Vaccine to pathogenic immune activation cells during infections

Abstract

In the present invention, the Applicant provides a novel method for preventing or treating an infectious disease in a subject in need thereof. In particular said method comprise the administration of a combination, pharmaceutical combination, medicament or kit-of-parts comprising a first part comprising a CD8 vaccine specific for at least one infectious disease-related antigen, optionally a second part comprising an interferon alpha blocking agent, and a third part comprising a type III interferon and/or an agent stimulating the production of type III interferon.

Claims

1. A method for prophylactically treating acquired immune deficiency syndrome (AIDS) in a subject in need thereof, comprising administering to the subject: 1) a CD8 therapeutic vaccine specific for at least one human immunodeficiency virus (HIV) antigen, 2) optionally interferon-alpha blocking agent, and 3) a type III interferon and/or an agent stimulating the production of type III interferon, wherein the type III interferon comprises at least one IFN-λ selected from the group consisting of IFN-λ1, IFN-λ2 IFN-λ3 and IFN-λ4, and wherein the agent stimulating the production of type III interferon comprises at least one TLR ligand, RIG-I ligand, and/or MDA5 ligand.

2. The method for prophylactically treating according to claim 1, wherein the interferon-alpha blocking agent is selected from the group of: an agent neutralizing circulating alpha interferon, an agent blocking interferon-alpha signaling, an agent depleting IFN-a producing cells, and/or an agent blocking IFN-a production, wherein the agent neutralizing circulating alpha interferon is selected from the group comprising active anti-IFN-a vaccine including antiferon or passive anti-IFN-a vaccine including anti-IFN-a antibodies or anti-IFN-a hyper-immune serum, 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, wherein the agent depleting IFN-a producing cells is an agent depleting plasmacytoid dendritic cells (pDCs), and wherein the agent blocking IFN-a production is an agent blocking the production of IFN-a by pDCs.

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

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

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

6. The method for prophylactically 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.

7. The method for prophylactically 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.

8. The method for prophylactically 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).

9. The method for prophylactically 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.

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

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

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

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

14. The method for prophylactically treating according claim 1, wherein the CD8 therapeutic vaccine is an active vaccine, and wherein the CD8 therapeutic vaccine is an ex vivo generated dendritic, natural killer or B cell population presenting MHC-1b/E-restricted and MHC-II restricted antigens, and wherein the MHC-lb/E-restricted antigen is an HIV antigen.

15. The method for prophylactically 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.sup.+ T cell population, and wherein the MHC-1b/E-restricted CD8.sup.+ T cell population recognizes an MHC-1b/E-restricted HIV antigen.

16. The method for prophylactically treating according to claim 1, wherein the HIV antigen is derived 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.

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

18. The method for prophylactically treating according to claim 1, wherein the HIV antigen is a 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) FIG. 1A. Antiviral activity of type I and type III interferons. Expression of ISGs in HepG2. HepG2 cells were treated with IFNα2a or IFNλ1-4 (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.−ΔΔct method as compared with non-treated cell control and using endogenous S14 mRNA level for normalization.

(2) FIG. 1B. Antiviral activity of type I and type III interferons. Antiviral activity of type I and III IFNs against EMCV. IFNα2a or IFNλ1/2/3/4 (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-α treatment without viral challenge was used as a baseline of the viability of the cells.

(3) FIG. 2. Anti-proliferative activity of type I and type III interferons against CD4.sup.+ T cells. CFSE-stained CD4+ T cells (10×10.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-α 2a, or IFNλ1 or IFNλ2 or IFNλ3 or IFNλ4. When indicated, anti-interferon type I receptor antibody was added. The percentage of CFSE dilution was evaluated by flow cytometry.

(4) FIG. 3. IFN-α2a but not IFN-type III induces the expression of ISGs in CD4.sup.+ T cells. CD4.sup.+ T cells were treated with IFNα2a or IFNλ1/2/3/4 (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.−ΔΔCt method as compared with non-treated cell control and using endogenous S14 mRNA level for normalization.

(5) FIG. 4. IFN-α2a 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 IFNλ-1, IFN-λ2, IFN-λ3, IFN-λ4, or IFN-α2a 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)

(6) FIG. 5. IFN-α2a but not IFN-type III increases CD38 expression in CD3/CD28 stimulated CD4.sup.+ T cells. CFSE-stained CD4.sup.+ T cells (4×10.sup.4/well) were cultured in 96 round-bottomed microwells in the presence of λCD3-feeder (4×10.sup.4/well) and plate-bound anti-CD3 mAb (2 μg/ml), soluble anti-CD28 mAb (2 μg/ml) with increasing dose of IFN-α2a 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.

(7) 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 μM 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+ T cells.

(8) FIG. 7. Schematic representation of the immunization protocol in macaques with RhCMV/SIV vectors.

(9) FIG. 8. Schematic representation of the oral immunization protocol in macaques with inactivated SIV and living Lactobacillus plantarum.

(10) FIG. 9. Schematic representation of the oral immunization protocol in BLT mice with inactivated HIV and living Lactobacillus plantarum.

(11) FIG. 10A. Comparison of CM CD8+ T cell distributions and serum IFN-α levels in HIV-1-infected subjects and critical pathogenic role of IFN-α in human HIV-1 infection. Comparison of CM CD8+ T cell distributions in HIV-1-infected subjects.

(12) FIG. 10B. Comparison of CM CD8+ T cell distributions and serum IFN-α levels in HIV-1-infected subjects and critical pathogenic role of IFN-α in human HIV-1 infection. Comparison of serum IFN-α levels in HIV-1-infected subjects.

(13) FIG. 10C. Comparison of CM CD8+ T cell distributions and serum IFN-α levels in HIV-1-infected subjects and critical pathogenic role of IFN-α in human HIV-1 infection. Relationship between CD8+CM frequency and serum IFN-α level in non-treated HIV patients (EC and pre-cART group).

(14) FIG. 11. Schematic representation of the oral immunization protocol in macaques with inactivated SIV and living Lactobacillus plantarum.

(15) FIG. 12. Schematic representation of the oral immunization protocol in BSLT mice with inactivated HIV and living Lactobacillus plantarum.

EXAMPLES

(16) 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

(17) Materials and Methods

(18) Human Cell Lines

(19) 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.

(20) Human Blood Sample

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

(22) Cell Purification and Culture

(23) 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 (ΔCD3 cells) are isolated from either fresh or frozen PBMCs. T cell-depleted accessory cells (ΔCD3 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 ΔCD3-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%.

(24) Freezing and Thawing of Cells

(25) 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

(26) Real-Time PCR for ISGs Detection

(27) HepG2 cells were seeded at a density of 2×10.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 μL. 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 ΔΔCt method. The geometric mean of the Ct values of the reference genes, S14, was used as a reference value.

(28) Virus Production

(29) 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 5,000×g for removal of cellular debris.

(30) Antiviral Assay

(31) Antiviral assays were done on HepG2 cells, which were seeded in DMEM supplemented with 10% FCS at a density of 1.5×10.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.

(32) Flow Cytometry Analysis

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

(34) 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).

(35) STAT1 Signaling Analysis:

(36) 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)

(37) CFSE Staining:

(38) CD4.sup.+ T cells were stained with 1 μM 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.

(39) 7-AAD Staining:

(40) Apoptosis of stimulated CFSE-labeled CD4.sup.+ T was determined using the 7-AAD assay. Briefly, cultured cells were stained with 20 μg/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.low/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.

(41) 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).

(42) Functional Assay

(43) T Cell Proliferation:

(44) 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.

(45) T Cell Activation:

(46) 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.

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

(48) CFSE-stained CD4.sup.+ T cells (5×10.sup.4/well) were cultured in 96 round-bottomed microwells in the presence of ΔCD3-feeder (1×10.sup.5/well) and plate-bound anti-CD3 Ab (2 μg/ml), soluble anti-CD28 mAb (2 μg/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.

(49) Allogeneic Mixed Lymphocyte Reaction:

(50) CFSE-stained CD4.sup.+ T cells (5×10.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.

(51) Stat1 Phosphorylation Analysis:

(52) CD4.sup.+ T cells were stimulated with IFN-λ1, IFN-λ2, IFN-λ3, IFN-λ4, or IFN-α2a (10 ng/ml) for 20 min, or were left unstimulated (control). Phosphorylated Stat1 levels was assessed by flow cytometry as described above.

(53) Results

(54) Type I interferons (IFN-α/β) and the more recently identified type III IFNs (IFN-λ) 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.

(55) 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).

(56) Antiviral Activities of Types I and III

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

(58) Briefly, the antiviral activity of type I and III was tested in HepG2 cells treated with IFN-α2a, IFNλ1, IFNλ2, IFNλ3 or IFNλ4 for 4 hours. Then the induction of the well-known interferon-stimulated genes (ISGs) MX1, IFIT1 and OASL was monitored by qPCR.

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

(60) 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.

(61) 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.

(62) As shown in FIG. 1B, IFN type III and IFN-α2a have intrinsic cellular antiviral activity and are able to fully protect HepG2 cells challenged with EMCV.

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

(64) 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.

(65) 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.

(66) As shown in FIG. 2, IFN-α2a 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.

(67) 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.

(68) Indeed, as shown in FIG. 3, ISGs are induced only in CD4.sup.+ T cells stimulated with IFN-α2a. Thus, IFN-α2a but not IFN-type III induce the expression of ISGs in CD4.sup.+ T cells.

(69) 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.

(70) As shown in FIG. 4, only IFN-α2a was able to stimulate the phosphorylation of Stat1 within CD4.sup.+ T cells. Therefore, IFN-α2a but not IFN-type III induces tyrosine phosphorylation of STAT1 in CD4.sup.+ T cells.

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

(72) Because chronic immune activation has been reasoned to be a significant contributor to disease progression in HIV-1-infected subjects, 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.

(73) As shown in FIG. 5, only IFN-α2a was able to enhance the expression of CD38 on stimulated CD4.sup.+ T cells.

(74) Collectively, these ex vivo experiments show that while exhibiting anti-viral activity, as does IFN-α, interferon type III, by contrast to the immunosuppressive IFN-α, 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-α2a.

(75) 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, interferons type III exert their antiviral effects in mucosal sites whereas IFN-α 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-α on activated CD4+ T cells.

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

(76) Materials and Methods

(77) Human Blood Sample.

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

(79) Cell Purification and Culture

(80) 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 (ΔCD3 cells) are isolated from either fresh or frozen PBMCs. T cell-depleted accessory cells (ΔCD3 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 ΔCD3-feeder). Naïve 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%.

(81) Freezing and Thawing of Cells

(82) 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

(83) Dendritic Cell Generation

(84) 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-1β (2 ng/ml) IL-6 (30 ng/ml), PGE2 (1 microg/ml) and TNF-α (10 ng/ml), LPS 250 ng/ml, Poly I:C (150 ng/ml).

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

(86) Matured DC, obtained as described above, are electroporated with 20 μg 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).

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

(88) TAP-inhibited mature DCs (TAP-mDC) were pulsed with 50 μg/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.

(89) Flow Cytometry Analysis

(90) T cells were transferred per v-bottomed 96-well, washed (300 g, 2 min) and stained in 100 μL 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.

(91) 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).

(92) Results

(93) 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 transfert 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-HLA-E pentamer (VMAPRTLVL, SEQ ID NO: 6) allows to assess specific T cell expansion.

(94) 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.

(95) 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 with Recombinant RhCMV/SIV Vectors

(96) The FIG. 7 is a schematic representation of the immunization protocol in macaques.

(97) Immunization Protocol (DNA Vaccination)

(98) The CD8 vaccine composition is in a form adapted to intramuscular administration and comprises RhCMV/SIV vectors (see Hansen et al. (2013) Science 24; 340(6135):1237874; Hansen et al. (2016) Science; 351(6274)). Chinese rhesus macaques receive 2 intramuscular (i.m.) injections of said CD8 vaccine composition at days 0 and 14.

(99) At days −7, −3, 3, 11, 38, 45, 52, 59, 66, 73 and 80, macaques receive (i.p.) injection of interferon lambda 1 (50-100 μg) and/or anti-IFN-α antibodies (PBL, 100 μg/kg).

(100) Pre-challenge

(101) Optionally, macaques receive intra-rectal injection of non-infectious dose of SIV or an attenuated SIV (e.g., SIV depleted in protein nef).

(102) Challenge

(103) At days 45, 52, 59, 66, 73, and 80 macaques receive intra-rectal injection of suboptimal dose of SIVmac 239.

(104) Acquisition of SIV infection is determined as a plasma viral load >30 copy eq/mL and/or development of an immune reaction to SIV Vif (i.e., an antigen not included in the RhCMV/SIV vector).

Example 4: Prophylactic Vaccine to SIV in Macaques with Inactivated SIV and Living Lactobacillus plantarum

(105) The FIG. 8 is a schematic representation of the oral immunization protocol in macaques.

(106) Oral Priming Immunization Protocol

(107) The CD8 vaccine composition is in a form adapted to intragastrically administration and comprises: 4×10.sup.7 copies/mL of an inactivated SIV and 3×10.sup.9 cfu/mL of living Lactobacillus plantarum in maltodextrin (20%) solution. Chinese rhesus macaques receive 30 mL of said CD8 vaccine composition and then 25 mL of the same composition every 30 min for 3 hours, at days 0, 1, 3, 5, 7 and 28, 29.

(108) At day −3 and before each series of immunizations at days 0, 28 and 29, macaques receive (i.p.) injections of a combination of different reagents according to the protocol as described hereinafter: Protocol 1: nothing Protocol 2: poly I:C (100 μg/kg) and interferon lambda 1 (50-100 μg) Protocol 3: polyclonal anti-IFN-α antibodies (PBL, 100 μg/kg) and poly I:C (100 μg/kg) Protocol 4: polyclonal anti-IFN-α antibodies (PBL, 100 μg/kg), poly I:C (100 μg/kg) and interferon lambda 1 (50-100 μg)

(109) At day 57, macaques receive injection of polyclonal anti-IFN-α antibodies (PBL, 100 μg/kg), poly I:C (100 μg/kg) and/or interferon lambda 1 (50-100 μg).

(110) Bosting Immunization (Pre-Challenge)

(111) At day 60, macaques receive intra-rectal injection of non-infectious dose of SIV or an attenuated SIV (e.g., SIV depleted in protein nef).

(112) Immune Response Analysis

(113) Anti-SIV immune response is analyzed at day 25, 57 and 80. The analysis of the anti-SIV immune response comprises the monitoring of: Plasma SIV IgM/IgG/IgA antibody titers, and/or SIV Gag specific CD8.sup.+ T cell and CD8.sup.+ T cell-mediated anti-viral activity.

(114) Challenge

(115) At day 90, macaques receive intra-rectal injection of an infectious dose of SIV only if an anti-SIV specific CD8.sup.+ suppressive T cells (as described in the present invention) is observed.

(116) After challenge, anti-SIV immune responses and plasma viremia are monitored every two weeks.

Example 5: Prophylactic Vaccine to HIV in BLT Mice

(117) The FIG. 9 is a schematic representation of the oral immunization protocol in BLT mice.

(118) BLT mice are valuable humanized models for the study of HIV infection. Indeed, BLT mice recapitulate important aspects of human immunity, including T cell immunity (Marshall E. Karpel et al., Curr Opin Virol. 2015 August; 13: 75-80).

(119) Oral Immunization Protocol

(120) The CD8 vaccine composition is in a form adapted to intragastrically administration and comprises: 4×10.sup.7 copies/mL of an inactivated HIV-1 and 3×10.sup.9 cfu/mL of living Lactobacillus plantarum in maltodextrin (20%) solution. BLT mice receive 0.2 mL of said CD8 vaccine composition and then 0.2 mL of the same composition every 30 min for 3 hours, at days 0, 1, 3, 5, 7 and 28, 29.

(121) At day −3 and before each series of immunizations at days 0, 28 and 29, BLT mice receive (i.p.) injections of a combination of different reagents according to the protocol as described hereinafter: Protocol 1: nothing Protocol 2: poly I:C (100 μg/kg) and interferon lambda 1 (50-100 μg) Protocol 3: polyclonal anti-IFN-α antibodies (PBL, 100 μg/kg) and poly I:C (100 μg/kg) Protocol 4: polyclonal anti-IFN-α antibodies (PBL, 100 μg/kg), poly I:C (100 μg/kg) and interferon lambda 1 (50-100 μg)

(122) At day 57, BLT mice receive injection of polyclonal anti-IFN-α antibodies (PBL, 100 μg/kg), poly I:C (100 μg/kg) and/or interferon lambda 1 (50-100 μg).

(123) Bosting Immunization (Pre-Challenge)

(124) At day 60, BLT mice receive intra-rectal injection of non-infectious dose of HIV-1 or an attenuated HIV-1 (e.g., HIV-1 depleted in protein nef).

(125) Immune Response Analysis

(126) Anti-HIV immune response is analyzed at day 25, 57 and 80. The analysis of the anti-SIV immune response comprises the monitoring of: Plasma HIV IgM/IgG/IgA antibody titers, and/or HIV Gag specific CD8.sup.+ T cell and CD8.sup.+ T cell-mediated anti-viral activity.

(127) Challenge

(128) At day 90, BLT mice receive intra-rectal injection of an infectious dose of HIV-1 only if an anti-HIV-1 specific CD8.sup.+ suppressive T cells (as described in the present invention) is observed.

(129) After challenge, anti-HIV-1 immune responses and plasma viremia are monitored every two weeks.

Example 6: Ex Vivo Generation and Expansion of Antigen (Ag) Specific CD8+ HLA-E Restricted T Cells Using HLA-E*01033-Transfected Derivative of K562 Cell Line

(130) Materials and Methods

(131) Human Blood Sample

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

(133) Cell Purification and Culture

(134) Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation on Ficoll-Hypaque (Pharmacia). PBMCs were used either as fresh cells or stored frozen in liquid nitrogen. Naïve CD8+ T cells were isolated from PBMCs by negative selection using a MACS system. T cell subsets were 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%.

(135) HLA-E*01033-transfected derivative of K562 (K562/HLA-E) cell line were maintained in RPMI 1640 medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 25 mM HEPES and antibiotics in presence of 0.4 mg/ml G-418 (Calbiochem, San Diego, Calif.). When used as antigen-presenting cells, pulsed K562/HLA-E cell line was irradiated at 80000 rad.

(136) Peptide-HLA Molecule Binding Assays

(137) Peptide binding was assessed by HLA-E stabilization assays using HLA-E*01033-transfected derivative of K562 (K562/HLA-E) cell lines. Briefly, cells were re-suspended in serum-free medium at 1×10.sup.6 cells/ml. Where appropriate, a peptide (see Table 1) was added. After overnight incubation at 26° C., cells were washed with PBS to remove free peptides. Next, HLA surface expression was monitored after staining with anti-HLA-E mAb. Analysis was done on a FACScalibur cytometer (BD Biosciences). Results are presented as the MFI of cells stained with anti-HLA-E mAb.

(138) Generation and Expansion of Peptide-Specific HLA-E Restricted CD8+ T Cell Lines

(139) Naïve CD8+ T were cultured in the presence of irradiated K562/HLA-E cell line as antigen-presenting cells (at 0.5×10.sup.6/ml) pulsed with the appropriate peptide (10 μM) in complete medium supplemented with IL-21 (30 ng/ml). After 3 days, half of medium was 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. Restimulation was done after 10 days with irradiated K562/HLA-E cells pulsed with the corresponding peptide 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. Then, cultures were stimulated weekly for 4-10 weeks. Peptide-specific expansion of T cells was monitored by flow cytometric analysis using a cytotoxic assay.

(140) Cytotoxicity Assay

(141) A cell-based Flow Cytometry assay was used to measured specific cytotoxic activity of the peptide-specific HLA-E restricted CD8+ T cell lines ex vivo generated. Briefly, CFSE labelled targets were incubated overnight at 26° C. (K562/HLA-E) in presence or absence of synthetic peptides and co-cultured with the CD8+ T cell lines for 5 hours at different ratios (10:1 and 1:1). Control tubes (target cells without CD8+ T cell lines) were also assayed to determine the spontaneous cell death. After 5 hours of co-culture, cells were stained with 7-AAD. For data analysis, the CFSE-positive target cells were examined for cell death by uptake of 7-AAD. CFSE and 7-AAD double positive cells were considered to be dead target cells. The percentage of specific cytotoxic activity was subsequently calculated using the following equation:
Cytotoxicity (%)=Target cell death−Spontaneous death×100−Spontaneous death×100
Results
HLA-E Expression on K562 Cell Line after Peptide Loading

(142) Untransfected K562 cells do not display surface expression of HLA-E as assessed by flow cytometry as well as HLA-E transfected K562 in the absence of peptide loading (see Table 1). After pulsing K562/HLA-E overnight at 26° C. with specific HLA-E peptide from different origin, HLA-E surface expression was induced (see Table 1).

(143) TABLE-US-00001 TABLE 1 Peptide-HLA molecule binding assay Cells Peptides MFI Untransfected Without peptide 1250 K562 HLA-E Transfected Without peptide 1400 K562 HLA-E Transfected HCMV derived peptide 6250 K562 VMAPRTLIL (SEQ ID NO: 5) HLA-E Transfected EBV derived peptide 5860 K562 SQAPLPCVL (SEQ ID NO: 14) HLA-E Transfected MBt derived peptide 7523 K562 VMATRRNVL (SEQ ID NO: 15) HLA-E Transfected HIV-1 Pol derived peptide 7320 K562 PEIVIYDYM (SEQ ID NO: 16) HLA-E Transfected HIV-1 Pol derived peptide 6580 K562 RIRTWKSLV (SEQ ID NO: 17) HLA-E Transfected HIV-1 Gag derived peptide 8240 K562 RMYSPVSIL (SEQ ID NO: 1) HLA-E Transfected HIV-1 Gag derived peptide 8530 K562 TALSEGATP (SEQ ID NO: 3)
Detection of Antigen Specific HLA-E Restricted CD8+ T Cells Using Cytotoxicity Assay

(144) Seeing cell proliferation in the long-term stimulated cultures, the inventors wanted to estimate their specific functional activity using cytotoxicity assay. K562/HLA-E peptide pulsed cells induced the activation and the expansion of antigen-specific CD8+ T cell lines, since cells that have proliferated exhibit a high cytotoxic response against the candidate antigens, while no significant activity against an irrelevant antigen (see Table 2).

(145) TABLE-US-00002 TABLE 2 specific cytotoxic activity of the expanded CD8+ T cells Peptide % cytotoxicity % cytotoxicity specific against the against an HLA-E relevant irrelevant restricted peptide peptide CD8+ T Ratio Ratio Ratio Ratio cell lines 10:1 1:1 10:1 1:1 HCMV derived 75 32 12 8 peptide VMAPRTLIL EBV derived 82 35 15 10 peptide SQAPLPCVL MBt derived 67 28 9 5 peptide VMATRRNVL HIV-1 Pol 87 35 17 8 derived peptide PEIVIYDYM HIV-1 Pol 73 28 16 10 derived peptide RIRTWKSLV HIV-1 Gag 82 33 15 9 derived peptide RMYSPVSIL HIV-1 Gag 86 32 17 10 derived peptide TALSEGATP

Example 7: Critical Pathogenic Role of IFN-α in Human HIV-1 Infection

(146) Material and Methods

(147) Cryopreserved PBMCs were thawed in RPMI 1640 with 10% fetal bovine serum (FBS) and washed in FACS buffer. Phenotypic staining was performed on 10.sup.6 cells by incubation with a viability marker (AmCyan live-dead kit from Invitrogen) and with antibodies conjugated to CD3, CD4, CD8, CD45RA, CCR7. Subsequently, cells were washed, fixed with 4% paraformaldehyde for 5 min, washed, and acquired with an AURORA cytometer (Cytek).

(148) Frozen serums were thawed at 4° C. and centrifuged at 4000 G for 10 min at 4° C. IFN-α serum concentrations were measured using the high sensitivity Simoa® technology (Digital ELISA technology) (Quanterix).

(149) Results

(150) Comparison of Central Memory (CM) CD8+ T Cell Distributions in HIV-1-Infected Subjects

(151) In study of chronically HIV-1-infected subjects, the following groups were studied:

(152) (i) elite controllers (EC) who naturally suppress HIV-1 in the absence of combined antiretroviral therapy treatment (c-ART)

(153) (ii) non-controllers before (pre-cART) and after cART (post-cART) treatment, and

(154) (iii) a cohort of age-matched healthy donor (HD) subjects.

(155) The relative frequencies of the CM populations within the CD8+ T cell compartments were evaluated in each of the subject groups.

(156) The gating strategy to define this subset is the following. Briefly, singlet cells were defined, followed by gating on lymphocytes and live cells. Among the live cells, CD3+T lymphocytes were identified, followed by the definition of CD8+ subpopulations. Subsequently, the expression of CD45RA and CCR7 was analyzed in the CD8+T lymphocytes. Central memory T cells (TCM) are CD45RA− CCR7+.

(157) FIG. 10A shows that the level of CM CD8+ cells was significantly lower in non-controllers before cART than in other groups. Moreover, combined antiretroviral therapy (cART) results in increase of CM CD8+ cells.

(158) Comparison of Serum IFN-α Levels in HIV-1-Infected Subjects

(159) Serum levels of IFN-α was measured in the 4 groups. FIG. 10B shows that the non-controller patients have significantly increased serum IFN-α levels before treatment compared with after treatment.

(160) IFN-α Inversely Correlates with the Percentage of CM CD8.sup.+ Cells in HIV-Infected Patients without Treatment

(161) In the population of HIV infected patients (EC pre-cART patients), the inventors explored the potential correlations between the level of CM CD8.sup.+ cells and the serum IFN-α levels. In this study, there was a significant negative correlation between the frequency of CM CD8.sup.+ cells and serum IFN-α levels (spearman correlation r=−0.667; p<0.005). This reflects the critical pathogenic effect of IFN-α on T cell proliferation in secondary organs (see FIG. 10C).

Example 8: Prophylactic Vaccine to SIV in Macaques with Inactivated SIV and Living Lactobacillus plantarum

(162) This protocol comprises two steps described below.

(163) FIG. 11 is a schematic representation of the protocol scheme of step 1.

(164) Step 1: Identification of the Most Efficient Regimen for Induction of Virus Specific CD8+ Suppressive T Cells in Rhesus Macaques (RM)

(165) Immunization protocol is based on the experimental work performed on Chinese rhesus macaques and described in Lu et al. (2012) Cell Rep. 2(6), 1736-46. This protocol is comprised of three steps:

(166) 1) oral priming with preparation containing inactivated SIVmac239, as activate principle, and living Lactobacillus plantarum (LP), as adjuvant,

(167) 2) oral boosting with the same preparation and

(168) 3) intrarectal boosting with non-infectious doses of living virus.

(169) Eight male RM, 2 RM of Chinese origin and 6 of Indian origin are included.

(170) Oral priming immunization is carried out on 5 consecutive days. Each day, monkeys are intragastrically administered 30 ml of a preparation containing 4×10.sup.7 copies/ml of inactivated SIV and 3×10.sup.9 cfu/ml of living LP in maltodextrin (20%) solution. Then they receive 25 ml of the same preparation intragastrically every 30 min for 3 hours.

(171) Oral boosting immunization is administered at day 28 and day 29 and if necessary at day 60 and day 61.

(172) Intrarectal boost is performed twice by a 2-week interval from day 90. Monkeys of Indian origin additionally receive an intraperitoneal (IP) injection of poly I:C and lambda IFN at day −2, day 3 during the oral priming and two days before each oral boosting, i.e. at day 26 and if necessary at day 58 and one day before IR boosts.

(173) The 6 RM of Indian origin are distributed in two groups (B and C) depending on when the anti-IFNα antibody is added during the immunization. For group B anti-IFNα antibody is administered at day 32, and if necessary at day 64, one day before each IR boosts. For group C, anti-IFNα antibody is administered only five days after the first IR boost. For all macaques, induction of virus specific CD8+ suppressive T cells is monitored at day 50 and if necessary at day 80 and at day 126. Plasma viral loads are followed weekly after the IR boosts.

(174) Step 2: Evaluation of the Vaccine Efficacy by Rectal Challenge with SIVmac239.

(175) Only monkeys who mount virus-specific CD8+ suppressive T cells are challenged intrarectally with high infectious dose of SIVmac239 (100,000 TCID50). Plasma viral loads are followed every week for 6 weeks.

Example 9: Prophylactic Vaccine to HIV in BSLT Mice

(176) This protocol comprises two steps described below.

(177) FIG. 12 is a schematic representation of the protocol scheme of step 1.

(178) Step 1: Identification of the Most Efficient Regimen for Induction of Anti-HIV-1 Specific CD8+ Suppressive T Cells in BSLT Mice

(179) Immunization protocol is based on the experimental work performed on Chinese rhesus macaques and described in Lu et al. (2012) Cell Rep. 2(6), 1736-46. This protocol is comprised of three steps:

(180) 1) oral priming via intragastric route with preparation containing inactivated HIV-1, as activate principle, and living Lactobacillus plantarum (LP), as adjuvant;

(181) 2) oral boosting via intragastric route with the same preparation;

(182) 3) intrarectal boosting with low doses of living virus.

(183) Four groups of 10 mice (A, B, C and D) are included. Group D is a control group to monitor the effectiveness of the challenge. Mice of this group D receive no immunization or is immunized with PBS.

(184) Oral priming immunization via intragastric route consists of daily oral intake of inactivated HIV-1 and living LP in maltodextrin (20%) solution over a period of 5 days. Each day, mice receive the same preparation intragastrically, 3 times every 30 minutes over 1 hour. Mice receive 200 mc1 of the preparation.

(185) Oral boosting immunization via intragastric route is administered at day 28 and day 29 and, if necessary, at day 60 and day 61.

(186) Intrarectal boost is performed twice by a 2-week interval at day 90 and day 104. Group B and C additionally receive an intraperitoneal injection of poly I:C and lambda IFN (100-500 mc1) at day −2, day 3 during the oral priming via intragastric route and two days before each oral boosting via intragastric route, i.e. at day 26 and, if necessary, at day 58 and one day before the IR boosts (day 89 and day 103).

(187) Group B and C differ depending on when the anti-IFNα antibody is added during the immunization. For group B anti-IFNα antibody is administered at day 32 during the oral boost via intragastric route and, if necessary, at day 64 during the oral boost via intragastric route. For group C anti-IFNα antibody is administered only five days after the IR boosts. For all mice, induction of virus-specific CD8+ suppressive T cells is monitored at day 50 and, if necessary, at day 80 and at day 126. Plasma viral loads are followed weekly after the IR boosts.

(188) Step 2: Evaluation of the Vaccine Effectiveness by Rectal Challenge with HIV-1

(189) Two months after the last antibody anti-IFNα administration, only mice which mount virus-specific CD8+ suppressive T cells are challenged intrarectally with high infectious dose of HIV-1 (100,000 TCID50). Plasma viral loads are followed every week for 6 weeks.