All experiments involving animals were approved by the Animal Care and Use Committee of Boston Children’s Hospital and Harvard Medical School (protocol numbers 15-11-3011 and 16-02-3130).

C57BL/6 and BALB/c mice were obtained from Taconic Biosciences or Charles River Laboratories and housed in specific pathogen-free conditions in the animal research facilities at Boston Children’s Hospital. For breeding purposes, mice were housed in couples, and cages checked daily to assess pregnancy status of dams and/or the presence of pups. When a new litter was discovered, that day was recorded as day of life (DOL) 0. Both male and female pups were used for experiments.

BMDCs were generated from newborn (5–7 days old) and adult (6–12 weeks old) C57BL/6 mice with an adaptation of previously described methods (50, 51). Briefly, mice were sacrificed and legs removed; bones were surgically cleaned from surrounding tissue, extremities of tibiae and femurs were trimmed with sterile scissors and bone marrow flushed through a 70-µm nylon mesh strainer (Corning Life Sciences). Cell number and viability was determined by trypan blue exclusion. Whole bone marrow cells were plated into non-tissue culture-treated 100 mm Petri dishes (Corning Life Sciences) at a density of 0.3 × 10⁶ cells/ml in 10 ml total volume/plate of complete culture medium (RPMI 1640 plus 10% heat-inactivated fetal bovine serum [FBS, GE Healthcare HyClone], 50 µM 2-mercaptoethanol, 2 mM l-glutamine, 100 U/ml penicillin/streptomycin [Gibco ThermoFisher Scientific]) supplemented with 20 ng/ml of recombinant murine GM-CSF (rmGM-CSF, R&D systems). Plates were incubated in humidified atmosphere at 37°C, 5% CO₂ for 6 days, with one supplement of 10 ml of complete culture medium and rmGM-CSF on day 3. On day 6, non-adherent and loosely adherent cells were harvested by washing the plate gently with culture medium. Adherent cells were discarded. For flow cytometry analysis, BMDCs were stained (20 min at 4°C) in PBS + FBS 2% + EDTA 2 mM, fixed with formaldehyde 4% [10 min at room temperature (RT)] and acquired on a BD LSRFortessa flow cytometer (BD Biosciences) or a Sony spectral analyzer SP6800 (Sony Biotechnology) and data were analyzed using FlowJo v.10 software (Tree Star). For a complete list of antibodies and fluorochromes used in the study, see Table S1 in Supplementary Material.

Rough (Salmonella Minnesota, R595) and smooth (Escherichia coli, O55:B5) lipopolysaccharide (LPS) were purchased from List Biological Laboratories. Aluminum hydroxide (Alhydrogel) and Aluminum phosphate (Adju-phos) were purchased from Brenntag Biosector. All remaining PRR agonists and adjuvants, as indicated in Table S2 in Supplementary Material, were purchased from Invivogen. All PRR agonists employed in the studies were chosen based on and verified endotoxin free as indicated by the manufacturers. For stimulation experiments, immature BMDCs generated from newborn and adult mice were plated in round bottom 96-wells non-tissue culture-treated plates at the density of 10⁵ cells/well in 200 µl of fresh complete culture medium with rmGM-CSF as described above, with the appropriate stimuli at the concentrations indicated in Table S2 in Supplementary Material. Cells were incubated at 37°C for 20–24 h, then supernatant harvested and TNF, IL-6, IL-1β, and IL-12p70 concentrations were measured by ELISA (R&D Systems). IFNβ was measured with a bioluminescent ELISA kit (LumiKine, Invivogen). Alternatively, BMDCs were stained and analyzed by flow cytometry as indicated above. For experiments involving blocking antibodies, BMDCs were pre-incubated for 20 min at 37°C with anti-mouse IFNAR1 (clone MAR1-5A3, 10 µg/ml, Biolegend) or anti-mouse TNF (clone MP6-XT22, 10 µg/ml, Biolegend) antibodies or an isotype control before stimulation.

Both neonate and adult mice were immunized intramuscularly (i.m.) in the right posterior thigh with 50 µl of the 2016–2017 formulation of the FluBlok vaccine (Protein Sciences Corp.) containing 0.33 µg of each of the following recombinant influenza virus hemagglutinins (rHA): A/Michigan/45/2015 (H1N1), A/Hong Kong/4801/2014 (H3N2), and B/Brisbane/60/2008. Mice were immunized with a single dose at DOL 7 or a prime-boost schedule (two injections 1 week apart, for newborn mice at DOL 7 and 14). As indicated for specific experimental groups, the vaccine was formulated with Aluminum hydroxide (100 µg, hereafter “alum”) with or without 2′3′-cGAMP (10 µg). Serum was collected at the indicated intervals for antibody detection. rHA-specific IgG, IgG1, IgG2c (for C57BL/6 mice), and IgG2a (for BALB/c mice) antibodies were quantified by ELISA. High binding flat bottom 96-well plates (Corning Life Sciences) were coated with 1 µg/ml rHA in carbonate buffer pH 9.6, incubated overnight at 4°C and blocked with PBS + BSA 1% (Sigma-Aldrich) for 1 h at RT. Then, sera from vaccinated mice were added with an initial dilution of 1:100 and 1:4 serial dilutions in PBS + BSA 1% and incubated for 2 h at RT. Plates were then washed and incubated for 1 h at RT with HRP-conjugated anti-mouse IgG, IgG1, IgG2c, or IgG2a (Southern Biotech). At the end of the incubation, plates were washed again and developed with tetramethylbenzidine (BD Biosciences) for 5 min, then stopped with 2 N H₂SO₄. The optical density was read at 450 nm Versamax microplate reader with SoftMax Pro Version 5 (both from Molecular Devices) and endpoint titers were calculated using as cutoff three times the optical density of the background.

For assessing antibody avidity, plates were incubated 15 min with ammonium thiocyanate 0.5 M before the addition of HRP-conjugated anti-mouse IgG antibodies. Avidity was expressed as the LogEC₅₀ ratio of corresponding plates treated with or without ammonium thiocyanate.

To quantify the extent of 2′3′-cGAMP adsorption to aluminum hydroxide (Alhydrogel) we mixed 100 µg/100 μl of 2′3′-cGAMP with 1000 µg/100 μl of alum (a 1:10 cGAMP:alum mass ratio) plus 300 µl of 0.9% saline. After vortexing for 10 s the sample was placed in a 37°C incubator. Every 15 min, the sample was vortexed for an additional 5 s and placed back into the incubator. Aliquots were taken at t = 0.25, 0.5, 1, 2, 4 and 24 h and centrifuged at 3,000 RPM (rcf = 664 g) to separate the alum from the supernatant. Supernatant was immediately removed and placed into an autosampler vial undiluted for analysis by reverse-phase high-performance liquid chromatography (RP-HPLC) to determine adsorption as a function of time. RP-HPLC samples were run on a Waters 2695 HPLC equipped with a 2996 photodiode array detector at a wavelength of 254 nm. A gradient was performed using a two mobile phase system of 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile, on an Agilent Zorbax Eclipse Plus C18, 4.6 × 150 mm, 5 µm column at 25°C. The response (peak area) of the samples were compared against a 50 µl 2′3′-cGAMP plus 200 µl 0.9% saline control and a separate 100 µl alum plus 400 µl saline control.

Splenocytes from immunized mice were harvested 10 days post-boost (DOL 24) as previously reported (25, 52, 53) and re-stimulated in vitro to assess cytokine production by flow cytometry. Spleens were mashed through a 70 µM strainer, washed with PBS, and erythrocytes were lysed with 2 min of incubation in ammonium chloride-based lysis buffer (BD Biosciences). Cells were then counted and plated 2 × 10⁶ per well (round bottom 96-well plate) in 200 µl of complete culture medium with or without rHA 10 µg/ml or rHA 10 µg/ml + anti-mouse CD28 2 µg/ml (BioLegend). Plates were incubated for 18 h at 37°C with the addition of Brefeldin A (BD Biosciences) for the last 6 h. Cells were stained against for surface antigens in (PBS + BSA 0.2% + NaN₃ 0.05%) for 20 min at 4°C, then fixed with formalin 2% (10 min at RT) and permeabilized with intracellular staining permeabilization wash buffer (BioLegend) for 20 min at 4°C. Finally, cells were stained with conjugated antibodies against IFNγ, IL- 2, IL-4, and IL-17. Data were acquired on a BD LSRFortessa flow cytometer (BD Biosciences) and analyzed using FlowJo v.10 software (Tree Star). For a complete list of antibodies and fluorochromes used in the study, see Table S1 in Supplementary Material.

Draining (inguinal) lymph nodes (dLNs) from immunized mice were harvested 10 days post-boost (DOL 24) as previously reported (25, 52, 53). To prepare a single-cell suspension, dLNs were pressed using the plunger end of a syringe. Then, cells were washed and stained with the following antibodies: for GC Tfh cells, anti-CD45, anti-B220, anti-CD3, anti-CD4, anti-programmed death-1 (CD279 or PD-1), anti-CXCR5; for GC B cells, anti-CD45, anti-B220, anti-CD3, anti-GL7, and anti-Syndecan-1 (CD138) (all from BioLegend). GC Tfh cells were defined as viable singlet CD45⁺ B220⁻ CD3⁺ CD4⁺ CXCR5⁺ PD-1⁺ cells. GC B cells were defined as viable singlet CD45⁺ B220⁺ CD3⁻ CD138⁻ GL-7⁺. Cells were acquired on a BD LSRFortessa (BD Biosciences) and data were analyzed using FlowJo v.10 software (Tree Star). Absolute number of cell subsets were determined using CountBright Absolute Counting Beads (ThermoFisher Scientific). For a complete list of antibodies and fluorochromes used in the study, see Table S1 in Supplementary Material.

Draining lymph nodes from immunized mice were harvested 3 days post-boost (DOL 17). Nitrocellulose 96-microwell plates (Millipore) were coated with 75 µl/well of anti-mouse IFNγ (10 µg/ml in PBS, clone R4-6A2, BD Pharmingen) overnight at 4°C, washed twice with wash buffer (PBS + Tween-20 0.05%) and once with distilled water. Wells were blocked with 200 µl of complete culture medium for 2 h at RT. Single-cell suspensions of dLNs in complete culture medium supplemented with recombinant mouse IL-2 (5 ng/ml, PeproTech) were added to the wells in the presence or absence of 10 µg/ml of Flublok and 2 µg/ml anti-mouse CD28 (Biolegend) and cultured for 18 h. Wells were then washed and incubated with 100 ml of biotinylated anti-mouse IFNγ (5 µg/ml in PBS + FBS 10%, clone XMG1.2, BD Pharmingen) for 2 h at RT, washed again and incubated with 100μl of streptavidin-alkaline phosphatase (1:1000 dilution in PBS + FBS 10%, MabTech) for 1 h prior to color development using BCIP/NBT substrate (Biorad) as per manufacturer’s protocol. Spots on air-dried plates were counted on an ImmunoSpot Analyzer.

Data were analyzed and graphed using Prism for MacIntosh v. 7.0 (GraphPad Software). Tests used for statistical comparisons are indicated in figure legends. p-value <0.05 was considered significant.

Murine BMDCs represent a widely used model to study DC function in vitro. Adult BMDCs represent a heterogeneous population composed of CD11c⁺ macrophage-like and DC-like cells with distinct phenotypic and functional profiles (54). However, murine neonatal BMDCs have never been characterized in depth. Therefore, we first sought to define the phenotypic and functional properties of neonatal BMDCs. Although the cell yield from neonatal bone marrow was lower compared to adult ones (Figures S1A,B in Supplementary Material), neonatal immature BMDCs generated from 7-day-old mice grew in culture similarly to adult cells (Figure S1C in Supplementary Material), and once fully differentiated they expressed similar levels of CD11c compared to adult cells but significantly lower levels of MHCII (Figures S1D,E in Supplementary Material). To further characterize phenotypic differences between newborn and adult BMDCs, we assessed by flow cytometry the expression of different macrophage and DC markers. As previously reported for adult BMDCs (54), neonatal BMDCs also comprised CD11c⁺ MHCII-low and CD11c⁺ MHCII-high cells. Of note, the percentage of MHCII-low cells was higher in neonatal BMDCs compared to adult BMDCs. Neonatal MHCII-low BMDCs also expressed higher levels macrophage-associated markers (CD64, CD115, CD11b, F4/80) compared to MHCII-high BMDCs, while this population expressed higher levels of CD117. No significant differences in surface marker expression were found between corresponding neonatal and adult MHCII-high and -low populations, except for neonatal MHCII-low BMDCs that expressed higher levels of F4/80 and neonatal MHCII-high BMDCs that expressed higher levels of CD117 compared to their adult counterparts (Figures S2A,B in Supplementary Material).

To characterize a functional response of newborn BMDCs, we next assessed cytokine production and upregulation of co-stimulatory molecules in response to the TLR4 agonist smooth LPS. While newborn BMDC production of IL-6 and TNF was, respectively, comparable or slightly lower than adult BMDCs, IL-12p70 production, albeit detectable, was markedly reduced compared to adult BMDCs (Figure S3A in Supplementary Material). The latter result might be consistent with a more macrophage-like phenotype of newborn BMDCs. As previously reported, both adult and newborn BMDCs produced IL-1β in response to rough but not smooth LPS (55), with newborn BMDCs producing slightly higher amounts of IL-1β (Figure S3B in Supplementary Material). Finally, newborn BMDCs expressed lower levels of MHCII, CD40, and CD86 in response to smooth LPS (Figures S3C,D in Supplementary Material).

Having characterized phenotypic and functional features of neonatal and adult BMDCs, we next assessed their response to a panel of PRR agonists and adjuvants (Table S2 in Supplementary Material). As readouts we measured cytokine production (TNF, IL-1β, IL-6, and IL-12p70) and surface expression of maturation markers (CD40, CD80, and CD86). At the most effective, non-toxic (as established in preliminary experiments, data not shown) concentration of each agonist (in bold in Table S2), neonatal BMDCs produced similar amounts of TNF, IL-6, and IL-1β compared to adult BMDCs in response to different TLR7/8 agonists, namely R848 (Resiquimod, imidazoquinoline), CL075 (thiazoloquinolone) or CL264 (9-benzyl-8 hydroxyadenine), but again failed to produce IL-12p70 (Figure 1A). Remarkably, the upregulation of surface maturation marker expression on neonatal BMDCs was much lower than adult BMDCs upon any PRR stimulation, with the exception of the STING agonist 2′3′-cGAMP (hereafter cGAMP) (Figure 1B). To assess in depth the response to STING and TLR7/8 agonists, we stimulated neonatal and adult BMDCs with different concentrations of cGAMP and R848. We confirmed that R848 induced higher production of TNF and IL-12p70 (the latter only in adult BMDCs), while cGAMP was more effective than R848 at upregulating the expression of surface maturation markers (Figure 1C). cGAMP also induced dose-dependent IFNβ production in both newborn and adult BMDCs (Figure 1C). Of note, the response of neonatal and adult BMDCs to cGAMP was comparable (Figure S4 in Supplementary Material). Using neutralizing antibodies against TNF or type I IFN receptor (IFNAR), we demonstrated that the expression of maturation markers by neonatal BMDCs mostly relies on type I IFN signaling (Figure S5 in Supplementary Material).

The in vitro results obtained so far supported further investigation of cGAMP as adjuvant candidate for early life immunization. Therefore, we proceeded to test this hypothesis in vivo. We immunized newborn (7-day old) and adult (8- to 10-week old) C57BL/6 mice using a prime-boost schedule (Figure 2A) and employing trivalent recombinant hemagglutinin (rHA) influenza vaccine Flublok as clinically relevant model antigen that is devoid of adjuvant, alone, or formulated with alum [Alhydrogel, Al(OH)3], cGAMP or (cGAMP + alum) (Figure 2B). Mice were bled 14, 21, 28, and 35 days post-prime (respectively, day of life (DOL) 21, 28, 35, and 42 for newborn mice) to assess the magnitude and kinetic of the antibody response. As expected, both alum and cGAMP increased anti-rHA IgG titers in adult mice. We also investigated the titers of the IgG subclasses IgG1 and IgG2c, respectively associated with type 2 and type 1 (IFNγ-driven) immunity (56, 57). In keeping with previously published data, alum preferentially increased anti-rHA IgG1 titers (median anti-rHA IgG1 titers at Day 35 post-prime: 5.02 × 10⁶ for alum, 0.77 × 10⁶ for cGAMP), while cGAMP was more effective than alum at enhancing anti-rHA IgG2c titers (median anti-rHA IgG2c titers at day 35 post-prime: 0.16 × 10⁶ for alum, 0.82 × 10⁶ for cGAMP). (cGAMP + alum) was as effective as alum at increasing anti-rHA IgG and IgG1 titers [median anti-rHA IgG and IgG1 titers at day 35 post-prime: respectively, 4.77 × 10⁶ and 4.46 × 10⁶ for (cGAMP + alum)], and even more effective than cGAMP alone at enhancing anti-rHA IgG2c titers [median anti-rHA IgG2c titers at day 35 post-prime: 3.27 × 10⁶ for (cGAMP + alum)] (Figure 2B, upper panels and Figure S6 in Supplementary Material). In newborn mice, we unexpectedly found that cGAMP was much less effective at increasing anti-rHA IgG, IgG1, and IgG2c titers [median anti-rHA IgG, IgG1, and IgG2c titers at day 35 post-prime (DOL 42): respectively, 20.57 × 10³, 24.51 × 10³, and 0.23 × 10³ for cGAMP]. Alum enhanced anti-rHA IgG and IgG1 titers, but in marked contrast from adult mice it did not induce anti-rHA IgG2c titers [median anti-rHA IgG, IgG1, and IgG2c titers at day 35 post-prime (DOL 42): respectively, 48.35 × 10³, 143.23 × 10³, and 0.00 × 10³ for alum]. Surprisingly, (cGAMP + alum) adjuvantation matched or exceeded alum at increasing anti-rHA IgG and IgG1 titers [median anti-rHA IgG and IgG1 titers at Day 35 post-prime (DOL 42): respectively, 329.19 × 10³ and 167.83 × 10³ for (cGAMP + alum)], and, remarkably, also induced relatively high titers of anti-rHA IgG2c as early as 14 days post-prime (DOL 21) [median anti-rHA IgG2c titers at day 14 (DOL 21) and day 35 post-prime (DOL 42): respectively, 0.14 × 10³ and 4.23 × 10³ for (cGAMP + alum)] (Figure 2B, lower panels and Figure S7 in Supplementary Material). Therefore, the addition of cGAMP to alum markedly enhanced anti-rHA antibody production (in particular IgG2c), with a more prominent effect in newborn than adult mice (~400 as compared to ~150-fold increase, respectively) (Figure 2C). Interestingly, newborn mice immunized at DOL 7 and 14 (as indicated in Figure 2B) with (cGAMP + alum) still display the highest anti-rHA IgG and IgG2c titers at DOL 90 compared to saline and alum groups (Figure S8 in Supplementary Material). Enhancement of anti-rHA IgG and IgG2a titers induced by (cGAMP + alum) was also demonstrable in the Th2-skewed mouse strain BALB/c (Figure S9 in Supplementary Material).

In light of the robust adjuvanticity of the (cGAMP + alum) formulation, we quantified cGAMP adsorption to alum by RP-HPLC (Table 1). We observed a rapid initial adsorption of cGAMP onto alum (63% of total cGAMP) within 15 min from the incubation. The adsorption rate dropped quickly, with the overall adsorption reaching a plateau (75.33% of total cGAMP) after 24 h of incubation. No significant degradation products were observed over this time window.

Altogether, our in vivo results demonstrate that (cGAMP + alum) is an effective formulation to enhance antigen-specific antibody titers (especially of the IgG2a/c subclass) for early life immunization.

IgG2a/c isotype switching is driven by IFNγ in vivo (58), and reduced in early life, since newborns display reduced IFNγ production and Th1 polarization to many stimuli (6, 7). Therefore, we investigated whether (cGAMP + alum) was able to modulate the polarization and cytokine production of antigen-specific T cells. Accordingly, newborn mice were immunized as indicated in Figure 2A with alum or (cGAMP + alum). Ten days post-boost, splenocytes were harvested, re-stimulated with rHA in the presence or absence of the co-stimulus αCD28, and cytokine production by CD4⁺ T cells was measured by flow cytometry (Figure 3A). While IL-2- and IL-4-producing cells were observed in both groups, IFNγ⁺ CD4⁺ T (Th1) cells were only detected among splenocytes isolated from mice immunized with (cGAMP + alum) [median percentages of IFNγ⁺ CD4⁺ T cells upon rHA re-stimulation: 0.000 for saline, 0.031 for alum, and 0.295 for (cGAMP + alum) groups; upon rHA + αCD28 re-stimulation: 0.009 for saline, 0.021 for alum, and 0.280 for (cGAMP + alum) groups]. No IL-17 production was observed in any of the tested conditions (Figure 3B). To corroborate this evidence, upon in vitro re-stimulation with rHA + αCD28 we found by ELISPOT a higher number of IFNγ-producing cells in the dLNs of mice immunized with (cGAMP + alum) 3 days post-boost (Figure 4).

T cell-dependent antibody generation is initiated in GCs and guided by Tfh cells (59, 60). Since GCs are major sites for isotype switching, we reasoned that immunization of newborn mice with (cGAMP + alum) might promote the GC reaction, thereby inducing IgG2a/c switching. To this aim, we assessed by flow cytometry the percentages and absolute numbers of GC Tfh and B cells (respectively, identified as viable singlet CD45⁺ B220⁻ CD3⁺ CD4⁺ CXCR5⁺ PD-1⁺ and CD45⁺ CD3⁻ B220⁺ GL-7⁺ CD138⁻ cells) in dLNs 10 days post-boost of newborn mice immunized with alum or (cGAMP + alum). Interestingly, we found a significant increase in the percentage [median: 0.275 for saline, 0.42 for alum, and 0.925 for (cGAMP + alum)] and absolute number [median: 1,360 for saline, 2,558 for alum, and 5,754 for (cGAMP + alum)] of GC Tfh cells and the percentage [median: 14.4 for saline, 19.7 for alum, and 27.35 for (cGAMP + alum)] and absolute number [median: 10,975 for saline, 19,878 for alum, and 42,524 for (cGAMP + alum)] of GC B cells only in the (cGAMP + alum) group (Figure 5A). Immunization with alum induced a small increase in the percentage (but not absolute number) of GC B cells, while only minor modifications of the percentages and absolute numbers of total CD4⁺ T cells and B cells were observed across different immunization groups (Figures 5A,B).

The GC is also the site where the processes of somatic hypermutation of antibody variable region genes and generation of high-affinity antibodies take place (60). To verify whether cGAMP modulates antibody affinity maturation, we measured rHA-specific IgG avidity of newborn mice immunized with alum or (cGAMP + alum) as indicated in Figure 2A. Although we observed a steep increase in antibody avidity 21 days post prime (DOL 28) which reached a plateau later on [28 (DOL 35) and 35 (DOL 42) days post-prime], no differences between the two groups were detected at any time point (Figure 6).

Overall, these results demonstrate that the addition of cGAMP to alum promoted the induction of IFNγ-producing T cells and appeared to foster the GC reaction, which might in turn drive IgG2a/c isotype switching in our early life immunization model.

The results obtained so far supported the efficacy of (cGAMP + alum) as an adjuvantation system in a prime/boost model of neonatal murine immunization. Of note, a single-dose immunization strategy capable of enhancing antigen-specific antibody titers would be highly desirable early in life. To this end, we immunized newborn mice with rHA formulated with alum, cGAMP, or (cGAMP + alum). Distinct from its effects in prime/boost immunization, cGAMP without alum did not induce detectable anti-rHA IgG, IgG1, and IgG2c titers. Alum and (cGAMP + alum) significantly increased anti-rHA IgG and IgG1 titers [median anti-rHA IgG and IgG1 titers: respectively, 26.74 × 10³ and 52.08 × 10³ for alum; respectively, 1.07 × 10⁶ and 1.48 × 10⁶ for (cGAMP + alum)]. Interestingly, only (cGAMP + alum) induced detectable levels of anti-rHA IgG2c (median: 571.9), albeit at lower levels compared to prime/boost immunization (Figure 7). Altogether, these results demonstrate that (cGAMP + alum) is an effective adjuvantation system also for single dose early life immunization.