Research ArticleDengue

Increased adaptive immune responses and proper feedback regulation protect against clinical dengue

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Science Translational Medicine  30 Aug 2017:
Vol. 9, Issue 405, eaal5088
DOI: 10.1126/scitranslmed.aal5088
  • Fig. 1. Characteristics of asymptomatic individuals and clinical dengue patients.

    (A) Viral load as measured by qRT-PCR (as viral RNA copies/ml plasma) and days of fever for all patients included in transcriptome analysis. (B to D) Percentages of cells subsets and ratio as determined by cell surface marker expression (ASD, n = 6; CD, n = 18). Bar represents median with IQR. P values were obtained with Mann-Whitney test.

  • Fig. 2. Transcriptional signatures discriminate between asymptomatic and clinical dengue patients.

    (A) Unsupervised hierarchically clustered heat map of the genes differentially expressed between asymptomatic dengue (n = 8) and clinical dengue patients (n = 25). (B) Top immunity-related molecular processes found as a result of GO enrichment analysis using GOrilla. The significance of the observed enrichment for GO molecular processes was estimated by the P value plotted on a log10 scale. The enrichment score reflects the strength of association between input gene expression and enriched molecular processes. (C) Immunity-related canonical pathways found as a result of pathway enrichment analysis using the IPA software. The significance of the association between gene expression and canonical pathways was estimated by the P value plotted on a log10 scale, and the ratio value reflects its strength. The z score reflects the activation state of the canonical pathway (activated in asymptomatic dengue: z score > 0; inhibited in asymptomatic dengue: z score < 0. (D) Venn diagram showing the number of overlapping significantly different genes between asymptomatic individuals versus clinical dengue patients, healthy controls versus clinical dengue patients, and viral load response.

  • Fig. 3. Serum cytokines related to innate immune response and inflammation are not associated with the clinical outcome of dengue infection.

    Serum concentrations of various cytokines measured by Luminex in asymptomatic dengue-infected individuals (n = 8) and clinical dengue patients (n = 58). Line represents median. P values were obtained with Mann-Whitney test.

  • Fig. 4. Asymptomatic viremic individuals show differentially regulated pathways and molecules related to antigen presentation.

    (A) Antigen-presentation pathway showing differentially expressed genes, adapted from IPA. Red, up-regulated in ASD; green, down-regulated in ASD; white, no change. (B) Representative histograms of HLA-DR and CD86 expression on CD14+ monocytes and LinCD11c+ dendritic cells of asymptomatic dengue-infected individuals (gray) and clinical dengue patients (white). Data are summarized on the right, where lines represent median and IQR (ASD, n = 6; CD, n = 18). MFI, mean fluorescence intensity. (C) Serum concentrations of IL-12 and IL-23 (pg/ml) measured by Luminex in asymptomatic dengue-infected individuals (n = 8) and clinical dengue patients (n = 58). Line represents median. P values were obtained with Mann-Whitney test.

  • Fig. 5. Increased T cell activation in asymptomatic viremic individuals.

    (A) PKCθ signaling in T lymphocytes, adapted from IPA. Red, up-regulated in ASD; green, down-regulated in ASD; white, no change. (B) Serum concentrations of IL-2 measured by Luminex in asymptomatic dengue-infected individuals (n = 8) and clinical dengue patients (n = 58). Line represents the median. (C) Representative dot plots of CD69 expression on both CD4+ and CD8+ T cells (ASD, n = 6; CD, n = 18). Data are summarized on the right, where lines represent the median and IQR. P values were obtained with Mann-Whitney test.

  • Fig. 6. Increased plasmablast differentiation in clinical dengue patients.

    (A) Ingenuity canonical pathways associated with B cell biology. Ratio indicates the strength of association between the pathway and the list of differentially expressed genes. The z score reflects the activation state of the canonical pathway (activated in asymptomatic dengue: z score > 0; inhibited in asymptomatic dengue: z score < 0). (B) Summary of genes associated with B cell biology. The adjusted P value was calculated using the Benjamini-Hochberg (BH) procedure. Log fold change (FC) indicates the log2 FC for the gene between asymptomatic dengue and clinical dengue infection. (C) Serum concentrations of IL-10 and IL-21 measured by Luminex in asymptomatic dengue-infected individuals (n = 8) and clinical dengue patients (n = 58). Line represents the median. (D) Left: Serum anti-DENV IgM as measured by MAC-ELISA, where optical density (OD) is reported. Right: HI test where the log2 of the maximum dilution preventing agglutination is shown. Patients are stratified according to immune status. Asymptomatic dengue-infected individuals (n = 8) and clinical dengue patients (n = 58). Sera were analyzed 8 ± 2 days after inclusion in the study. Line represents median. P values were obtained with Mann-Whitney test.

  • Table 1. Demographic data and clinical parameters of the studied populations.

    Patients are characterized according to the WHO 1997 criteria. DENV serotype and viral load were determined by qRT-PCR. Primary or secondary infection was determined by an HI test on acute and convalescent samples. N/A, not applicable; CD, clinical dengue; ASD, asymptomatic dengue-infected individuals; IQR, interquartile range; DF, dengue fever; DHF, dengue hemorrhagic fever; DSS, dengue shock syndrome.

    Asymptomatic dengueClinical dengue
    Total cohort
    n976
    Age (mean, SD)9 ± 48 ± 3
    Days of fever at inclusion (median, range)N/A4 (1–9)
    WHO 1997
    DFN/A28
    DHFN/A29
    DSSN/A19
    Viral load (median, IQR)9.5 × 103 (2.0 × 102–3.3 × 104)2.2 × 105 (2.6 × 104–4.6 × 107)
    Infecting dengue serotype
    DENV-189%88%
    DENV-20%11%
    DENV-30%0%
    DENV-411%1%
    Secondary infection44%88%
    Analysis 1: Gene expression (ASD versus CD)
    n825
    Age (mean, SD)9 ± 48 ± 3
    Days of fever at inclusion (median, range)N/A4 (1–9)
    WHO 1997
    DFN/A7
    DHFN/A7
    DSSN/A11
    Viral load (median, IQR)1.5 × 104 (3.7 × 102–3.6 × 104)3.9 × 104 (6.3 × 103–1.6 × 105)
    Infecting dengue serotype
    DENV-1100%100%
    DENV-20%0%
    DENV-30%0%
    DENV-40%0%
    Secondary infection50%100%
    Analysis 2: Serum cytokine and antibody concentrations
    n858
    Age (mean, SD)9 ± 48 ± 3
    Days of fever at inclusion (median, range)N/A3 (1–9)
    WHO 1997
    DFN/A21
    DHFN/A22
    DSSN/A15
    Viral load (median, IQR)1.5 × 104 (3.7 × 102–3.6 × 104)2.0 × 105 (2.5 × 105–2.0 × 107)
    Infecting dengue serotype
    DENV-1100%98%
    DENV-20%2%
    DENV-30%0%
    DENV-40%0%
    Secondary infection50%86%
    Analysis 3: Cellular phenotyping
    n618
    Age (mean, SD)8 ± 49 ± 3
    Days of fever at inclusion (median, range)N/A4 (1–6)
    WHO 1997
    DFN/A7
    DHFN/A7
    DSSN/A4
    Viral load (median, IQR)5.1 × 103 (1.3 × 102–1.8 × 105)1.4 × 106 (5.1 × 104–1.5 × 108)
    Infecting dengue serotype
    DENV-183%55%
    DENV-20%39%
    DENV-30%0%
    DENV-417%6%
    Secondary infection50%94%
    Analysis 4: Gene expression (viral load)
    n836
    Age (mean, SD)9 ± 48 ± 3
    Days of fever at inclusion (median, range)N/A3 (1–9)
    WHO 1997
    DFN/A13
    DHFN/A11
    DSSN/A12
    Viral load (median, IQR)1.5 × 104 (3.7 × 102–3.6 × 104)1.5 × 105 (1.6 × 104–3.6 × 106)
    Infecting dengue serotype
    DENV-1100%100%
    DENV-20%0%
    DENV-30%0%
    DENV-40%0%
    Secondary infection50%100%

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/405/eaal5088/DC1

    Fig. S1. Gating strategy identifying cell subsets.

    Fig. S2. Viral load in asymptomatic and clinical dengue patients.

    Fig. S3. Serum cytokines related to innate immune response and inflammation are not associated with the clinical outcome of DENV infection.

    Fig. S4. Serum cytokines related to innate immune responses and inflammation are not associated with the clinical outcome of dengue infection in individuals undergoing secondary DENV infection.

    Fig. S5. Asymptomatic viremic individuals undergoing secondary DENV infection show differentially regulated pathways and molecules related to antigen presentation.

    Fig. S6. Increased T cell activation in asymptomatic viremic individuals undergoing secondary DENV infection.

    Table S1. Differentially expressed genes obtained by LIMMA.

    Table S2. GO enrichment analysis using GOrilla.

    Table S3. Ingenuity canonical pathways analysis of pathways significantly enriched in asymptomatic and clinical dengue infection.

  • Supplementary Material for:

    Increased adaptive immune responses and proper feedback regulation protect against clinical dengue

    Etienne Simon-Lorière, Veasna Duong, Ahmed Tawfik, Sivlin Ung, Sowath Ly, Isabelle Casadémont, Matthieu Prot, Noémie Courtejoie, Kevin Bleakley, Philippe Buchy, Arnaud Tarantola, Philippe Dussart, Tineke Cantaert,* Anavaj Sakuntabhai*

    *Corresponding author. Email: anavaj.sakuntabhai{at}pasteur.fr (A.S.); tcantaert{at}pasteur-kh.org (T.C.)

    Published 30 August 2017, Sci. Transl. Med. 9, eaal5088 (2017)
    DOI: 10.1126/scitranslmed.aal5088

    This PDF file includes:

    • Fig. S1. Gating strategy identifying cell subsets.
    • Fig. S2. Viral load in asymptomatic and clinical dengue patients.
    • Fig. S3. Serum cytokines related to innate immune response and inflammation are not associated with the clinical outcome of DENV infection.
    • Fig. S4. Serum cytokines related to innate immune responses and inflammation are not associated with the clinical outcome of dengue infection in individuals undergoing secondary DENV infection.
    • Fig. S5. Asymptomatic viremic individuals undergoing secondary DENV infection show differentially regulated pathways and molecules related to antigen presentation.
    • Fig. S6. Increased T cell activation in asymptomatic viremic individuals undergoing secondary DENV infection.
    • Legends for tables S1 to S3

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Differentially expressed genes obtained by LIMMA.
    • Table S2 (Microsoft Excel format). GO enrichment analysis using GOrilla.
    • Table S3 (Microsoft Excel format). Ingenuity canonical pathways analysis of pathways significantly enriched in asymptomatic and clinical dengue infection.

    [Download Tables S1 to S3]

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