Abstract
Squalene-based adjuvants are widely utilized in various vaccines because of their effectiveness in enhancing immune responses. Among these, A-910823 and AS03 are recognized squalene-based adjuvants containing α-tocopherol. Although α-tocopherol-containing squalene-based adjuvants are known to enhance humoral immune responses and inflammatory cytokine production, their underlying mechanisms remain unclear. Here, we found that A-910823 regulated IL-1 signaling pathway gene expression and IL-1α and IL-1β protein expressions in an α-tocopherol-dependent manner. While the source of IL-1α was mainly from eosinophils, IL-1β was from a wide range of myeloid cells. Mechanistically, IL-1β/CD11c+ cell-IL-1R1/MyD88 axis mediated adjuvant efficacy, however, systemic reactogenicity was induced by the IL-1β/IL-1R1/MyD88/IL-6/cyclooxygenase 2 axis, which was an unexpectedly distinct mechanism from the local reactogenicity that was mediated by the eosinophil-derived IL-1α/IL-1R1/MyD88 axis. These findings demonstrate that adjuvant efficacy and reactogenicity are regulated by distinct pathways and cell types. This study thus provides novel insights into the mechanisms of adjuvants, providing valuable information to support the future development of effective and safe vaccines.
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Introduction
Vaccines are essential in safeguarding public health by markedly reducing the incidence of infectious diseases globally. Vaccines act as a key means of achieving herd immunity and managing outbreaks to ensure the preservation of individual and community health. Pathogen complexity and variability in host immune responses often limit the immunogenicity of isolated antigens or subunit vaccines; therefore, adjuvants are commonly added across vaccine platforms, including inactivated whole‑pathogen, recombinant subunit, and viral‑vector vaccines, to enhance the magnitude, durability, and breadth of antigen‑specific immunity. Adjuvants come in various forms, including aluminum hydroxide (alum), nucleic acid derivatives, saponin-based substances, and squalene-based adjuvants, all of which are widely utilized in many vaccines1.
A-910823, AS03, and MF59 are notable squalene-based adjuvants that have been approved for use in humans. MF59 does not contain α-tocopherol, but A-910823 and AS03 are oil-in-water type adjuvants composed of α-tocopherol, squalene, and polysorbate 80. A-910823 has been employed in clinical settings in a novel vaccine against COVID-19, COVGOZE®, which uses the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein as its antigen. This vaccine induces broad-spectrum, potent neutralizing antibodies with high safety and tolerability2,3,4,5,6,7,8. Regarding the clinical significance of α-tocopherol, the efficacy and reactogenicity of AS03 have been suggested to depend on its α-tocopherol content9. In addition, A-910823 was demonstrated to induce high levels of neutralizing antibodies and T follicular helper (Tfh) cells in an α-tocopherol-dependent manner in nonclinical studies10,11.
Several studies have investigated the mechanism of action of squalene-based adjuvants. From the perspective of adjuvant components, the adjuvant activity of MF59 has been shown to depend on each of its constituents, and the inclusion of α-tocopherol in A-910823 and AS03 improved the induction of cytokines and neutralizing antibodies10,11,12. From a biological molecular perspective, studies using MF59 and its mimic, AddaVax, showed that the induction of humoral immunity depended on myeloid differentiation primary response protein 88 (MyD88) and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)13,14. In addition, receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-dependent macrophage cell death was shown to contribute to the induction of cellular immunity, and transient ATP production was also involved in adjuvant activity15,16. Recent evidence also suggested that AddaS03TM, a mimic of AS03, enhanced humoral immune responses in a MyD88-dependent manner, and AS03 induced cytokines in an endoplasmic reticulum stress-dependent manner17,18.
Despite extensive research on squalene-based adjuvant mechanisms, however, the specific roles of their components and the related cellular and molecular pathways remain poorly defined, and the precise molecular mechanism by which α-tocopherol, present in A-910823 and AS03, contributes to the induction of neutralizing antibodies and Tfh cells, along with the specific cell types involved in this process, remains obscure. Furthermore, previous studies have primarily focused on the mechanisms that enhance vaccine efficacy, and the molecular mechanisms underlying both local and systemic reactogenicity remain poorly understood. It is essential to clarify these mechanisms to support the development of vaccines with high efficacy and minimal reactogenicity.
Here, we explore the mechanism of action of A-910823, alongside other squalene-based adjuvants such as AddaS03 and AddaVax, focusing on responses induced in an α-tocopherol-dependent manner. Specifically, we investigated both the efficacy-enhancing mechanisms and the pathways leading to reactogenicity, thereby addressing two critical aspects of squalene-based adjuvants.
Results
α-Tocopherol in A-910823 controlled IL-1 pathway-related genes
We conducted comprehensive gene expression analysis to elucidate the molecular mechanisms of the immune responses induced by the squalene-based adjuvant A-910823. Mice were immunized intramuscularly (IM) with full‑length recombinant SARS‑CoV‑2 S protein (Ag), with or without A‑910823. Muscle tissue from the injection site and the iliac LNs, collected as the draining LNs, were harvested 30 min and 4 h post-immunization for RNA sequencing. In order to assess the contribution of α‑tocopherol, an essential component of A‑91082311, we also analyzed samples from mice immunized with Ag and A‑910823 lacking α‑tocopherol (Ag + A‑910823 w/o α‑Toc).
The principal component analysis (PCA) revealed clear differences in global gene expression among treatment groups and time points (Fig. 1A). At 4 h post‑immunization, the presence or absence of α‑tocopherol affected gene expression in both LNs and muscle. The number of differentially expressed genes (DEGs) was greater at 4 h than at 30 min and was higher in LNs than in muscle (Figure S1A). α‑Tocopherol had little effect on DEG numbers in muscle but increased those in LNs compared with the Ag group. These findings indicate that α‑tocopherol in A‑910823 has a stronger impact on LNs than on muscle and contributes importantly to its adjuvant activity.
Mice were immunized IM with antigen (Ag), Ag combined with A-910823 (Ag + A-910823), or Ag combined with A-910823 without α-tocopherol (Ag + A-910823 w/o α-Toc), and the draining LNs and muscle tissues were harvested 30 min or 4 h post-immunization for gene expression analysis via RNA sequencing. The data set for each group comprised three independent pooled biological replicates. A Principal component analysis (PCA) of gene expression in the indicated tissues and groups. Each dot represents a biological replicate. B Volcano plots showing differentially expressed genes in the indicated tissues at 4 h after immunization. Red and blue dots represent upregulated and downregulated genes, respectively, in the Ag + A‑910823 group compared with the Ag group. C Heatmaps showing Z-score normalized expression of differentially expressed genes associated with cytokines, including chemokines, ILs, IFNs, TNF, TGF-β, and their respective receptors in the indicated tissues at 4 h post-immunization. Value in parentheses represents number of differentially expressed genes. D Over-representation analysis using KEGG signaling pathways with upregulated genes in the Ag + A-910823 group compared with the Ag group in the indicated tissues at 4 h post-immunization. E Relative expression of genes associated with IL-1 signaling in draining LNs at 4 h post-immunization. Red and blue dots represent differentially upregulated and downregulated genes, respectively, in the Ag + A-910823 group compared with the Ag + A-910823 w/o α-Toc group. Statistical significance calculated using Wilcoxon’s matched-pairs signed rank test. ***P < 0.001.
We analyzed gene expression changes in LNs and muscle at 4 h post-immunization, when the largest number of genes was altered. Cytokine- and chemokine-encoding genes, including Il1b, Il6, Csf3, and Cxcl1, were significantly upregulated in the Ag + A-910823 group compared with the Ag group in both tissues (Fig. 1B). Given these shared changes, we next analyzed a broader cytokine gene set—including chemokines, ILs, IFNs, TNF, TGF-β, and their receptors—and found distinct expression patterns in the Ag + A-910823 group at 4 h post-immunization (Fig. 1C).
Pathway analysis of genes significantly upregulated in the Ag + A-910823 group at 4 h post-immunization revealed enrichment of pathways associated with leukocyte migration (Fig. S1B), and IL-1 signaling was strongly enriched in both tissues, with the highest enrichment observed in LNs (Fig. 1D).
To evaluate the α-tocopherol dependency of these responses, we compared gene expression profiles among the Ag, Ag + A-910823, and Ag + A-910823 without α-tocopherol groups in LNs at 4 h post-immunization (Fig. S1C). Of the 543 genes upregulated in the Ag + A-910823 group relative to Ag, 524 showed further increased expression compared with the α-tocopherol-deficient group. Similarly, of the 970 downregulated genes, 965 genes were further decreased in the presence of α-tocopherol. Focusing on IL-1 signaling, nine of 26 genes showed significantly higher expression in the Ag + A-910823 group than in the α-tocopherol-deficient group (Figs. S1D and 1E). These results suggest that, at least in LNs, most of the genes induced by A‑910823 immunization were α‑tocopherol‑dependent and that IL‑1 signaling may play a crucial role in its adjuvant activity.
IL-1α and IL-1β induction depended on α-tocopherol in squalene-based adjuvant
IL-1 signaling is triggered by IL-1α or IL-1β binding to its receptor (IL-1 receptor 1, IL-1R1), and we therefore examined the expression of these genes. Transcriptomic analysis of LN cells revealed no changes at 30 min post-administration, but both Il1a and Il1b were significantly upregulated in the Ag + A-910823 group compared with the Ag alone group at 4 h (Fig. 2A, B). However, only Il1b induction was significantly decreased by excluding α-tocopherol (Fig. 2B).
Relative expression levels of Il1a (A) and Il1b (B) in draining LNs, from RNA-seq results in Fig. 1. C–H C57BL/6J mice were injected IM with SARS-CoV-2 S-protein S-910823 (Ag) alone, Ag mixed with A-910823, A-910823 w/o α-Toc, AddaS03, or AddaVax. C Kinetics of immune cell infiltration in draining LNs: numbers of eosinophils, neutrophils, monocytes, and dendritic cells (DCs) at 2, 6, and 14 h post‑administration (n = 3/group). Mean fluorescence intensity (MFI) of IL‑1α (D) and IL‑1β (E) in eosinophils, neutrophils, monocytes, and DCs at 6 h post‑administration (n = 3/group). F MFI of IL‑1α and G IL‑1β at 6 h (A‑910823 vs. A‑910823 without α‑tocopherol; AddaS03 vs. AddaVax) (n = 5/group). Bars represent means; error bars indicate standard error of the mean. Statistical significance determined using two-way ANOVA. ns, P ≥ 0.05, *P < 0.05, **P < 0.01, ****P < 0.0001. H IL-1β and β-actin levels in draining LNs 6 h post-administration by western blotting.
To extend the Il1a and Il1b transcript findings to the protein level, we performed intracellular staining (ICS) and flow cytometry (FCM) on LN cells collected at 2, 6, and 14 h after IM administration (Fig. S2A). Adjuvant-dependent increases in cell numbers showed distinct kinetics: neutrophils and monocytes were significantly increased at 6 h after A-910823 administration, whereas eosinophil accumulation and sustained neutrophil increases were most pronounced at 14 h (Fig. 2C). Notably, eosinophils showed a small, non-significant increase at 6 h following A-910823 administration that became statistically significant by 14 h (Fig. 2C). In contrast, induction of IL-1α and IL-1β protein (expressed as MFI) was transient and restricted to the 6 h time point: IL-1α expression was induced predominantly in eosinophils at 6 h, while IL-1β expression was elevated in multiple myeloid populations including eosinophils, neutrophils, and monocytes at 6 h but not at 2 h or 14 h (Fig. 2D, E). These data indicate that cellular infiltration and cytokine induction follow distinct temporal patterns, and that evaluating IL-1α and IL-1β at 6 h captures the peak of cytokine induction.
To assess whether the α-tocopherol-dependent pattern of IL-1 induction is observed with other squalene-based formulations, we examined AddaS03 (an AS03 mimic containing α-tocopherol) and AddaVax (an MF59-like adjuvant; unlike AS03, it does not contain α-tocopherol) at the 6 h time point. In comparisons within each formulation set, we observed a consistent α-tocopherol-associated effect on cytokine expression: IL-1α expression in eosinophils and IL-1β expression in eosinophils and monocytes were significantly higher when α-tocopherol was present (A-910823 vs. A-910823 without α-tocopherol; AddaS03 vs. AddaVax) (Figs. 2F, G and S2B). Complementing the MFI results, the numbers of IL-1α-positive eosinophils and IL-1β-positive neutrophils were also consistently and significantly greater in the α-tocopherol-containing formulations in these within-formulation comparisons (Fig. S2C, D). These concordant changes in both per-cell expression (MFI) and positive-cell counts indicate that the α-tocopherol-dependent induction of IL-1 family cytokines is reproducible across squalene-based adjuvants. Furthermore, since IL-1β is activated after the cleavage of pro-IL-1β by caspase119, we found that A-910823 induced the activated form of IL-1β via cleavage, and this induction was depended on α-tocopherol (Fig. 2H). In parallel, serum cytokines were measured by Bio-Plex on independent biological replicates; Bio-Plex confirmed increased IL-1α and IL-1β and additionally detected elevated IL-6 and G-CSF (encoded by Csf3), consistent with the RNA-seq inflammatory signature (Fig. S2E). These orthogonal protein measurements strengthen the concordance between transcriptomic, cellular, and systemic cytokine responses following A-910823 immunization.
Taken together, these results suggest that the squalene-based adjuvant administration induces IL-1α and IL-1β induction in draining LNs via the presence of α-tocopherol in their formulation.
A-910823 directly induced IL-1α and IL-1β
We next investigated the mechanism by which A-910823 induced IL-1α and IL-1β using fluorescently labeled A-910823. We chemically synthesized α-tocopherol covalently bonded to the fluorescent molecule, BODIPY (Fig. 3A), and replaced 0.1% of the α-tocopherol content with this fluorescent-labeled α-tocopherol and emulsified it to create BODIPY-labeled A-910823 (A-910823-BODIPY). The physicochemical properties of A-910823 and A-910823-BODIPY were analyzed by dynamic light scattering, which showed that both molecules had similar particle sizes (151 and 158 nm, respectively) and polydispersity index values (0.08 and 0.03, respectively) (Fig. S3A). Furthermore, fluorescence spectrum measurements revealed fluorescence signals with a single peak at 515 nm in A-910823 containing fluorescently labeled α-tocopherol (A-910823-BODIPY) (Fig. 3B), confirming the successful preparation of fluorescently labeled A-910823. To determine if A-910823-BODIPY retained equivalent adjuvant activity to A-910823, we administered Ag with A-910823 or A-910823-BODIPY to mice twice at 2-week intervals and quantified the serum neutralizing antibody titers, Tfh cells, and germinal center B (GCB) cells induced in the draining LNs 2 weeks later, as described previously11. A-910823-BODIPY induced equivalent neutralizing antibody titers, Tfh cells, and GCB cells to A-910823, showing that A-910823-BODIPY was equivalent to A-910823 in terms of its physical properties and its ability to enhance vaccine immunogenicity (Fig. S3B–E).
A Chemical structure of α-tocopherol covalently bound with BODIPY. B Fluorescence intensity of A-910823 and A-910823 containing fluorescently labeled α-tocopherol (A-910823-BODIPY) confirmed by fluorescence spectrum. C57BL/6 J mice were injected IM with Ag mixed with A-910823 or A-910823-BODIPY (n = 3/group). C Representative BODIPY luminance of eosinophils, neutrophils, monocytes, and (DCs) 6 h post-administration. Mean fluorescence intensity (MFI) of D IL-1α and E IL-1β in BODIPY-positive and -negative cells, respectively. Bars represent means; error bars indicate standard error of the mean. Statistical significance determined using two-way ANOVA. ns, P ≥ 0.05, *P < 0.05, ***P < 0.001, ****P < 0.0001.
To identify the cells that take up the adjuvant, we administered Ag with A-910823 or A-910823-BODIPY IM to mice and evaluated draining LN cells 6 h later by FCM. BODIPY-positive (i.e., adjuvant-positive) cells were detected among eosinophils, neutrophils, monocytes, and DCs (Fig. 3C). To determine if the induction of IL-1α and IL-1β was due to direct action of the adjuvant on cells, we compared the expression levels of IL-1α and IL-1β in BODIPY-positive and -negative cells. Expression levels of IL-1α in eosinophils and IL-1β in eosinophils, monocytes, and DCs were higher in BODIPY-positive than in BODIPY-negative cells (Fig. 3D, E), while neutrophils also showed higher IL-1β expression in BODIPY-positive cells, although the difference was not significant. These results suggest that A-910823 induced IL-1α and IL-1β directly in an α-tocopherol-dependent manner.
A-910823 regulated vaccine efficacy and reactogenicity via the IL-1R1/MyD88 signaling pathway and enhanced humoral immune responses through IL-1R1/MyD88 signaling in CD11c+ cells
Previous studies demonstrated that squalene-based adjuvants, such as AS03, rely on MyD88, a downstream adapter of the IL-1R, to mediate humoral immune responses18. To investigate the mechanism by which a squalene-based adjuvant enhanced immune efficacy, we immunized mice twice at 2-week intervals with Ag plus A-910823 and compared appropriate control groups (wild-type, heterozygous, or Cre-negative littermates as applicable) and multiple genetically modified strains; serum neutralizing antibody titers, Tfh cells, and GCB cell induction in the draining LNs were measured two weeks after the final immunization (Fig. 4A). The exact genotypes and group assignments are listed in the Methods and in the Fig. 4 legend. Consistent with previous findings14,18, the induction of neutralizing antibody titers, Tfh cells, and GCB cells were significantly reduced when Ag and A-910823 were administered to Myd88 KO mice, compared with control mice (Fig. 4B). In contrast, when antigen-specific T cell responses were evaluated via ELISpot analysis using splenocytes, the numbers of IFN-γ- and IL-4-producing cells were comparable in control and Myd88 KO mice (Fig. S4A), suggesting that MyD88 dependency did not extend to T cell response induction. These findings demonstrate that A-910823, similar to AS03, enhanced humoral immune responses in a MyD88-dependent manner.
A Study schedule. Mice were injected IM with Ag mixed with A-910823 on days 0 and 14 (n = 4–10/group), using Myd88 (B), Il1r1 (C), and Il1rn KO mice (D), as well as Myd88flox/flox (f/f) and Cd11ccre (E) or LysMcre mice (F). B–F Serum titers of neutralizing antibodies against SARS-CoV-2 on day 28. Bars represent geometric mean titer (GMT); error bars indicate 95% confidence intervals; circles represent neutralizing antibody titers in individual mice. Percentages of T follicular helper (Tfh; PD1+CXCR5+) cells in TCRb+CD4+ cells and percentages of germinal center B (GCB; FAS+GL7+) cells in CD19+ cells in draining LNs on day 28. Bars represent means; error bars indicate standard error of the mean; circles represent percentages of Tfh and GCB cells in individual mice. Injection-site swelling scores on day 15. Bars represent means; error bars indicate standard error of the mean; circles represent swelling scores in individual mice. Average body temperature was assessed on Day 12 (08:00–19:59) and on Day 15 (08:00–19:59). Vaccination at day 14 was performed at 18:00. Bars represent means; error bars indicate standard error of the mean. The data were derived from independent experiments: (1) Neutralizing titer/Tfh/GCB measured in the same cohort, (2) Swelling measured in an independent cohort, and (3) Fever measured in an independent cohort. Statistical significance determined using two-tailed Student’s t-test for two-group comparisons or two-way ANOVA. ns, P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
We also investigated their mechanism of reactogenicity induction. Mice were administered antigen with one of four adjuvants (A-910823, A-910823 without α-tocopherol, AddaS03, or AddaVax) twice at 2-week intervals. Local reactogenicity was assessed by swelling score at the injection site, and systemic reactogenicity was evaluated by changes in body temperature pre- and post-immunization (Fig. 4A).
Injection-site swelling was scored quantitatively on a three-point scale. Temporal scoring revealed that squalene-based adjuvants induced peak swelling at 24 h after administration, in an α-tocopherol-dependent manner (Fig. S4B). These findings indicate that squalene-based adjuvants promoted localized swelling via an α-tocopherol-dependent pathway. To ensure the accurate assessment of swelling at the injection site, we measured the size of the limb at the injection site and examined the histological changes. The administration site was photographed with a 3D scanner coordinate-measuring machine (Fig. S4C). Both the width and cross-sectional area of the injection site were significantly increased in the Ag + A-910823 group compared with the Ag group. Furthermore, histopathological evaluation revealed that Ag + A-910823 resulted in edema, characterized by fibrin exudation and erythrocyte leakage (Fig. S4D, E). In contrast, the frequency and severity of edema were reduced in mice treated with Ag + AddaVax or Ag + A-910823 without α-tocopherol compared with the Ag + A-910823-treated group. The differences in width and cross-sectional area, together with the histopathological evidence, suggest that the three-point swelling score was an objective and reliable method for assessing swelling.
We assessed systemic reactogenicity by measuring body temperature fluctuations using a nano-tag® device, integrating a temperature sensor and an accelerometer, implanted IP in mice. Prior to vaccine administration, mice in all the groups exhibited a diurnal pattern, with a lower body temperature during the daytime than at night. Moreover, variations in body temperature were correlated with levels of physical activity, suggesting that temperature changes were influenced by mouse behavior. To minimize the impact of activity-driven temperature variations, we analyzed body temperature during daytime periods, when mice exhibited reduced activity levels, as the parameter for fever assessment (Fig. S4F). Mice were administered Ag and various adjuvants at 18.00. Mice treated with Ag + A-910823 demonstrated significant elevations in daytime average body temperature (08:00–19:59) on the calendar day after vaccination (first post-vaccination daytime window; 14–26 h after the 18:00 vaccination) compared with the daytime average on the calendar day 2 days prior to vaccination. In contrast, there was no observable increase in body temperature relative to pre-vaccination levels following the administration of Ag, Ag + A-910823 without α-tocopherol, or Ag + AddaVax (Fig. S4F, G). These findings indicate that squalene-based adjuvants induced fever in an α-tocopherol-dependent manner.
Using the established systems for assessing systemic and local reactogenicity, we found that both the injection-site swelling score and fever induction by A-910823 were MyD88-dependent (Fig. 4B), indicating that MyD88 was involved in enhancing the immunogenic efficacy of A-910823 and in its reactogenicity induction.
We subsequently examined the involvement of IL-1 signaling in A-910823-mediated humoral immune response enhancement and reactogenicity induction using Il1r1 and Il1rn KO mice. To facilitate the evaluation of a potentially heightened response in Il1ra KO mice compared with control mice, the concentration of A-910823 was set at 10% v/v, while A-910823 was administered at 50% v/v in Il1r1 KO mice, consistent with other experiments. Serum neutralizing antibody titers and GCB cells were significantly reduced in Il1r1 KO mice compared with control mice, following administration of Ag + A-910823, with a trend toward decreased Tfh cell levels (Fig. 4C). In addition, both injection-site swelling score and fever induction were notably reduced in these mice (Fig. 4C). Consistent with these findings, serum neutralizing antibody titers, Tfh cells, and GCB cells were significantly increased in Il1rn KO mice compared with control mice (Fig. 4D), and swelling and fever were more strongly induced in Il1rn KO mice (Fig. 4D). These results collectively demonstrated that the IL-1R1/MyD88 signaling pathway played a critical role in both humoral immune response induction and reactogenicity caused by A-910823.
MyD88 is known to act downstream of membrane proteins, such as IL-1R family and TLR family members20,21. We investigated the potential contribution of TLR signaling to neutralizing antibody production and swelling induction by A-910823 using various Tlr KO mice. There were no significant differences in neutralizing antibody titers or swelling induction in Tlr2, Tlr4, Tlr7, and Tlr9 KO mice compared with control mice, indicating that TLR2, TLR4, TLR7, and TLR9 were not involved in humoral immune response induction or reactogenicity caused by A-910823 (Fig. S4H–J).
We further investigated which cell populations expressing MyD88 were involved in the efficacy and reactogenicity induction by A-910823 using two types of Myd88 conditional KO mice generated by crossing Myd88flox/flox (f/f) and Cd11ccre or LysMcre mice. The selective KO of MyD88 in specific cell types was confirmed by isolating CD11c+ cells and CD11b+ Ly6G+ cells from splenocytes using FCM sorting, followed by western blot analysis to detect MyD88 expression (Fig. S4K, L). The exon 3 region of Myd88 flanked by flox sites was excised in the presence of Cre, and MyD88 expression was thus evaluated using an antibody targeting the epitope within exon 3.
Ag + A-910823 administration significantly reduced serum neutralizing antibody titers and Tfh cell levels in Cd11ccre-Myd88f/f mice compared with control mice (Fig. 4E), but there were no changes in swelling score or fever induction (Fig. 4E). Conversely, there were no significant differences in serum neutralizing antibody titers, Tfh cell induction, GCB cell induction, swelling score, or fever induction in LysMcre-Myd88f/f mice compared with control mice (Fig. 4F).
These findings suggest that A-910823 enhanced humoral immunity via the IL-1R1/MyD88 signaling pathway in CD11c+ cells, such as DCs. Notably, the results also imply that the pathways responsible for reactogenicity induction were distinct from those driving immune enhancement.
A-910823 induced local reactogenicity originating from IL-1α in eosinophils, and systemic reactogenicity driven by the IL-1β/IL-6/ cyclooxygenase 2 (COX2) pathway
We further evaluated the contribution of IL-1R1/MyD88 signaling pathway molecules to the enhancement of vaccine efficacy and reactogenicity induced by A-910823 using IL-1β-neutralizing antibody-treated mice and Il1a KO mice. Administration of an IL-1β-neutralizing antibody 24 h prior to vaccination significantly reduced neutralizing antibody titers, Tfh cells, and GCB cells in mice treated with Ag + A-910823 compared with control mice (Fig. 5A, B). Regarding reactogenicity, there was no change in swelling induction, but fever induced by Ag + A-910823 was significantly suppressed (Fig. 5B). These results suggest that IL-1β contributed to enhanced vaccine efficacy and fever induction, but not to swelling, mediated by A-910823.
A Mice were injected IM with Ag mixed with A-910823 on days 0 and 14 (n = 4, 5/group), using IL-1β-neutralizing antibody-treated mice (B), Il1a KO mice (C), eosinophil-depleted mice pretreated with anti-CCR3 antibodies, and neutrophil-depleted mice pretreated with anti-Ly6G antibodies (D). B–D Serum titers of neutralizing antibodies against SARS-CoV-2 on day 28. Bars represent GMT; error bars indicate 95% confidence intervals; circles represent neutralizing antibody titers in individual mice. Percentages of Tfh (PD1+CXCR5+) cells in TCRb+CD4+ cells and percentages of GCB (FAS+GL7+) cells in CD19+ cells in draining LNs on day 28. Bars represent means; error bars indicate standard error of the mean; circles represent percentages of Tfh and GCB cells in individual mice. Injection-site swelling scores on day 15. Bars represent means; error bars indicate standard error of the mean; circles represent swelling scores in individual mice. Average body temperature was assessed on Day 12 (08:00–19:59) and on Day 15 (08:00–19:59). Vaccination at day 14 was performed at 18:00. Bars represent means; error bars indicate standard error of the mean. The data were derived from independent experiments: (1) Neutralizing titer/Tfh/GCB/Swelling measured in the same cohort and (2) Fever measured in an independent cohort. Statistical significance determined using two-tailed Student’s t-test for two-group comparisons or two-way ANOVA. ns, P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
The IL-1β/IL-6/COX2 axis has been reported to play a role in fever induction in models such as LPS administration22,23. Elevated serum IL-6 levels induced by squalene-based adjuvants in an α-tocopherol-dependent manner were accordingly suppressed by IL-1β-neutralizing antibody administration (Fig. S5A, B). We further investigated the roles of IL-6 and COX2 in fever induction by A-910823 using IL-6-neutralizing antibody-treated mice and mice treated with the COX2 inhibitor diclofenac. IL-6-neutralizing antibodies were administered 24 h prior to vaccination, and diclofenac was administered 14 h post-vaccination. Ag + A-910823 had no effect on neutralizing antibody titers, Tfh cells, GCB cells, or swelling score in IL-6-neutralized or diclofenac-treated mice, compared with control mice (Fig. S5C, D), but fever induction was significantly suppressed (Fig. S5C, D). These findings indicate that A-910823 induced fever via activation of the IL-6/COX2 pathway mediated by α-tocopherol-dependent IL-1β induction. The fact that neither IL-6-neutralizing antibodies nor COX2 inhibitors affected vaccine efficacy markers such as neutralizing antibody titers suggests that the IL-6/COX pathway did not contribute to vaccine efficacy.
We further investigated the role of IL-1R1 ligands using Il1a KO mice. Administration of Ag + A-910823 had no significant effect on neutralizing antibody titers, Tfh cells, GCB cells, or fever induction in Il1a KO mice compared with control mice (Fig. 5C); however, in contrast to the results in IL-1β-neutralized mice, swelling at the injection site was significantly suppressed (Fig. 5C). Given that IL-1α was induced in eosinophils in an α-tocopherol-dependent manner following administration of squalene-based adjuvants (Fig. 2C, D, F), we hypothesized that eosinophils were involved in the induction of local reactogenicity. We then tested this hypothesis by generating eosinophil-depleted mice by administering anti-C-C chemokine receptor type 3 (CCR3) antibodies, and control neutrophil-depleted mice by administering anti-Ly6G antibodies. Both antibodies were administered 24 h before vaccination, and successful depletion of the target cell populations was confirmed by measuring cell counts in the draining LNs (Fig. S5E). Administration of Ag + A-910823 had no significant effect on neutralizing antibody titers, Tfh cells, GCB cells, or fever induction in eosinophil-depleted mice pretreated with anti-CCR3 antibodies compared with mice treated with isotype control antibodies; however, swelling at the injection site was significantly suppressed (Fig. 5D). There were no significant differences in any of the measured parameters in neutrophil-depleted mice pretreated with anti-Ly6G antibodies compared with mice treated with isotype control antibodies (Fig. 5D).
Overall, these findings delineate the distinct molecular and cellular pathways underlying squalene-based adjuvant-induced vaccine efficacy and reactogenicity. The results suggest that the IL-1β/CD11c+ cell-mediated IL-1R1/MyD88 axis drives vaccine efficacy, whereas reactogenicity is governed by separate mechanisms, with systemic responses such as fever driven by the IL-1β/IL-1R1/MyD88/IL-6/COX2 cascade, and local responses, including swelling, involving eosinophil-derived IL-1α signaling, through the IL-1R1/MyD88 pathway.
Squalene-based adjuvants induced IL-1α production in human eosinophils in an α-tocopherol-dependent manner
We identified eosinophils as key inducers of local reactogenicity in mice. Eosinophils expressing IL-1α were notably induced by squalene-based adjuvants, while slight induction was observed with saponin-based adjuvants such as AS01 and minimal induction with alum adjuvants, suggesting that IL-1α-expressing eosinophils were primarily associated with squalene-based adjuvants (Fig. S5F).
We further investigated whether the α-tocopherol-dependent induction of IL-1α production observed in murine eosinophils was also applicable to human eosinophils. Peripheral blood samples from five healthy donors were treated by hemolysis and stimulated with adjuvants in vitro (Fig. 6A). Eosinophils were defined as cells that were positive for the granulocyte marker CD66b, exhibited high side-scatter properties, and were negative for CD16 (which is highly expressed in neutrophils) (Fig. 6B). IL-1α expression was quantified by ICS-FCM. Supplementation with A-910823 or AddaS03 resulted in concentration-dependent induction of IL-1α (Fig. 6C). In contrast, treatment with Ag + A-910823 without α-tocopherol or Ag + AddaVax resulted in no clear induction of IL-1α production, even at the highest treatment concentrations. Similarly, stimulation with LPS or IL-5 also failed to induce significant IL-1α expression. These findings indicate that α-tocopherol-dependent induction of IL-1α by squalene-based adjuvants is conserved between mouse and human eosinophils.
A Study schedule. B Gating strategy for eosinophils. C MFI in human peripheral blood-derived eosinophils. Bars represent means; error bars indicate standard error of the mean. Statistical significance determined using one-way ANOVA, ns, P ≥ 0.05, ***P < 0.001, ****P < 0.0001).
Discussion
In this study, we utilized A-910823 to elucidate the mechanism of action of squalene-based adjuvants, focusing on α-tocopherol. Although previous research has established that α-tocopherol contained in squalene-based adjuvants contributes to the enhancement of humoral immune responses, the precise mechanism underlying its action remains unclear. In addition, the relationship between α-tocopherol and MyD88, which is known to contribute to the adjuvant activity of squalene-based adjuvants, as well as the role of α-tocopherol in reactogenicity induction and the mechanisms underlying this process, is also poorly understood. The current results demonstrated that α-tocopherol in A-910823 impacted the IL-1 signaling pathway. Similarly, comparative analysis using the AS03 mimic AddaS03 and MF59 mimic AddaVax confirmed the α-tocopherol-dependent induction of IL-1α and IL-1β. This mechanism thus represents a common pathway for squalene-based adjuvants. Moreover, evaluations using KO mice and mice treated with neutralizing antibodies targeting IL-1 pathway molecules revealed distinct cellular and molecular pathways involved in squalene-based adjuvant activity and reactogenicity. Specifically, we demonstrated that the IL-1β/CD11c+ cell IL-1R1/MyD88 axis mediated the immune efficacy, while the IL-1β/IL-1R1/MyD88/IL-6/COX2 axis drove the systemic reactogenicity, and the eosinophil IL-1α expression/IL-1R1/MyD88 axis governed the local reactogenicity. The novelty of this study lies in its elucidation of the mechanisms of immunogenicity and reactogenicity induction by α-tocopherol-containing squalene-based adjuvants, demonstrating the distinct separation of these processes.
IL-1 is a potent pro-inflammatory cytokine comprising two independently regulated molecules, IL-1α and IL-1β, both of which play integral roles in host defense responses to infection by promoting the expression of immune and inflammatory response genes. Conversely, IL-1R antagonists act as competitive inhibitors, binding to IL-1Rs to mitigate these pro-inflammatory actions and maintain immune homeostasis24,25. IL-1α is constitutively expressed in many cell types and acts as an ‘alarmin’ by alerting the immune system in response to tissue damage, while IL-1β is produced in response to inflammatory stimuli. In the context of vaccines, IL-1α and IL-1β production depend on various adjuvants, such as alum24. The current findings highlight the contribution of IL-1β to both vaccine efficacy and systemic reactogenicity, while IL-1α was involved in local reactogenicity. Although neutrophils were the major IL-1β-expressing population in draining LNs at 6 h (Figs. 2 and S2), functional data argue they are not the principal mediators of A-910823’s adjuvant effect. Neutrophil depletion with anti-Ly6G did not alter neutralizing antibody titers, Tfh or GCB responses, nor swelling or fever (Fig. 5D), whereas systemic IL-1β neutralization reduced humoral responses and fever (Fig. 5B). These findings suggest that other myeloid populations (for example, CD11c+ cells or monocytes) or a small, functionally critical subset of IL-1β-producing cells provide the key signal for vaccine potentiation. We acknowledge limitations of the depletion approach and propose that cell-type-specific IL-1β ablation or conditional depletion will be required to definitively identify the functionally relevant source. In addition, we revealed that IL-1α was consistently induced in eosinophils through an α-tocopherol-dependent mechanism in both mice and humans, highlighting comparable results across species. Although clinical trials have demonstrated that COVGOZE®, which includes A-910823 as an adjuvant, is generally well-tolerated, there have been some observations of local reactogenicity4,5. The results suggest that these occurrences may be associated with fluctuations in eosinophil-derived IL-1α, indicating its potential utility as a predictive marker for local reactogenicity associated with α-tocopherol-containing squalene-based adjuvants. In contrast to squalene-based adjuvants, alum and saponin-based adjuvants did not induce IL-1α expression in eosinophils; however, the existing literature suggests that the IL-1 signaling pathway may play a role in modulating both the efficacy and reactogenicity of vaccines, impacting both their effectiveness and the nature of any associated systemic and local events26,27,28,29. For instance, the addition of mRNA-lipid nanoparticles induced the production of IL-1β and IL-6 by human peripheral blood mononuclear cells, and serum IL-6 induction following mRNA-lipoplex administration was abrogated in Il1r1 KO mice29. Other adjuvants and vaccines may thus also have the potential to induce systemic reactogenicity via the IL-1 signaling pathway, similar to squalene-based adjuvants. Building upon these insights, the utilization of recently established adjuvant databases may provide a broader understanding of the commonality in mechanisms across different adjuvants30.
Further research is required to understand how α-tocopherol induces IL-1 ligands and how IL-1β induces Tfh cells. Previous studies have shown that selective knockout of Il1r1 in CD4+ cells resulted in a reduction in Tfh cells induced by vaccines containing alum as an adjuvant, compared with control mice31. The effect of IL-1β on T cells may thus also be considered in the induction of Tfh. Furthermore, AS03 has been shown to induce endoplasmic reticulum stress both in vitro and in vivo17, which has recently been found to contribute to the induction of IL-1β32. This suggests that stress induction by α-tocopherol could be involved in activation of the IL-1 pathway, resulting in the induction of both vaccine efficacy and reactogenicity. Most previous studies of α-tocopherol, as a form of vitamin E, have been conducted in vitro using low concentrations or in vivo by oral administration because of its insolubility in water33. In contrast to these experimental conditions, however, α-tocopherol is present at extremely high concentrations in oil-in-water emulsions like A-910823 and AS03, indicating the need for more detailed studies of the function of α-tocopherol under these conditions.
Previous studies using squalene-based adjuvants showed that both AS03 and MF59 depended on MyD88 for the induction of humoral immunity14,18. Consistent with this, the current study found that the induction of humoral immunity was strongly inhibited in Myd88 KO mice administered A-910823, more notably than the inhibition of humoral immunity induction observed in Il1r1 KO mice and IL-1β neutralized mice. These results suggest that the enhancement of humoral immunity induction by α-tocopherol in squalene-based adjuvants depends on IL-1R1, while humoral immunity induction by components other than α-tocopherol might involve an IL-1R1-independent and MyD88-dependent pathway. The current experiments using AddaVax, together with previous studies of MF59, indicated that the induction of IL-1β was hardly confirmed, even with the use of squalene-based adjuvants without α-tocopherol, but humoral immune induction by MF59 is known to depend on MyD88 and ASC13,14. It is therefore possible that another IL-1 family member not verified in this study, such as IL-18, might be involved in the induction of humoral immunity by components other than α-tocopherol.
This study had several limitations. First, we could not elucidate the mechanism of induction of cellular immunity. We found no significant difference in the induction of cellular immunity in the KO mice used in this study, including Myd88 KO mice. A previous study using MF59 showed that RIPK3-dependent cell death of macrophages was involved in the induction of cellular immunity, and it is plausible that a similar mechanism could exist for A-91082316. Second, we lacked spatial and cell-type resolution to link adjuvant uptake, Il1 transcription, and intracellular protein expression. Notably, bulk RNA-seq showed Il1a induction irrespective of α-tocopherol (Fig. 2A), whereas ICS-FCM detected IL-1α protein mainly in eosinophils and only with α-tocopherol at 6 h (Fig. 2D); this likely reflects bulk averaging, post-transcriptional regulation, and timing differences. Single‑cell and spatial transcriptomic/proteomic approaches with finer temporal sampling, together with in vivo distribution studies such as immunohistochemistry using fluorescently-labeled adjuvant, will be needed to resolve these discrepancies. Third, although recent studies have revealed that eosinophils can be categorized into several subsets, the subset responsible for inducing reactogenicity remains unclear34. The identification and characterization of these eosinophil subsets, as well as the downstream mechanisms of reactogenicity induction via IL-1α, are thus important topics for future investigations. Finally, further studies are also needed to identify the specific cells responsible for the production of IL-1β, which is involved in efficacy and fever induction, as well as the cells producing IL-6.
Despite the above limitations, the results of this study represent an important advancement in vaccine science, offering a novel perspective on the relationship between reactogenicity and efficacy. Historically, reducing vaccine reactogenicity has been considered to necessarily compromise efficacy, while the current findings disrupt this traditional view by indicating that these elements can be differentiated at both a molecular and a cellular level. This represents a paradigm shift that challenges longstanding beliefs and proposes that the separation of efficacy and reactogenicity is not only possible but also practical. This finding has substantial impacts for the field of adjuvant research, paving the way for the development of adjuvants that are both highly effective and possess superior safety profiles, with the potential to minimize adverse reactions and enhance immune responses, with significant implications for public health by improving vaccine safety and efficacy. By demonstrating the feasibility of dissociating reactogenicity from efficacy, this study lays the foundation for future advances in vaccine technology, guiding the development of vaccines that are not only safer and more effective but which are also tailored to meet the diverse needs of global populations.
Methods
Antigen and adjuvants
Full-length recombinant SARS-CoV-2 S protein from Pango lineage A (S-910823; Ag) was expressed using a baculovirus expression vector system, as described previously35,36,37. Briefly, the S-910823 protein was extracted from baculovirus-infected cells and purified by affinity and hydrophobic interaction chromatography. Host-cell DNA and baculoviruses were removed by Q membrane chromatography. Oil-in-water emulsion adjuvant A-910823 and A-910823 excluding α-tocopherol (A-910823 without α-tocopherol) were prepared at Shionogi & Co., Ltd. (Osaka, Japan) by high-pressure homogenization, as described previously11,38,39. BODIPY-labeled A-910823 (A-910823-BODIPY), containing 0.1% BODIPY-labeled α-tocopherol of the total α-tocopherol content, was prepared by Shionogi & Co., Ltd. BODIPY-labeled α-tocopherol was synthesized by covalently modifying the hydroxyl groups of α-tocopherol using 6-(Boc-amino) hexyl bromide (#89171, Sigma-Aldrich, Burlington, MA, USA) and BDP FL NHS ester (#D5555, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). BODIPY-labeled α-tocopherol was added to the oil phase of squalene, α-tocopherol, and polysorbate 80, and the subsequent manufacturing process was carried out as described previously11. The average diameter and polydispersity index were measured as described previously11. The fluorescence spectrum of A-910823-BODIPY was measured using a microplate reader with 480 nm-excitation.
Animals and immunization procedures
C57BL/6JJcl mice (6–8 weeks old) were purchased from CLEA Japan, Inc. (Tokyo, Japan). Myd88 KO mice, Tlr2 KO mice, Tlr4 KO mice, and Tlr9 KO mice on a C57BL/6 background were obtained from Oriental Bioservice (Kyoto, Japan). Il1a KO mice were obtained from the Center for Animal Disease Models, Tokyo University of Science40. Il1r1 KO mice were obtained as described previously41. Tlr7 KO mice were provided by Dr. Shizuo Akira (Osaka University, Suita, Japan). Il1rn KO mice, Myd88flox mice, Cd11ccre transgenic mice, and LysMcre transgenic mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Breeding colonies were maintained by mating Myd88f/f with Cd11ccre-Myd88f/f mice or LysMcre-Myd88f/f mice and the Myd88f/f littermates were used as control mice42. All mouse experiments were performed in accordance with the appropriate laws and guidelines approved by the Institute of Medical Science at the University of Tokyo (Tokyo, Japan; approval no. PA21-46) and the Institutional Animal Care and Use Committee of Shionogi & Co., Ltd (approval no. S19017D). After anesthetization, mice received an IM injection of the following into the gastrocnemius muscle: Ag (1 μg) in 50 μL PBS solution (antigen-alone group); Ag (1 μg) in 25 μL PBS solution mixed with A-910823 (25 μL); Ag (1 μg) in 25 μL PBS solution mixed with A-910823 without α-tocopherol (25 μL); Ag (1 μg) in 25 μL PBS solution mixed with AddaS03 (25 μL; InvivoGen, San Diego, CA, USA); Ag (1 μg) in 25 μL PBS solution mixed with AddaVax (25 μL; InvivoGen); Ag (1 μg) in 2.5 μL PBS solution mixed with AS01 (47.5 μL; GlaxoSmithKline K. K., UK); and Ag (1 μg) in alum (250 μg; InvivoGen). Il1rn KO mice received Ag (1 μg) in 45 μL PBS solution mixed with A-910823 (5 μL) by IM injection into the gastrocnemius muscle. This reduction in dosage was made in anticipation of stronger adverse reactions in Il1rn KO mice compared to control mice. Treatments were administered in the right hind limb on days 0 and 14 to assess neutralizing antibody titers, Tfh cells, GCB cells, IFN-γ and IL-4 positive cells, swelling score, and body temperature, and dissections were conducted on day 28. Other experiments involved a single administration, and dissections were carried out at the times described in the Methods, Results, and Figure legends. A summary of the immunization schemes, injection and endpoint time points, and the organs/tissues collected for each experiment is provided in Table 1.
RNA-seq
Mice were administered Ag alone, Ag with A-910823, and Ag with A-910823 without α-tocopherol IM, and the muscles and the iliac LNs (collected as the draining LNs) were collected at 30 min and 4 h later. The muscles were homogenized in QIAzol Lysis Reagent (Qiagen, Hilden, Germany) using a TissueRuptor II (Qiagen), and the LNs were homogenized using a BiomusherII (Nippi Research Institute of Biomatrix, Ibaraki, Japan), followed by the addition of chloroform. The samples were then centrifuged at 12,000 × g for 15 min, the aqueous phase was transferred to a new tube, and isopropanol was added and mixed. The mixture was centrifuged at 12,000 × g for 10 min, the supernatant was aspirated, ethanol was added, and the mixture was centrifuged again at 12,000 × g for 5 min. The supernatant was removed, and the RNA pellet was air-dried. The RNA was then purified using an RNeasy Plus kit (Qiagen) following the manufacturer’s instructions, and the concentration and purity were measured using a NanoDrop one (Thermo Fisher Scientific, Waltham, MA, USA). The sequence library was prepared using SMART-Seq® mRNA LP (Takara) with purified RNA, following the manufacturer’s instructions, and sequenced on a NextSeq 2000 (Illumina) with 61-bp paired-end reads. The sequence reads were mapped to GRCm39 (Release M34 from the GENCODE project) using STAR43 after trimming of adapters, polyG, polyX, and trimming one base from the tail of the sequences using fastp44 to generate the sequence alignment map files. The raw read counts for genes were quantified using TPMCalculator45 with the alignment files. The calculation of normalized tag counts, differential gene expression analysis, and PCA were performed using the DESeq2 package46 in R. Differentially expressed genes were defined by the criteria log2 fold change ≥1 or ≤−1 and false discovery rate <0.05. Heatmaps illustrating the gene expression pattern were generated using the ComplexHeatmap package47 in R. The gene list of chemokines, cytokines, INFs, ILs, TGF-β, TNF, and their receptors was obtained from the Gene Lists section of ImmPort48. These genes were then converted to their mouse homologs and used in the analysis. Over-representation analysis of signaling pathways from the Gene Ontology biological process49,50 and Kyoto Encyclopedia of Genes and Genomes (KEGG) gene sets51 in the Molecular Signatures Database52,53 was performed using the clusterProfiler package54 in R. For the over-representation analysis, the input mouse genes were converted to their human homologs, because the reference signaling pathways consist of human genes. The gene ratio was obtained by dividing the number of input genes shared with genes in the signaling pathway by the total number of input genes, and adjusted p-values were calculated using the default settings of the package. Conversion of gene homologs between humans and mice was conducted using the Human and Mouse Homology Classes report at the Vertebrate Homology section of the Mouse Genome Informatics (https://www.informatics.jax.org/homology.shtml). To obtain sufficient RNA input and to reduce inter‑individual variability, total RNA for RNA‑seq was prepared by pooling tissues from three mice. This pooling procedure was performed independently three times per experimental group and time point, resulting in three independent pooled biological replicates per condition (n = 3), with a total of nine mice used per group. RNA‑seq libraries were prepared independently from each pooled sample and sequenced once per library.
Specimen preparation
Mice were euthanized, and whole blood, iliac LNs (collected as the draining LNs), muscles, and spleens were obtained. Serum was prepared from the blood samples and stored at −80 °C for subsequent assays. Spleens were dissociated into cell suspensions using a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). RBCs were lysed using Red Blood Cell Lysis Buffer (Roche, Basel, Switzerland), and splenocytes were suspended in RPMI-1640 medium (Nacalai Tesque Inc., Kyoto, Japan) supplemented with 10% FBS (Sigma-Aldrich). Draining LNs were collected, and single-cell suspensions were prepared using BiomusherII (Nippi Research Institute of Biomatrix).
Flow cytometry
Draining LNs were collected on day 28 and at 2, 6, and 14 h post-first immunization to analyze Tfh and GCB cells, and eosinophils, neutrophils, monocytes, and DCs, respectively. Draining LN cells were then washed and incubated with anti-CD16/32 (clone 2.4G2; BD Pharmingen, San Diego, CA, USA) and then stained for viability using a Zombie NIR™ Fixable Viability Kit (BioLegend, San Diego, CA, USA). Cells were surface-stained with the antibody panels listed in Table 2 and then fixed with Fixation Buffer (BioLegend) for intracellular staining of IL-1α and IL-1β. After surface staining, cells were fixed with Fixation Buffer (BioLegend) and stained to detect IL-1α and IL-1β, using a BD LSR II flow cytometer (BD Bioscience, San Jose, CA, USA) or NovoCyte 3000 flow cytometer (Agilent Technologies, Inc., Santa Clara, CA, USA). Data were analyzed using FlowJo (Tree Star, Ashland, OR, USA) or NovoExpress 1.4.0 software (Agilent Technologies, Inc.). The operational phenotype (gating) used to define each population is provided in the final column of Table 2; representative gating strategies are shown in Figs. 6B, S2A, and S3D.
Western blotting and cell sorting
Sorted cells were obtained from one whole spleen per mouse; the entire spleen was processed and stained for sorting. Splenocytes were incubated with anti-CD16/32 (clone 2.4G2; BD Pharmingen) and stained for viability using a Zombie NIR™ Fixable Viability Kit (BioLegend). The cells were then immunostained with the antibodies outlined in Table 2 and the target cells were sorted in a two-way sorting mode and collected into non-coated tubes using a BD Influx cell sorter (BD Biosciences). To ensure high purity of the sorted fraction, a 1.0 drop purity mode with an extra coincidence scale of six was selected. From one spleen we collected at least 200,000 target cells for each sorted fraction; these sorted splenocyte fractions were lysed directly in RIPA buffer (Nacalai Tesque Inc.) containing protease inhibitor cocktail (Nacalai Tesque Inc., 100-fold dilution) for 30 min on ice and used for MyD88 and β-actin detection. The sorted splenocyte lysates were loaded at 100,000 cells per lane for electrophoresis. For IL-1β and β-actin detection from lymphoid tissue, cells obtained from one whole lymph node per mouse were lysed in RIPA buffer and prepared separately; these lymph node lysates were quantified by BCA assay (Thermo Fisher Scientific) and loaded at 10 μg protein per lane. Following the removal of insoluble material by centrifugation, cellular proteins were resolved by 4%–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Thermo Fisher Scientific) under reducing conditions and transferred to polyvinylidene difluoride membranes (Thermo Fisher Scientific). The membranes were then blocked with 5% BSA (Sigma-Aldrich) in Tris-buffered saline containing 0.05% Tween 20 (Sigma-Aldrich) and incubated with anti-β-actin (#3700, 1:5000, Cell Signaling Technology, MA, USA), anti-MyD88 (#4283, 1:1000, Cell Signaling Technology), or anti-IL-1β (#12507, 1:1000, Cell Signaling Technology), followed by peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibody (Cell Signaling Technology). Luminol-based detection was conducted using Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation, Billerica, MA, USA) according to the manufacturer’s instructions, and signals were analyzed using a LAS-4000 mini image analyzer (GE Healthcare, Little Chalfont, UK).
SARS-CoV-2 neutralization assay
Serum levels of neutralizing antibodies were analyzed against ancestral SARS-CoV-2 WK-521 using transmembrane serine protease 2-expressing VeroE6 (VeroE6/TMPRSS2) cells (Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan), as described previously11,37,55. In brief, VeroE6/TMPRSS2 cells were maintained in DMEM (Thermo Fisher Scientific) with heat-inactivated FBS (Corning Inc., Corning, NY, USA) and G418 (Thermo Fisher Scientific). The WK-521 strain of SARS-CoV-2, isolated from a Wuhan traveler (National Institute of Infectious Diseases, Tokyo, Japan)55, was propagated on these cells to prepare virus stocks. Titers were calculated using tissue culture infectious dose 50 (TCID50) assays as previously described on VeroE6/TMPRSS2 cells37. Heat-inactivated serum samples were serially diluted two-fold. Each sample was mixed with an equal volume of viral suspension (2000 TCID50/mL) and incubated for approximately 1 h at room temperature for neutralization. After neutralization, 100 µL of the sample/virus mixtures were dispensed in duplicate into 96-well culture plates. The samples were then incubated with 100 µL of VeroE6/TMPRSS2 cell suspension (3 × 104 cells/well) to obtain a virus titer of 100 TCID50/well. The virus and cell suspension were added to the virus control wells, whereas only the cell suspension was added to the cell control wells. The samples were incubated at 37 °C with 5% CO2 for 3 days. Cell viability was evaluated using CellTiter-Glo 2.0 (Promega, Madison, WI, USA). After removing 100 μL of supernatant from each well, 100 μL of CellTiter-Glo was added, and the samples were incubated at room temperature for approximately 30 min under light-shielded conditions. Next, 100 μL of the mixture was transferred to measurement plates, and the luminescence intensity was measured using an EnSpire 2300 microplate reader (PerkinElmer, Waltham, MA, USA).
ELISpot assay
Antigen-specific cytokine production was measured using ELISpotPLUS kits for mouse IFN-γ and IL-4 (Mabtech, Cincinnati, OH, USA), as described previously11. In brief, ELISpot plates were washed with PBS and blocked with RPMI-1640 medium supplemented with 10% FBS for at least 1 h at room temperature. Spike protein overlapping peptides PepTivator SARS-CoV-2 Prot_S, PepTivator® SARS-CoV-2 Prot_S1, and PepTivator® SARS-CoV-2 Prot_S+ (all from Miltenyi Biotec, Bergisch Gladbach, Germany) were dissolved in distilled water and diluted with RPMI-1640 medium supplemented with 10% FBS at a concentration of 180 pmol/mL per peptide. After removing the blocking solution, 100 µL of peptide solution was added to the ELISpot plates. Distilled water in RPMI-1640 medium supplemented with 10% FBS served as a peptide negative (−) control. Next, 100 µL of splenocyte suspension (3 × 106 cells/mL) was added to each well, and the plates were incubated at room temperature for approximately 30 min, followed by incubation at 37 °C with 5% CO2 overnight. Following five washes with PBS, the samples were incubated with 100 µL of detection antibody solution for 2 h. Following a further five washes with PBS, the samples were incubated with 100 µL of diluted alkaline phosphatase-conjugated streptavidin solution for at least 1 h. Finally, the sample was incubated with 100 µL of the chromogen 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium, which was used as a chromogen. The color development was stopped by washing the sample with tap water. The spots were counted using an immunospot S6 UNIVERSAL-V ELISPOT reader (Cellular Technology Limited, Beachwood, OH, USA). The mean spot-forming cell value was calculated for each group. The antigen-specific cytokine production was evaluated by comparing spot-forming cell values of peptide (+) samples with those of peptide (−) control wells.
Evaluation of swelling score at the administration site
Injection-site swelling was scored on a three-point scale as follows: 0, no visible swelling at the injection site or abnormal hind limb movement (Supplementary Movie 1A); 1, visible or palpable swelling at the injection site, accompanied by abnormal hind limb movement (Supplementary Movie 1C) (specifically, the hind limb can still be extended when pulled, but with resistance during extension); 2, more severe condition, characterized by pronounced swelling at the injection site and significant abnormalities in hind limb movement (Supplementary Movie 1B) (extending the hind limb becomes difficult). The swelling score at the administration site was evaluated in vaccinated mice at 6, 12, 24, 48, 72, 96, and 168 h post-administration (Fig. S4A). The administration site was photographed using a 3D scanner coordinate-measuring machine (VL-800; Keyence, Osaka, Japan) (Fig. S4B, C). The X, Y, and Z-axes were defined, and the cross-sectional area and width of the administration site were measured. The Y-axis was defined as the straight line connecting the anterior part of the ankle and the patella, and the Z-axis was defined as the straight line connecting the heel, the base of the middle toe, and the anterior part of the ankle.
Body temperature measurement
Mice were implanted under anesthesia with a nano-tag® (KISSEI COMTEC, Nagano, Japan) equipped with an accelerometer and temperature sensor into the abdominal cavity 1 week prior to vaccine administration. Measurements were conducted at 1-min intervals for 48 h before and after vaccine administration. The vaccination regimen consisted of two intramuscular injections given 2 weeks apart; body temperature analyses focused on the period surrounding the second (day 14) vaccination. Vaccines were administered at 18:00. Activity data from the accelerometer indicated pronounced diurnal variation and activity-dependent temperature fluctuations; therefore, to minimize activity-related noise, we used daytime averages (08:00–19:59) for fever assessment. For baseline control, daytime averages (08:00–19:59) from 2 days before vaccination were calculated and used for pre‑vaccination comparisons. Because vaccination occurred at 18:00, the daytime average on the first calendar day after vaccination (08:00–19:59) spans approximately 14–26 h post-vaccination. Where elapsed-time labels (e.g., 12, 24, and 48 h) are used elsewhere in the manuscript for other assays, they denote hours after vaccination; for body temperature reporting, however, the daytime averages defined above are presented unless an instantaneous time-point is explicitly specified. Some mice received the COX inhibitor diclofenac (30 mg/kg, IP), and control mice received an IP injection of saline at 07:30 on the day after vaccination. Fever induction by A-910823 and its α‑tocopherol dependence measured with the nano‑tag system were confirmed in three independent experiments, yielding consistent quantitative results; other nano‑tag experiments with extremely low throughput (for example, those involving KO mice) were performed once due to practical constraints but produced comparable outcomes after both the first and second vaccine doses.
Histology
Tissue samples from the gastrocnemius muscle at the injection sites were harvested and subsequently fixed with 10% neutral buffered formalin (Mildform 10 N; FUJIFILM Wako Pure Chemical, Osaka, Japan) for 24 h for histopathological analysis. The fixed tissues were then embedded in paraffin to create blocks. These blocks were sectioned into slices with a thickness of 3 µm using a microtome. The resultant sections were then stained with hematoxylin and eosin for visualization. Histopathological changes at the injection sites, including infiltration of inflammatory cells, edema, and necrosis of muscle fibers, were evaluated independently. Edema was scored as follows: 0, no observable changes; 1, presence of lesions in the perimysium or epimysium, along with localized and small lesion area; 2, lesions spreading into the endomysium and presence of plasma components in the epimysium, along with localized but large lesion area, slightly spreading to surrounding tissues; 3, lesions not localized and spreading extensively to surrounding tissues, but with a low frequency; and 4, lesions diffuse and with a high frequency.
In vivo administration of antibodies targeting CCR3, Ly6G, IL-6, and IL-1β
In vivo depletion of eosinophils and neutrophils and neutralization of IL-6 and IL-1β were achieved by IP injection of 200 µg purified mAbs 24 h prior to vaccination. CCR3 (Bio X Cell, Lebanon, NH, USA; clone: 6S2-19-4, Rat IgG2b, λ) and Ly6G (BioLegend; clone: 1A8, Rat IgG2a, κ) mAbs were used to selectively deplete murine eosinophils and murine neutrophils, respectively, and IL-1β (BioLegend; clone: B122, Armenian Hamster IgG) and IL-6 (BioLegend; clone: MP5-20F3, Rat IgG1, κ) mAbs were used to neutralize IL-1β and IL-6, respectively. Control mice were injected IP with an isotype control antibody (Bio X Cell, BioLegend) for an equal duration.
Measurement of cytokine levels
Serum cytokine levels were measured using a Bio-Plex Pro Mouse Cytokine 23-plex Assay kit (Bio-Rad, Hercules, CA, USA), following the manufacturer’s instructions. This study specifically focused on detectingIL-1α, IL-1β, IL-6, and G-CSF among the 23 cytokines provided by the kit. Fluorescence signals were detected using a Bio-Plex system, allowing for precise measurement of cytokine levels. Assay-specific limits of detection (LOD) were 2.17 pg/mL for IL-1α, 2.19 pg/mL for IL-1β, 0.82 pg/mL for IL-6, and 4.61 pg/mL for G-CSF. No measurements exceeded the assay upper limit for any analyte. Values below the LOD were assigned LOD/2 for statistical analyses.
Human whole blood cells
Blood samples were collected from healthy participants following approval by the Institutional Review Board of the Institute of Medical Science, The University of Tokyo (IMSUT) of Medicine (approval no. 2024-15-0620). All participants provided written informed consent. Peripheral blood was collected into sodium-EDTA tubes (TERUMO, Tokyo, Japan) by venous puncture. Following hemolysis with ammonium chloride, cells were seeded into 96-well plates and cultured for 2.5 h in the presence of various adjuvants (0.1%–10% v/v), LPS (1 μg/mL), and IL-5 (10 ng/mL). Eosinophil IL-1α was detected by FCM. Staining with antibodies against cell surface markers was carried out according to Table 2. Following cell surface staining, cells were fixed with Fixation Buffer (BioLegend) and subjected to ICS with IL-1α antibody (Table 2).
Reproducibility and statistics
Differences between two groups were analyzed by two-tailed Student’s t tests, and differences among multiple groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test; multiple groups with two influencing factors were compared using two-way ANOVA. For RNA-seq analyses, differential expression was assessed using DESeq2 based on three independent pooled biological replicates per group. For selected comparisons within predefined gene sets, paired data from RNA-seq analyses were additionally assessed using Wilcoxon’s matched-pairs signed rank test. Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software Inc, San Diego, CA, USA). Most key experiments were independently repeated 2–3 times with comparable results. Data are presented as mean ± SEM unless otherwise stated. Total RNA for RNA-seq was prepared by pooling tissues from three mice, and this pooling procedure was independently repeated three times per group to generate pooled biological replicates (n = 3). RNA-seq analyses were intended to provide an exploratory overview of transcriptional and pathway-level responses. Key transcriptional changes identified by RNA‑seq (for example, upregulation of Il1a, Il1b, Csf3, Il6) were validated on independent biological replicates at the protein level using Bio-Plex assays (see Fig. S2). Fever induction by the adjuvant and its α-tocopherol dependence measured with the nano-tag system were confirmed in three independent experiments, yielding consistent quantitative results; other nano-tag experiments with extremely low throughput (for example, those involving KO mice or mice receiving neutralizing antibody treatment) were performed once due to practical constraints but produced comparable outcomes after both the first and second vaccine doses.
Data availability
Sequence data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE306134. Differentially expressed genes with DESeq2 of this study are provided as a supplementary material file (see Supplementary_Gene_List.txt). Data from other experiments presented in this study are available from the corresponding author upon reasonable request.
References
Pulendran, B., Arunachalam, P. S. & O’Hagan, D. T. Emerging concepts in the science of vaccine adjuvants. Nat. Rev. Drug Discov. 20, 454–475 (2021).
Iwata, S. et al. A phase 3 randomized controlled trial of a COVID‑19 recombinant vaccine S‑268019‑b versus ChAdOx1 nCoV‑19 in Japanese adults. Sci. Rep. 14, 9830 (2024).
Iwata, S. et al. Phase 1/2 clinical trial of COVID‑19 vaccine in Japanese participants: interim findings. Vaccine 40, 3721–3726 (2022).
Shinkai, M. et al. Immunogenicity and safety of booster dose of S‑268019‑b or BNT162b2 in Japanese participants: a phase 2/3, randomized, observer‑blinded, non‑inferiority study. Vaccine 40, 4328–4333 (2022).
Sonoyama, T. et al. Safety and immunogenicity of a booster dose of S‑268019‑b: interim findings of a phase 3, open‑label clinical study in Japan. Vaccin. X 15, 100390 (2023).
Takano, T. et al. Heterologous SARS‑CoV‑2 spike protein booster elicits durable and broad antibody responses. Nat. Commun. 14, 1451 (2023).
Dinh Thiem, V. et al. A SARS-CoV-2 recombinant spike protein vaccine (S-268019-b) for COVID-19 prevention during the Omicron-dominant period: a phase 3 randomized, placebo-controlled trial. Vaccine 42, 3699–3709 (2024).
Fujitani, M. et al. Longitudinal analysis of immune responses to SARS‑CoV‑2 recombinant vaccine S‑268019‑b in a phase 1/2 prime‑boost study. Front. Immunol. 16, 1550279 (2025).
Hastie, A. et al. Immunogenicity and safety of AS03‑adjuvanted H7N9 influenza vaccine in adults: a phase 1/2 randomized, placebo‑controlled trial. Influenza Other Respir. Viruses 18, e70020 (2024).
Morel, S. et al. Adjuvant system AS03 containing α‑tocopherol modulates innate immune response and improves adaptive immunity. Vaccine 29, 2461–2473 (2011).
Yoshioka, Y. et al. A‑910823, a squalene‑based emulsion adjuvant, induces T follicular helper cells via α‑tocopherol. Front. Immunol. 14, 1116238 (2023).
Calabro, S. et al. The adjuvant effect of MF59 is due to the oil‑in‑water formulation. Vaccine 31, 3363–3369 (2013).
Ellebedy, A. H. et al. Inflammasome‑independent role of ASC in MF59 adjuvanticity. Proc. Natl. Acad. Sci. USA 108, 2927–2932 (2011).
Seubert, A. et al. MF59 adjuvanticity is independent of Nlrp3 but requires MyD88. Proc. Natl. Acad. Sci. USA 108, 11169–11174 (2011).
Vono, M. et al. MF59 induces ATP release from muscle to boost vaccine response. Proc. Natl. Acad. Sci. USA 110, 21095–21100 (2013).
Kim, E. H. et al. Squalene emulsion adjuvants stimulate CD8 T cells via a RIPK3‑dependent pathway. Elife 9, e52687 (2020).
Givord, C. et al. AS03 activates ER stress sensor IRE1α contributing to immunostimulation. NPJ Vaccines 3, 20 (2018).
Nakkala, J. R. et al. Differential regulation of DC function by two squalene emulsion adjuvants. Vaccines 12, 531 (2024).
Martinon, F. & Tschopp, J. Inflammatory caspases and inflammasomes. Cell Death Differ. 14, 10–22 (2007).
Kawai, T. & Akira, S. Pattern‑recognition receptors in innate immunity. Nat. Immunol. 11, 373–384 (2010).
Zheng, C. et al. Inflammatory role of TLR–MyD88 signaling in multiple sclerosis. Front. Mol. Neurosci. 12, 314 (2020).
Li, S. et al. COX‑2 mediates febrile response to IL‑1β in mice. Brain Res. 910, 163–173 (2001).
Rummel, C. et al. IL‑6 induces fever through STAT3‑linked COX‑2 activation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 291, R1316–R1326 (2006).
Muñoz‑Wolf, N. & Lavelle, E. C. IL‑1 family cytokines in adjuvanticity. FEBS J. 285, 2377–2401 (2018).
Mantovani, A. et al. Interleukin‑1 in inflammation and immunity. Immunity 50, 778–795 (2019).
Alameh, M. G. et al. Lipid nanoparticles enhance mRNA and protein vaccine efficacy. Immunity 54, 2877–2892.e7 (2021).
Kobiyama, K. & Ishii, K. J. Innate sense of mRNA vaccine adjuvanticity. Nat. Immunol. 23, 474–476 (2022).
Li, C. et al. Mechanisms of immune responses to BNT162b2 vaccine. Nat. Immunol. 23, 543–555 (2022).
Tahtinen, S. et al. IL‑1 and IL‑1ra regulate inflammatory response to RNA vaccines. Nat. Immunol. 23, 532–542 (2022).
Natsume‑Kitatani, Y. et al. Adjuvant database for preclinical vaccine evaluation. Cell Chem. Biol. 7, S2451–945600228‑4 (2025).
Belbezier, A. et al. IL‑1 regulates follicular T cells in germinal centers. Front. Immunol. 15, 1393096 (2024).
Sasaki, I. et al. IRE1α is required for exotoxin‑induced IL‑1β production. Cell Rep. 43, 113981 (2024).
Han, S. N. & Meydani, S. N. Vitamin E and immune function. Expert Rev. Clin. Immunol. 2, 561–567 (2006).
Gurtner, A. et al. Active eosinophils regulate host defense in colitis. Nature 615, 151–157 (2023).
Cox, M. M. & Hashimoto, Y. Fast‑track influenza virus vaccine produced in insect cells. J. Invertebr. Pathol. 107, S31–S41 (2011).
Contreras‑Gómez, A. et al. Protein production using baculovirus–insect cell system. Biotechnol. Prog. 30, 1–18 (2014).
Hashimoto, M. et al. Immunogenicity of SARS‑CoV‑2 S‑protein vaccine S-268019-b in monkeys. Vaccine 40, 4231–4241 (2022).
Fox, C. B. et al. Oil composition influences adjuvant emulsion activity. Vaccine 29, 9563–9572 (2011).
Adlington, K. et al. Molecular design of squalene countertypes for vaccine adjuvants. Biomacromolecules 17, 165–172 (2016).
Horai, R. et al. Mice deficient in IL‑1 family genes reveal role of IL‑1β in fever. J. Exp. Med. 187, 1463–1475 (1998).
Kuroda, E. et al. Inhaled fine particles induce IL‑1α release. Immunity 45, 1299–1310 (2016).
Lee, M. S. J. et al. B cell–intrinsic MyD88 signaling controls class switching. Eur. J. Immunol. 49, 1433–1440 (2019).
Dobin, A. et al. STAR: ultrafast RNA‑seq aligner. Bioinformatics 29, 15–21 (2013).
Chen, S. et al. fastp: an ultra‑fast FASTQ preprocessor. Bioinformatics 34, i884–i890 (2018).
Alvarez, R. V. et al. TPMCalculator for quantifying mRNA abundance. Bioinformatics 35, 1960–1962 (2019).
Love, M. I., Huber, W. & Anders, S. DESeq2 for RNA‑seq analysis. Genome Biol. 15, 550 (2014).
Gu, Z., Eils, R. & Schlesner, M. Complex heatmaps for genomic data. Bioinformatics 32, 2847–2849 (2016).
Bhattacharya, S. et al. ImmPort for immunology data reuse. Sci. Data 5, 180015 (2018).
Ashburner, M. et al. Gene ontology unifies biology. Nat. Genet. 25, 25–29 (2000).
Gene Ontology Consortium. The Gene Ontology knowledgebase 2023. Genetics 224, iyad031 (2023).
Kanehisa, M. & Goto, S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27–30 (2000).
Subramanian, A. et al. Gene set enrichment analysis. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).
Liberzon, A. et al. MSigDB 3.0. Bioinformatics 27, 1739–1740 (2011).
Yu, G., Wang, G., Han, Y. & He, Q. clusterProfiler: comparing biological themes. OMICS 16, 284–287 (2012).
Matsuyama, S. et al. Enhanced isolation of SARS‑CoV‑2 by TMPRSS2‑expressing cells. Proc. Natl. Acad. Sci. USA 117, 7001–7003 (2020).
Acknowledgements
We acknowledge Hideo Negishi and Burcu Temizoz for critical discussions and comments that have improved this study. We also thank Susan Furness, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. This work was funded by Shionogi & Co., Ltd. This research was supported by AMED under Grant Numbers JP223fa627001, JP223fa727001, and JP223fa727002 to K.I.
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Y.Yo., A.N.-Y., K.K., T.H., Y.K., Y.Y., J.K., K.T., Y.A., N.K., A.K., H.O., A.Y., M.O., T.N., S.I., S.O., M.N., C.C., and K.I. conceived and designed the study. Y.Yo., A.N.-Y., T.H., Y.K., Y.Y., J.K., K.T., Y.A., N.K., and H.O. performed experiments and analyzed the data. Y.Yo., A.N.-Y., Y.K., and K.I. drafted the manuscript. All authors contributed to the critical revision of the article for important intellectual content and approved the final version to be submitted.
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Yuya Yoshioka, Anri Nishinaka-Yoshioka, Yujiro Kidani, Yosuke Yanagida, Junpei Kasahara, Yoshiji Asaoka, Kenichiro Tsujii, Norikazu Kuroda, Akira Kugimiya, Hiroaki Osawa, Akimasa Yoshimura, Motoyasu Onishi, Takayuki Nakagawa, Satoru Ishida, Shinya Omoto, and Morio Nagira are employees of Shionogi & Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential competing interest.
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Yoshioka, Y., Nishinaka-Yoshioka, A., Kobiyama, K. et al. IL-1 delineates squalene-based adjuvant efficacy and reactogenicity in a cell-type-specific manner. npj Vaccines 11, 67 (2026). https://doi.org/10.1038/s41541-026-01420-0
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DOI: https://doi.org/10.1038/s41541-026-01420-0
