Vaccine to pathogenic immune activation cells during infections

Abstract

A novel method for preventing or treating an infectious disease in a subject in need thereof. In particular the method includes the administration of a combination, pharmaceutical combination, medicament or kit-of-parts having a first part including a CD8 vaccine specific for at least one infectious disease-related antigen, a second part including an agent neutralizing circulating alpha interferon and/or an agent blocking interferon alpha signaling, and/or a third part including a type III interferon and/or an agent stimulating the production of type III interferon.

Claims

1. A method for treating acquired immune deficiency syndrome (AIDS) in a subject in need thereof, comprising administering to the subject: a) a CD8 therapeutic vaccine specific for at least one human immunodeficiency virus (HIV) antigen, b) an agent neutralizing circulating alpha interferon and/or an agent blocking interferon alpha signaling, and c) a type III interferon and/or an agent stimulating the production of type III interferon.

2. The method for treating according to claim 1, wherein the type III interferon comprises at least one IFN- selected from the group of IFN-1, IFN-2, IFN-3 and IFN-4; wherein the agent stimulating the production of type III interferon comprises TLR ligands, RIG-I ligands, and MDA5 ligands; and wherein the agent stimulating the production of type III interferon can stimulate the production of type I interferon.

3. The method for treating according to claim 1, wherein the agent neutralizing circulating alpha interferon is selected from the group comprising active anti-IFN- vaccine including antiferon or passive anti-IFN- vaccine including anti-IFN- antibodies or anti-IFN- hyper-immune serum; and wherein the blocking agent of interferon alpha signaling is selected from the group of anti-type I interferon R1 or R2 antibodies or from interferon alpha endogenous regulators including SOSC1 or aryl hydrocarbon receptors.

4. The method for treating according to claim 1, wherein the CD8 therapeutic vaccine elicits or comprises suppressor MHC-1b/E-restricted CD8+ T cells.

5. The method for treating according to claim 1, wherein the CD8 therapeutic vaccine elicits or comprises suppressor MHC-1b/E-restricted CD8+ T cells, and wherein the suppressor MHC-1 b/E-restricted CD8+ T cells are generated by ex vivo or in vivo induction of HLA-1a-deprived dendritic cells.

6. The method for treating according to claim 1, wherein the CD8 therapeutic vaccine elicits or comprises suppressor MHC-1b/E-restricted CD8+ T cells, wherein the suppressor MHC-1b/E-restricted CD8+ T cells are generated by ex vivo or in vivo induction of HLA-1a-deprived dendritic cells, and wherein the HLA-1a-deprived dendritic cells are obtained by an agent inhibiting TAP expression or activity.

7. The method for treating according to claim 1, wherein the CD8 therapeutic vaccine is an active vaccine, wherein the CD8 therapeutic vaccine is a live viral vector comprising at least one HIV antigen, and wherein the live viral vector is selected from the group of cytomegalovirus, lentivirus, vaccinia virus, adenovirus or plasmid.

8. The method for treating according to claim 1, wherein the CD8 therapeutic vaccine is an active vaccine, and wherein the CD8 therapeutic vaccine is a cytomegalovirus (CMV) vector comprising: a first nucleic acid sequence encoding at least one HIV antigen, optionally a second nucleic acid sequence comprising a first microRNA recognition element (MRE) operably linked to a CMV gene that is essential or augmenting for CMV growth, wherein the MRE silences expression in the presence of a microRNA that is expressed by a cell of endothelial lineage; and wherein the CMV vector does not express an active UL128 protein or ortholog thereof; does not express an active UL130 protein or ortholog thereof; does not express an active UL146 or ortholog thereof; does not express an active UL147 protein or ortholog thereof, and wherein the CMV vector expresses at least one active UL40 protein or an ortholog thereof; expresses at least one active US27 protein or an ortholog thereof and/or expresses at least one active US28 protein or an ortholog thereof.

9. The method for treating according to claim 1, wherein the CD8 therapeutic vaccine is a cytomegalovirus (CMV) vector, and wherein the CMV vector is a human CMV (hCMV).

10. The method for treating according to claim 1, wherein the CD8 therapeutic vaccine is an active vaccine, and wherein the CD8 therapeutic vaccine comprises at least one HIV antigen and a non-pathogenic bacterium.

11. The method for treating according to claim 1, wherein the CD8 therapeutic vaccine comprises at least one HIV antigen and a non-pathogenic bacterium, wherein the infectious disease-related antigen is selected from the group of inactivated virus, virus particles, virus-like particles, recombinant virus particles, conjugate viral proteins and concatemer viral proteins, and wherein said virus particles or said recombinant virus particles are inactivated.

12. The method for treating according to claim 1, wherein the CD8 therapeutic vaccine comprises at least one HIV antigen and a non-pathogenic bacterium, wherein the bacterium is living, and wherein said bacterium is selected from attenuated or inactivated pathogenic bacteria.

13. The method for treating according to claim 1, wherein the CD8 therapeutic vaccine comprises at least one HIV antigen and a non-pathogenic bacterium, and wherein the bacterium is a Lactobacillus bacterium.

14. The method for treating according to claim 1, wherein the CD8 therapeutic vaccine comprises at least one HIV antigen and a non-pathogenic bacterium, and wherein the bacterium is Lactobacillus plantarum.

15. The method for treating according claim 1, wherein the CD8 therapeutic vaccines is an active CD8 therapeutic vaccine, and wherein the CD8 therapeutic vaccine is an ex vivo generated dendritic cell population presenting MHC-1b/E-restricted and MHC-II restricted antigens, and wherein the MHC-1b/E-restricted antigen is an HIV antigen.

16. The method for treating according to claim 1, wherein the CD8 therapeutic vaccine is a passive vaccine, and wherein the CD8 therapeutic vaccine is an ex vivo generated autologous MHC-1b/E-restricted CD8+ T cell population, and wherein the MHC-1b/E-restricted CD8+ T cells population recognizes an MHC-1b/E-restricted HIV antigen.

17. The method for treating according to claim 1, wherein the HIV antigen is selected from any HIV strain, and wherein the HIV antigen is selected from the group consisting of HIV gag, HIV env, HIV rev, HIV tat, HIV nef, HIV pol, and HIV vif antigens.

18. The method for treating according to claim 1, wherein the HIV antigen is an HIV-derived HLA-E-binding antigen.

19. The method for treating according to claim 1, wherein the HIV antigen is an HIV-derived HLA-E-binding antigen, and wherein the HIV-derived HLA-E-binding antigen is selected from the antigens of SEQ ID NO: 1 to SEQ ID NO: 4.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A and 1B Antiviral activity of type I and type III interferons. FIG. 1A shows expression of ISGs in HepG2. HepG2 cells were treated with IFN2a or IFN1-4 (10 ng/ml). After 4 h of stimulation, qRT-PCR were used to examine the mRNA levels of the interferon-induced genes, IFIT1, MX1 and OASL and fold-changes was calculated by 2.sup.Ct method as compared with non-treated cell control and using endogenous S14 mRNA level for normalization. FIG. 1B shows antiviral activity of type I and III IFNs against EMCV. IFN2a or IFN1/2/3/4 (10 ng/ml) were added to HepG2 cells 24 h prior to challenge with EMCV. Forty-eight after infection with EMCV, cells were assayed for viability with a bioassay. A570 values were directly proportional to cell viability and therefore antiviral activity of the respective IFNs. IFN- treatment without viral challenge was used as a baseline of the viability of the cells.

(2) FIG. 2. Anti-proliferative activity of type I and type III interferons against CD4.sup.+ T cells. CFSE-stained CD4.sup.+ T cells (1010.sup.4/well) were stimulated for 5 days in 96 round-bottomed microwells with allogeneic poly I:C matured DC in absence (control) or presence of 10 ng/ml of IFN- 2a, or IFN1 or IFN2 or IFN3 or IFN4. When indicated, anti-interferon type I receptor antibody was added. The percentage of CFSE dilution was evaluated by flow cytometry.

(3) FIG. 3. IFN-2a but not IFN-type III induces the expression of ISGs in CD4.sup.+ T cells. CD4.sup.+ T cells were treated with IFN2a or IFN1/2/3/4 (10 ng/ml). After 4 h of stimulation, qRT-PCR were used to examine the mRNA levels of the interferon-induced genes, IFIT1, MX1 and OASL and fold-changes was calculated by 2.sup.Ct method as compared with non-treated cell control and using endogenous S14 mRNA level for normalization.

(4) FIG. 4. IFN-2a but not IFN-type III stimulates the phosphorylation of Stat1 in CD4.sup.+ T cells. CD4.sup.+ T cells were stimulated with 10 ng ml.sup.1 of IFN-1, IFN-2, IFN-3, IFN-4, or IFN-2a for 20 min, or were left unstimulated (control). Increases in pSTAT1 were evaluated as a ratio of induction over baseline levels (MFI fold change=MFI cytokine-stimulated/MFI untreated cells)

(5) FIG. 5. IFN-2a but not IFN-type III increases CD38 expression in CD3/CD28 stimulated CD4.sup.+ T cells. CFSE-stained CD4.sup.+ T cells (410.sup.4/well) were cultured in 96 round-bottomed microwells in the presence of CD3-feeder (410.sup.4/well) and plate-bound anti-CD3 mAb (2 g/ml), soluble anti-CD28 mAb (2 g/ml) with increasing dose of IFN-2a or IFN type III. CD38 Median Fluorescence Intensity (MFI) was measured by flow cytometry in CD3.sup.+ 7-AAD-CFSE.sup.+ stimulated CD4.sup.+ T cells at the end of the culture.

(6) FIG. 6. Generation and expansion of peptide-specific CD8 HLA-E restricted by peptide-loaded m-DCs. TAP-inhibited mDCs was pulsed with peptides (10 M for 1 h) and co-cultured with autologous naive CD8.sup.+ T cells at a 1:10 ratio. Peptide positive CD8.sup.+ T cells was monitored at day 0 and one week after the last stimulation by flow cytometric analysis using MHC-peptide pentamers. Data are expressed as percentage of tetramer-positive cells among CD8.sup.+ T cells.

(7) FIG. 7. Schematic protocol of the combination that can be used for a prophylactic vaccination to SIV in macaques.

EXAMPLES

(8) The present invention is further illustrated by the following examples.

Example 1: Effects of Type I and Type III Interferons on Innate and Adaptative Immune Responses

(9) Materials and Methods

(10) Human Cell Lines

(11) HCC HepG2 and normal kidney epithelial Vero cell lines were obtained from ATCC. Cells were grown in Dulbecco's Modified Eagle Medium supplemented with 10% heat-inactivated Fetal Bovine Serum, 2 mM L-glutamine, 1% penicillin and streptomycin solution in hypoxia 2%. Cancer cell lines were grown to 70-100% confluency, subsequently passaged for a maximum of 5 times and freshly thawed thereafter. Cells were detached by means of accutase, resuspended in FBS-containing medium and collected by means of centrifugation (300 g, 3 min). Cell numbers were determined by means of trypan blue.

(12) Human Blood Sample

(13) Blood samples from healthy individuals originated from Etablissement Francais du Sang (EFS, Paris). Blood cells are collected using standard procedures.

(14) Cell Purification and Culture

(15) Peripheral blood mononuclear cells (PBMCs) are isolated by density gradient centrifugation on Ficoll-Hypaque (Pharmacia). PBMCs are used either as fresh cells or stored frozen in liquid nitrogen. T-cell subsets and T cell-depleted accessory cells (CD3 cells) are isolated from either fresh or frozen PBMCs. T cell-depleted accessory cells (CD3 cells) are isolated by negative selection from PBMCs by incubation with anti-CD3-coated Dynabeads (Dynal Biotech) and are irradiated at 3000 rad (referred to as CD3-feeder). CD4.sup.+ T cells are negatively selected from PBMCs with a CD4.sup.+ T-cell isolation kit (Miltenyi Biotec), yielding CD4.sup.+ T-cell populations at a purity of 96-99%. T cell subsets are cultured either in IMDM supplemented with 5% SVF, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES (IMDM-5 media) in hypoxia 2%.

(16) Freezing and Thawing of Cells

(17) Cells were frozen in FBS containing 10% DMSO. Cryo tubes were placed in CoolCell (Biocision) freezing containers and incubated at 80 C. After 2 days tubes were transferred to liquid nitrogen and stored until required. Thawing of cells was performed by placing cryo tubes in a 37 C. water bath for approximately 30 seconds. Next, cell suspension was mixed with equivalent volume of pre-warmed media and subsequently transferred to falcon tubes containing the same medium. Cells were pelleted by centrifugation (300 g, 3 min) to remove DMSO. The cell pellet was resuspended in cell culture medium

(18) Real-Time PCR for ISGs Detection

(19) HepG2 cells were seeded at a density of 210.sup.5 cells per well in 12-well plates and incubated for 24 h. Then, fresh media was added with the indicated interferons. The cells were incubated for 4 h and then lysed, and RNA was purified using an extraction kit (Qiagen), according to the manufacturer's instructions. Synthesis of cDNA was performed using the PrimeScript RT Reagent kit (TAKARA). Quantitative PCR was carried out using the Power SYBR Green PCR Master Mix (Applied Biosystems) on a LightCycler 480 instrument (Roche). Each reaction was carried out in duplicate in a total volume of 100 L. Primers were designed to be intron-spanning using Primer3 or Primer Express v3.0 software (Applied Biosystems). To measure the cellular transcriptional response to IFN stimulation, 3 ISG targets, MXI, OASL and ISG15, were selected based on published results investigating the transcriptional response in IFN-stimulated PBMCs (see, for example, Waddell et al. (2010) PLoS One. 5(3):e97532). For gene induction assays, fold change values were calculated using the Ct method. The geometric mean of the Ct values of the reference genes, S14, was used as a reference value.

(20) Virus Production

(21) The virus used EMCV (FA strain) was grown on monolayers of Vero cells to complete cytopathic effect or until all cells were affected by the infection as determined by microscopy and prepared by two cycles of freezing and thawing, followed by centrifugation for 30 min at 5,000g for removal of cellular debris.

(22) Antiviral Assay

(23) Antiviral assays were done on HepG2 cells, which were seeded in DMEM supplemented with 10% FCS at a density of 1.510.sup.4 in 96-well plates and left to settle. The cells were incubated with indicated doses of IFNs for 24 h before challenge with EMCV. The cells were incubated with virus for 48 h. The medium was removed between each step. The viability of the cells was analyzed by a bioassay based on the dehydrogenase system; this system in intact cells will convert the substrate, MTT, into formazan (blue), which in turn can be measured spectrophotometrically. Briefly, the cells were given MTT and incubated for 2 h. An extraction buffer (containing 6 to 11% sodium dodecyl sulfate and 45% N,N-dimethylformamide) was added to the cells, and the cells were then incubated overnight at 37 C. Subsequently, the absorbance at 570 nm was determined employing the extraction buffer as the blank probe. A570 was directly proportional to antiviral activity.

(24) Flow Cytometry Analysis

(25) CD3.sup.+ T Cells Staining:

(26) anti-CD4 (SK3)-APC, anti-CD3 (UCHT1)-FITC, anti-CD8 (RPA-T8)-BV421 are from Becton Dickinson. Cells are stained for surface markers (at 4 C. in the dark for 30 min) using mixtures of Ab diluted in PBS containing 3% FBS, 2 mM EDTA (FACS buffer).

(27) STAT1 Signaling Analysis:

(28) Flow cytometry analysis of STAT1 phosphorylation (pSTAT1) was conducted in CD4.sup.+ T cells by using BD Phosflow technology according to the manufacturer's instructions (BD Bio-sciences, San Jose, Calif.). CD4.sup.+ T cells were stimulated by incubation with interferon type I and Type III at 37 C. for 20 min or left untreated. Activation was stopped by fixation using BD Phosflow Lyse/Fix Buffer (BD Biosciences) and cells were permeabilized with BD Perm Buffer III (BD Biosciences). Cells were stained with antibody recognizing specific phosphorylated STAT tyrosines: p-STAT1 (Y701)-PE. In multiparametric immunophenotyping experiments, cells were simultaneously stained with anti-CD3-FITC and 7-AAD. Increases in pSTAT1 were assayed as a ratio of induction over baseline levels (MFI fold change=MFI cytokine-stimulated/MFI untreated cells)

(29) Cfse Staining:

(30) CD4.sup.+ T cells were stained with 1 M CFSE (CellTrace cell proliferation kit; Molecular Probes/Invitrogen) in PBS for 8 min at 37 C. at a concentration of 1.107 cells/ml. The labeling reaction was stopped by washing twice the cell with RPMI-1640 culture medium containing 10% FBS. The cells were then re-suspended at the desired concentration and subsequently used for proliferation assays.

(31) 7-AAD Staining:

(32) Apoptosis of stimulated CFSE-labeled CD4.sup.+ T was determined using the 7-AAD assay. Briefly, cultured cells were stained with 20 g/mL nuclear dye 7-amino-actinomycin D (7-AAD; Sigma-Aldrich, St-Quentin Fallavier, France) for 30 minutes at 4 C. FSC/7-AAD dot plots distinguish living (FSC.sup.high/7-AAD.sup.) from apoptotic (FSC.sup.high/7-AAD.sup.+) cells and apoptotic bodies (FSC.sup.low/7-AAD.sup.+) and debris ((FSC.sup.low/7-AAD.sup.). Living cells were identified as CD3.sup.+ 7-AAD-FSC.sup.+ cells.

(33) Appropriate isotype control Abs are used for each staining combination. Samples are acquired on a BD LSR FORTESSA flow cytometer using BD FACSDIVA 8.0.1 software (Becton Dickinson). Results are expressed in percentage (%) or in mean fluorescence intensity (MFI).

(34) Functional Assay

(35) T Cell Proliferation:

(36) T cell proliferation was assessed with CFSE-dilution assays. For CFSE-dilution assay, at coculture completion, stimulated CFSE-labeled CD4.sup.+ T cells were harvested, co-stained with anti-CD3 mAb and 7-AAD, and the percentage of proliferating cells (defined as CFSE low fraction) in gated CD3.sup.+ 7-AAD.sup. cells was determined by flow cytometry.

(37) T Cell Activation:

(38) CD38 Median Fluorescence Intensity (MFI) of CD38 expression was measured by flow cytometry in CD3.sup.+ 7-AAD-CFSE.sup.+ stimulated CD4.sup.+ T cells at the end of the culture.

(39) CD4.sup.+ T Cell Polyclonal Stimulation:

(40) CFSE-stained CD4.sup.+ T cells (510.sup.4/well) were cultured in 96 round-bottomed microwells in the presence of CD3-feeder (110.sup.5/well) and plate-bound anti-CD3 Ab (2 g/ml), soluble anti-CD28 mAb (2 g/ml). CD4.sup.+ T cell proliferation was evaluated with CFSE dilution assays as described above by flow cytometry. Cells were stimulated in presence of different amounts of recombinant cytokines.

(41) Allogeneic Mixed Lymphocyte Reaction:

(42) CFSE-stained CD4.sup.+ T cells (510.sup.4/well) were cultured in 96 round-bottomed microwells in the presence of allogeneic mature DC. Proliferation of allo-activated CD4.sup.+ T cells with CFSE dilution assays as described above by flow cytometry. Cells were stimulated in presence of different amounts of recombinant cytokines.

(43) Stat1 Phosphorylation Analysis:

(44) CD4.sup.+ T cells were stimulated with IFN-1, IFN-2, IFN-3, IFN-4, or IFN-2a (10 ng/ml) for 20 min, or were left unstimulated (control). Phosphorylated Stat1 levels was assessed by flow cytometry as described above.

(45) Results

(46) Type I interferons (IFN-/) and the more recently identified type III IFNs (IFN-) function as the first line of defense against virus infection, and regulate the development of both innate and adaptive immune responses. Type III IFNs were originally identified as a novel ligand-receptor system acting in parallel with type I IFNs, but subsequent studies have provided increasing evidence for distinct roles for each IFN family.

(47) The inventors aimed to evaluate the effects of type I and type III interferons on both innate (antiviral) and adaptive immune response (CD4.sup.+ T cell proliferation).

(48) Antiviral Activities of Types I and III

(49) The ability of IFN type I and III to induce the expression of interferon-stimulated genes (ISGs) was analyzed by qPCR.

(50) Briefly, the antiviral activity of type I and III was tested in HepG2 cells treated with IFN-2a, IFN1, IFN2, IFN3 or IFN4 for 4 hours. Then the induction of the well-known interferon-stimulated genes (ISGs) MX1, IFIT1 and OASL was monitored by qPCR.

(51) As shown in FIG. 1A, all five interferons clearly induced all three ISGs.

(52) Since the investigated ISGs are functionally related to an antiviral defense, the inventors further evaluate the capacity of both IFN to protect HepG2 cells from EMCV-induced cytopathogenic effect.

(53) Briefly, cells were seeded in a 96-well microtiter plate and treated with the indicated amount of IFNs for 24 h and then challenged with EMCV for 20 h. Cell survival was measured by an MTT coloring assay.

(54) As shown in FIG. 1B, IFN type III and IFN-2a have intrinsic cellular antiviral activity and are able to fully protect HepG2 cells challenged with EMCV.

(55) Anti-Proliferative Activity of Type I and Type III Interferons Against CD4.sup.+ T Cells Proliferation

(56) The effect of IFN-type I and IFN type III on CD4.sup.+ T cells proliferation in response either to polyclonal or to allogeneic stimulation was evaluated in a mixed lymphocyte reaction (MLR) assay.

(57) Briefly, CFSE labelled CD4.sup.+ T cells were first stimulated with poly I:C matured allogeneic dendritic cells in presence of different dose of IFNs. At 5 days post activation, the CFSE fluorescence dilution was analyzed.

(58) As shown in FIG. 2, IFN-2a inhibits the proliferation of stimulated CD4.sup.+ T cells, while IFN type III exhibits no ability to suppress their proliferation. Of note, when the MLR was performed in the presence of anti-interferon type I receptor antibody, CD4.sup.+ T cells exhibit a greater proliferation. Thus, IFN-type I but not IFN type III inhibit the proliferation of allo-activated CD4.sup.+ T cells.

(59) Moreover, the analysis of mRNA levels of the interferon-induced genes (ISG), IFIT1, MX1 and OASL in IFNs treated CD4.sup.+ T cells confirmed the lack or minimal sensitivity of CD4.sup.+ T cells to interferon type III.

(60) Indeed, as shown in FIG. 3, ISGs are induced only in CD4.sup.+ T cells stimulated with IFN-2a. Thus, IFN-2a but not IFN-type III induce the expression of ISGs in CD4.sup.+ T cells.

(61) Because the Jak-STAT1/2 pathway being the major regulators of the transcription of ISG, the inventors have analyzed the phosphorylation levels of Stat1 proteins in response to IFN-type I, or interferon type III within CD4.sup.+ T cells.

(62) As shown in FIG. 4, only IFN-2a was able to stimulate the phosphorylation of Stat1 within CD4.sup.+ T cells. Therefore, IFN-2a but not IFN-type III induces tyrosine phosphorylation of STAT1 in CD4.sup.+ T cells.

(63) Induction of Chronic Immune Activation in Presence of Type I and III Interferons.

(64) Because chronic immune activation has been reasoned to be a significant contributor to disease progression in HIV-1-infected patients, it is possible to monitor disease progression by measuring the expression of activation markers on CD4.sup.+ T cell surface. Thus, the inventors have evaluated, by flow cytometry, the capacity of both IFNs to increase the CD38 expression on stimulated CD4.sup.+ T cells.

(65) As shown in FIG. 5, only IFN-2a was able to enhance the expression of CD38 on stimulated CD4.sup.+ T cells.

(66) Collectively, these ex vivo experiments show that while exhibiting anti-viral activity, as does IFN-, interferon type III, by contrast to the immunosuppressive IFN-, have no effect on CD4.sup.+ T cell activation and proliferation. Indeed interferon type III do not inhibit the initiation of the adaptative immune reaction as do IFN-2a.

(67) In conclusion, while interferon type I and type III are induced by the same viral stimulating factors and exhibit similar signature profiles, their biological activity appears not redundant but rather complementary. Indeed, following viral infection, during the innate phase of the immune response, interferon type III exert their antiviral effects in mucosal sites whereas IFN- act more systemically in the whole organism. Furthermore, the subsequent adaptive immune reaction is inhibited at its initiation level by the immunosuppressive effect of the IFN- on activated CD4.sup.+ T cells.

Example 2: Ex Vivo Generation and Expansion of Antigen (Ag) Specific CD8+ HLA-E Restricted T Cells

(68) Materials and Methods

(69) Human Blood Sample.

(70) Blood samples from healthy individuals originated from Etablissement Francais du Sang (EFS, Paris). Blood cells are collected using standard procedures.

(71) Cell Purification and Culture

(72) Peripheral blood mononuclear cells (PBMCs) are isolated by density gradient centrifugation on Ficoll-Hypaque (Pharmacia). PBMCs are used either as fresh cells or stored frozen in liquid nitrogen. T-cell subsets and T cell-depleted accessory cells (CD3 cells) are isolated from either fresh or frozen PBMCs. T cell-depleted accessory cells (CD3 cells) are isolated by negative selection from PBMCs by incubation with anti-CD3-coated Dynabeads (Dynal Biotech) and are irradiated at 3000 rad (referred to as CD3-feeder). Nave CD8.sup.+ T cells were isolated from PBMCs by negative selection using a MACS system. CD14.sup.+ monocytes are isolated from PBMCs by positive selection using a MACS system. T cell subsets are cultured either in IMDM supplemented with 5% SVF, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES (IMDM-5 media) in hypoxia 2%.

(73) Freezing and Thawing of Cells

(74) Cells were frozen in FBS containing 10% DMSO. Cryo tubes were placed in CoolCell (Biocision) freezing containers and incubated at 80 C. After 2 days tubes were transferred to liquid nitrogen and stored until required. Thawing of cells was performed by placing cryo tubes in a 37 C. water bath for approximately 30 seconds. Next, cell suspension was mixed with equivalent volume of pre-warmed media and subsequently transferred to falcon tubes containing the same medium. Cells were pelleted by centrifugation (300 g, 3 min) to remove DMSO. The cell pellet was resuspended in cell culture medium

(75) Dendritic Cell Generation

(76) Monocytes were cultured in RPMI supplemented with 10% heat-inactivated Fetal Bovine Serum, 2 mM L-glutamine, 1% penicillin and streptomycin solution (RPMI medium), in presence of IL-4 (20 ng/ml) and GM-CSF (20 ng/ml). At day 6, DC were matured overnight in different cocktails: a (IL-1 (2 ng/ml) IL-6 (30 ng/ml), PGE2 (1 microg/ml) and TNF- (10 ng/ml), LPS 250 ng/ml, Poly I:C (150 ng/ml).

(77) In Vitro Generation of TAP-Inhibited Stimulator Cells for MLR Assay

(78) Matured DC, obtained as described above, are electroporated with 20 g of RNA synthesized from the pGem4Z vector containing the UL49.5 gene from BHV-1 (see, for example, Lampen et al. (2010) J Immunol. 185(11):6508-17).

(79) Induction and Expansion of Human Ag-Specific CD8 T Cells HLA-E Restricted

(80) TAP-inhibited mature DCs (TAP-mDC) were pulsed with 50 g/ml synthesized peptide. Then DCs were mixed with naive CD8 T cells at a ratio 1:10. IL-21 (30 ng/ml) was immediately added after the culture was initiated. After 3 days, half of medium were exchanged and 30 ng/ml IL-21, 20 ng/mL interleukin 15 (IL-15) and 500 ng/mL soluble, Fc-fused IL15-Receptor alpha (sIL15Ra-Fc, R&D Systems) were added. After 10 days of coculture, T cells were restimulated with peptides pulsed TAP-inhibited mature DCs in presence of IL-21, IL-15 and Fc-fused IL15-Receptor alpha. IL-2 (50 IU/ml) and IL-7 (10 ng/ml) were added 1 day after the second stimulation to further facilitate expansion of activated Ag-specific T cells. Peptide-specific expansion of T cells was monitored by flow cytometric analysis using MHC-peptide pentamer.

(81) Flow Cytometry Analysis

(82) T cells were transferred per v-bottomed 96-well, washed (300 g, 2 min) and stained in 100 L FACS buffer (PBS, 3% FBS, 2 mM EDTA) containing respective peptide-MHC pentamers (1:10, ProImmune) for 1 hour at 4 C. Cells were washed three times in FACS buffer and subjected to flow cytometric analysis.

(83) Appropriate isotype control Abs are used for each staining combination. Samples are acquired on a BD LSR FORTESSA flow cytometer using BD FACSDIVA 8.0.1 software (Becton Dickinson). Results are expressed in percentage (%) or in mean fluorescence intensity (MFI).

(84) Results

(85) Recent advances in the field of SIV vaccinology have highlighted the role of MHC-1b/E-restricted CD8.sup.+ T cell responses in controlling SIV infection in rhesus macaques, thereby raising the possibility that the adoptive transfer of HLA-E-restricted CD8.sup.+ T cells could be beneficial in controlling HIV-1 infection. The inventors thus established an experimental procedure to generate and expand autologous CD8.sup.+ T cell lines directed to peptide presented by HLA-E, using as HLA-E peptide, the CMV UL40-derived peptide (VMAPRTLIL (SEQ ID NO: 5)) and as stimulator cells, a TAP-inhibited mature DC. The use of VMAPRTLVL (SEQ ID NO: 6)-HLA-E pentamer allow to assess specific T cell expansion.

(86) As shown in FIG. 6, following two rounds of stimulation, 72% CD8.sup.+ T cells in culture were tetramer positive, suggesting that the inventors have developed a culture system that facilates the expansion and the generation of Ag-specific CD8.sup.+ T cells HLA-E restricted.

(87) Such ex vivo expanded cellular material represent per se an example of active principle for adoptive T cell therapy.

Example 3: Prophylactic Vaccine to SIV in Macaques

(88) The FIG. 7 is a s Schematic protocol of the combination that can be used for a prophylactic vaccination to SIV in macaques.

(89) Said protocol comprises: DNA vaccination: 2 intramuscular (i.m.) injections separated by two weeks of RhCMV/SIV vector (Hansen et al. (2013) Science 24; 340(6135):1237874; Hansen et al. (2016) Science; 351(6274), 714-20), Administration (i.v.) of pegylated IFN- and antibodies neutralizing IFN-, Rectal challenge: weekly rectal injection of suboptimal dose of SIVmac 239 (Hansen et al. (2013) Science 24; 340(6135):1237874; Hansen et al. (2016) Science; 351(6274), 714-20) up to acquisition of SIV infection determined as a plasma viral load of >30 copy eq/ml and/or development of an immune reaction to SIV Vif (Ag not included in the RhCMV/SIV vector).