Mathieu Surenaud

Long-Term Specific Immune Responses Induced in Humans by a Human Immunodeficiency Virus Type 1 Lipopeptide Vaccine: Characterization of CD8+-T-Cell Epitopes Recognized

ABSTRACT We studied the effect of booster injections and the long-term immune response after injections of an anti-human immunodeficiency virus type 1 (HIV-1) lipopeptide vaccine. This vaccine was injected alone or with QS21 adjuvant to 28 HIV-uninfected volunteers. One month later, after a fourth injection of the vaccine, B- and T-cell anti-HIV responses were detected in >85% of the vaccinated volunteers. One year after this injection, a long-term immune response was observed in >50% of the volunteers. At this point, a positive QS21 effect was observed only in the sustained B-cell and CD4+-T-cell responses. To better characterize the CD8+-T-cell response, we used a gamma interferon enzyme-linked immunospot method and a bank of 59 HIV-1 epitopes. For the six most common HLA molecules (HLA-A2, -A3, -A11, -A24, -B7 superfamily, and -B8), an average of 10 (range, 3 to 15) HIV-1 epitopes were tested. CD8+-T-cell responses were evaluated according to the HLA class I molecules of the volunteers. Each assessment was based on 18 HIV-1 epitopes in average. We showed that 31 HIV-1 epitopes elicited specific CD8+-T-cell responses after vaccination. The most frequently recognized peptides were Nef 68-76 (-B7), Nef 71-79 (-B7), Nef 84-92 (-A11), Nef 135-143 (-B7), Nef 136-145 (-A2), Nef 137-145 (-A2), Gag 259-267 (-B8), Gag 260-268 (-A2), Gag 267-274 (-A2), Gag 267-277 (-B7), and Gag 276-283 (A24). We found that CD8+-T-cell epitopes were induced at a higher number after a fourth injection (P < 0.05 compared to three injections), which indicates an increase in the breadth of HIV CD8+-T-cell epitope recognition after the boost.

Immunogenicity and safety of an HIV-1 lipopeptide vaccine in healthy adults: a phase 2 placebo-controlled ANRS trial

Background:French National Agency for Research on AIDS and Viral Hepatitiss HIV-LIPO-5 vaccine includes five HIV-1 peptides, containing multiple CD8+ and CD4+ T-cell epitopes and coupled to a palmitoyl tail. Whether HIV-LIPO-5 immunogenicity varies with the dose is unknown. Methods:HIV-negative volunteers were randomized to receive HIV-LIPO-5 vaccine at 50 μg/lipopeptide (N = 32), 150 μg/lipopeptide (N = 32), 500 μg/lipopeptide (N = 33) or placebo (N = 34) at weeks 0, 4, 12 and 24. HIV-1-specific CD8+ (interferon-γ ELISpot on peripheral blood mononuclear cells cultured for 12 days) and CD4+ responses (peripheral blood mononuclear cell lymphoproliferation) were assessed at baseline, after each injection and at week 48. Results:Local reactions were dose-dependent but no differences in systemic reactions appeared between groups. Sustained (at least on two separate occasions) CD8+ response rates to at least one given HIV-1 pool were obtained in 22 of 32 (69%), 21 of 33 (64%) and 21 of 34 (62%) individuals for LIPO-5 50, 150 and 500 groups, respectively (P ≤ 0.0001 for all comparisons to the placebo). Cumulative CD4+ response rates were obtained in 15 of 32 (47%), 18 of 33 (55%) and 15 of 34 (44%) individuals (P < 0.0001 for all comparisons to placebo). At week 48, CD8+ responses persisted in 47 of 91 (52%) HIV-LIPO-5 recipients. Conclusion:Doses of 50, 150 and 500 μg of French National Agency for Research on AIDS and Viral Hepatitiss HIV-LIPO-5 vaccine were able to elicit HIV-specific sustained CD8+ and CD4+ T-cell responses in healthy adults. Safety is good and all doses appear appropriate in further ‘prime-boost’ trials.

Cellular immune responses induced with dose-sparing intradermal administration of HIV vaccine to HIV-uninfected volunteers in the ANRS VAC16 trial.

Objective The objective was to compare the safety and cellular immunogenicity of intradermal versus intramuscular immunization with an HIV-lipopeptide candidate vaccine (LIPO-4) in healthy volunteers. Methodology A randomized, open-label trial with 24 weeks of follow-up was conducted in France at six HIV-vaccine trial sites. Sixty-eight healthy 21– to 55–year-old HIV-uninfected subjects were randomized to receive the LIPO-4 vaccine (four HIV lipopeptides linked to a T-helper–stimulating epitope of tetanus-toxin protein) at weeks 0, 4 and 12, either intradermally (0.1 ml, 100 µg of each peptide) or intramuscularly (0.5 ml, 500 µg of each peptide). Comparative safety of both routes was evaluated. CD8+ T-cell immune responses to HIV epitopes (ELISpot interferon-γ assay) and tetanus toxin-specific CD4+ T-cell responses (lymphoproliferation) were assessed at baseline, two weeks after each injection, and at week 24. Results and Conclusion No severe, serious or life-threatening adverse events were observed. Local pain was significantly more frequent after intramuscular injection, but local inflammatory reactions were more frequent after intradermal immunization. At weeks 2, 6, 14 and 24, the respective cumulative percentages of induced CD8+ T-cell responses to at least one HIV peptide were 9, 33, 39 and 52 (intradermal group) or 14, 20, 26 and 37 (intramuscular group), and induced tetanus toxin-specific CD4+ T-cell responses were 6, 27, 33 and 39 (intradermal), or 9, 46, 54 and 63 (intramuscular). In conclusion, intradermal LIPO-4 immunization was well tolerated, required one-fifth of the intramuscular dose, and induced similar HIV-specific CD8+ T-cell responses. Moreover, the immunization route influenced which antigen-specific T-cells (CD4+ or CD8+) were induced. Trial Registration ClinicalTrials.gov NCT00121121

Long-term CD4(+) and CD8(+) T-cell responses induced in HIV-uninfected volunteers following intradermal or intramuscular administration of an HIV-lipopeptide vaccine (ANRS VAC16).

BACKGROUNDnWe have shown that the intradermal (ID) administration of an HIV-1 lipopeptide candidate vaccine (LIPO-4) is well tolerated in healthy volunteers, with one fifth the IM dose delivered by this route inducing HIV-1-specific CD8(+) T-cell responses of a magnitude and quality similar to those achieved by IM administration. In this long-term follow-up, we aimed to investigate the sustainability and epitopic breadth of the immune responses induced.nnnMETHODSnIn a prospective multicentre trial, 68 healthy volunteers were randomised to receive, at weeks 0, 4 and 12, either a 0.5 ml IM (500 μg of each lipopeptide; 35 volunteers) dose or a 0.1 ml ID (100 μg of each lipopeptide; 33 volunteers) dose of the LIPO-4 vaccine, in the deltoid region of the non-dominant arm. All 68 volunteers received the first two vaccinations, and 44 volunteers in the ID group and 22 in the IM group received the third. We describe here the long-term CD8(+) and CD4(+) T-cell immune responses, up to 48 weeks after the first immunisation.nnnRESULTSnResponse frequency was highest at week 14 for CD4(+) T cells, at 85% (28/33) for the IM group and 61% (20/33) for the ID group (p=0.027), and at week 48 for CD8(+) T cells, at 36% (12/33) for the ID group and 31% (11/35) for the IM group (p=0.67). Response rates tended to be lower for volunteers receiving the third vaccination boost, whether IM or ID. Finally, we also observed a striking change in the specificity of the CD8(+) T-cell responses induced shortly (2 weeks) or several months (48 weeks) after LIPO-4 vaccination.nnnCONCLUSIONnLipopeptide vaccines elicited sustainable CD4(+) and CD8(+) T-cell responses, following IM or ID administration. CD8(+) T-cell responses had shifted and expanded to different epitopes after one year of follow-up. These results should facilitate the design of the next generation of prime-boost trials with repeated doses of lipopeptide vaccines.

Cytokine and gene transcription profiles of immune responses elicited by HIV lipopeptide vaccine in HIV-negative volunteers.

Objective:To dissect the biological mechanisms involved in the cellular responses to a candidate vaccine containing 5 HIV peptides coupled to a palmytoil tail (HIV-LIPO-5) in healthy volunteers, by using extensive immunogenicity assessments with different stimulation durations. Design:Immunogenicity substudy of a randomized phase II prophylactic HIV vaccine trial (ANRS VAC 18). Methods:HIV-LIPO-5 or placebo was administered at W0, W4, W12 and W24. Peripheral blood mononuclear cells from a subset of participants at W0 and W14 were stimulated with HIV-LIPO-5, Gag peptides contained in the vaccine and control peptides. ELISpot, lymphoproliferation, intracellular cytokine staining (ICS), cytokine multiplex and transcriptomic analyses were performed. Different time points and stimulation conditions were compared, controlling for test multiplicity. Results:Cultured ELISpot and lymphoproliferation responses were detected at W14. Ex-vivo ICS showed mainly interleukin (IL)-2-producing cells. Secretion of interferon (IFN)-&ggr;, tumour necrosis factor (TNF)-&agr;, IL-5 and IL-13 increased significantly after culture and Gag stimulation at W14 compared to W0. Metallothionein genes were consistently overexpressed after HIV-LIPO-5 stimulation at W0 and W14. At W14, significant probes increased substantially, including IFN-&ggr;, CXCL9, IL2RA, TNFAIP6, CCL3L1 and IL-6. Canonical pathway analyses indicated a role of interferon signalling genes in response to HIV-LIPO-5. Conclusion:HIV-LIPO-5 vaccination elicited Th1 and Th2 memory precursor responses and a consistent modulation in gene expression. The response profile before vaccination suggests an adjuvant effect of the lipid tail of HIV-LIPO-5. Our combined immunogenicity analyses allowed to identify a specific signature profile of HIV-LIPO-5 and indicate that HIV-LIPO-5 could be further developed as a prime in heterologous prime-boost strategies.

Memory CD8(+) T cells elicited by HIV-1 lipopeptide vaccines display similar phenotypic profiles but differences in term of magnitude and multifunctionality compared with FLU- or EBV-specific memory T cells in humans.

Differentiation marker, multifunctionality and magnitude analyses of specific-CD8(+) memory T cells are crucial to improve development of HIV vaccines designed to generate cell-mediated immunity. Therefore, we fully characterized the HIV-specific CD8(+) T cell responses induced in volunteers vaccinated with HIV lipopeptide vaccines for phenotypic markers, tetramer staining, cytokine secretion, and cytotoxic activities. The frequency of ex vivo CD8(+) T cells elicited by lipopeptide vaccines is very rare and central-memory phenotype and functions of these cells were been shown to be important in AIDS immunity. So, we expanded them using specific peptides to compare the memory T cell responses induced in volunteers by HIV vaccines with responses to influenza (FLU) or Epstein Barr virus (EBV). By analyzing the differentiation state of IFN-γ-secreting CD8(+) T cells, we found a CCR7(-)CD45RA(-)CD28(+int)/CD28(-) profile (>85%) belonging to a subset of intermediate-differentiated effector T cells for HIV, FLU, and EBV. We then assessed the quality of the response by measuring various T cell functions. The percentage of single IFN-γ T cell producers in response to HIV was 62% of the total of secreting T cells compared with 35% for FLU and EBV, dual and triple (IFN-γ/IL-2/CD107a) T cell producers could also be detected but at lower levels (8% compared with 37%). Finally, HIV-specific T cells secreted IFN-γ and TNF-α, but not the dual combination like FLU- and EBV-specific T cells. Thus, we found that the functional profile and magnitude of expanded HIV-specific CD8(+) T precursors were more limited than those of to FLU- and EBV-specific CD8(+) T cells. These data show that CD8(+) T cells induced by these HIV vaccines have a similar differentiation profile to FLU and EBV CD8(+) T cells, but that the vaccine potency to induce multifunctional T cells needs to be increased in order to improve vaccination strategies.

Absence of the Adaptor Protein PEA-15 Is Associated with Altered Pattern of Th Cytokines Production by Activated CD4+ T Lymphocytes In Vitro, and Defective Red Blood Cell Alloimmune Response In Vivo.

TCR-dependent and costimulation signaling, cell division, and cytokine environment are major factors driving cytokines expression induced by CD4+ T cell activation. PEA-15 15 (Protein Enriched in Astrocyte / 15kDa) is an adaptor protein that regulates death receptor-induced apoptosis and proliferation signaling by binding to FADD and relocating ERK1/2 to the cytosol, respectively. By using PEA-15-deficient mice, we examined the role of PEA-15 in TCR-dependent cytokine production in CD4+ T cells. TCR-stimulated PEA-15-deficient CD4+ T cells exhibited defective progression through the cell cycle associated with impaired expression of cyclin E and phosphoRb, two ERK1/2-dependent proteins of the cell cycle. Accordingly, expression of the division cycle-dependent cytokines IL-2 and IFNγ, a Th1 cytokine, was reduced in stimulated PEA-15-deficient CD4+ T cells. This was associated with abnormal subcellular compartmentalization of activated ERK1/2 in PEA-15-deficient T cells. Furthermore, in vitro TCR-dependent differentiation of naive CD4+ CD62L+ PEA-15-deficient T cells was associated with a lower production of the Th2 cytokine, IL-4, whereas expression of the Th17-associated molecule IL4I1 was enhanced. Finally, a defective humoral response was shown in PEA-15-deficient mice in a model of red blood cell alloimmunization performed with Poly IC, a classical adjuvant of Th1 response in vivo. Collectively, our data suggest that PEA-15 contributes to the specification of the cytokine pattern of activated Th cells, thus highlighting a potential new target to interfere with T cell functional polarization and subsequent immune response.

Serum suppression of tumorigenicity 2 level is an independent predictor of all-cause mortality in HIV-infected patients

OBJECTIVEnTo evaluate the predictive value of Soluble Suppression of Tumorigenicity 2 (sST2), a decoy receptor of IL-33 involved in several inflammatory and immune diseases, for death in HIV infection.nnnDESIGNnPatients enrolled in the ANRS CO3 Aquitaine Cohort, a prospective hospital-based cohort of HIV-1-infected patients, who had a plasma sample available in the bio-bank were systematically eligible.nnnMETHODSnsST2, sCD14 and IL6 were measured using Luminex® multiplex bead-based technology (R&D Systems) and a Bio-Plex 200 instrument (BioRad). Predictive capacities of sST2, sCD14, IL6 and of the Veteran Aging Cohort Study (VACS) clinical score at baseline on overall mortality were compared using multivariable Cox proportional hazards models.nnnRESULTSnDuring a median follow-up of 7.2 years (IQR: 6.0; 7.9), 93 deaths from all causes (incidence rate 9.9 per 1000 patient-years; 95% confidence interval 7.9; 11.9) were reported in 1,414 patients. The median sST2 baseline concentration was 22.9u200ang/mL (IQR: 17.7; 30.3) and was higher (30.8u200ang/mL, IQR: 21.5; 42.1) in patients who died as compared to those who stayed alive (22.6u200ang/mL; IQR: 17.5; 29.6) (pu200a<u200a10). An increased risk of death of 21% for a concentration 10.0u200ang/mL higher of sST2 remained after adjustment for sCD14, IL6 and VACS score (adjusted hazard ratio: 1.21; pu200a<u200a10). The predictive capacity of sST2 was confirmed in a validation cohort (nu200a=u200a386, 31 deaths) with an improved area c-index from 0.804 without sST2 to 0.811 with sST2.nnnCONCLUSIONnsST2 is a new valuable biomarker to evaluate the risk of all-cause mortality in HIV disease.Objective: To evaluate the predictive value of soluble suppression of tumorigenicity 2 (sST2), a decoy receptor of IL-33 involved in several inflammatory and immune diseases, for death in HIV infection. Design: Patients enrolled in the ANRS CO3 Aquitaine Cohort, a prospective hospital-based cohort of HIV-1-infected patients, who had a plasma sample available in the biobank were systematically eligible. Methods: sST2, soluble CD14 (sCD14) and IL-6 were measured using Luminex multiplex bead-based technology (R&D Systems) and a Bio-Plex 200 instrument (BioRad). Predictive capacities of sST2, sCD14, IL-6 and of the Veterans Aging Cohort Study clinical score at baseline on overall mortality were compared using multivariable Cox proportional hazards models. Results: During a median follow-up of 7.2 years [interquartile range (IQR): 6.0; 7.9], 93 deaths from all causes (incidence rate 9.9 per 1000 patient-years; 95% confidence interval 7.9–11.9) were reported in 1414 patients. The median sST2 baseline concentration was 22.9u200ang/ml (IQR: 17.7; 30.3) and was higher (30.8u200ang/ml, IQR: 21.5; 42.1) in patients who died as compared with those who stayed alive (22.6u200ang/ml; IQR: 17.5; 29.6) (Pu200a<u200a10−4). An increased risk of death of 21% for a concentration 10.0u200ang/ml higher of sST2 remained after adjustment for sCD14, IL-6 and Veterans Aging Cohort Study score (adjusted hazard ratio: 1.21; Pu200a<u200a10−4). The predictive capacity of sST2 was confirmed in a validation cohort (nu200a=u200a386, 31 deaths) with an improved area c-index from 0.804 without sST2 to 0.811 with sST2. Conclusion: sST2 is a new valuable biomarker to evaluate the risk of all-cause mortality in HIV disease.