Research ArticleVACCINES

Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production

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Science Signaling  12 Nov 2019:
Vol. 12, Issue 607, eaav4717
DOI: 10.1126/scisignal.aav4717
  • Fig. 1 HF enhances antigen-specific T cell responses in vivo.

    (A) Experimental outline of HF or vehicle control DMSO treatment and DENV-2 envelope domain III (DENVrEDIII) protein immunization. i.p., intraperitoneally; s.c., subcutaneously. (B) Proliferation analysis by [3H]thymidine incorporation in antigen-specific T cells from splenocytes of mice 28 days after immunization that were restimulated with DENVrEDIII protein. Data are means ± SEM of 12 mice per treatment group from two independent experiments.(C to J) Flow cytometry analysis of DENVrEDIII-specific CD8+ (C to F) and CD4+ (G to J) T cell responses in blood (D and H), spleen (E and I), and lymph node (F and J) after treatment and immunization, as indicated. Fluorescence-activated cell sorting (FACS) plots (C and G) are representative of two independent experiments. Quantification of the percentage of IFN-γ–producing T cells (D to F and H to J) are means ± SEM of 12 mice per treatment group from two experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-tailed unpaired Student’s t test (B) and Mann-Whitney U test (D to F and H to J).

  • Fig. 2 HF increases the frequency of antigen-specific polyfunctional T cells in mice.

    (A) Flow cytometry analysis of antigen-specific CD8+IFN-γ+ and CD4+IFN-γ+ T cell kinetics in PBMCs of mice treated with DMSO or HF and immunized with DENV-2 envelope domain III after 14 and 28 days. Data are means ± SEM of 12 mice per group from two independent experiments. (B to D) Flow cytometry analysis of DENVrEDIII-specific polyfunctional CD8+ (B) and CD4+(C) T cells in blood, spleen, and lymph node of immunized mice. The frequency of double cytokine (IFN-γ and IL-2)–producing cells with means (bar) ± SEM (B and C) and pie charts of the frequency of all cytokine-producing T cells (D) are from 12 mice per group from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-way analysis of variance (ANOVA) with Bonferroni post hoc between DENVrEDIII + DMSO– and DENVrEDIII + HF–immunized groups (A), and Mann-Whitney U test (B and C).

  • Fig. 3 HF enhances secretion of multiple cytokines after DENVrEDIII immunization.

    Enzyme-linked immunosorbent assay (ELISA) analysis of the amounts of IFN-γ, IL-12p40, and TNF-α produced by splenocytes and lymph node cells from immunized mice after restimulation in vitro with DENVrEDIII for 72 hours. Spleen and lymph nodes were collected from mice at the 28th day after secondary immunization. Data are means ± SEM from 12 mice per treatment group from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-tailed unpaired Student’s t test.

  • Fig. 4 HF-mediated AAR activation augments the antibody responses against DENVrEDIII.

    (A) ELISA analysis of DENVrEDIII-specific total IgG, IgG2a, IgG2b, and IgG1 in the serum of mice 14 days after primary immunization and 28 days after secondary immunization. Data are means ± SEM of 12 mice per group from two independent experiments. OD, optical density; ns, not significant. (B and C) BIAcore SPR analysis of DENVrEDIII protein binding by pooled serum samples from mice preconditioned with HF or DMSO 28 days after immunization. Sensogram trace of the DENVrEDIII-specific antibody-binding affinity (B) and correlation of the maximal response unit (RUmax) with the dissociation constant (C) (top) are representative of two independent experiments. Quantified avidity scores (C) (bottom) are means ± SEM from two independent experiments on pooled serum samples from 10 mice per group assayed in triplicate. (D) DENV-2 virus neutralization assay on serum samples collected from mice 28 days after immunization. The 50% focus reduction neutralization titer (FRNT50) data are means ± SEM of 10 mice from two independent experiments assayed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA with Bonferroni post hoc test (A), two-tailed unpaired Student’s t test (C), and Mann-Whitney U test (D).

  • Fig. 5 HF enhances the antibody response to a tetravalent DENV.

    (A) ELISA of total IgG specific for all four DENV serotypes in the serum of mice immunized with a tetravalent combination of DENVrEDIII protein 14 days after primary immunization and 28 days after secondary immunization. Data are means ± SEM of 10 mice per group from two independent experiments. (B) DENV serotype neutralization assay on serum from tDENVrEDIII-immunized mice. The FRNT50 data are means ± SEM of 10 mice from two independent experiments. *P < 0.05 and **P < 0.01 by one-way ANOVA with Bonferroni post hoc test (A) and Mann-Whitney U test (B).

  • Fig. 6 HF pretreatment in DENVrEDIII-immunized mice enhances GC formation.

    (A and B) Confocal microscopy imaging of the GL7+ (red), B220+ (green), and IgG+ (blue) GC B cells in lymph node sections from mice treated with HF or DMSO and immunized with DENVrEDIII protein. Images (A) are representative of two independent experiments. Quantified data (B) are means ± SEM of eight mice per condition from all experiments. (C) Flow cytometry analysis of lymph node GC–B cell frequency in DENVrEDIII-immunized mice at the indicated time points. Data are means ± SEM of eight mice per group at each time point from two independent experiments. (D) ELISA analysis of IL-10 production by splenocytes and lymph node cells from mice 28 days after secondary immunization that were restimulated with DENVrEDIII. Data are means ± SEM of 10 mice per group from two independent experiments. (E) qRT-PCR analysis of the indicated gene expression in lymph node cells restimulated with DENVrEDIII for 24 hours. Heat maps of statistically significant changes are from the analysis of 10 biological replicates from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-tailed unpaired Student’s t test (B and D) and two-way ANOVA with Bonferroni post hoc test (C).

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/607/eaav4717/DC1

    Fig. S1. Expression of dengue envelope protein domain III (DENVrEDIII) from all four DENV serotypes.

    Fig. S2. HF activated the AAR in immunized mice.

    Fig. S3. HF increased the frequency of IL-2–producing CD4+ and CD8+ T cells in DENVrEDIII-immunized mice.

    Fig. S4. HF augments antigen-specific antibody responses to OVA antigen.

    Fig. S5. HF-mediated augmentation in antigen-specific antibody responses is GCN2 dependent.

    Fig. S6. HF enhances affinity of DENVrEDIII antigen–specific antibodies.

    Fig. S7. HF enhances GC formation in mice immunized with DENVrEDIII.

    Table S1. Gene-specific primers.

  • This PDF file includes:

    • Fig. S1. Expression of dengue envelope protein domain III (DENVrEDIII) from all four DENV serotypes.
    • Fig. S2. HF activated the AAR in immunized mice.
    • Fig. S3. HF increased the frequency of IL-2–producing CD4+ and CD8+ T cells in DENVrEDIII-immunized mice.
    • Fig. S4. HF augments antigen-specific antibody responses to OVA antigen.
    • Fig. S5. HF-mediated augmentation in antigen-specific antibody responses is GCN2 dependent.
    • Fig. S6. HF enhances affinity of DENVrEDIII antigen–specific antibodies.
    • Fig. S7. HF enhances GC formation in mice immunized with DENVrEDIII.
    • Table S1. Gene-specific primers.

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