[show abstract][hide abstract] ABSTRACT: Many candidate vaccine strategies against human immunodeficiency virus (HIV) infection are under study, but their clinical development is lengthy and iterative. To accelerate HIV vaccine development optimised trial designs are needed. We propose a randomised multi-arm phase I/II design for early stage development of several vaccine strategies, aiming at rapidly discarding those that are unsafe or non-immunogenic.
We explored early stage designs to evaluate both the safety and the immunogenicity of four heterologous prime-boost HIV vaccine strategies in parallel. One of the vaccines used as a prime and boost in the different strategies (vaccine 1) has yet to be tested in humans, thus requiring a phase I safety evaluation. However, its toxicity risk is considered minimal based on data from similar vaccines. We newly adapted a randomised phase II trial by integrating an early safety decision rule, emulating that of a phase I study. We evaluated the operating characteristics of the proposed design in simulation studies with either a fixed-sample frequentist or a continuous Bayesian safety decision rule and projected timelines for the trial.
We propose a randomised four-arm phase I/II design with two independent binary endpoints for safety and immunogenicity. Immunogenicity evaluation at trial end is based on a single-stage Fleming design per arm, comparing the observed proportion of responders in an immunogenicity screening assay to an unacceptably low proportion, without direct comparisons between arms. Randomisation limits heterogeneity in volunteer characteristics between arms. To avoid exposure of additional participants to an unsafe vaccine during the vaccine boost phase, an early safety decision rule is imposed on the arm starting with vaccine 1 injections. In simulations of the design with either decision rule, the risks of erroneous conclusions were controlled <15%. Flexibility in trial conduct is greater with the continuous Bayesian rule. A 12-month gain in timelines is expected by this optimised design. Other existing designs such as bivariate or seamless phase I/II designs did not offer a clear-cut alternative.
By combining phase I and phase II evaluations in a multi-arm trial, the proposed optimised design allows for accelerating early stage clinical development of HIV vaccine strategies.
[show abstract][hide abstract] ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) infection is characterized by chronic immune activation and suppressed T-lymphocyte functions. Here we report that CD73, both a coactivator molecule of T cells and an immunosuppressive ecto-enzyme through adenosine production, is only weakly expressed by CD8(+) T cells of HIV-infected patients and only partially restored after successful antiviral treatment. CD73 expression on CD8(+) T cells correlates inversely with cell activation both ex vivo and in vitro. However, CD8(+) T cells from HIV controllers (HICs), which spontaneously control HIV replication, express CD73 strongly, despite residual immune activation. Finally, we demonstrate that CD73 is involved in the HIV-specific CD8(+) T-cell expansion. Thus, we show that CD73 is central to the functionality of HIV-specific CD8(+) T cells and that the preservation of HIV-specific CD73(+)CD8(+) T cells is a characteristic of HICs. These observations reveal a novel mechanism involved in the control of viral replication.
The Journal of Infectious Diseases 12/2013; · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: MicroRNAs (miRNAs) have emerged as critical regulators of gene expression within cells. One particular miRNA, miR-155 is highly expressed within lymphocytes (both B and T cells) and mediates a number of important roles. These include shaping the transcriptome of lymphoid cells that control diverse biological functions vital in adaptive immunity. The use of mice engineered to be deficient in miR-155, as well as the identification of endogenous targets of miR-155 in T-cells by transcriptome-wide analysis, has helped unravel the crucial role this miRNA plays in fine tuning the regulation of lymphocyte subsets such as B cells, CD8(+) and CD4(+) T cells ranging from Th1, Th2, Th17 and regulatory T cells. In this review, we summarize what we have learned about miR-155 in the regulation of lymphocyte responses at the cellular and molecular level and in particular, we focus on the recent findings that demonstrate miR-155 shapes the balance between tolerance and immunity. This article is protected by copyright. All rights reserved.
[show abstract][hide abstract] ABSTRACT: The heterogeneity of human regulatory T cells (Tregs) may explain the discrepancies between studies on Tregs in physiology and pathology. Contrasting effects of IL-7 on the expansion and survival of human Tregs were reported. Therefore, we investigated the effects of IL-7 on the phenotype and function of well-characterized populations of human Tregs. We show that IL-7 signals via the CD127 receptor on naive, memory, and activated memory Tregs sorted from the blood of healthy donors, but it does not affect their proliferation. In contrast, IL-7 affects their suppressive capacities differently. This effect was modest on naive Tregs but was dramatic (90%) on memory Tregs. We provide evidence that IL-7 exerts a synergistic effect through downmodulation of the ectoenzyme CD39, which converts ATP to ADP/AMP, and an increase in ATP receptor P2X7. Both effects lead to an increase in the ATP-mediated effect, tipping the balance to favor Th17 conversion. Using an IL-7 therapeutic study, we show that IL-7 exerts the same effects in vitro and in vivo in HIV-infected individuals. Globally, our data show that IL-7 negatively regulates Tregs and contributes to increase the number of tools that may affect Treg function in pathology.
The Journal of Immunology 08/2013; · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: We 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.
In a prospective multicentre trial, 68 healthy volunteers were randomised to receive, at weeks 0, 4 and 12, either a 0.5ml IM (500μg of each lipopeptide; 35 volunteers) dose or a 0.1ml ID (100μg 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.
Response 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.
Lipopeptide 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.
[show abstract][hide abstract] ABSTRACT: Background: Natural Killer (NK) cells are the major antiviral effector cell population of the innate immune system. It has been demonstrated that NK cell activity can be modulated by the interaction with dendritic cells (DC). The vaccine candidate Modified Vaccinia Ankara encoding an HIV polypeptide (MVAHIV), developed by the French National Agency for Research on AIDS (ANRS), has the ability to infect DC and to prime T cells. However, whether or not MVAHIV-infected DC are able to induce anti-HIV specific NK cell activity remains undetermined.
Methods: DC were infected by MVAHIV, or MVAWT vector as control, and co-cultured with autologous NK cells for 4 days. Then, NK cells were transferred to a plate containing recently infected DC or CD4+ T cells, and p24 production was determined at days 7 and 10 post-infection by ELISA test and flow cytometry. The implication of NK cell receptors NKG2D and NKp46, and membrane-bound IL-15 (mbIL-15) on MVA-infected DC, during the priming of NK cells was determined by using blocking mAbs.
Results: We found that NK cells primed by MVAHIV-infected DC are significantly better at controlling HIV-1 replication in autologous DC and CD4+ T cells as compared to those primed by MVAWT-infected DC. The specificity of anti-HIV NK cell activity was determined by measuring the NK cell activity against CMV-infected DC and target cells. In depth analysis of the priming showed that blockade of NKG2D and NKp46 during the priming induced decreased and increased anti-HIV-1 NK cell activity, respectively. Blockade of NKG2D during priming of NK cells by MVAHIV-infected DC endowed NK cells with a particular NK cell receptor repertoire, with a marked decreased expression of activating receptors, and resulted in lower expression of mbIL-15 on MVA-infected DC; whereas blockade of NKp46 resulted in increased expression of mbIL-15 on MVA-infected DC.
Conclusions: These data demonstrate that the MVAHIV vaccine candidate is able to induce a specific anti-HIV-1 NK cell activity following their interaction with MVAHIV-infected DC, and that the acquisition of such antiviral activity relies on a modulated crosstalk involving NKG2D and NKp46 on NK cells and the regulation of mbIL-15 on infected DC.
7th IAS Conference on HIV Pathogenesis, Treatment and Prevention; 07/2013
[show abstract][hide abstract] ABSTRACT: The Agence National de Recherche sur le SIDA et les hepatitis Lipo5 vaccine is composed by five long fragments of HIV proteins and was recently shown to induce in seronegative volunteers a CD4 T cell response largely dominated by the G2 fragment. To understand this response profile, we submitted the five HIV fragments to HLA-DR-binding assays and evaluated the frequency of naive Lipo5-specific CD4 T lymphocytes in the blood of 22 healthy individuals. We enumerated the Lipo5-specific T cell lines induced in vitro by weekly rounds of specific stimulation. Four peptides and hence not only G2 exhibited a broad specificity for HLA-DR molecules. In contrast, most of the T cell lines specific for Lipo5 reacted with G2, revealing a G2-specific T cell repertoire superior to 2 cells per million, whereas it is close to 0.4 for the other peptides. We also found good cross-reactivity of all the peptides with clade B and C variants and that G2 and P1 are able to recruit T cells that recognize HIV-infected cells. We therefore mainly observed very good concordance between the frequency to individual Lipo5 peptides among vaccinees in a large-scale vaccine trial and the distribution of peptide specificity of the in vitro induced T cell lines. These findings underline the role of the size of the epitope-specific naive repertoire in shaping the CD4 T cell response after vaccination and highlight the value of evaluating the naive repertoire to predict vaccine immunogenicity.
The Journal of Immunology 05/2013; · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: OBJECTIVE:: Targeting HIV antigens directly to dendritic cells (DCs) using monoclonal antibodies against cell surface receptors has been shown to evoke potent cellular immunity in animal models. The objective of this study was to configure an anti-human CD40 antibody fused to a string of 5 highly conserved CD4 and CD8 T cell epitope-rich regions of HIV-1 Gag, Nef and Pol (αCD40.HIV5pep), and then to demonstrate the capacity of this candidate therapeutic vaccine to target these HIV peptide antigens to human DCs to expand functional HIV-specific T cells. METHODS:: Antigen-specific cytokine production using intracellular flow cytometry and multiplex bead-based assay, and suppression of viral inhibition, were used to characterize the T cells expanded by αCD40.HIV5pep from HIV-infected individual PBMC and DC/T cell co-cultures. RESULTS:: This candidate vaccine expands memory CD4 and CD8 T cells specific to multiple epitopes within all 5 peptide regions across a wide range of MHC haplotypes from HIV-infected patient PBMC and DC/T cell co-cultures. These in vitro-expanded HIV antigen-specific CD4 and CD8 T cells produce multiple cytokines and chemokines. αCD40.HIV5pep-expanded CD8 T cells have characteristics of cytotoxic effector cells and are able to kill autologous target cells and suppress HIV-1 replication in vitro. CONCLUSION:: Our data demonstrate the therapeutic potential of this CD40-targeting HIV candidate vaccine in inducing a broad repertoire of multifunctional T cells in patients.
AIDS (London, England) 04/2013; · 4.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: The mechanisms by which Regulatory T cells suppress IL-2 production of effector CD4+ T cells in pathological conditions are unclear. A subpopulation of human Treg expresses the ectoenzyme CD39, which in association with CD73 converts ATP/ADP/AMP to adenosine. We show here that Treg/CD39+ suppress IL-2 expression of activated CD4+ T-cells more efficiently than Treg/CD39-. This inhibition is due to the demethylation of an essential CpG site of the il-2 gene promoter, which was reversed by an anti-CD39 mAb. By recapitulating the events downstream CD39/adenosine receptor (A2AR) axis, we show that A2AR agonist and soluble cAMP inhibit CpG site demethylation of the il-2 gene promoter. A high frequency of Treg/CD39+ is associated with a low clinical outcome in HIV infection. We show here that CD4+ T-cells from HIV-1 infected individuals express high levels of A2AR and intracellular cAMP. Following in vitro stimulation, these cells exhibit a lower degree of demethylation of il-2 gene promoter associated with a lower expression of IL-2, compared to healthy individuals. These results extend previous data on the role of Treg in HIV infection by filling the gap between expansion of Treg/CD39+ in HIV infection and the suppression of CD4+ T-cell function through inhibition of IL-2 production.
[show abstract][hide abstract] ABSTRACT: Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella β 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella β 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8(+) T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4(+) and CD8(+) T cell responses including cross-presentation by different human DC subsets. Brucella β 1,2 cyclic glucans increased the memory CD4(+) T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.
[show abstract][hide abstract] ABSTRACT: NK cells become activated during viral infection in response to cytokines or to engagement of NK cell activating receptors. However, the identity of cells sensing viral particles and mediating NK cell activation has not been defined. Here, we show that local administration of a Modified Vaccinia virus Ankara vaccine in mice results in the accumulation of NK cells in the subcapsular area of the draining lymph node and their activation, a process that is strictly dependent on type I IFN signaling. NK cells located in the subcapsular area exhibited reduced motility and were found associated with CD169(+) positive subcapsular sinus (SCS) macrophages and collagen fibers. Moreover, depletion of SCS macrophages using clodronate liposomes abolished NK cell accumulation and activation. Our results identify SCS macrophages as primary mediators of NK cell activation in response to lymph born viral particles suggesting that they act as early sensors of local infection or delivery of viral-based vaccines.
[show abstract][hide abstract] ABSTRACT: OBJECTIVE:: In immunocompromised patients, alternative schedules more immunogenic than the standard influenza vaccine regimen are necessary to enhance and prolong vaccine efficacy. We previously reported that the AS03A-adjuvanted 2009 A/H1N1 v vaccine yielded a higher short term immune response than the unadjuvanted one in HIV-1-infected adults. This study reports the long term persistence of the immune response. DESIGN AND METHODS:: In a prospective, multicenter, randomized, patient-blinded trial, two doses of AS03A-adjuvanted H1N1 v vaccine containing 3.75 μg hemagglutinin (n = 155; group A) or non-adjuvanted H1N1 v vaccine containing 15 μg hemagglutinin (n = 151; group B), were administered 21 days apart.Hemagglutination inhibition (HI) and neutralizing antibodies were assessed 6 and 12 months after vaccination. RESULTS:: In group A and B, the seroprotection rates were 83.7% and 59.4% at month 6, 70.4% and 49.3% at month 12, respectively. In a multivariate analysis, persistence of seroprotection 12 months after vaccination was negatively associated with current smoking (odds ratio [OR] = 0.6, p = 0.03) and positively related with the AS03A-adjuvanted H1N1 v vaccine (OR = 2.7, p = 0.0002). CONCLUSION:: In HIV-1-infected adults, two doses of adjuvanted influenza vaccine induce long term persistence of immune response up to one year after vaccination.
AIDS (London, England) 09/2012; · 4.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Activating mutations in NOTCH1, an essential regulator of T cell development, are frequently found in human T cell acute lymphoblastic leukemia (T-ALL). Despite important advances in our understanding of Notch signal transduction, the regulation of Notch functions in the nucleus remains unclear. Using immunoaffinity purification, we identified NOTCH1 nuclear partners in T-ALL cells and showed that, beyond the well-characterized core activation complex (ICN1-CSL-MAML1), NOTCH1 assembles a multifunctional complex containing the transcription coactivator AF4p12, the PBAF nucleosome remodeling complex, and the histone demethylases LSD1 and PHF8 acting through their demethylase activity to promote epigenetic modifications at Notch-target genes. Remarkably, LSD1 functions as a corepressor when associated with CSL-repressor complex and as a NOTCH1 coactivator upon Notch activation. Our work provides new insights into the molecular mechanisms that govern Notch transcriptional activity and represents glimpse into NOTCH1 interaction landscape, which will help in deciphering mechanisms of NOTCH1 functions and regulation.
[show abstract][hide abstract] ABSTRACT: The role of the thymus in the depletion or restoration of T-cell pool in HIV infection is still debatable. Studies are hampered by the lack of valuable tools to investigate thymic activity.
We have evaluated thymic activity using (18)F-fluorodeoxyglucose-positron emission tomography/computed tomography and molecular and phenotypic analyses of thymic precursors. Longitudinal analyses were performed in HIV-infected patients either treatment naive with indication to initiate combination antiretroviral therapy (c-ART) (n = 11) or stable under c-ART (n = 9).
Thymic standardized uptake value was significantly lower in c-ART-treated patients as compared with historical age-matched HIV-negative controls. In c-ART-naive patients, baseline thymic standardized uptake value correlated with T-cell repector excision circle levels and naive CD4+ T cells. These patients exhibited a high metabolic lymph node activity positively correlated to the percentage of activated HLA-DR+CD38+ T cells. Basal metabolic thymic activity predicts the gain in CD4+ T cells after c-ART initiation. A decrease of thymic activity, which paralleled circulating plasma IL-7 levels, was noted after c-ART initiation.
A metabolic thymic activity is detectable in c-ART naive and correlates with indirect phenotypic and molecular markers of thymic output. This activity may participate to the pool of peripheral naive CD4+ T cells and predicts the magnitude of T-cell reconstitution under treatment.
[show abstract][hide abstract] ABSTRACT: Notch proteins play an important role in embryonic development and cell-fate decisions. Notch influences also the activation and differentiation of peripheral T cells. Here, we investigated whether Notch signaling modulates the response of effector T cells to regulatory T (Treg) cells. Pre-exposure of CD4(+) CD25(-) effector T cells to the Notch ligands Delta-4 and Jagged-1, but not Delta-1, increases significantly effector T-cell sensitivity to Treg cell-mediated suppression through upregulation of TGF-βRII expression and increased levels of the phosphorylated form of the Smad 3 protein. This effect is relieved by anti-TGF-β Abs. We demonstrate that HES (hairy and enhancer of split), the main transcription factor downstream of Notch, induces strong transactivation of TGF-ßRII by binding the TGF-βRII promoter through its DNA-binding domain. Thus, the crosstalk between Notch and the TGF-β pathway leads to potentiation of the suppressive effect of Treg cells.
European Journal of Immunology 05/2012; 42(7):1796-803. · 4.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: The immune deficiency of human immunodeficiency virus (HIV) infection is not fully corrected with ARV therapy. Interleukin-7 (IL-7) can boost CD4 T-cell counts, but optimal dosing and mechanisms of cellular increases need to be defined.
We performed a randomized placebo-controlled dose escalation (10, 20 and 30 µg/kg) trial of 3 weekly doses of recombinant human IL-7 (rhIL-7) in ARV-treated HIV-infected persons with CD4 T-cell counts between 101 and 400 cells/µL and plasma HIV levels <50 copies/mL. Toxicity, activity and the impact of rhIL-7 on immune reconstitution were monitored.
Doses of rhIL-7 up to 20 µg/kg were well tolerated. CD4 increases of predominantly naive and central memory T cells were brisk (averaging 323 cells/µL at 12 weeks) and durable (up to 1 year). Increased cell cycling and transient increased bcl-2 expression were noted. Expanded cells did not have the characteristics of regulatory or activated T cells. Transient low-level HIV viremia was seen in 6 of 26 treated patients; modest increases in total levels of intracellular HIV DNA were proportional to CD4 T-cell expansions. IL-7 seemed to increase thymic output and tended to improve the T-cell receptor (TCR) repertoire in persons with low TCR diversity.
Three weekly doses of rhIL-7 at 20 µg/kg are well tolerated and lead to a dose-dependent CD4 T-cell increase and the broadening of TCR diversity in some subjects. These data suggest that this rhIL-7 dose could be advanced in future rhIL-7 clinical studies. Clinical Trials Registration: NCT0047732.
[show abstract][hide abstract] ABSTRACT: To determine the relationship between erythrocyte and plasma ribavirin concentrations in hepatitis C virus (HCV)/HIV-coinfected patients, and to correlate ribavirin exposure with early and sustained virological response (EVR and SVR) and haemoglobin level reductions.
Clinical and biological data from 68 HCV/HIV-coinfected patients were recorded at baseline, week 4 (W4), week 12 and at 24 weeks after completion of treatment. Plasma and erythrocyte ribavirin concentrations were determined 12 h after the final ribavirin dose (C(min)).
Erythrocyte ribavirin concentrations were 100-fold higher than plasma concentrations, with a significant relationship between them (P < 0.05). In patients with HCV genotype 1 or 4, a plasma ribavirin C(min) threshold of 1.95 mg/L at W4 tended to predict EVR [sensitivity 44%; specificity 87%; AUC 0.67 (95% CI 0.50-0.84)] and was predictive of SVR [sensitivity 58%; specificity 84%; AUC 0.71 (95% CI 0.51-0.90)]. Among patients with these HCV genotypes, an erythrocyte ribavirin C(min) threshold of 146 mg/L at W4 was found to be the best value for discriminating between responders and non-responders for both EVR [sensitivity 67%; specificity 75%; AUC 0.58 (95% CI 0.24-0.93)] and SVR [sensitivity 50%; specificity 80%; AUC 0.70 (95% CI 0.39-1.01)]. We also demonstrated a significant relationship between reduced haemoglobin levels and plasma ribavirin C(min) at W4 (P = 0.05).
Therapeutic drug monitoring may be useful for the management of anti-HCV treatment in HCV/HIV-coinfected patients.
Journal of Antimicrobial Chemotherapy 03/2012; 67(6):1449-52. · 5.34 Impact Factor