compared to control cultures [Levy JA, Mackewicz CE, Barker E: Controlling HIV pathogenesis: the role of the noncytotoxic anti-HIV response of CD8+ T cells. Immunol Today 1996, 17: 217-224]. In assays which use CD8+ T cell supernatants, a one-fourth dilution of supernatant is the

highest level permissible for the detection of 50% suppression of viral replication. This has been taken as an evidence that CAF activity is present in very low amount in CD8+ T cell supernatants. Clearly, the low sensitivity of this type of assay represents a major technical pitfall in the isolation and characterization of CAF. On the other hand, CD8+/CD4+ cell coculture assays do not discriminate between CAF activity and other CD8+ T cell functions like direct cytotoxicity, unless cocultures are run in separate transwell chambers. In addition, since all standard assays of CAF activity are based on the use of peripheral blood T cells as indicator cells, results are difficult to standardize due to donor to donor variations. Moreover, current CAF assays are time consuming, have a low sensitivity and do not allow a clear quantification of the suppressive activity.

It is therefore an object of the present invention to develop a cell line-based assay for the quantification of CAF in culture supernatants of CD8+ cells.

The present inventors have therefore developed a new assay which directly measures CAF activity on a human cell line which is stably transfected with CD4, CXCR4 and a HIV-1 long terminal repeat (LTR)-IacZ construct inducible by the HIV-1 transactivator Tat.

Accordingly, the present invention provides a rapid quantitative assay for the detection of an anti-viral factor, produced by an in vitro cell culture. Advantageously, using the method of the present invention, it is possible to detect suppression of viral replication which can be measured on both TCLA and primary X4 virus isolates, as little as 48 hours after the virus inoculum, by the addition of a preferably chromogenic substrate. The assay can be used to more precisely quantitate the CD8+ T cell-derived CAF activity.

Preferably, the quantitative assay is colourimetric. For example, the cell line is preferably capable of producing a reporter analyte, the level of the reporter analyte being indicative of the level of the anti-viral factor activity within the cells. Preferably, the reporter analyte is an enzyme, such as (3-galactosidase.

The term "reporter analyte" as used herein is intended to indicate any chemical, biochemical, biological, micro-biological or genetic moiety which is detectable using rapid assay technique such as colourimetry, spectrophotometry, HPLC or ELISA.

Preferably the cell line is a human cell line, grown under suitable in vitro conditions. More preferably, the cell line is derived from human peripheral blood mononuclear cells (PBMC), especially a cell line capable of expressing CD4 and CXCR4. Ideally, for the preferred embodiment of the invention the cells are glioma cell derived cell lines such as U373MG-CD4-CXCR4 construction of which will be described hereinafter.

Preferably, the anti-viral factor to be detected is CD8+ cell anti-viral factor (CAF) although the method of the present invention may be modified to detect other anti-viral factors, neutralizing antibodies, chemokines or interferons.

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, of which, Figure 1A is a graph showing the kinetics of HIV infection of total (plain line) and CD8+ cells-depleted PBMC (dotted line) from two donors. HIV-1 p24 antigen production measured in cell-free supernatants is shown. Cells were obtained from healthy donors d#3 (left panels) and d#6 (right panels). Cells were infected with 0,5 ng p24 of NL4-3 per million cells, and Figure 1B is a graph showing inhibition of PBMC infection by supernatants of CD3/CD28-stimulated CD8+ cells. Control PHA-stimulated PBMC depleted of CD8+ cells were infected in the absence (AL) or presence of 1/2 dilution of day 10 supernatants of CD3/CD28- stimulated CD8+ cells from donors d#3 (0) or d#6 (0). Cells were infected with 2 ng p24 per million cells of TCLA NL4-3 strain. HIV-1 p24 antigen production was quantified in cell-free supernatants and the living cells counted by trypan blue exclusion (ng p24 antigen per million living cells) Figure 2a is a series of graphs showing titration of HIV suppressive activity in supernatants of CD8+ cells from donors d#3 and d#6. U373MG-CD4-CXCR4

cells were infected with 1 ng p24 of the TCLA strain NL4-3 in the presence of serial dilutions of supernatants of CD8+ cells collected at different times after CD3ICD28 costimulation (day 3, day 6 , day 10 and day 12). CD8+ cells were obtained from donors d#3 (0) and d#6 (0): percentages of viral inhibition, calculated as described in methods, are the mean of duplicate determinations standard deviation.

Figure 3 is a histogram showing 50% Inhibitory Titer of supernatants from CD8+ cells of d#3 and d#6 donors: inter-assay mean and standard deviation. U373MG-CD4-CXCR4 cells were infected with 1 ng p24 of the TCLA strain NL4- 3 in the presence of serial dilutions of supernatants from CD3/CD28-stimulated CD8+ cells of donors d#3 (g)and d#6 (0). 50% Inhibitory Titer were calculated as described in methods and results are the mean standard deviation of two independent experiments, and Figure 4 is a histogram showing 50% Inhibitory Titer of CD8+ cell supernatants from HIV-1-exposed uninfected (ExU) individuals and healthy donors (d#3 and d#6): inter-assay mean and standard deviation. U373MG-CD4- CXCR4 cells were infected with 2ng p24 of the TCLA strain NL4-3 in the presence of serial dilutions of supernatants from CD3/CD28-stimulated CD8+ cells . 50% Inhibitory Titer were calculated as described in methods and the results are the mean standard deviation of two independent experiments (maximum 50% inhibitory titers are shown).

Materials and Methods Cell purification.

Peripheral blood mononuclear cells (PBMC), obtained from healthy uninfected donors (d#3 and d#6) were isolated by Ficoll-Paque PLUS (Pharmacia Biotech, Orsay, France) gradient centrifugation. CD8+ cells were positively purified with Dynabeads M-450 CD8 (Dynal France SA., Compiegne, France). The remaining CD4+ cells were further depleted of CD8+ cells with

OKT8 (CD8 monoclonal antibody (mAb), Ortho Diagnostic System, Issy les Moulineaux, France) and Dynabeads M-450 Sheep anti-mouse IgG (Dynal) ( < l% CD3+CD8+ cells remaining). Cell phenotype was analysed by four-colour flow cytometry.

Flow cytometry Cell phenotype was determined by four-colour acquisition with a FACSxalibur and analysis with Cell Quest software (Becton Dickinson, le Pont de Ciaix, France).

Lymphocytes stained with anti-CD45-PerCP/anti-CD3-FITC/anti-CD4- APC/anti-CD8-PE were gated using side-scatter versus CD45 expression. Isotypically-matched conjugated mouse antibodies were used as negative controls. MAbs and isotypically-matched mouse antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinal chlorophyll protein (PerCP) or allophycocyanin (APC) were purchased from Becton Dickinson.

CD8+ cells activation.

CD3/CD28 immunobeads were prepared by incubating M-450 Sheep anti- mouse IgG (Dynal) with equal amounts of anti-CD3 (UCH-T-1 SeraLab, Crawley Down, Sussex, England) and anti-CD28 (CD28-2, Immunotech/Beckman Coulter, Marseille, France) at 0.5 pg mAbs per 10' beads. Purified CD8+ cells were cultivated at 1 x 106 cells/ml with CD3/CD28-coated beads at a ratio of 5 beads per cell in complete-11-2-Medium : RPMI 1640-Glutamax supplemented with 10% heat-inactivated FCS (D. Dutcher S.A., Brumath, France), 5001U/ml recombinant human interleukin-2 (rIL2, Proleukin, Chiron, Suresnes, France), 50Ng/ml of gentamicin (Gibco BRL Life Technologies, Cergy Pontoise, France), 1 IU/ml sheep polyclonal anti-interferon a antibody (Valbiotech, Chatillon, France) and 2 pg/ml polybrene (Sigma, St. Louis, MO). At day 3, 6, 10 and 12, cell-free culture supernatants were collected, aliquoted and kept at -20 C until used. At each passage, cells were resuspended at 1 x 106 cells/ml in fresh medium.

Virus stocks and infections.

HIVA NL4-3 [Adachi A, Gendelman HE, Koenig S, et al.: Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 1986, 59: 284-291] was produced by transfection of 293 T cell line with full- length provirus molecular clone. The viral stock supernatant was harvested at the peak of production of viral p24 antigen, aliquoted and kept at -80 C until used.

Infectivity assays with PBMC Infections of PHA-activated total PBMC or CD4+ T cells (PBMC depleted of CD8+ cells) were performed for 60 min at 37 C with an inoculum corresponding to 0.5 ng or 2 ng p24 of the viral stock per 1 x 106 cells. The inoculum was then removed and the cells washed twice in RPMI 1640-Glutamax medium. Cells were resuspended at 1 x 106 cells/ml in 24-well trays in complete-IL2-Medium. Medium was replaced twice weekly and the living cells counted by trypan-blue exclusion. Cultures were monitored for production of viral p24 antigen assayed in cell-free culture supernatants using HIVA p24 core profile ELISA as instructed by the manufacturer (NEN life Science Products, les Ulis, France). For inhibition experiments, 1/2 supernatant from CD8+ cells costimulated with CD3/CD28 immunobeads was added at the time of infection and when medium was replaced.

Titration of suppressive activity of CD8+ cell supernatants Adherent U373MG-CD4-CXCR4 cells [Labrosse B, Brelot A, Heveker N, et al.: Determinants for sensitivity of human immunodeficiency virus coreceptor CXCR4 to the bicyclam AMD3100. J Virol 1998, 72: 6381-6388.] cultivated in Dulbecco's modified Eagle's Glutamax medium supplemented with 4.5 g/L of glucose, 10% heat-inactivated FCS and antibiotics (penicillin and streptomycin or gentamicin) were distributed at 0.005 x 106 cells/well in 96-flat bottom well trays. The next day, cultures were inoculated with optimum dose of

virus in the presence of 2 Ng/ml polybrene, in the absence or presence of 1I4 and successive 10-fold dilution of cell-free CD8+ cell supernatants. Duplicate determinations were performed. Medium was removed 48 h after infection and the cells were lysed in 50 ul of buffer containing 0.125% of NP-40 and 50mM (3- mercaptoethanol. The accumulation of P-galactosidase in cell lysates was measured by colorimetry at 560 nm after addition of 50 Nl of a 6 mM solution of the chromogenic substrate CPRG (chlorophenol red-beta-D-galactopyranoside, Roche Diagnostics, Meylan, France) [Eustice DC, Feldman PA, Colberg-Poley AM, Buckery RM, Neubauer RH: A sensitive method- for the detection of beta- galactosidase in transfected mammalian cells. Biotechniques 1991, 11: 739- 740, 742-733]. The percentage of viral inhibition was calculated as 100-{100/(0D infected cells-OD uninfected cells) x (OD infected cells with CD8+ cell supernatant-OD uninfected cells)). The titer of CD8+ cell supernatant suppressive activity was determined by regression analysis as the dilution giving 50% inhibition of viral infection (1T50).

Results Selection of two donors according to the HIV suppressive activity of their CD8+ T cells.

CAF activity is produced by CD8+ T cells of HIV-infected, HIV-exposed but also non-infected and non-exposed individuals. For these experiments two non- infected non-exposed control individuals were initially selected (donor d#3 and d#6) who displayed different levels of CAF activity. As a first CAF assay the difference between total peripheral blood mononuclear cells (PBMC) and CD8+ cell-depleted PBMC of d#3 and d#6 in their capacity to support the replication of an exogenous virus was measured. PBMC obtained from d#3 and d#6 were depleted or not of CD8+ cells, activated with PHA and then infected with the HIV- 1 X4 strain NL4-3. Viral replication was followed for a period of 24 days by

measurement of HIV-1 p24 antigen. For donor d#3 the kinetics of viral replication in PBMC cultures depleted or not of CD8+ cells were equivalent. In contrast, for donor d#6 viral production by total PBMC was reduced 3 fold and the peak of viral replication was delayed of 7 days compared to the CD8+ cells depleted population, indicating that CAF activity was higher in d#6 compared to d#3 (Fig. 1A). This difference in the kinetic of viral replication between the two donors could not be accounted for by differences in the size of the CD8+ cell populations since flow cytometry analysis of CD45+ gated PBMC from d#3 and d#6 showed equivalent percentage of CD8+ and CD4+ cells (34.3% of CD8+ cells and 43.1% of CD4+ cells for d#3 and 31.1% of CD8+ cells and 43.7% of CD4+ cells for d#6).

A different CAF assay was then used which measures the capacity of supernatants of purified CD8+ cells to suppress viral replication in control CD4+ T cells. CD8+ cells of d#3 and d#6 were purified by CD8 magnetic beads and costimulated with CD3/CD28 immunobeads in the presence of exogenous IL-2. The purity of the cell populations was tested by flow-cytometry four-color analysis (CD3/CD4/CD8/CD45) of 5,000 to 10,000 events. No CD4+CD8- cells were detected on freshly purified populations and at any time point during culture. Supernatants of d#3 and d#6 CD8+ cells were collected at day 10 after costimulation and tested on control PHA-activated CD4+ cells for the capacity to suppress the replication of NL4-3. Supernatants from both d#3 and d#6 suppressed the replication of the virus (Fig. 1B). The CAF activity of d#6 supernatant was more pronounced than that of d#3, particularly at day 12 after infection. Based on these experiments d#3 and d#6 were classified respectively as "low" and "high" CAF producers.

Establishment of an in vitro assay of HIV suppressive activity. Selection of cell lines.

It has been reported that the control of HIV replication by CAF takes place at the level of HIV long terminal repeat (LTR)-driven transcription [Chen CH,

Weinhold KJ, Bartlett JA, Bolognesi DP, Greenberg ML: CD8+ T lymphocyte- mediated inhibition of HIVA long terminal repeat transcription: a novel antiviral mechanism. AIDS Res Hum Retroviruses 1993, 9: 1079-1086], [Mackewicz CE, Blackbourn DJ, Levy JA: CD8+ T cells suppress human immunodeficiency virus replication by inhibiting viral transcription. Proc Natl Acad Sci U S A 1995, 92: 2308-2312], [Tomaras GD, Lacey SF, McDanal CB, Ferrari G, Weinhold KJ, Greenberg ML: CD8+ T cell-mediated suppressive activity inhibits HIVA after virus entry with kinetics indicating effects on virus gene expression. Proc Natl Acad Sci U S A 2000, 97: 3503- 3508]. To assay for CAF activity two cell lines (HeLa-P4 and U373MG-CD4- CXCR4) were initially selected which are stably transfected with a HIVA LTR- LacZ construct inducible by the HIV transactivator Tat and allow a colourimetric determination of HIV infection (induction of (3-galactosidase activity).

U373MG-CD4-CXCR4 cells are U373MG-CD4 glioma-derived cells which express endogenous CD4 and are stably transfected with an expression vector encoding CXCR4 [Labrosse B, Brelot A, Heveker N, et al.: Determinants for sensitivity of human immunodeficiency virus coreceptor CXCR4 to the bicyclam AMD3100. J Virol 1998, 72: 6381-6388.]. The HeLa-P4 cells are HeLa cells which express endogenous CXCR4 and are stably transfected with the human CD4 gene [Charneau P, Mirambeau G, Roux P, Paulous S, Buc H, Clavel F: HIVA reverse transcription. A termination step at the center of the genome. J Mol 8iol 1994, 241: 651-662]. Both cell lines were preliminary tested for their sensitivity to the toxic effect of CD8+ cell-derived supernatants. U373MG-CD4-CXCR4 cells infected with the primary HIVA X4-isolate proliferated equally well in the presence of either DMEM medium alone, complete-IL-2 medium or supernatant from CD3/CD28-stimulated CD8+ cells from d#6. In contrast, proliferation of infected HeLa-P4 cells was reduced by the addition of CD8+ cells supernatants (data not shown). Thus, for further experiments the U373MG-CD4-CXCR4 cells were selected.

The TCLA NL4-3 were then titrated on U373MG-CD4-CXCR4 cells and the optimum infectious dose determined. As 5.0, 2.5 and 1.25 ng p24 resulted in equivalent induction of (3-galactosidase activity, we chose 1 or 2 ng p24 for subsequent experiments on CAF activity.

Quantification of the HIV suppressive activity Purified T cells from d#3 and d#6 were activated with CD3/CD28 immunobeads and supernatants collected at days 3, 6, 10 and 12 were tested on U373MG-CD4-CXCR4 cells for the presence of suppressive activity against NL4- 3. U373MG-CD4-CXCR4 cells were infected in the presence of serial dilutions of CD8+ cell supernatants and a-galactosidase activity measured 48 hours later. The percentage reduction of viral replication in the presence of CAF was calculated as 100-{100/(0D infected cells-OD uninfected cells) x (OD infected cells with CD8+ cell supernatant-OD uninfected cells)}. The titration curves of HIV suppressive activity of d#3 and d#6 CD8+ cell supernatants are shown in Fig. 2. Both d#3 and d#6 supernatants suppressed HIV replication in a dose- dependent fashion. The HIV suppressive titers were determined by regression analysis as the dilution giving 50% inhibition of infection (50% inhibitory titer: IT50) (shown in table I). Maximum suppressive activity was detected at day 6 after stimulation for d#6 (1I618) and at day 3 for d#3 (1/154). The difference between the two donors was consistent and reproducible at each day of supernatant collection. Fig. 3 shows the mean IT50 obtained in two independent experiments with d#3 and d#6 CD8+ cell supernatants.

HIV suppressive activity of CD8+ cells derived from exposed uninfected individuals.

HIV suppressive activity is elevated in individuals who remain seronegative despite repetitive exposure to HIV (exposed uninfected: ExU). As a preliminary test of the potential clinical applications of the cell line-based assay supernatants

derived from CD8+ cells of 11 ExU individuals were titrated and their IT50 against NL4-3 determined. Supernatants were collected at different time points after CD3/CD28 costimulation and tested in two independent experiments. The IT50 obtained at the day of peak suppressive activity are shown in Fig. 4. Ten out of 11 ExU individuals tested displayed IT50 which was above 1/200 (range: 1/317 to 1/2.176). One ExU displayed an IT50 (1I102) similar to that of the "low" CAF producer d#3 (1/176). Thus, the majority of ExU CD8+ supernatants displayed IT50 which were equivalent or higher than that of the "high" CAF producer d#6 (1/446).

Discussion CD8+ T cells can control viral replication by several distinct mechanisms including direct killing of virus infected cells (CTLs), production of C-C chemokines, CAF and production of other HIV suppressive cytokines. Abundant clinical evidence suggest that CTLs are a key protective immune response [Yang 00, Kalams SA, Trocha A, et al.: Suppression of human immunodeficiency virus type 1 replication by CD8+ cells: evidence for HLA class I-restricted triggering of cytolytic and noncytolytic mechanisms. J Virol 1997, 71: 3120- 3128]. On the other hand, non-cytotoxic T cell suppression of HIV replication is also associated to favorable clinical outcome. However, it is still unclear whether C-C chemokines or other soluble factors or both play a critical role in these immune responses. A major limitation to these type of studies is the use of assays which employ mixed cell populations and cannot discriminate between different HIV suppressive factors. In addition, the sensitivity of current assays of CD8+ T cell suppressive activity is too low to allow a clear quantification of CAF activity. This is of particular importance since production of CAF appears not to be restricted to HIV-infected or to HIV-exposed individuals but can also be detected in non-exposed and non-infected individuals. Therefore, a quantitative

assay is needed to better analyze the HIV suppressive activity of individuals in different clinical conditions.

The present inventors have enabled the use of a cell line U373MG-CD4- CXCR4 to quantitate a HIV suppressive activity which is not related to [3- chemokines. The presence of an integrated LTR-LacZ construct allows the rapid (48 hours) determination of the level of viral replication in these cells. The assay can be used with both TCLA and primary viruses (data not shown).

Two non-infected non-exposed donors were initially selected for the study. Of the two donors, d#6 displayed a stronger CAF activity compared to d#3 when studied with conventional CAF assays. Based on the results of these assays, the present inventors classified the two donors as " high " and " low " CAF producers. When tested in the cell line-based assay, donor d#6 displayed HIV inhibitory titers significantly higher than those of donor d#3. As an example, d#6 CD8+ cell supernatants collected at day of maximum CAF activity gave a 50% inhibitory titer (1T50) against HIV-1 NL4-3 which was 3 fold higher than that of the equivalent supernatant collected from d#3. When the present inventors tested CD8+ cells from 11 exposed-uninfected individuals, 10/11 displayed inhibitory titers comparable to d#6 which ranged from 1/317 to 1I2.176. These data confirm previous observations on high risk seronegative individuals who display elevated levels of CAF activity [Stranford SA, Skurnick J, Louria D, et al.: Lack of infection in HIV-exposed individuals is associated with a strong CD8(+) cell noncytotoxic anti-HIV response. Proc Natl Acad Sci U S A 1999, 96: 1030-1035].

The sensitivity of the cell line-based assay is superior to that of PBMC-based assays since we obtained 50% inhibitory titers which were above 1I2,000. As a comparison, a 1/4 dilution of supernatants is the highest permissible in PBMC assays. It should be noted that these values were obtained using supernatants from polyclonally-activated CD8+ cells, i.e. in conditions of maximum T cell stimulation. Data obtained with supernatants from CD8+ cell stimulated with

syngeneic HIV-1 antigen-activated cultures (not reported) indicate that CAF activity can be detected and titrated also in these conditions and that the cell line-based assay can be used for analysis of the antigen-specific cell response.

The experiments described herein do not address the question of the nature of the suppressive activity detected in the cell line-based assay. The present inventors believe that it is possible to rule out the involvement of P-chemokines since all experiments were run using X4 viruses. In addition, preincubation of CD8+ cell supernatants with neutralizing antibodies specific for MIP-1a, MIP-1(3, and RANTES did not modify the inhibitory effect on viral replication (data not shown). Furthermore, we have tested the possibility that the C-X-C chemokine SDF-1 a. may account for the CAF activity by adding exogenous recombinant human SDF-1 a to the U373MG-CD4-CXCR4 cells before virus inoculum. The inventors obtained a maximum 20% reduction of HIV replication with a SDF-1a concentration as high as 1 NM indicating that this chemokine cannot account for the CD8+ cell suppressive activity detected in our assay. Finally, in all our experiments CD8+ cell supernatants were produced in the presence of a neutralizing polyclonal anti-IFNa antibody, thereby excluding the involvement of this cytokine in CAF activity. Although these experiments only partially address the issue of the molecular nature of the suppressive activity, the high sensitivity of the assay may be in the future exploited for the molecular characterization of CAF.

Table I : 50% Inhibitory Titers of X4-HIV-1 by supernatants of CD31CD28-stimulated CD8+ cells CD8+ cells Supernatant IT50 d#3 IT50 d#6 Day 3 154 305 Day 6 113 618 Day 10 53 230 Day 12 53 141 Table 1: HIV-1 suppressive activity titers of CD8+ cell supernatants from donors d#3 and d#6. CD8+ cells were stimulated with CD3lCD28 immunobeads and the cell-free supernatants harvested at day 3, 6, 10 and 12 of costimulation. U373MG-CD4-CXCR4 cells were infected with 1 ng p24 of the TCLA strain NL4- 3 in the presence of serial dilutions of supernatants. 50% inhibitory titers of HIV-1 by CD8+ cell supernatants were calculated as described in methods and are the mean of duplicate determinations (the corresponding inhibition curves are represented in Fig. 2).

Claims

CLAIMS 1. A rapid quantitative assay for the detection of an immune response factor to a preselected micro-organism produced by an in vitro cell culture exposed to said preselected micro-organism by detection of the suppression of replication of said micro-organism. 2. An assay according to claim 1 the cell line produces a reporter analyte indicative of the suppression of replication of said micro-organism. 3. An assay according to claim 1, in which the reporter analyte produces a colorimetric or fluorimetric signal indicative of suppression of replication of said micro-organism. 4. An assay according to claim 2 or claim 3, in which the reporter analyte is an enzyme or a nucleic acid. 5. An assay according to claim 4, in which the reporter analyte is (3- galactosidase. 6. An assay according to any previous claim in which the in vitro cell line is a human cell line. 7. An assay according to claim 7 in which the human cell line is derived from peripheral blood mononuclear cells. $. An assay according to claim 6 or claim 7 in which the cell line is derived from glioma cells.

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9. An assay according to claim 8 on which the cell line is U373MG-CD4- CXC R4. 10. An assay according to any previous claim in which the micro-organism is a virus. 11. An assay according to claim 10 in which the virus is the HIV virus. 12. An assay according to any preceding claim in which the immune response factor detected is a protein, a nucleic acid, a lipid or a sugar. 13. An assay according to any preceding claim, in which the immune response factor is a cytokine, antibody or chemokine. 14. An assay according to claim 13 in which the immune response factor is CD8+ cell anti-viral factor (CAF). 15. An assay substantially as described herein with reference to and as illustrated by figures 1 to 4 of the present invention.