Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production

Abstract

Specific reduction in the intake of proteins or amino acids (AAs) offers enormous health benefits, including increased life span, protection against age-associated disorders, and improved metabolic fitness and immunity. Cells respond to conditions of AA starvation by activating the amino acid starvation response (AAR). Here, we showed that mimicking AAR with halofuginone (HF) enhanced the magnitude and affinity of neutralizing, antigen-specific antibody responses in mice immunized with dengue virus envelope domain III protein (DENVrEDIII), a potent vaccine candidate against DENV. HF enhanced the formation of germinal centers (GCs) and increased the production of the cytokine IL-10 in the secondary lymphoid organs of vaccinated mice. Furthermore, HF promoted the transcription of genes associated with memory B cell formation and maintenance and maturation of GCs in the draining lymph nodes of vaccinated mice. The increased abundance of IL-10 in HF-preconditioned mice correlated with enhanced GC responses and may promote the establishment of long-lived plasma cells that secrete antigen-specific, high-affinity antibodies. Thus, these data suggest that mimetics of AA starvation could provide an alternative strategy to augment the efficacy of vaccines against dengue and other infectious diseases.

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. (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 2 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. FACS plots (C and G) are representative of 2 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 2 experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two tailed unpaired student t test (B) and Mann-Whitney U test (D, E, F, H, I and 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⁺ andCD4⁺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/group from 2 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/group from 2 independent experiments.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001by 2-way ANOVA with Bonferroni post-hoc between DENVrEDIII+DMSO and DENVrEDIII+HF immunized groups(A), Mann-Whitney U test(B and C)

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

ELISA analysis of the amounts of IFN-γ, IL-12p40 and TNF-α produced bysplenocytes and lymph node cells from immunized mice after re-stimulation in vitro with DENVrEDIII for 72hr. Spleen and lymph nodes were collected from mice at 28^th day post-secondary immunization. Data are means ± SEM from 12 mice per treatment group from 2 independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two tailed unpaired student t test.

Fig. 4.. HF mediated AAR activation augments of antibody responses against DENVrEDIII.

(A) ELISA analysis of DENVrEDIII specific total IgG, IgG2a, IgG2b, and IgG1 in the serum of mice14 days after primary immunization and 28 days after secondary immunization. Data are means ± SEM of 12 mice/group from 2 independent experiments. (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 (RU_max) with the dissociation constant (C, upper) are representative of 2 independent experiments. Quantified avidity scores(C, lower) are means ± SEM from 2 independent experiments on pooled serum samples from 10 mice/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 titre (FRNT₅₀) data are means ± SEM of 10 mice from 2 independent experiments assayed in triplicate.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, by one-way ANOVA with Bonferroni post-hoc test (A), two tailed unpaired student t test(C) and Mann-Whitney U test (D).

Fig. 5.. HF enhances the antibody response to a tetravalent DEN vaccine.

(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 ± SEMof10 mice/group from 2 independent experiments. (B)DENV serotype neutralization assay on serum from tDENVrEDIII immunized mice. The 50% focus reduction neutralization titre (FRNT₅₀) data are means ± SEM of 10 mice from 2 independent experiments. *P<0.05, **P<0.01 by one-way ANOVA with Bonferroni post-hoc test (A) and Mann-Whitney U test (B).

Fig. 6.. HF pre-treatment in DENVrEDIII immunized mice enhances GC formation.

(A and B) Confocal microscopy imaging of the GL-7⁺(red), B220⁺(green) 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 2 independent experiments. Quantified data (B) are means ± SEM of 8 mice/condition from all experiments. (C) Flow cytometry analysis of lymph node GC-B cells frequency in DENVrEDIII immunized mice at the indicated time-points. Data are means ± SEM of 8 mice per group at each time-point from 2 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/group from 2 independent experiments. (E) qRT-PCR analysis of the indicated gene expression in lymph node cells restimulated with DENVrEDIII for 24hr. Heatmap of statistically significant changes are from the analysis of 10 biological replicates from 2 independent experiments. *P<0.05, **P< 0.01, ***P<0.001, ****P<0.0001by two tailed unpaired student t test (B and D) and 2-way ANOVA with Bonferroni Post-hoc test (C).