WO2004069272A2

WO2004069272A2 - Adjuvant combination for use in the immunization of a mamal comprising il2 and il12

Info

Publication number: WO2004069272A2
Application number: PCT/IB2004/000864
Authority: WO
Inventors: Jean-Marie Andrieu; Louis Wei-Lu
Original assignee: Movecare Ltd
Priority date: 2003-02-05
Filing date: 2004-02-05
Publication date: 2004-08-19
Also published as: WO2004069272A3

Abstract

A method of lowering blood glucose in a mammal includes administering orally or by injection or inhalation a therapeutically effective amount of crystallized dextran microparticles and insulin to the mammal to lower blood glucose of the mammal. The composition may be a one phase or a structured multi-phase composition for controlled release of insulin or other therapeutic agents.

Description

ADJUVANT COMBINATION FOR USE IN THE IMMUNIZATION OF A MAMMAL COMPRISING IL2 AND IL12.

Field of the invention The invention pertains to the field of methods and compositions for the treatment of a chronic infectious disease or a cancer.

The present invention relates more particularly to an adjuvant combination for use in the immunization of a mammal (a human patient) against a chronic infectious disease or a cancer, said combination comprising two cytokines . Therefore the invention also relates to the use of a combination of IL2 and IL12 for the preparation of (a) pharmaceutical composition (s) for enhancing proliferation and differentiation of T and B lymphoid cells directed to at least one antigen specific for a chronic infectious disease or cancer in a mammal (a human patient) .

Background of the invention

When a pathogen (such as virus, bacteria, or other parasites) invades the body via topologically external epithelial sites (such as skin epidermis and the mucosae of the respiratory, gastrointestinal and urogenital tracts) , a non-specific inflammatory response is generated to attract local immature dendritic cells (DCs) which are characterized by their capacities of uptake and processing of foreign antigens via a number of mechanisms (including phagocytosis, macropinocytosis, or receptor-mediated endocytosis) .

Such foreign antigen-encountered DCs migrate subsequently via lymph or blood into T-cell rich areas (TRA) in secondary lymphoid tissues where they differentiate into a mature stage, characterized by i) expression of the maturation-associated markers DC-LAMP and CD83; ii) upregulation of MHC, adhesion and costimulatory molecules (including CD40, CD80, and CD86) . Mature DCs are specialized to interact with TRA CD4+ and CD8+ T cells inducing pathogen-specific humoral (antibody) and/or cellular (cytotoxic T lymphocyte) immunity, resulting in the eradication of pathogens or the control of infection. However, pathogens can also suppress DC function through multiple mechanisms including induction of apoptosis, inhibition of maturation, abrogation of cytokine production, loss of migratory capacity and inhibition of DC-T-cell interaction, resulting in a status of immune tolerance (i.e. chronic infection). It has been thought that when tumor cells occur, an over-expression of "self antigens" within tumor cells (referred as tumor-associated antigens, TAA) will be captured by local immature DCs. Such TAA- sensitized DCs migrate to secondary lymphoid tissues and then stimulate anti -tumor cellular immunity by cross-presentation. TAA-specific CTLs will in turn migrate to tumor sites, infiltrate into tumor tissues, and eliminate tumor cells at the early stage of tumor- development. However, neoplastic diseases could be developed when local tissue DCs and DC function are decreased or impaired by exposure to physical radiation

(such as UN-induced skin tumors) , chemical molecules

(such as tobacco-associated oral, and lung), and viral infections (such as EBV-associated B lymphoma, HBV/HCV- associated liver carcinoma, HPV-associated cervical cancers) as well as aging-associated chronic inflammations (such as prostate cancers) .

In order for autologous DC-replacing therapies to be successfully implemented in the settings of chronic infections and neoplastic diseases, it will be necessary to obtain adequate numbers of DCs from individual patients. The ability now exists to culture large numbers of DCs from blood or bone marow precursors using simple culture methods and recombinant cytokines. Cultured DCs may be derived from multi-step differentiation (one-month culture) of CD34+ DC precursors (obtained from mobilized peripheral blood or bone marrow) or easily from single-step differentiation (one-week culture) of blood monocytes. DCs are readily obtained from apheresis-collected peripheral blood monocytes using simple techniques, such as adherence or CD14-positive selection by immune beads, and a 5-day culture in cytokines such as GM-CSF and IL-4 or IL-13. In several studies, peripheral blood monocytes (M) - derived and CD34+ precursor (CD34 ) -derived DCs exhibited similar capacity to present peptide antigen. However, M-DCs presented soluble protein and stimulated allogeneic cells in a mixture reactive culture (MRC) better than did CD34-DCs. Thus, both M-DC and CD34 DC have potent immunostimulatory properties in vitro.

DC therapies are currently under investigation in the setting of cancer, and they appear to be safe and to induce anti-tumor effects in vivo in preliminary studies. To date, a single pilot study involving the administration of antigen-loaded DCs to HIV-infected persons has been performed. This phase I clinical trial, carried out by Drs Kundu and Merigan at the Center for AIDS Research at Stanford University Medical Center, assessed the safety and antigen- presenting properties of allogeneic or autologous DCs pulsed with recombinant gpl60 or synthetic peptides administered to six HIV-infected volunteers. Peripheral blood DCs (2-32 million cells without knowing their status of maturation) were collected from leukapheresed PBMCs from recipient (one case) or HLA-matched siblings (five cases) using stepwise centrifugation and a short culture period with antigen, without exogenous cytokines. DCs were incubated with HIV-1 gpl60 or synthetic peptides corresponding to HLA-A2 -restricted CTL epitopes (in env, gag, pol of HIV-1) and infused intravenously to HLA-A2+ HIV- infected subjects six to nine times at monthly intervals. Study subjects had CD4 counts ranging from 300 to 700/μl at the initiation of the study and viral loads ranging from 3 to 5 loglO plasma HIV-1 RNA copies/ml. Following infusions, no clinically significant adverse effects were seen in any patient, and viral load and CD4+ T-cell count were stable during a 40-week study period. No enhancement in HIV-specific immunity was noted in the three subjects with starting CD4 counts of < 410/μl, but the three subjects with higher starting CD4 counts displayed evidence of enhanced HIV-specific memory CTLs and/or lymphoproliferative responses. The small size of this pilot study using enriched in vivo peripheral blood DCs

(which are known to be functionally impaired in HIV- infected patients) precluded any conclusions as to clinical benefit of this approach, but the infusions were well-tolerated and enhanced HIV-specific immune responses were seen in 50%.

Summary of the indention

The present invention provides a method of treatment and/or prophylaxis of a chronic infectious disease or a cancer, comprising the administration to a mammal (a human patient) suffering of said affection of

(i) a therapeutic vaccine against said chronic infection disease or cancer, and (ii) an adjuvant combination of two cytokines which are IL2 and IL12 , analogs thereof, nucleic acid molecules expressing them or cells transformed with said nucleic acid molecules, said two cytokines being administrated, sequentially or simultaneously, in an amount effective for enhancing proliferation and differentiation of T and B lymphoid cells directed to at least one antigen of said therapeutic vaccine. In a first embodiment, said therapeutic vaccine comprises mature dendritic cells from the mammal activated in vitro.

In a second embodiment, said therapeutic vaccine is an antigenic composition comprising at least one antigen specific for said chronic infectious disease or a cancer.

Advantageously, said two cytokines are administrated, sequentially or simultaneously, two or more times within a period of 1 day to about more than 15 days.

The method of the present invention is especially directed to chronic infectious selected from the group comprising the infection by HIV, HCV, HBV, HPV.

The present invention also provides an adjuvant combination for use in the immunization of a mammal (a human patient) against a chronic infectious disease or a cancer, said combination comprising two cytokines for at least one, sequential or simultaneous, administration, which are IL2 and IL12 , analogs thereof, nucleic acid molecules expressing them or cells transformed with said nucleic acid molecules, in an amount effective for enhancing proliferation and differentiation of T and B lymphoid cells directed to at least one antigen specific for said chronic infection disease or cancer.

The present invention is also directed to the use of a combination of IL2 and IL12 for the preparation of (a) pharmaceutical composition (s) for enhancing proliferation and differentiation of T and B lymphoid cells directed to at least one antigen specific for a chronic infectious disease or cancer in a patient wherein IL2 and IL12 are comprised in one or in a first and a second pharmaceutical composition (s) , respectively, said first and second pharmaceutical compositions being applicable simultaneously or sequentially.

As indicated above, said one or first and second pharmaceutical composition (s) , (is) are administrated in two or more times within a period of one day to more than about 15 days, The antigen (s) may be known or unknown antigens specific for said chronic infectious disease or a cancer.

Brief description of the figures

Figure 1. IFN-gamma-secreting T cells (STCs or spot forming cells [SFCs] ) primed by HIV-pulsed DCs in the presence of IL-2 and IL-12

Figure 2. HIV-specific tetramer⁺ T cells primed by HIV-pulsed DCs in the presence of IL-2 and IL-12.

Figure 3. HIV-specific lysis of T cells primed by HIV-pulsed DCs in the presence of IL-2 and IL-12. Figure 4. Antiviral activity of T cells primed by inactivated HIV-pulsed DCs in the presence of IL-2 and IL-12.

Figure 5. IFN-gamma-secreting T cells (STCs or SFCs) primed by tumor cell lysate-pulsed DCs in the presence of IL-2 and IL-12.

Figure 6. Tumor-specific cytolytic activity of T cells primed by tumor cell lysate-pulsed DCs in the presence of IL-2 and IL-12. Figure 7. Spontaneous ex vivo IFN-gamma- secreting T cells (STCs or SFCs) in the presence of IL- 2 and IL-12.

Figure 8. Spontaneous ex vivo cytolytic activity of T cells in the presence of IL-2 and IL-12. Figure 9. IFN-gamma-secreting T cells (STCs or SFCs) stimulated by inactivated-HIV- or tumor-cell- lysate-pulsed DCs in the presence of IL-2 and IL-12.

Figure 10. HIV- or tumor-specific cytolytic activity of T cells stimulated by inactivated-HIV- or tumor-cell-lysate-pulsed DCs in the presence of IL-2 and IL-12.

Detailed description

Dendritic cells (DCs) are bone marrow- derived professional antigen presenting cells that are poised by virtue of their locations (such as skin epidermis and the mucosae of the respiratory, gastrointestinal and urogenital tracts) to encounter pathogens upon their entry into the body. The all DC functions (including capture, transport, and presentation of foreign antigens) are necessary for making and amplifying the antiviral weapons (including virus-specific cytotoxic T killer cells and neutralizing antibodies) , which will control or eradicate viral infections.

The lack of functional virus- or tumor- specific T killer cells is a key immunogical feature in individuals with chronic human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections (1-5) or neoplastic diseases (6) . It has been also shown that DCs are multi-functionally deficient in individuals with various chronic infections (7-10) . Such an in vivo DC deficiency was thought to determine the characteristic features of human and simian AIDS: high level of viral loads and progressive decrease of peripheral blood CD4 count; - absence of neutralizing antibodies; - absence of antiviral activity of cytotoxic T lymphocytes (CTL) ; - and destruction of lymph node (LN) follicular dendritic cell network with disappearance of LN germinal center.

Tumor-associated antigens (TAAs) are essentially "self antigens" which are frequently over- expressed during the development of neoplastic diseases. Since the phenomenon of tumor infiltrating lymphocytes (TILs) has been well characterized in most tumors in situ (11-16) , TAAs can be readily primed in vivo to mount anti-TAA immune memories. However, such immune memories are not functionally effective, probably due to the presence of immune- suppressive regulatory cells such as CD25⁺CD4⁺ T cells (17) . Nonetheless, numerous studies have recently demonstrated that in vitro activated DCs fused with tumor cells or loaded with TAA, or DNA or mRNA encoding TAA, or tumor lysates are capable of inducing effective anti-tumor immunity in vitro as well as in vivo in mice models, suggesting promising therapeutic vaccines for human cancers (18) . However, the apparent success in mice stands in contrast to the current situation in the clinic, wherein only a minority of patients have thus far benefited from DC-based anti-tumor therapeutic vaccination approaches (19) . To resolve the in vivo DC deficiency in the setting of chronic viral infections, we seek at first, in previous studies conducted on the blood samples taken from HIV- infected patients, to isolate peripheral blood monocytes, the precursors of DCs, which remain functionally intact. In the laboratory, monocytes were transformed into DCs with the help of biotechnology products. Having demonstrated that such monocyte- derived (MD) DCs loaded with 2 , 2 ' -dithiodipyridine or aldrithiol-2 (AT-2) (20) -inactivated HIV were capable of stimulating potent antiviral CTL activity in vitro

(21, 22), we used macaque MD-DCs loaded with AT-2- inactivated SIV to vaccinate 10 SIV-infected monkeys by

5 subcutaneous injections made at two-week intervals.

As short as 5 weeks after the first immunization, a 1000-fold decrease of plasma SIV RNA and a 50-fold decrease of blood cellular SIV DNA, associated with a peak increase of circulating anti-SIV CTLs, were observed in the 10 vaccinated animals. On the other hand, CD4⁺ T-cell counts, the principal surrogate marker of the immune system capacities, increased significantly as from week 13. Moreover, neutralizing antibodies appeared in the peripheral blood of all vaccinated animals. In contrast, in the 4 infected but unvaccinated animals, viral loads and CD4⁺ T-cell counts remained unchanged and neutralizing antibodies were not detected during the 300 days of the follow up. LN examination confirmed the permanent killing of SIV- infected cells by SIV-specific CTLs in the LN of vaccinated animals. Importantly, the vaccinated animals remained free of any destruction of their LN follicular dendritic cell network, a histopathologic sign associated with the development of AIDS, which was indeed seen in 2 out of the 4 infected but non- vaccinated animals. Three hundreds days after the first vaccination, the anti-SIV immunity remained strong and stable in 7 animals while it decreased progressively in the other 3 animals (23) .

Although it has been demonstrated that an effective and durable SIV-specific cellular and humoral immunity is elicited by a vaccination with chemically inactivated SIV-pulsed DCs, the virus still persisted in the body and did re- increase with time in 3 out of the 10 vaccinated animals. We thus pursued further to determine whether IL-2 (the well-known second signal cytokine) and/or IL-12 (a recently identified third signal cytokine (24)) could be helpful in amplifying the anti-virus (HIV) or anti-tumor immunity elicited by inactivated virus (or viral antigens) or tumor-lysate (or TAA) -pulsed DCs (which deliver the first signal allowing the activation of T cells) .

Examples

Methods

1) Donors and samples

We studied the peripheral monocyte-derived dendritic cells (DCs) taken from 1) 10 individuals with asymptomatic HIV-1 infection (7 men and 3 women) who were naive for antiretroviral therapies and were positive for HLA-A*0201 by a PCR-based assay (LiPA HLA- A, Innogenetics, Gent, Belgium) . Plasma HIV-1 RNA load of the 10 HIV-1-infected patients ranged from 5,531 to 26,037 copies/ml (median, 13,400 copies/ml) and their CD4⁺ T-cell count ranged from 274 to 928 cells/μl (median, 341 cells/μl) ; 2) 5 patients with metastatic cancers (1 neuroendocrine pancreas carcinoma, 2 melanoma, and 2 renal cell carcinoma) . HIV gag (SLYNTVATL) - and pol (ILKEPVHGV) -HLA-A*0201⁺ CD8⁺ T cells were assessed by flow-cytometry analysis using a commercial product (iTAg MHC Tetramer, Tetramer-PE/CD8- FITC, Beckman Coulter, San Diego, CA, USA) . Institutional ethical approval and informed consent were obtained from all patients before blood donation. EDTA-treated whole blood was collected from the above donors and transported to our laboratory within 2 hours .

2) Dendritic cells culture Peripheral blood mononuclear cells (PBMCs) were isolated from 20 ml of fresh EDTA-treated whole blood using Ficoll -Hypaque density gradient centrifugation. After three washes with Hank's balanced salt solution (Hank's buffer), PBMCs were suspended in 5 x 10^ε/ml of RPMI 1640 medium (Eurobio, Les Ulis, France) containing 0.5% of bovine serum albumin (Sigma, St Louis, Missouri, USA) and were then subjected to plastic adherence at a density of 10⁶ cells/cm². After 30-minute incubation at 37 °C in 5% C0₂, non-adherent cells were removed by rinsing 3 times with Hank's buffer. The plastic-adherent cells were cultured for 7 days in AIM-V medium (Life Technologies, Grand Island, NY, USA) supplemented with 100 ng/ml GM-CSF (Schering- Plough, Brinny, Ireland) and 50 ng/ml IL-4 (R&D system, Minneapolis, MN, USA) . At day 5, a cocktail made of 20 ng/ml TNF- (R&D System) , 10 ng/ml IL-lβ (R&D System) , and 100 ng/ml IL-6 (R&D System) was added to the culture for the last 2 days. At this time point, monocyte-derived dendritic cells (DCs) were normally differentiated as shown by their mature morphology and phenotype (CD83⁺) with increased expression of costimulatory molecules (CD40, CD80, CD86) and HLA-DR.

3) Flow cytofluorometry DCs and CD8⁺ T-cell subsets were assessed or sorted by four-color flow-cytometry (FacsVantage, Beσkton Dickinson [BD] , San Jose, CA, USA) using a panel of direct fluorescence- labeled anti-human monoclonal antibodies and their corresponding isotype controls: anti-CDla-FITC (BD Biosciences) , CD8-PerCP (BD) , CDllc-FITC (BD) , CD14-PE (BD) , CD28-FITC (BD) , CD40-FITC (BD) , CD45RA-APC and CD45RO-APC (Dako S.A., Trappes, France), CD80-FITC (BD) , CD83-PE (BD) , CD86-PE (BD) , CD209-FITC (BD) , and HLA-DR-PE (BD) . Pure anti- CCR7 monoclonal antibody (BD) was coupled with the use of biotin-labeled anti-mouse antibody first and then with PE-labeled (BD) or PE-Cy5 - labeled (Dako) streptavidin.

4) ELISPOT assay The human IFN-γ ELISPOT assay was performed in the cultured or uncultured T cells (2, 1, and 0.5 x 10^s) using a commercial kit (Autoimmun Diagnostika [AID] GmbH, Straβberg, Germany) . AT-2-inactivated autologous HIV-pulsed or tumor-lysate-pulsed (at day 5) autologous DCs (2xl0⁴) were used as virus- or tumor- specific antigens stimulators (23) . They were added to their matched T cells in the absence or the presence of IL-2 and/or IL-12 for a 16-hour stimulation according to the instruction of the kit's manufacturer. Unstimulated T cells alone or stimulated with 100 ng/ml of anti-CD3 antibody (BD) served as negative or positive control. The data were read with an automated ELISPOT reader (AID) . The number of HIV- or tumor- specific spot-forming cells (SFCs) were calculated by subtracting the nonspecific SPCs in the presence of non-pulsed autologous DCs with the use of a build-in software (Elispot 2.9, AID).

5) Viral quantitation assays HIV RNA in the culture supernatants was quantified by a previously described quantitative assay (26) .The detection threshold of the assay was 10 copies/ml. Cell-associated HIV DNA was quantified by a recently improved assay (21) . The sensitivity of the HIV DNA assays reached 5 HIV DNA per 10^e cells.

6) Cytolytic and antiviral assays

HIV- or tumor-specific cytolytic activity was performed by measuring the percentage of specific lysis (at an effector/target ratio of 10:1) of inactivated HIV-pulsed or tumor- lysate-pulsed autologous DCs by unexpanded memory T cells or naϊve CD8 cells expanded with AT-2- inactivated HIV-pulsed or tumor-lysate-pulsed (at day 5) autologous DCs (at a stimulator/responder ratio of 1:3) for 30 days in the presence or the absence of recombinant IL-2 (20 U/ml and/or IL-12 (0.2 ng/ml). Antiviral activity of T cells was assessed using purified patients CD4 cells super- infected in vitro with autologous virus as targets and non-pulsed DC-expanded or HIV-pulsed-DC-expanded T cells as effectors (at an effect/target ratio of 1:1) (21) . The frequency of cells carrying HIV DNA was determined by limiting dilution assay (27) .

7) Statistical analysis

Paired data under different treatments from the same group of donors samples were compared by the Wilcoxon test.

Results 1) Effects of exogenous IL-2 and IL-12 on the priming of naive CD8 T cells

The naive (CD28⁺CD45RA⁺) CD8 T cells were first collected from 10 HIV⁺ patients (Table 1) peripheral blood lymphocytes (PBLs) by cell sorting.

Table 1

Sorted cells were cocultured with AT-2- inactivated HIV-loaded autologous DCs in the absence or the presence of IL-2 and/or IL-12 for 30 days. Fresh inactivated virus-loaded DCs were added at days 10 and 20. Cultured cells were then subjected to an ELISPOT assay designed to determine the relative number of HIV antigen-specific T cells which secrete interferon-γ (IFN-γ) when stimulated with inactivated HIV- loaded autologous DCs. Naϊve CD8 cells primed in the presence of IL-2 and IL-12 showed 45-, 25-, or 27-fold increases in the frequency of T cells secreting IFN-γas compared to those primed by inactivated HIV-loaded DCs alone (P < 0.01), plus IL-2 alone (P < 0.01) or IL-12 alone (P < 0.01) respectively (figure 1). HIV gag- or pol-specific tetramer⁺ cells increased 7, 3, or 4 fold in the cells primed in the presence of IL-2 and IL-12 compared to those primed by inactivated HIV-loaded DCs alone (P < 0.01) or plus IL-2 (P < 0.01) alone or IL-12 alone (P < 0.01) respectively (figure 2).

To study the effector function of HIV⁺ patients CD8 cells primed in vitro with inactivated HIV-loaded autologous DCs in the presence of IL-2 and/or IL-12, the cytolytic and antiviral activities of the primed CD8⁺ T cells were examined in 30-day- cultured cells using an HIV-gag-specific CTL assay and an antiviral activity assay ²¹. HIV-gag-specific lysis was 10-, 3-, or 4-fold higher (P < 0.01) (figure 3) and the number of HIV DNA⁺ CD4⁺ T cells was 200- , 10-, or 20-fold lower (P < 0.01) (figure 4) by the cells expanded by AT-2-inactivated HIV-loaded DCs in the presence of IL-2 and IL-12 as compared to the cells stimulated by inactivated HIV-loaded DCs alone, in the presence of IL-2 alone or IL-12 alone respectively.

Next, we sorted naϊve CD8 cells from PBLs of 5 patients with metastatic cancers (Table 2) .

Table 2

Sorted cells were then primed by autologous

DCs loaded with the lysates of their own tumor cells that had been obtained from metastiatic tumor biopsies. Similarly, around 40-, 25-, or 30-fold increases were observed in the tumor-specific IFN-γ-secreting cells

(figure 5) and about 8, 3, or 4-fold increases were demonstrated in the tumor-specific killing cells

(figure 6) primed in the presence of IL-2 and IL-12, as compared to the cells primed by tumor-lysate-loaded DCs alone, in the presence of IL-2 or IL-12 respectively.

2) Effects of exogenous IL-2 and IL-12 on the differentiation of memory CD8 T cells Having demonstrated that exogenous IL-2 and

IL-12 are highly synergic to enhance DC-mediated priming of naϊve T cells, we examined further whether IL-2 had a synergy with IL-12 to stimulate the proliferation and/or differentiation of memory T cells. IL-2 and IL-12 had a synergy to stimulate ex vivo the spontaneous secretion of IFN-γ by the in vivo activated effector memory cells (CD28^"CCR7^~D45RO⁺) pre-existing in uncultured PBLs taken from chronically HIV- infected individuals or patients with metastatic cancers (figure 7) . Moreover these cytokines promote the differentiation of these effector memory cells into terminally differentiated effector cells (CD28^~CCR7^~ D45RA⁺) with virus- or tumor-specific cytolytic activity (figure 8) ; on the other hand, IL-2 and IL-12 failed to induce the proliferation of effector memory T cells in the absence of antigen-loaded DCs (data not shown) .

In contrast, stimulation with inactivated HIV-loaded DCs or tumor-lysate-loaded DCs in the presence of IL-2 and IL-12 up-regulated both proliferation (data not shown) and IFN-γ secretion

(figure 9) of virus- or tumor-specific central memory cells (CD28⁺CCR7⁺CD45RO⁺ or CD28⁺CCR7^"CD45RO⁺) existing in the uncultured PBLs, resulting in the expansion of terminally differentiated killer cells (CD28^~CCR7^~

CD45RA⁺) with optimum virus- or tumor-specific cytolytic activity (figure 10) . On the other hand, PBLs under DC-mediated stimulation alone or in the presence of IL-2 or IL-12 showed relatively low levels of IFN-γ secreting cells and antiviral or anti-tumor cytolytic activity despite an equivalent expansion of effector killer cells was achieved (data not shown) .

Here we have shown that exogenous IL-2 and IL-12, coupled with HLA-restricted TCR/CD3 ligation and CD28 costimulation provided by viral or tumor antigen- pulsed autologous DCs, could prime efficiently the naϊve CD8 cells and expand optimally the memory T cells to produce a large number of virus- or tumor-specific CTLs. This strategy has allowed the optimization of three immunologic events which are necessary for the initiation of a dynamic immune response: i) optimization of the first signal by generating, loading, and activating DCs in vitro; ii) optimization of the second signal by providing exogenous IL-2; and iii) optimization of the third signal by providing at the same time exogenous IL-2 and IL-12. Since virus or "tumor (or self) antigens" are well tolerated in the body of people with chronic viral infections or neoplastic diseases, the control or even the cure of these diseases by immunotherapy requires to alter such an immune tolerant situation by manipulating DC (or APC) with the adequate target antigens. Now, we have proved that the addition of IL-2 and IL-12 results in the dramatic increase of the killing of virus-infected cells as well as tumor cells by DC-primed naϊve CD8 cells and DC-stimulated memory CD8 cells. Such findings should have soon clinical applications to better control or cure chronic infectious diseases and cancers in human.

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Claims

1) Method of treatment and/or prophylaxis of a chronic infectious disease or a cancer, comprising the administration to a mammal (a human patient) suffering of said affection of (i) a therapeutic vaccine against said chronic infection disease or cancer, and (ii) an adjuvant combination of two cytokines which are IL2 and IL12, analogs thereof, nucleic acid molecules expressing them or cells transformed with said nucleic acid molecules, said two cytokines being administrated, sequentially or simultaneously, in an amount effective for enhancing proliferation and differentiation of T and B lymphoid cells directed to at least one antigen of said therapeutic vaccine.

2) The method of claim 1 wherein said therapeutic vaccine comprises mature dendritic cells from the mammal activated in vitro.

3) The method of claim 1 wherein said therapeutic vaccine is an antigenic composition comprising at least one antigen specific for said chronic infectious disease or a cancer.

4) The method according to anyone of claims 1 to 3 wherein said two cytokines are administrated, sequentially or simultaneously, two or more times within a period of 1 day to about more than 15 days.

5) The method according to anyone of claims 1 to 4 wherein the chronic infectious is selected from the group comprising the infection by HIV, HCV, HBV, HPV, and EBV.

6) Adjuvant combination for use in the immunization of a mammal (a human patient) against a chronic infectious disease or a cancer, said combination comprising two cytokines for at least one, sequential or simultaneous, administration, which are IL2 and IL12, analogs thereof, nucleic acid molecules expressing them or cells transformed with said nucleic acid molecules, in an amount effective for enhancing proliferation and differentiation of T and B lymphoid cells directed to at least one antigen specific for said chronic infection disease or cancer.

7) The adjuvant of claim 6 wherein the chronic infectious is selected from the group comprising the infection by HIV, HCV, HBV, HPV and EBV.

8) The use of a combination of IL2 and IL12 for the preparation of (a) pharmaceutical composition (s) for enhancing proliferation and differentiation of T and B lymphoid cells directed to at least one antigen specific for a chronic infectious disease or cancer in a patient wherein IL2 and IL12 are comprised in one or in a first and a second pharmaceutical composition (s) , respectively, said first and second pharmaceutical compositions being applicable simultaneously or sequentially.

9) The use of claim 8 wherein said one or first and second pharmaceutical composition (s) , is/are administrated in two or more times within a period of one day to more than about 15 days . 10) The use according to anyone of claims 8 or 9 wherein the chronic infectious is selected from the group comprising the infection by HIV, HCV, HBV, HPV, and EBV.