Identification of early gene expression profiles associated with long-lasting antibody responses to the Ebola vaccine Ad26.ZEBOV/MVA-BN-Filo

Summary: Ebola virus disease is a severe hemorrhagic fever with a high fatality rate. We investigate transcriptome profiles at 3 h, 1 day, and 7 days after vaccination with Ad26.ZEBOV and MVA-BN-Filo. 3 h after Ad26.ZEBOV injection, we observe an increase in genes related to antigen presentation, sensing, and T and B cell receptors. The highest response occurs 1 day after Ad26.ZEBOV injection, with an increase of the gene expression of interferon-induced antiviral molecules, monocyte activation, and sensing receptors. This response is regulated by the HESX1, ATF3, ANKRD22, and ETV7 transcription factors. A plasma cell signature is observed on day 7 post-Ad26.ZEBOV vaccination, with an increase of CD138, MZB1, CD38, CD79A, and immunoglobulin genes. We have identified early expressed genes correlated with the magnitude of the antibody response 21 days after the MVA-BN-Filo and 364 days after Ad26.ZEBOV vaccinations. Our results provide early gene signatures that correlate with vaccine-induced Ebola virus glycoprotein-specific antibodies

HIV Controllers Have Low Inflammation Associated with a Strong HIV-Specific Immune Response in Blood

International audienceHIV controllers (HIC) maintain control of HIV replication without combined antiretroviral treatment (cART). The mechanisms leading to virus control are not fully known. We used gene expression and cellular analyses to compare HIC and HIV-1-infected individuals under cART. In the blood, HIC are characterized by a low inflammation, a downmodulation of natural killer inhibitory cell signaling, and an upregulation of T cell activation gene expression. This balance that persists after stimulation of cells with HIV antigens was consistent with functional analyses showing a bias toward a Th1 and cytotoxic T cell response and a lower production of inflammatory cytokines. Taking advantage of the characterization of HIC based upon their CD8+ T lymphocyte capacity to suppress HIV-infection, we show here that unsupervised analysis of differentially expressed genes fits clearly with this cytotoxic activity, allowing the characterization of a specific signature of HIC. These results reveal significant features of HIC making the bridge between cellular function, gene signatures, and the regulation of inflammation and killing capacity of HIV-specific CD8+ T cells. Moreover, these genetic profiles are consistent through analyses performed from blood to peripheral blood mononuclear cells and T cells. HIC maintain strong HIV-specific immune responses with low levels of inflammation. Our findings may pave the way for new immunotherapeutic approaches leading to strong HIV-1-specific immune responses while minimizing inflammation.IMPORTANCE A small minority of HIV-infected patients, called HIV controllers (HIC), maintains spontaneous control of HIV replication. It is therefore important to identify mechanisms that contribute to the control of HIV replication that may have implications for vaccine design. We observed a low inflammation, a downmodulation of natural killer inhibitory cell signaling, and an upregulation of T-cell activation gene expression in the blood of HIC compared to patients under combined antiretroviral treatment. This profile persists following in vitro stimulation of peripheral blood mononuclear cells with HIV antigens, and was consistent with functional analyses showing a Th1 and cytotoxic T cell response and a lower production of inflammatory cytokines. These results reveal significant features of HIC that maintain strong HIV-specific immune responses with low levels of inflammation. These findings define the immune status of HIC that is probably associated with the control of viral load

P2X7 Receptor Inhibition Improves CD34 T-Cell Differentiation in HIV-Infected Immunological Nonresponders on c-ART

<div><p>Peripheral CD4+ T-cell levels are not fully restored in a significant proportion of HIV+ individuals displaying long-term viral suppression on c-ART. These immunological nonresponders (INRs) have a higher risk of developing AIDS and non-AIDS events and a lower life expectancy than the general population, but the underlying mechanisms are not fully understood. We used an <i>in vitro</i> system to analyze the T- and B-cell potential of CD34+ hematopoietic progenitor cells. Comparisons of INRs with matched HIV+ patients with high CD4+ T-cell counts (immune responders (IRs)) revealed an impairment of the generation of T-cell progenitors, but not of B-cell progenitors, in INRs. This impairment resulted in the presence of smaller numbers of recent thymic emigrants (RTE) in the blood and lower peripheral CD4+ T-cell counts. We investigated the molecular pathways involved in lymphopoiesis, focusing particularly on T-cell fate specification (Notch pathway), survival (IL7R-IL7 axis) and death (<i>Fas</i>, <i>P2X7</i>, <i>CD39/CD73</i>). <i>P2X7</i> expression was abnormally strong and there was no <i>CD73</i> mRNA in the CD34+ cells of INRs, highlighting a role for the ATP pathway. This was confirmed by the demonstration that <i>in vitro</i> inhibition of the P2X7-mediated pathway restored the T-cell potential of CD34+ cells from INRs. Moreover, transcriptomic analysis revealed major differences in cell survival and death pathways between CD34+ cells from INRs and those from IRs. These findings pave the way for the use of complementary immunotherapies, such as P2X7 antagonists, to restore T-cell lymphopoiesis in INRs.</p></div

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

International audienceOBJECTIVE: 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)-γ, tumour necrosis factor (TNF)-α, 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-γ, 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

Early initiation of combined antiretroviral therapy preserves immune function in the gut of HIV-infected patients

International audienceMassive loss of lamina propria CD4+ T cells, changes in the lymphatic architecture, and altered intestinal epithelial barrier leading to microbial translocation are the common features of HIV-1 infection and are not fully restored under combined antiretroviral therapy (cART). To better understand determinants of gut mucosal restoration, we have performed phenotypic and gene expression analyses of the gut from HIV-infected patients, naive or treated with cART initiated either at the early phase of the primary infection or later during the chronic phase. We found a depletion of T helper type 22 (Th22) and interleukin-17-producing cells in naive patients. These populations, except Th22 cells, were not restored under cART. Regulatory T cells/Th17 ratio was significantly increased in HIV-infected patients and was inversely correlated to the restoration of CD4+ T cells but not to gut HIV DNA levels. Gene profile analysis of gut mucosal distinguished two groups of patients, which fitted with the timing of cART initiation. In their majority early, but not later treated patients, exhibited conserved intestinal lymphoid structure, epithelial barrier integrity and dendritic cell maturation pathways. Our data demonstrate that early initiation of cART helps to preserve and/or restore lymphoid gut mucosal homeostasis and provide a rationale for initiating cART during the acute phase of HIV infection

Immune activation and inflammation in HIV-uninfected individuals and HIV-infected patients.

<p>(A) Percentage of CD38^high cells among CD8+ T lymphocytes (HIV-, <i>n</i> = 18; IRs, <i>n</i> = 16; INRs, <i>n</i> = 16). (B, C, D) Plasma concentrations of IL-6 (B), CRP (C) and sCD14 (D) in HIV-uninfected (HIV-, <i>n</i> = 3) and HIV-positive individuals (IRs, <i>n</i> = 15; INRs, <i>n</i> = 16). Bars indicate the mean and standard error. The Kruskal-Wallis test was used to assess differences between groups. NS for <i>P</i>>0.05, *<i>P</i><0.05, **<i>P</i><0.01.</p

Limiting dilution assays (LDAs) to determine the T-cell and B-cell differentiation potential of CD34+ cells.

<p>(A) LDA design. Conditions for determining the potential of CD34+ cells to generate T cells ^a and B cells ^b are shown. For further details, see the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005571#sec011" target="_blank">materials and methods</a>. (B, C) Cell cultures on D21 positive for T-cell precursors (B) defined as CD45RA^highCD7+CD5+CD1a+ cells, and B cells (C) defined as CD79a^intra+ cells. (D) Analysis of the T-cell potential of CD34+ cells. Each point on the graph represents the mean value from three independent experiments. (E) The presence of T-cell precursors and B cells was assessed with the ELDA webtool, applying the maximum likelihood method to the Poisson model. Mean values (min-max) for three experiments are indicated for each group and each set of conditions. NS for <i>P</i>>0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Analysis of IL7R polymorphisms and Notch activation in CD34+ cells from IRs and INRs.

<p>(A) <i>IL7RA</i> polymorphisms in HIV-infected patients. The allelic frequency of each SNP is shown. Fisher’s exact test was used to compare the distribution of SNPs between IRs (<i>n</i> = 9) and INRs (<i>n</i> = 10). NS for <i>P</i> >0.05. (B) Expression of the Notch target gene <i>HES1</i> in purified CD34+ cells from HIV-uninfected subjects (<i>n</i>≥3), IRs (<i>n</i> = 4) and INRs (<i>n</i> = 6) exposed overnight to IgG1-Fc (IgG), Delta-like 4 (DLL-4), and DLL-4 plus hIL-7 (DLL-4+IL7) (5 μg/mL for DLL-4 and 5 ng/mL for IL-7). The mean and standard error are shown. Kruskal-Wallis and Friedman tests were used to compare differences in mRNA levels between groups, for each set of conditions (unpaired data), and between sets of conditions for the same group (paired data), respectively. *<i>P</i><0.05, **<i>P</i><0.01.</p

Characteristics of the subjects included in the study.

<p>Characteristics of the subjects included in the study.</p

Transcriptomic analysis of CD34+ cells in HIV-infected IRs and INRs.

<p>(A) Genes differentially expressed in the CD34+ cells of IRs (<i>n</i> = 7) and INRs (<i>n</i> = 10) are shown. Each column represents an individual sample, and each row, an individual gene, with normalized expression level indicated on a color scale (red = upregulation, green = downregulation). (B) The top 5 biological functions in Ingenuity analysis, based on activation <i>z</i>-score, an algorithm predicting the degree of activation or inactivation of the genes of the group concerned. Rank in the top 5 is indicated by the number after the #. <i>P</i>-values are shown. Biological functions upregulated (positive <i>z</i>-score) in IR patients are shown in red, and biological functions downregulated in IR patients (negative <i>z</i>-score) are shown in green.</p