Identification and Characterization of Stimulator of Interferon Genes As a Robust Adjuvant Target for Early Life Immunization

Abstract

Immunization is key to preventing infectious diseases, a leading cause of death early in life. However, due to age-specific immunity, vaccines often demonstrate reduced efficacy in newborns and young infants as compared to adults. Here, we combined in vitro and in vivo approaches to identify adjuvant candidates for early life immunization. We employed newborn and adult bone marrow-derived dendritic cells (BMDCs) to perform a screening of pattern recognition receptor agonists and found that the stimulator of interferon genes ligand 2'3'-cGAMP (hereafter cGAMP) induces a comparable expression of surface maturation markers in newborn and adult BMDCs. Then, we utilized the trivalent recombinant hemagglutinin (rHA) influenza vaccine, Flublok, as a model antigen to investigate the role of cGAMP in adult and early life immunization. cGAMP adjuvantation alone could increase rHA-specific antibody titers in adult but not newborn mice. Remarkably, as compared to alum or cGAMP alone, immunization with cGAMP formulated with alum (Alhydrogel) enhanced newborn rHA-specific IgG2a/c titers ~400-fold, an antibody subclass associated with the development of IFNγ-driven type 1 immunity in vivo and endowed with higher effector functions, by 42 days of life. Highlighting the amenability for successful vaccine formulation and delivery, we next confirmed that cGAMP adsorbs onto alum in vitro. Accordingly, immunization early in life with (cGAMP+alum) promoted IFNγ production by CD4⁺ T cells and increased the proportions and absolute numbers of CD4⁺ CXCR5⁺ PD-1⁺ T follicular helper and germinal center (GC) GL-7⁺ CD138⁺ B cells, suggesting an enhancement of the GC reaction. Adjuvantation effects were apparently specific for IgG2a/c isotype switching without effect on antibody affinity maturation, as there was no effect on rHA-specific IgG avidity. Overall, our studies suggest that cGAMP when formulated with alum may represent an effective adjuvantation system to foster humoral and cellular aspects of type 1 immunity for early life immunization.

Keywords: T follicular helper cells; adjuvants; antibodies; antigen-presenting cells; germinal centers; newborn; stimulator of interferon genes; vaccines.

Figure 1

Screening of pattern recognition receptor (PRR) agonists on neonatal and adult BMDCs. (A–C) Newborn (N) and adult (Ad) BMDCs were stimulated with the indicated PRR agonists or adjuvants for 20–24 h. Cytokine production (A,C) and MFI of surface marker expression (B,C) were, respectively, assessed by ELISA and flow cytometry. (A,B) Color intensities of the heatmaps are proportional to (A) mean cytokine levels (expressed as pg/ml) or (B) mean co-stimulatory molecule levels (expressed as fold change of median fluorescence intensity over CTRL) of 5–6 (A) or 3 (B) independent experiment. (C) Results are expressed as mean + SEM of 4–5 (cytokine production) or 3 (surface marker expression) independent experiments. *p < 0.05, **p < 0.01 determined by repeated measures two-way ANOVA with Sidak post hoc test.

Figure 2

Immunization with recombinant hemagglutinin (rHA) formulated with cGAMP and alum induces distinct antibody profiles in adult and newborn mice. (A) Schematic representation of the immunization schedule for adult (day post-priming is indicated) and newborn [day of life (DOL) is indicated] mice. (B) Adult (top) and newborn (bottom) mice were immunized i.m. with saline (black line), rHA (brown line), (rHA + alum) (blue line), (rHA + cGAMP) (orange line) or (rHA + cGAMP + alum) (red line), and antibody titers for rHA-specific IgG, IgG1, and IgG2c were determined by ELISA in serum samples collected at the reported timepoints. (C) Fold change of median Ab titers over (rHA + alum) group. White bars, newborn mice. Black bars, adult mice. Results are shown as median of 9–10 (adult) or 7–8 (newborn) mice per group. *, ⁺, ^#p < 0.05, **, ⁺⁺, ^##p < 0.01 of groups indicated by the corresponding color, respectively, vs. saline, rHA, and (rHA + alum) groups determined by Kruskal–Wallis with Dunn’s post hoc test.

Figure 3

Immunization with (cGAMP + alum) induces Th1 polarization in early life. Newborn mice were immunized with alum or (cGAMP + alum) as indicated in Figure 2A. Ten days after boost [day of life (DOL) 24] splenocytes were harvested, re-stimulated for 18 h with recombinant hemagglutinin (rHA) in the presence or absence of the co-stimulus αCD28, and cytokine production by CD4⁺ T cells was assessed by intracellular flow cytometry. (A) Representative gating strategy. CD4⁺ T cells were defined as viable singlet CD3⁺ CD4⁺ cells. (B) Results are shown as the median, the 25th and 75th percentiles (boxes) and the 5th and 95th percentiles (whiskers) of 9–10 mice per group. **p < 0.01 of in vitro CTRL vs. rHA vs. rHA + αCD28, ⁺⁺p < 0.01 of respective in vitro conditions compared to in vivo saline group, ^#p < 0.05 and ^##p < 0.01 of respective in vitro conditions compared to in vivo alum group, determined by two-way ANOVA with Tukey’s post hoc test.

Figure 4

Immunization with (cGAMP + alum) induces IFNγ-producing cells in draining lymph nodes (dLNs) of newborn mice. Newborn mice were immunized with alum or (cGAMP + alum) as indicated in Figure 2A. 3 days after boost [day of life (DOL) 17] cells were isolated from dLNs, re-stimulated for 18 h with rHA + αCD28, and the number of IFNγ-producing cells per LN was assessed by ELISPOT. Results are shown as the median, the 25th and 75th percentiles (boxes) and the fifth and 95th percentiles (whiskers) of 4–5 mice per group. **p < 0.01 of in vitro CTRL vs. rHA + αCD28, ⁺⁺p < 0.01 of respective in vitro conditions compared to in vivo saline group, ^##p < 0.01 of respective in vitro conditions compared to in vivo alum group, determined by two-way ANOVA with Sidak’s post hoc test.

Figure 5

Immunization with (cGAMP + alum) fosters the germinal center (GC) reaction. Newborn mice were immunized with alum or (cGAMP + alum) as indicated in Figure 2A. Ten days after boost [day of life (DOL) 24] cells were isolated from draining lymph nodes and the percentages and absolute numbers of CD4⁺ T cells, B cells, GC Tfh, and B cells were assessed by flow cytometry. [(A,B) top panels] Representative gating strategies. CD4⁺ T cells were defined as viable singlet CD45⁺ B220⁻ CD3⁺ CD4⁺ cells. GC Tfh cells were defined as viable singlet CD45⁺ B220⁻ CD3⁺ CD4⁺ CXCR5⁺ PD-1⁺ cells. B cells were defined as viable singlet CD45⁺ B220⁺ CD3⁻ cells. GC B cells were defined as viable singlet CD45⁺ B220⁺ CD3⁻ GL-7⁺ CD138⁻ cells. (B) Results are shown as the median, the 25th and 75th percentiles (boxes) and the 5th and 95th percentiles (whiskers) of 9–10 mice per group. *p < 0.05, **p < 0.01 determined by one-way ANOVA with Holm–Sidak’s post hoc test.

Figure 6

Immunization with (cGAMP + alum) does not modulate recombinant hemagglutinin (rHA)-specific IgG avidity. Newborn mice were immunized with rHA formulated with alum or (cGAMP + alum) and serum samples were collected as indicated in Figure 2. Avidity of rHA-specific IgG was measured by ELISA and expressed as the ratio between the LogEC50 values obtained with and without ammonium thiocyanate treatment (0.5 M). Results are shown as median (left panel) or as the median, the 25th and 75th percentiles (boxes) and the 5th and 95th percentiles (whiskers) (right panels) of 7–8 newborn mice per group. *p < 0.05, **p < 0.01 determined by two-way ANOVA with Sidak’s post hoc test (left panel) or Mann-Whitney test (right panels).

Figure 7

Single-dose immunization of newborn mice with (cGAMP + alum) significantly increases anti-recombinant hemagglutinin (rHA) IgG2c titers. Newborn mice were immunized i.m. with saline, rHA alone or formulated with alum, cGAMP or (cGAMP + alum) and antibody titers for rHA-specific IgG, IgG1, and IgG2c were determined by ELISA in serum samples collected 28 days after boost [day of life (DOL) 35]. Results are shown as the median, the 25th and 75th percentiles (boxes), and the 5th and 95th percentiles (whiskers) of 9–13 mice per group. **p < 0.01 determined by Kruskal–Wallis with Dunn’s post hoc test.