WO1992017193A1 - Medicament containing an agent inhibiting in vivo pathological apoptosis and applications thereof - Google Patents

Abstract

A medicament containing an agent inhibiting in vivo pathological apoptosis may contain: an agent capable of blocking the reception or transduction of signals leading to cell suicide, or an agent providing or generating a co-signal enabling normal cell differentiation and multiplication, or an agent which, alone, causes cell proliferation and differentiation, and generates cells which have lost the suicide capability in pathological conditions, or an agent capable of inhibiting the signals which make a cell capable of responding to activation, by pathological apoptosis. Said medicaments may be used in treating retrovirus related diseases, and in particular diseases related to the lentiviruses such as the HIV in humans.

Description

 "Drug containing an agent inhibiting in vivo pathological apoptosis and applications".

 The present invention relates to the treatment of apoptosis of human cells characterized by the dysfunction or deficiency of certain cell populations.

 It also relates to a method of selecting agents for the treatment of apoptosis in its pathological manifestations.

 It also relates to methods for determining the capacity of cells of individuals affected by an HIV virus to respond to aggression by an infectious agent.

 The term apoptosis is synonymous with the expressions "programmed cell death" or "activation-induced death".

 In the present description, it will essentially be a question of apoptosis.

 Apoptosis is a suicide mechanism of cells which has the particularity of being physiologically active. It is involved in the regeneration of tissues thus found in the involution during embryogenesis and adult life and the maturation of the embryonic immune system.

 As a result, apoptosis differs from necrosis which is a response to attacks by the cell.

 We also know that apoptosis is always linked to the reception or non reception by the cell of a given physiological signal.

Apoptosis has pathological consequences that are all the greater since it can occur in cell populations that the organism is unable to regenerate in their adult form. differentiated, in particular memory lymphocyte cells and neurons.

 In the event of apoptosis, the capacity for cellular regeneration of the organism can also be exceeded from a quantitative point of view.

 In the present description the word "pathological" also relates to phenomena such as cell degeneration or aging involving an involution.

 Apoptosis only concerns cells in the process of disappearing, and does not cause effects on the tissue and cellular environment: it is therefore not an apparent phenomenon, unless we really try to bring it into evidence.

 It has already been shown that, in the case of immature thymocytes, the mobilization of the T cell receptor (TCR) leads to apoptosis. This phenomenon plays an essential role in the selection of the T cell repertoire (Blackman et al. Science, 248; 1335-1341; 1990).

 We also know that certain retroviruses are responsible for the dysfunction and collapse of the immune system as well as the dysfunction and involution of certain tissues.

 They are in particular lentiviruses causing pathological disorders resulting in an abnormal appearance of cellular suicides.

 By way of example of such lentiviruses, mention may be made of viruses SIV in monkeys, FIV in felines, BIV in cattle, VISNA in sheep, CAEV in goats and HIV in humans.

It has also been shown that individuals infected with an HIV virus show early deficiencies in the response of a family of T cells: CD4 helper (TH) T cells. These qualitative gaps precede quantitative reductions in this cell population and are characterized by a selective loss of proliferation in response to antigens of the major class II histo-compatibility complex (MHC-II) and to an extracted mitogen. of Phytolacca americana, called mitogen pockeweed or PWM in the rest of the text, see in particular Lane et al. (N. Engl. J. Med. 313, 79-84, 1985) and Van Noesel et al. (J. Clin. Invest , 86, 293-299, 1990).

The latter authors have also shown that the anti-CD28 antibody increases the proliferation of T cells in patients infected with the HIV _j virus, and thus allows a proliferative response of immature thymocytes to anti-CD3, and to two antibodies. anti-CD2.

 In addition, it has recently been shown that normal human and murine adult T cells can also undergo apoptosis under specific conditions (Newell, et al. Nature 347, 286-289 (1990); Janssen O. et al. J Immunol., 146, 35-39, 1991). In this case, stimulation induced apoptosis differs from that observed in immature thymocytes.

 In adult human T cells carrying the gamma-delta TCR receptor and stimulated by the anti-CD3 antibody, the addition of Interleukin-2 induces apoptosis instead of preventing it (Janssen et al. J. Immunol., 146, 35-39, (1991).

The results forming part of the state of the art relate only to cell cultures in vitro and do not mention any therapeutic application. In addition, the publications known to

Applicants do not mention the existence and even less the role of the phenomenon of apoptosis in the loss of T cell proliferation from patients infected with an HIV virus, and give no explanation as to the selective loss of response from infected patients. by HIV viruses vis-à-vis certain diseases or vis-à-vis certain antigens.

 The applicants therefore set out to find an effective therapeutic agent, in particular with regard to diseases linked to the HIV virus.

 As part of their work, they also provided an explanation for the phenomenon of loss of response with regard to certain antigens or certain diseases encountered in particular in the case of infections by HIV viruses, with a view to therapeutic applications. and preventive against these diseases. They have thus shown that, surprisingly, the mechanism responsible for this loss of response is of the apoptosis type.

 They have also shown, just as surprisingly, that the mechanisms involved in pathological apoptosis of cells from patients infected with an HIV virus are different from the mechanisms of apoptosis not linked to HIV viruses, such as apoptosis induced by a T receptor stimulation of normal human thymocytes or normal mature human T lymphocyte lines.

 According to the invention, it has been found that pathological apoptosis can be inhibited in vivo.

The present invention therefore relates to a medicament containing an agent which in vivo inhibits pathological apoptosis. Such an agent can be selected by a process comprising at least the steps:

 contacting at least two cell preparations of a patient infected with a retrovirus with a cell activating agent, such as pokeweed mitogen (PWM), respectively in the presence and in the absence of said agent, and

 - comparison of proliferation and / or mortality in the two cell preparations.

 It can also be selected by a process comprising at least:

- a first presentation of an antigen by monocytes preincubated with a retrovirus, to a cell line of CD4 ⁺ T lymphocytes,

a second presentation of the antigen by monocytes, possibly preincubated with a retrovirus, to the CD4 ⁺ T cell cell line, and

 - a step of estimating the death and / or proliferation of lymphocytes.

 Preferably, the retrovirus is an HIV. Such a method makes it possible to select an agent which directly prevents the induction of apoptosis.

 The virus in the presence of an agent according to the invention cannot teach cells apoptosis. This method, unlike the previous one, directly concerns the virus.

 An agent according to the invention will be chosen from those capable of:

either block the reception or transduction of signals leading to cell suicide, and in particular capable of inhibiting the activation of a nuclear endonuclease causing fragmentation of the cell genome, such as cyclosporine, or a protein synthesis inhibitor such as cycloheximide;

 - either to provide or generate a co-signal allowing the cell to differentiate and multiply normally such as an anti-CD28 antibody or a combination of an agent causing the stimulation of phosphokinase C such as phorbol ester, with interleukin 1;

 - either to cause cell proliferation and differentiation alone and to generate cells which have lost the capacity to commit suicide under pathological conditions, for example, phytohemagglutinin (PHA), irradiated allogeneic cells, an anti- CD2, or an anti-CD3 antibody.

 - or to inhibit the signals which make a cell capable of responding to an activation by pathological apoptosis.

 Allogeneic irradiated cells are cells from an individual genetically different in terms of their HLA system.

 Such a medicament is in particular intended for the treatment of diseases linked to retroviruses and in particular linked to lentiviruses, such as viruses.

HIV in humans and SIV, FIV, BIV, VISNA and CAEV in animals.

 Another subject of the present invention is the use of an agent as defined above for obtaining a medicament intended for the treatment of the previously mentioned diseases.

It also relates to a pharmaceutical composition containing a pharmaceutically effective amount of an agent as defined previously in combination with one or more pharmaceutically acceptable diluents or adjuvants. Such a composition is intended for the treatment of diseases linked to retroviruses and in particular linked to lentiviruses.

 The treatment of patients affected by these diseases will be done in a manner known per se and preferably by parenteral or intravenous route.

 The present invention also relates to a method for determining the qualitative deficiency of the immunological repertoire of individuals vis-à-vis an infectious agent comprising at least the steps of:

 - in vitro stimulation of lymphocytes in a medium containing at least one anti-CD28 antibody in the presence of one or more antigens specific for the infectious agent, and

 - measurement of the kinetics of proliferation of lymphocytes.

 The object of the present invention is also a method for determining the capacity of an individual's lymphocytes to respond to aggression by an infectious agent comprising at least the steps of:

 - in vitro stimulation of lymphocytes in a medium containing at least one anti-CD28 antibody in the presence of one or more antigens specific for the infectious agent, and

 - measurement of the kinetics of proliferation of lymphocytes.

The present invention further relates to a method of in vitro stimulation of lymphocytes from patients suffering from a disease linked to retrovirus by treatment of the lymphocytes isolated with an anti-CD28 antibody.

 Finally, the present invention relates to a method for selecting an agent which in vivo inhibits pathological apoptosis comprising at least one step of bringing cells infected with a lentivirus such as an HIV virus into contact with an antigen, respectively in the presence and in the absence of said agent and consequently of stimulation by said antigen.

 A difference in response between the cells cultured in the presence and in the absence of said agent would constitute an index of the inhibitory activity of this agent.

 The anti-CD28 antibody preferably used for the implementation of the present invention is the mouse monoclonal antibody derived from the clone CLB-402 (catalog number JANSSEN 1989

26.683.08) sold by the JANSSEN Company.

 The present invention is illustrated without however being limited by the following application examples, in which:

FIG. 1a is a diagram illustrating the proliferation of cells of patients infected with the HIV ₁ virus and of healthy individuals in the presence of phytohemagglutinin (PHA), of PWM, of staphylococcal enterotoxin B super-antigens (SEB) or of tetanus toxin (TT).

FIG. 1b is a diagram showing the effect of the anti-CD28 antibody on the proliferation of cells of patients infected with the HIV ₁ virus, in the presence of PWM, SEB or TT.

FIG. 2a illustrates the mortality of peripheral blood mononuclear cells (PBMC) of patients infected with the HIV ^ virus (columns 1) and healthy individuals (columns 2), in the presence of cycloheximide, cyclosporin A (CSA), anti-CD28 antibodies, PWM, or SEB.

FIG. 2b illustrates the cell mortality of PBMC cells of patients affected by the HIV ₁ virus (columns 1) and of healthy individuals (columns 2) from which CD4 + T cells or CD8 + T cells have been eliminated, in the presence or in the absence anti-CD28, PWM or SEB antibodies.

 FIG. 3 illustrates the results obtained after incorporation of tritiated thymidine in cells of patients affected by an HIV virus in the presence of PWM, or of PHA.

 FIG. 4 is an autoradiogram representing the hybridization of total RNA of PBMC cells of patients affected by the HIV virus, cultured in the presence of PWM or of PHA, with a DNA probe complementary to the interleukin-2 receptor.

FIG. 5 is a snapshot of a DNA electrophoresis gel of PBMC cells from patients infected with the HIV ₁ virus, cultured in the presence of PMW or SEB and of healthy individuals.

FIGS. 6a and 6b respectively represent photographs by electron microscopy of PBMC cells of healthy individuals and of patients affected by the HIV ₁ virus.

Figures 7a and 7b are histograms corresponding respectively to the proliferation and cell death of cells cultured in a simple medium (column 1), in a medium supplemented with IL-2 (10 units / ml - column 2) and activated with bound CD5 antibody as a control (column 3) or by bound CD3 antibody (column 4 to 8) in the presence of recombinant IL-1 (50 units / ml) column 5, recombinant IL-2 (10 units / ml column 6), anti-IFNgamma antibody (serum dilution 1/800 - column 7) or cycloheximide (50 μ / ml - column 8).

 FIG. 7c is an electrophoresis gel of DNA extracted from medullary thymocytes cultured in a simple medium (column 1) or stimulated by CD3 (columns 4, 5 and 7) in the presence of recombinant Interleukin (column 5) or d anti-IFNgamma antibody (column 7).

 FIG. 7d represents a dot blot analysis of RNA of medullary thymocytes stimulated by simple medium (column 1) with recombinant IL-1

(column 2) by bound CD3 (column 3) or by CD3 and recombinant IL-1 (column 4).

 FIG. 8 represents a gel for electrophoresis of DNA from T cells of chimpanzees infected with the HIV virus (columns 1 to 4) or of control chimpanzees

(columns 5 and 6) after in vitro stimulation with the super antigen SEB.

 FIG. 9 represents a gel for electrophoresis in agarose of DNA of control macaque T cells stimulated with SEB (columns 1 and 2) of T cells of macaques infected with the SIV virus incubated in a simple medium (columns 3, 5 and 7) and T cells of macaques infected with the SIV virus, incubated with SEB (columns 4, 6 and 8).

FIG. 10 represents a gel for electrophoresis of DNA of T cells of macaques infected with the SIV virus incubated in simple medium (columns 1 and 3) or of medium + SEB columns 2 and 4), of T cells of green monkeys infected with SIV and incubated with SEB (columns 5 to 7) and green monkey T cells incubated with SEB (columns 8 and 9).

 EXAMPLE 1:

In vivo proliferation of T lymphocytes from asymptomatic patients infected with the HIV ₁ virus.

 a) Stimulation by polyclonal activators.

 The proliferation of T lymphocytes from 38 patients infected with the HIV-1 virus and from 20 healthy individuals was tested in response to the polyclonal activators phytohemagglutinin, concanavalin A, mitogenic pockweed (PWM), anti-CD3 monoclonal antibody, and in response to the antigen stimulation of the tetanus toxin TT.

 Since the TT-specific memory T cells are rare, and may have been eliminated in patients infected with the HIV virus, the response to the mobilization of T cell receptors by the use of enterotoxin B superantigens Staphylococcus (SEB) was also tested.

 SEBs bind to MHC-II molecules and act with Vβ molecules of T cell receptors (TCR) expressed by up to 30% of normal T cells, and induce the proliferation of mature normal T cells (Marrack and Kappler, J Science 248, 705-711, 1990) and apoptosis in immature thymocytes (Genkinson et al. J. Immun. 19, 2175-20177, 1989).

To assess the intensity of the response, measurements were made after incorporation of the tritiated thymidine into the cells. (Figure 1a). Patients infected with HIV included 26 men and 12 women, 25 patients being at the stage of CDC II A (no biological abnormality, CD4 cells> 500 / mm ³ , and on average 856 / mm ³ ) and 13 of them were at CDC IIB stage (biological abnormality, CD4 cells <500 / mm ³ and 345 / mm ³ ).

 HIV infection was attributed to 15 of the patients to homosexual relationships, to 14 of the patients to heterosexual relationships, to 7 of the patients to intravenous drug use and to 2 of the patients to blood transfusion.

The peripheral blood mononuclear cells (PBMC) were obtained on Ficoll-Hypaque and were cultured as described by Reed et al. (Proc. National Acad. Sci. USA 83, 3982-3986 (1986)). The PBMCs are incubated at a cell density of 2.5 × 10 ⁵ / ml for 3 days with culture medium without adjuvant, medium containing phytohemagglutinin (PHA) at 10 μg / ml, pokeweed mitogen (PWM) at 10 μ / ml , staphylococcal enterotoxin B superantigen (SEB) at 1 μg / ml, or for 6 days with the antigen (TT) at 10 μg / ml.

 The proliferation of T cells in HIV + patients with Concanavalin A (10 μg / ml), with the monoclonal antibody CD3 (Groux et al. Nature, 339, 152-154, 1989) is only slightly reduced (result not shown in the figure ) as well as proliferation in the presence of PHA.

 In the figure, each circle represents the average of cultures carried out in triplica, the horizontal bars representing the average of the results for each treatment.

These results show that, while T cells of control individuals proliferate in response to all stimuli, T cells of HIV infected patients respond less to stimulation by PWM, SEB or TT antigen (Figure la).

 b) Stimulation in the presence of an anti-CD28 antibody.

 Trials have also been carried out by incorporating tritiated thymidine into T cells of 19 of the HIV infected patients (FIG. 1 b) in response to proliferation by PWM, SEB or TT, in the absence or in the presence of anti CD28 antibody.

 In this example as well as in those which follow the anti-CD-28 antibody is provided by the company JANSSEN (mouse monoclonal antibody derived from the clone CLB-402).

 Cells were prepared and used as described for FIG. La and the anti-CD28 antibody was added at a concentration of 10 μg / ml.

FIG. 1 _b shows that the anti-CD28 antibody increases the proliferation of control T cells in the presence of PWM or SEB.

 On the other hand, the antibodies CD5 (A50, gamma 1, CD43 (B1B6, gamma 1), CD44 (P245, gamma 2a), and CD45R (ALB11, gamma 1, Immunotech) at concentrations of 10 μg / ml do not stimulate proliferation T cells from HIV + patients (results not shown in the figure).

 EXAMPLE 2:

 Mortality of T cells and mononuclear cells of peripheral blood of patients infected with the HIV virus after in vitro culture in the presence of PWM and SEB.

1) FIG. 2 a) illustrates the cell death of PBMC of patients infected with the HIV virus as well as controls, after in vitro culture in the presence of PWM and SEB.

The results mentioned in FIG. 2a) represent the average plus or minus the standard deviation of experiments carried out on 20 different patients infected with the HIV virus (columns 1) and 20 uninfected individuals (column 2). Cell mortality is demonstrated by the permeability to trypan blue. PBMCs are cultured with an initial cell density of 2.5 10 ⁵ / ml in

48 hours in culture medium without adjuvant, medium containing PWM (10 mg / ml) or SEB

(Iμg / ml) in the absence or presence of cycloheximide

(50 μg / ml) of cyclosporine A (CsA) at 200 ng / ml or of anti-CD28 monoclonal antibody (10 μg / ml).

 The addition of PWM or SEB to the PBMC cells of patients infected with the HIV virus (columns 1) is followed after 48 hours of culture by respectively up to 60 and 30% of cell deaths, while no cell mortality is observed at 48 hours for the non-stimulated PBMCs of patients infected with the HIV virus (columns 2), as well as for the stimulated control PBMCs (columns 2) which show up to 95% of viability (FIG. 2 a).

 Culturing cells in the presence of CD3 monoclonal antibody does not cause T cell mortality (results not shown in the figures).

 After 48 hours of culture in an adjuvant-free medium, the basic cell mortality of the PBMC cells of 40 patients infected with the HIV virus never exceeds 10% (on average 5.3%).

2) Figure 2b concerns similar experiments. PBMC cells from an infected patient with the HIV virus (columns 1) and an uninfected individual (columns 2) were purified by elimination of CD4 + T cells (CD 8 cells) or CD8 + T cells (CD 4 cells) by incubation with respectively IOT4 or IOT8 antibody followed by incubation with magnetic particles coated with goat anti-mouse IgG antibody.

 The quality of the elimination is measured by cytofluorometry (Epix Coulter Clone) and shows that the cell contamination is less than 1%. The cells are cultured as described for FIG. 1 a). The results represent the average of three measurements of cell mortality after 48 hours. The proliferation is measured by incorporation of the tritiated thymidine after 3 days.

 CD4 cells of uninfected individuals proliferate in response to PWM (5250 + 651 CPM) and SEB (21.642 + 2.317 CPM), while CD8 control cells and CD4 and CD8 cells from patients infected with HIV do not proliferate after 5 days.

 The addition of anti-CD28 antibodies to the CD4 cells of patients infected with the HIV virus restores the capacity of these cells to proliferate during stimulation with PWM and SEB, but has no effect on the cells of patients. infected with unstimulated HIV or on control CD8 cells.

3) FIG. 3 illustrates the proliferation of PBMC cells of patients infected with the HIV virus and of healthy individuals after addition of PWM or of PHA, after incorporation of tritiated thymidine in the T cells of 3 patients infected with the HIV virus (ABC) and B ') and a healthy individual (D) in response to stimulation by PMW at 10 μg / ml (columns A, B and C, and column D) or by PHA at 10 μm / ml (column B ').

 Columns B and B * represent the results obtained with PBMCs from the same patient infected with the HIV virus. All the results are the average (+/- standard deviation) of cultures carried out in triplicate.

EXAMPLE 3:

 Synthesis of messenger RNAs encoding the Interleukin-2 receptor.

 FIG. 4 illustrates the expression of the messenger RNAs coding for the interleukin 2 receptor (IL-2R) of PBMC of patients infected with the HIV virus and of healthy individuals after addition of PWM or of PHA. The same patients (A, B, C, B 'and D) as in Figure 3 were repeated. This expression is measured on the same cells after 2 hours, 6 hours and 16 hours. These messenger RNAs were not detected before stimulation.

The experiments were carried out by extraction of the total cellular RNAs from 10 ⁷ PBMCs with RNAzol (Cinna-Biotex, Friendwood, USA) according to the manufacturer's instructions. The total RNA samples (approximately 10 μg / ml) are fractionated according to their size in 1% agarose gels containing formaldehyde and are transferred to HYBOND-N filters (Amersham). The filters are then hybridized with cDNA probes labeled with phosphate 32 (human IL-2R) and washed with a 0.1 SSC, 0.1% SDS solution at 55ºC. The relative sizes of the IL-2R mRNAs which hybridize with the probe are in agreement with the values of 3.5 kb previously published.

EXAMPLE 4: Degradation of cellular DNA.

 FIG. 5 represents the ectrophoresis on DNA agarose gel extracted from PBMC of patients infected with the HIV virus or of healthy individuals after culture overnight in a culture medium with or without adjuvants.

 These gels were obtained by lysis in 4 ml of a hypotonic lysis buffer (5 mM Tris pH 7.4, 5 mM

EDTA, 0.5% Triton X100) of 15.10 ⁶ cells centrifuged at 200 g for 10 min. The lysates are then centrifuged at 13,000 g for 15 minutes. The

DNA from the supernatants is concentrated by precipitation at

-20ºC in 50% isopropanol, 130mM NaCl. After centrifugation at 13,000 g, the precipitates are air dried and resuspended in 10mM Tris pH 8, ImM EDTA.

 All samples are deproteinized by phenol / chloroform extraction and double extraction with chloroform / isoamyl alcohol (24: 1). After electrophoresis on fine 2% agarose gels for 12 hours at 30 V, the DNA is visualized by staining with ethidium bromide.

 Well 1 in Figure 5 corresponds to the molecular weight marker. Well 2 corresponds to the DNA of PBMC cells from healthy individuals incubated with PWM.

 Wells 3 to 11 correspond to the DNA of PBMC cells of 7 patients infected with the HIV virus incubated respectively with medium without adjuvant (well 3), medium comprising PMW (wells 4 to 6, 8 and 9), and medium comprising SEB (wells 10 and 11).

Wells 3 and 4 correspond to the same patient. The figure shows that the DNAs from wells 4 to 6 and 8 to 11 have a degradation profile characteristic of apoptosis, the degraded DNA bands being multiples of 200 base pairs.

 The degradation of these DNAs is suppressed by adding CsA to the medium containing the PWM (column 7 corresponding to the same patient as column 6).

EXAMPLE 5

Cellular state of PBMCs of patients infected with HIV.

 FIG. 6a represents a photograph by electron microscopy of PBMC of a patient infected with the HIV virus after an incubation of 24 hours in a medium without adjuvant.

 FIG. 6b represents the same cells incubated in a medium containing 5 μg / ml of PWM.

 Cells with different degrees of color concentration can be seen in this figure and are characteristic of apoptosis. The arrows in this figure designate them.

EXAMPLE 6

 Effect of anti-CD28 antibody on restoring the ability of lymphocytes to proliferate.

 1º) Recovery of proliferation by co-stimulation with tetanus antigen and influenza antigen, in the presence of anti-CD28.

Patient cells are stimulated either by tetanus toxin (TT) or by influenza antigen (GP) in the presence or absence of the anti-CD28 antibody. The results obtained are collated in Table I below.

 These results show that in the case of tetanus toxin, the presence of the anti-CD28 antibody allows the lymphocyte cultures of said patients to restore the capacity to proliferate.

 These results are found in the case of seven out of ten patients with influenza antigen (GP).

 Since the influenza antigen is a commonly encountered antigen, it is possible that the three patients whose lymphocytes do not proliferate in the presence of anti-CD28 antibodies have already encountered it, and that the memory cells directed against this antigen have been eliminated thereby .

 2º) Demonstration of the need for the presence of the anti-CD28 antibody during the first stimulation for cell rehabilitation.

 Lymphocytes were stimulated (OJ) after purification, by tetanus toxin (TT) by phytohemagglutin (PHA) and by the mitogen pockeweed (PWM).

 The proliferation is measured by the incorporation of tritiated thymidine after 6 days (day 6).

 At the same time, the cells are cultured in the presence of tetanus toxin or the superantigen of staphylococcal B enteroxin (SEB) with or without anti-CD28 antibody.

 After 10 days of culture, when the cells have returned to rest, they are restimulated by the same stimuli as on D 0.

It is observed when the first stimulation of lymphocytes from patients infected with the HIV virus was carried out in the presence of TT or SEB without CD28, that it is not possible to re-proliferate, even if these lymphocytes are restimulated by the anti-CD28 antibody.

 On the other hand, the cells precultivated in the presence of the anti-CD28 antibody proliferate in the presence of the TT or SEB antigens.

 The results are summarized in Table II.

 3 ") Restoration of the capacity of lymphocytes to proliferate in response to PWM and SEB after a first stimulation with PHA.

 Lymphocytes from patients infected with the HIV virus are stimulated either directly (JO) with phytohememaglutinin (PHA), the mitogen pockeweed

(PWM) or staphylococcal enterotoxin B (SEB), or pre-simulated by PHA for 10 days.

 The cells are restimulated after 10 days under the same conditions as in the OJ.

 The results are collated in Table III.

 It is observed that stimulation of the cells with an adjuvant such as PHA, PWM or SEB allows restimulation after 10 days in the presence of PHA. EXAMPLE 7:

 Restoration of the capacity of lymphocytes to proliferate by stimulation with allogenic cells.

 The restoration of the capacity of lymphocytes to proliferate after a first stimulation with allogenic cells and in response to PWM and SEB was tested.

 In parallel, we tested the capacity to proliferate after a first stimulation by PHA.

Patients who have undergone a first stimulation with PHA or with cells allogenic (RLM or mixed lymphocyte reaction) are restimulated after ten days of culture (D10).

 Table IV below illustrates the results obtained.

 This table therefore shows that compared to the control, the cells which have undergone a first stimulation with allogenic cells exhibit a proliferation equivalent to those which have been stimulated by PHA.

EXAMPLE 8:

 Comparison of the mechanisms of aptotosis in normal cells and in cells of individuals infected with an HIV virus.

 1) Study of the apoptosis of cells of individuals not infected with the HIV virus.

The response of human total medullary thymocytes fixed by the monoclonal antibody CD3 and of subpopulations of mature medullary thymocytes CD4 ⁺ CD8 ~ or CD8 ⁺ CD4- was tested.

 In the three populations, the monoclonal antibody CD3 does not induce the proliferation of thymocytes and leads after 48 hours to the death of approximately 50% of the thymocytes, as indicated in Table V below.

In Table V, the responses to TCR mobilization of total human thymocytes (A), CD4 ⁺ CD8 ~ thymocytes (B), CD8 ⁺ CD4- thymocytes (C) and immature CD4- CD8- CD3- thymocytes ( D) have been tested.

The cells were stained with a mixture of CD4-FITC antibody (LFL1 green fluorescence) and CD8-RD antibody (LFL2 red fluorescence) to perform a flow cytofluorometry analysis (Coulter Epies Profile II). Cell proliferation after mobilization of TCRs with CD3 antibodies bound on a dish, in the presence or absence of IL-2 (10 units per ml), was determined by incorporation of tritiated thymidine ( ³ H-TdR). Thymidine was added in the last twelve hours of the third day. The results represent the average of triplicate culture or a representative experiment among the three. Cell death was determined 48 hours after stimulation by exclusion of trypan blue. The results and the percentages of cell death are chosen from three experiments.

 The method used was as follows. Thymus were obtained from children less than two months after cardiac surgery. The thymocytes were purified on a nylon sieve and were frozen until used. The medullary thymocytes were isolated from a population of total thymocytes by negative selection with magnetic beads covered with a CD1 antibody (D47, IgG1 at lμ.ml), according to the manufacturer's instructions (Biosys, France).

The CD4 ⁺ CD8- or CD8 ⁺ CD4- thymocytes were purified from medullary thymocytes by additional negative selection using respectively the CD8 monoclonal antibody (L533 IgG1 at 1 μ / ml) or the CD4 antibody (0516, IgG1, at 1 μ / ml). Proliferation tests with fixed monoclonal antibodies (OKT3, IgG2a) or CD5 (A50, IgG2a) as controls were carried out. To determine cell death, the cells were harvested after 48 hours by pipetting and diluted in half with 0.1% trypan blue in PBS buffers. Living cells and dead cells were counted in a hemocytometer. The results are expressed as the percentage of dead cells.

 As previously described (Nieto et al. J. Immunol. 145, 1364-1368,1990), the addition of IL-2 prevents cell death and allows proliferation of thymocytes in the presence of CD3 antibodies.

 The medullary thymocytes having been isolated by a negative selection, using the antibody CDla in all cases, and the antibodies CD46 and CD8 in the additional purifications, the three cell populations were contaminated by approximately 20% of immature thymocytes CD4- CD8- CD3-.

 As shown in Table V, these immature thymocytes when purified do not die or proliferate in response to the CD3 antibody, in the absence or presence of IL-2.

 The cell mortality due to CD3 of total medullary thymocytes is associated with a DNA fragmentation characteristic of apoptosis as shown in Figure 7 C.

 This figure is an agarose electrophoresis gel of DNA extracted from medullary thymocytes after different treatments. The spinal cord thymocytes were cultured in a simple medium (column 1) or were stimulated by the bound CD3 antibody (columns 4, 5 and 7) in the presence of recombinant IL-1 (rIL-1) (TO U / ml, column 5), or in the presence of anti-IFN gamma (serum dilution 1/800, column 7).

This cell death is an active process which can be prevented by cycloheximine as shown in Figure 7b and cyclosporine A as shown in Table VI. l As shown in Table VI and in FIG. 7d, apoptosis due to CD3 of medullary thymocytes is associated with the expression of the messenger RNA IFNgamma, with the secretion of IFNgamma, with the expression of the messenger RNA of the P55 chain of the IL-2 receptor, the absence of expression of the IL-2 gene and the absence of secretion of IL-2.

 FIG. 7d represents the dot blot analysis of RNA of medullary thymocytes stimulated with a simple medium (column 1), a medium containing rILlσ (50 units / ml, column 2), of the bound CD3 antibody (column 3) and CD3 antibody linked with rILlα (column 4). The IL-2 autoradiogram was performed after 6 hours. IFNgamma, IL-2 receptor and S-actin autoradiograms were performed after 6 p.m. The probes were tested on a Northern Blot of RNA of thymocytes and each gives a specific signal without cross hybridization. The relative sizes of the messenger RNAs which hybridize to the probes are in agreement with the previously published values.

The secretion of IFNgamma at high levels in the absence of secretion of IL-2 in response to the antibody CD3 has also been observed in medullary thymocytes CD4 ⁺ CD8- and CD8 ⁺ CD4- as shown in the table VI.

 Addition of anti-IFNgamma antibodies prevents thymocyte death due to CD3 (Figure 7b) and DNA fragmentation (Figure 7c), but does not restore thymocyte proliferation to the CD3 antibody

(Figure 7a).

In contrast, IL-2 prevents thymocyte death (Figure 7b) and allows proliferation thymocytic in response to the CD3 antibody (Figure 7a).

 As previously observed with cells

T-lymphocyte, the addition of IL-1 to medullary thymocytes stimulated with CD3 allows the expression of messenger RNAs of IL-2 (Figure 7d) and the secretion of IL-2 (Table VI).

 This is associated with the prevention of cell death (Figure 7b) or the fragmentation of DNA (Figure 7c) and thymocytic proliferation (Figure 7a).

 Curiously, IL-1 does not affect the expression of IFNgamma messenger RNAs (Figure 7d) or the secretion of IFNgamma (Table VI) in response to stimulation by CD3.

 Neither IL-1 nor IL-2 induce cell proliferation in the absence of CD3 (Figure 7a). IFNgamma does not induce cell death in the absence of CD3.

The addition of anti-IFNgamma antibodies to IL-2 and to CD4 ⁺ CD8- and CD8 ⁺ CD4- medullary thymocytes stimulated by CD3 have the same effect as in the case of total medullary thymocytes (Table VI). In the case of purified CD8 ⁺ CD4- thymocytes, IL-1 does not induce secretion of IL-2 and does not prevent cell death in response to CD3. This suggests that the preventive effect of IL-1 in total medullary thymocytes is linked to the secretion of IL-2 by CD4 ⁺ CD8- thymocytes.

The respective roles of IFNgamma and IL-2 in CD3 thymocyte proliferation and death were further tested in experiments using ciclosporin A CsA, which inhibits thymocyte apoptosis and inhibits secretion of IL-2 and 1 * IFNgamma (Table VI). As shown in Table VI, IFNgamma and / or IL-2 are added to thymocytes activated by CD3 and treated with CsA. Under these conditions, IFNgamma induces thymocyte death, while IL-2 induces thymocyte proliferation. When IFNgamma and IL-2 are added together, no cell death is observed and the thymocytes proliferate.

 These results indicate that mobilization by CD3 leads in the case of human medullary thymocytes, cell death which is dependent on IFNgamma, but which is inhibited by IL-2 even in the presence of IFNgamma.

Table VII shows, for its part, that such a cell death program is not restricted to the stages of development of T cells, but is also present in the case of peripheral T cells, activated and mature humans. A mature CD4 ⁺ T cell line specific for the tetanus toxin antigen behaves like human medullary thymocytes when stimulated by the CD3 antibody in the absence of other cells. As also shown in Table VII, the addition of IFNgamma and / or IL-2 to peripheral mononuclear cells activated by CD3 and treated with CsA leads to the same results with the antigen-dependent T cell lines and the medullary thymocytes.

 2) Apoptosis in cells of individuals infected with an HIV virus.

 Comparable experiments were carried out with CD4 T lymphocytes from a subject infected with the HIV virus.

Table VIII below shows that neither the addition of IL-2 nor the addition of anti- Interferon gamma are not capable, unlike the antibody CD28, of preventing apoptosis of these lymphocytes, nor of restoring their proliferation in response to stimuli. IL-1 is also devoid of effect.

 The comparison of these results therefore shows that the mechanism involved in the apoptosis of subjects infected with the HIV virus is distinguished from the apoptosis of cells of patients not infected with the HIV virus.

EXAMPLE 9

 In vitro induction in normal lymphocyte lines specific for antigens of apoptosis equivalent to apoptosis in cells of patients infected with an HIV virus.

An antigen (tetanus, influenza) is presented to a T-CD4 ⁺ lymphocyte line by monocytes which have been preincubated with an HIV virus. The presentation of the antigen takes place in the presence of AZT to avoid infection of T-CD4 ⁺ lymphocytes. A normal lymphocyte proliferative response ensues.

On the other hand, when the antigen is represented a second time to T-CD4 ⁺ lymphocytes by monocytes, even those not infected with the HIV virus, the lymphocytes die of apoptosis instead of proliferating.

 These results are presented in Tables IX and X below.

Like the apoptosis of the lymphocytes of subjects infected with the HIV virus, the apoptosis observed during the second stimulation is prevented by the antibody CD28, but neither by the IL-2 nor by the anti-gamma interferon antibodies. These results therefore show that contact between normal T-CD4 + lymphocytes and monocytes infected with an HIV virus initiates in T-CD4 ⁺ lymphocytes, in the absence of infection by an HIV virus, a program of apoptosis in response to subsequent stimulation.

 This cellular model allows:

 a) to study in a very large number of normal T-CD4 + lymphocytes the mechanisms of induction of a phenomenon of apoptosis comparable to the apoptosis observed in subjects infected with an HIV virus.

 b) to study the therapeutic agents making it possible to correct this phenomenon of apoptosis;

 c) identify the viral protein (s) involved in the initiation of the apoptosis program;

 d) to find therapeutic agents which make it possible not only to prevent already abnormal cells from dying but to prevent the lymphocytes from learning to die in response to a subsequent stimulation.

 EXAMPLE 10:

 Comparison of apoptosis of human cells induced by the HIV virus and apoptosis of simian cells induced by the SIV virus.

 In order to compare the apoptosis induced by the HIV virus in T lymphocytes, with other viral infections, different models, host virus, have been studied in monkeys.

 These models are:

- chimpanzees, experimentally infected with the HIV virus; these animals do not develop diseases; - rhesus macaques, experimentally infected with the HIV virus (Simian Immuno Deficiency virus); these animals develop AIDS;

 African green monkeys naturally infected with the SIV virus; these animals do not develop disease.

 FIG. 8 represents a gel for electrophoresis of DNA from T cells of chimpanzees infected with the HIV virus and incubated with SEB (columns 1 to 4) and of control chimpanzees, that is to say chimpanzees not infected with the HIV virus but whose T cells have been incubated with SEB (columns 5 and 6).

 This figure shows that apoptosis, which is characterized by DNA degradation, was not detected in any of the chimpanzees studied.

 FIG. 9 corresponding to gels made with the DNA of T cells of control macaques stimulated with SEB (columns 1 and 2), of macaques infected with the SIV virus and whose cells were incubated in simple media (columns 3 , 5 and 7) and of macaques infected with the SIV virus and whose cells were incubated with SEB (columns 4, 6 and 8) shows that there is degradation of DNA and therefore apoptosis in the cells of macaques infected with the SIV virus when incubated with SEB.

Figure 10 shows that the African green monkey infected with the SIV virus does not develop apoptosis. In this figure, columns 1 to 4 correspond to the DNA of T cells of macaques infected with the SIV virus incubated with simple medium (columns 1 and 3) or with SEB (columns 2 and 4) while columns 5 to 7 correspond to T cells of African Green monkeys infected with SIV virus, incubated with SEB and columns 8 and 9 correspond to control African green monkey T cells, not infected with SIV, incubated with SEB.

 Only macaques infected with the SIV virus reproduce a phenomenon comparable to that observed in humans with the HIV virus.

 This animal is therefore an interesting model for selecting therapeutic agents capable of preventing apoptosis and the onset of the disease. This animal is all the more interesting since, unlike humans, where AIDS is declared in ten years, the macaque infected with the SIV virus has the particularity of developing AIDS in six months to a year and a half.

 CONCLUSION:

 An essential feature of apoptosis is that cell death depends on cell activation, gene transcription and protein synthesis in dying cells. The activation of T cells of patients infected with the HIV virus after addition of PWM is shown by the expression of mRNA p55 corresponding to the interleukin-2 receptors at equivalent levels and with kinetics equivalent to that observed in cells. T of healthy individuals (Figure 4).

 In addition, the addition of a protein synthesis inhibitor (cycloheximide) or cyclosporin A prevents apoptosis of T cells from patients infected with the HIV virus stimulated by PMW or SEB (FIG. 2a and FIG. 3).

It should be noted that among the stimuli which induce apoptosis in normal immature thymocytes, and among the co-signals which prevent this process, only a limited number have the same effect on T cells of patients infected with HIV.

 In immature thymocytes, most of the stimuli inducing the proliferation of normal adult T cells, including the CD3 antibody, lead to apoptosis, and the addition of co-signals such as interleukin-1, interleukin- 2 or phorbol esters not only avoids apoptosis but also allows proliferation in response to the stimuli.

 In the T cells of patients infected with the HIV virus, the CD3 antibody induces cell proliferation and among the various co-signals (Interleukin 1, Interleukin 2, Interferon gamma, CD5 antibody, CD28, CD 43, CD44 and CD 45R or phorbol ester) which have been added to the T cells of patients infected with the HIV virus, only the CD28 antibody prevents death induced by PWM and SEB and restores proliferation to PMW, SEB and TT (FIG. 1b and Figure 2a).

Without this interpretation being considered as a restriction on the present invention, the set of results seems to indicate that the CD4 T _H cells of patients infected with the HIV virus are programmed in vivo for a suicide process by apoptosis after activation by defined stimulation, including the mobilization of T cell receptors (TCR) by the molecules presented by the major class II hiscompatibility complex.

Since anti-CD28 antibodies restore the possibility of proliferation after stimulation with the TT antigen, tetanus-specific T cells appear to be present in patients infected with the HIV virus. Although such cells are too rare to allow their death to be demonstrated, the induction of apoptosis seems to be the most likely mechanism to explain their lack of response in vitro.

In patients with HIV infection, less than 0.1% of CD4 T cells in peripheral blood are infected with HIV, which excludes the possibility that apoptosis is directly due to infection of the cells.

Claims

 1. Medicament containing an agent in vivo inhibiting pathological apoptosis, said agent being able to be selected by a process comprising at least the steps of:  contacting at least two cell preparations of a patient infected with a retrovirus with a cell activating agent, such as pokeweed mitogen, respectively in the presence and in the absence of said agent, and  - comparison of the proliferation and / or mortality of the two cell preparations.  2. Medicament according to claim 1, characterized in that the agent is capable of blocking the reception or the transduction of signals leading to suicide of the cells.  3. Medicament according to claim 2, characterized in that the agent is capable of inhibiting the activation of a nuclear endonuclease resulting in the fragmentation of the genome of the cell.  4. Medicament according to claim 1, characterized in that the agent provides or generates a cosignai allowing the cell to differentiate and multiply normally.  5. Medicament according to claim 1, characterized in that the agent alone leads to cell proliferation and differentiation and generates cells which have lost the ability to commit suicide under pathological conditions. 6. Medicament according to claim 1, characterized in that the agent is capable of inhibiting the signals which make a cell capable of responding by activation pathological apoptosis. 7. Medicament according to claim 2, containing cyclosporine, or an inhibitor of protein synthesis such as cycloheximide.  8. The medicament of claim 4 containing an anti-CD28 antibody or a combination of an agent causing stimulation of phosphokinase C, such as a phorbol ester, with interleukin I.  9. The medicament according to claim 5 containing a mitogenic agent such as phytohemagglutinin (PHA), irradiated allogenic cells, an anti-CD2 antibody or an anti-CD3 antibody.  10. Medicament according to one of claims 1 to 9, characterized in that it is intended for the treatment and prevention of diseases linked to retroviruses and in particular linked to lentiviruses, such as the HIV viruses in humans and SIV , IVF, BIV, VISNA and CAEV in animals.  11. Pharmaceutical composition characterized in that it contains a pharmaceutically effective amount of an agent as defined in one of claims 1 to 9 in combination with one or more pharmaceutically acceptable diluents or adjuvants.  12. Pharmaceutical composition according to claim 11, characterized in that it is intended for the treatment and prevention of diseases linked to retroviruses and in particular linked to lentiviruses, such as the HIV viruses in humans and SIV, FIV, BIV , VISNA and CAEV, in animals. 13. Use of an agent as defined in one of claims 1 to 9 for obtaining a medicament intended for the treatment and prevention diseases linked to retroviruses and in particular linked to lentiviruses, such as the HIV viruses in humans and SIV, FIV, BIV, VISNA and CAEV in animals.  14. Method for determining the qualitative deficiency of the immunological repertoire of an individual vis-à-vis an infectious agent comprising at least the steps of:  - in vitro stimulation of lymphocytes in a medium containing at least one anti-CD28 antibody in the presence of one or more antigens specific for the infectious agent, and  - measurement of the kinetics of proliferation of lymphocytes.  15. Method for determining the capacity of an individual's lymphocytes to respond to aggression by an infectious agent, comprising at least the steps of:  - in vitro stimulation of lymphocytes in a medium containing at least one anti-CD28 antibody in the presence of one or more antigens specific for the infectious agent, and  - measurement of the kinetics of proliferation of lymphocytes.  16. Method of in vitro stimulation of lymphocytes from patients suffering from a disease linked to retroviruses by treatment of the lymphocytes isolated with an anti-CD28 antibody. 17. Method for selecting an agent which in vivo inhibits pathological apoptosis comprising at least one step of bringing cells infected with a lentivirus, such as an HIV virus, into contact with an antigen respectively in the presence and in the absence of said agent and , following stimulation by said antigen. 18. Method for selecting an agent which in vivo inhibits pathological apoptosis, comprising at least: - a first presentation of an antigen by monocytes preincubated with a retrovirus, to a cell line of CD4 ⁺ T lymphocytes, - a second presentation of the antigen by monocytes, possibly preincubated with a retrovirus, to the CD4 ⁺ T cell cell line,  - estimation of the death and / or proliferation of lymphocytes.