Manganese Pyrophosphate Mineralized DNA Nanovaccine Elicits Potent Humoral and Cellular Immunity

ABSTRACT

Subunit vaccines offer safety and precision but require adjuvants to overcome weak immunogenicity, particularly for the induction of cellular immunity. Herein, we develop a manganese pyrophosphate mineralized DNA bi‐adjuvant‐based subunit vaccine platform via biomineralization, which demonstrated the capability to elicit robust antigen‐specific cellular immune responses. The Mn/CpG bi‐adjuvant modulated the balance between Th1 and Th2 immune responses. More importantly, the synergistic activation of Toll‐like receptor 9‐like (TLR9) signaling pathways by CpG and cGAS‐STING pathways mediated by Mn(II) ions robustly enhanced cellular immunity, which was a magnitude of enhancement over commercial aluminum‐based adjuvants both in vitro and in vivo. Furthermore, the mechanistic studies revealed that the Mn(II)‐based nanoadjuvant effectively promoted Th1‐biased cellular immune response, as evidenced by an elevated IgG2a/IgG1 ratio and enhanced Th1‐associated cytokine secretion, alongside a potential reduction in regulatory T cell activity. These insights established critical design principles essential for the development of next‐generation manganese‐derived adjuvant systems, thereby advancing the frontiers of vaccinology and immunology.

The manganese/DNA (Mn/DNA) composite nanovaccine (MnCA + RBD) simultaneously activates Toll‐like receptor 9 (TLR‐9) and the cyclic GMP‐AMP synthase‐stimulator of interferon genes (cGAS‐STING) signaling pathway.

1. Introduction

Viral infectious diseases pose major global health threats, as evidenced by the COVID‐19 pandemic causing billions of cases and persistent Long‐COVID symptoms. Due to insufficient innate immunity against viruses, vaccination remains the most effective prevention strategy, significantly reducing infection rates and mortality. Vaccine administration can initiate the activation and maturation of dendritic cells (DCs), during which antigen‐presenting major histocompatibility complex (MHC) molecules on DCs surfaces engage with T cell receptors, triggering T lymphocyte activation and subsequently facilitating B lymphocyte proliferation and differentiation [1, 2, 3, 4]. Subunit vaccines include only specific components of the pathogen, eliminating the risk of virulence reversion inherent in traditional vaccines (live‐attenuated or inactivated). However, current subunit vaccine exhibits limited immunogenicity, necessitating the incorporation of adjuvants as essential immunopotentiators in vaccine formulations. These adjuvants serve critical functions in enhancing vaccine efficacy by augmenting antigen‐specific immune responses, reducing the required antigen dose, and potentiating both the magnitude and duration of antibody‐mediated immunity in vivo [5, 6, 7]. Since 1920, aluminum salt‐based adjuvants (abbreviated as Alum) have been dominantly utilized in vaccine formulations [8]. Alum adjuvants demonstrate the capacity of potentiating humoral immunity through promotion of CD4⁺ T helper cell differentiation, particularly toward Th2 phenotype [9, 10]. Nevertheless, Alum demonstrates limitation in eliciting robust cellular immunity, particularly in the induction of antigen‐specific Th1‐type immune responses in clinical application. This limited immunostimulatory profile significantly impairs their ability to establish durable and potent antiviral immunity against intracellular pathogens [11]. Therefore, the development of adjuvant systems that can effectively stimulate comprehensive cellular immune responses while maintaining an acceptable safety profile represents a critical priority in vaccinology [5].

Manganese (Mn) plays a critical role in diverse physiological processes. Recent investigations revealed its significant immunomodulatory function through the potent activation of the cyclic GMP‐AMP synthase (cGAS)‐stimulator of interferon genes (STING) signaling pathway [12, 13, 14, 15]. Following viral infection, Mn^{2 +} are released from intracellular organelles (mitochondria/Golgi) into the cytosol and enhance dsDNA recognition by increasing receptor affinity [16, 17]. In 2018, Jiang et al. reported that Mn^{2 +} functions as a critical modulator of the DNA sensor cGAS, enabling cGAS‐mediated synthesis of the secondary messenger cyclic GMP‐AMP (cGAMP) even at suboptimal dsDNA concentrations. This can amplify the cGAS‐STING signaling cascade through enhanced cGAMP‐STING binding affinity, ultimately eliciting a potent host antiviral defense response [18]. Moreover, investigation revealed that Mn^{2 +} orchestrates a dual immunomodulatory mechanism through simultaneous activation of both the cGAS‐STING signaling axis and the NLRP3 inflammasome pathway. This synergistic activation cascade was shown to promote the differentiation and proliferation of adaptive immune cells, particularly CD4⁺ T helper cells and CD8⁺ cytotoxic T lymphocytes, thereby eliciting a comprehensive immune response that enhances both humoral and cell‐mediated immunity [19]. These findings provide compelling evidence that Mn^{2 +} exhibit significant potential as immunoadjuvants, particularly through their capacity to potentiate cellular immune responses.

DNA can function as an immunoadjuvant to enhance the immune response to vaccines or other immunogenic substances. Among them, CpG oligodeoxynucleotides, serving as specific Toll‐like receptor 9 (TLR‐9) agonists, have been shown to preferentially induce Th1‐polarized immune responses. This immunomodulatory property enables CpG to effectively counterbalance Th2‐dominant immune polarization, thereby preventing potential Th2‐mediated immunopathological responses [20]. Additionally, CpG significantly upregulates the expression of major histocompatibility complex (MHC) molecules and co‐stimulatory markers (CD40 and CD86) on plasmacytoid dendritic cells (pDCs), thereby enhancing both antigen processing efficiency and presentation capacity [21, 22]. However, the immune response induced by DNA adjuvants alone remains insufficient to meet practical application requirements.

Here, we developed a biosynthetic manganese pyrophosphate (Mn₂PPi)‐mineralized DNA nano‐adjuvant platform through precisely controlled rolling circle amplification (RCA). This process strategically integrated the synergistic effects of Mn^{2 +} immunomodulation with nucleic acid adjuvanticity to potentiate in vivo Th1‐biased cellular immunity for enhanced viral prophylaxis [23]. Conventional RCA employs Mg^{2 +} as enzymatic cofactor to form magnesium pyrophosphate‐DNA complexes through dNTP‐derived PPi^{4 −} precipitation, generating floriform mesoporous nanoparticles. In this work, the substitution of Mn²⁺ for Mg²⁺ not only facilitated the formation of Mn₂PPi complexes through coordination with PPi⁴⁻ byproducts but also activated the cGAS‐STING pathway. Meanwhile, we employed the trimeric receptor‐binding domain (RBD) protein of SARS‐CoV‐2 as a model antigen, and thus incorporated the complementary sequences of high‐affinity aptamers (Apt) specifically targeting RBD epitopes in the DNA template alongside CpG sequence. This molecular design strategy yielded an elongated DNA scaffold containing repetitive functional units of both immunostimulatory CpG and RBD‐specific Apt. The Mn²⁺, driven by electrostatic interactions with the CpG/Apt‐enriched DNA backbone, underwent controlled biomineralization, resulting in the formation of an advanced Mn‐incorporated DNA nanoadjuvant, designated as MnCA (Scheme 1A).

SCHEME 1.

Design of Mn‐based DNA nanovaccine for eliciting cellular immune response. (A) Schematic illustration of the synthetic process of MnCA nanoadjuvant via rolling circle amplification. The arrow represents the direction of the DNA sequence from 5’ to 3’. (B) Mechanism of MnCA+RBD nanovaccine for enhancing RBD antigen‐specific immune response. The nanovaccine was taken up by antigen‐presenting cells (APCs) in lymph nodes, which induced DC maturation and effective immune responses by activating both the TLR‐9 pathway and the cGAS‐STING pathway.

Capitalizing on its intrinsic porous architecture and RBD‐specific aptamer functionalization, the Mn‐based DNA nanoadjuvant demonstrated efficient adsorption and high‐affinity binding to trimeric RBD antigens, ultimately forming a potent Mn/DNA nanovaccine platform (designated as MnCA + RBD). Mechanistically, the nanovaccine exhibited multi‐modal synergistic immune enhancement in an animal model: (1) The aptamer sequence was amplified and precisely integrated into the Mn/DNA nanovaccine, ensuring optimal antigen recognition, uptake and presentation capabilities by APCs to potentiate humoral immunity [24, 25]. (2) The Mn₂PPi‐mineralized surface of the nanovaccine imparted the property of pH‐responsive disassembly, enabling the simultaneous release of RBD antigens and activation of dual adjuvant systems in the weakly acidic lysosomal compartment following endocytic uptake. (3) The CpG within the Mn/DNA nanovaccine activated TLR9, initiating innate immune responses through the MyD88‐dependent signaling pathway. (4) Concurrently, dsDNA from nanovaccine degradation and Mn²⁺‐mediated activation of the cGAS‐STING signaling pathway significantly promoted CD4⁺ T cell differentiation (Scheme 1B). Together, these synergistic mechanisms established a potent Th1‐type cellular immune response, effectively compensating for the Th1 response limitations associated with traditional Alum‐based adjuvants [26, 27].

2. Results and Discussion

2.1. Synthesis and Characterization of Mn‐Based DNA Nanovaccine

The Mn‐based DNA nanovaccine was prepared via RCA, comprising two main adjuvant ingredients, CpG/Apt‐containing DNA long‐chain and Mn₂PPi deposited on the DNA long‐chain [28]. To optimize the synthesis parameters, we systematically investigated the influence of Mn²⁺ concentration (ranging from 2 to 10 mM) and reaction duration on both DNA polymerization efficiency and nanoparticle morphology using 12% polyacrylamide gel electrophoresis (PAGE) and transmission electron microscopy (TEM) analysis. PAGE results demonstrated that Mn²⁺ concentration within the tested range had negligible impact on DNA chain elongation efficiency (Figure S1). However, TEM analysis revealed significant morphological variations, with 4 mM Mn²⁺ yielding nanoparticles exhibiting an ideal porous, flower‐like architecture. Higher Mn²⁺ concentrations resulted in progressively amorphous inorganic frameworks, compromising structural integrity (Figure 1A). Considering that high concentration of Mn²⁺ was beneficial for immune activation [18], the optimal concentration for the synthesis of MnCA was set as 4 mM.

FIGURE 1.

Synthesis and characterization of MnCA+RBD. (A) TEM images of MnCA at different concentrations of MnCl₂. (B) TEM images of MnCA with different synthesis times at 4 mM MnCl₂. (C) Statistical average particle size of MnCA from (B) calculated by ImageJ software. Data are shown as mean ± SEM, n = 30. (D) SEM/EDS mapping images of MnCA. (E) SDS‐PAGE analyzing the adsorption of RBD on MnCA at different mass ratios of MnCA to RBD. (F) TEM images of MnCA and MnCA + RBD. (G) Hydrodynamic diameters of MnCA and MnCA + RBD. (H) Zeta potential of MnCA and MnCA + RBD. Data are shown as mean ± SEM, n = 3.

Furthermore, we optimized the synthesis time to determine the particle size of MnCA. The TEM images and statistical analysis indicated that the size of MnCA increased with the prolongation of synthesis time, and the particle size of MnCA was approximately 500 nm at a Mn²⁺ concentration of 4 mM and synthesis time of 6 h (Figure 1B,C), which was chosen as the synthesis condition. The scanning electron microscope (SEM)/energy‐dispersive x‐ray spectroscopy (EDS) mapping images confirmed that MnCA contained nitrogen (N), phosphorus (P), manganese (Mn), oxygen (O) and carbon (C) elements, being consistent with the theoretical compositions of MnCA, which provided further evidence for the successful synthesis of MnCA (Figure 1D, Figure S2, and Table S2). The resulting MnCA exhibited excellent stability, with negligible Mn²⁺ release in PBS buffer and only 1–2% leakage observed over a period of 3 months (Figure S3).

To evaluate the antigen‐binding capability of MnCA, we selected the RBD of SARS‐CoV‐2 as a model vaccine antigen, which mediates viral entry through its interaction with human angiotensin‐converting enzyme 2 (hACE2). A series of mass ratios of MnCA to RBD was established, and Mn‐based DNA nanoadjuvant containing non‐aptamer sequences (MnCA_nA) served as a control. The SDS‐PAGE results demonstrated that when the m(MnCA):m(RBD) ranged from 2:4 to 4:4, over 50% of RBD incubated with MnCA remained free, and 60%–80% of RBD was not adsorbed by MnCA_nA (Figure S4). Although the adsorption capacity of MnCA containing Apt was marginally better than that without Apt, the MnCA inevitably had a certain degree of RBD adsorption due to its porous morphology. To further improve the adsorption efficiency of MnCA to RBD, the mass ratio of MnCA to RBD was adjusted to 8:1–13:1. Under these circumstances, the adsorption efficiency of MnCA for RBD reached ∼75%, while there was only less than 20% adsorption for HRP, a control protein with an isoelectric point (pI) equivalent to RBD, which proved the specificity of aptamers and the selective adsorption of MnCA for RBD (Figure 1E and Figure S5). The MnCA nanoadjuvant maintained a porous morphology after adsorbing RBD antigen (Figure 1F). Additionally, the increases in hydration particle size and decrease in Zeta potential of MnCA nanoadjuvant following the adsorption of RBD antigen further confirmed the successful synthesis of MnCA+RBD nanovaccine (Figure 1G,H). The polydispersity index (PDI) values indicated that both MnCA and MnCA post‐RBD adsorption exhibited acceptable dispersibility (Table S3).

2.2. In Vitro Immune Activity of Mn‐Based DNA Nanovaccine

To systematically evaluate the immune response elicited by Mn‐based DNA nanovaccine, in vitro experiments were conducted. Initially, the cytocompatibility of MnCA nanoadjuvant was assessed by incubating various concentrations of MnCA with DC2.4 cells for 24 h. As illustrated in Figure S6, the viability of DC2.4 cells remained above 80% when treated with 0–300 µg/mL of MnCA. In addition, the cellular uptake of MnCA nanoadjuvant by DC2.4 was evaluated by fluorescence microscopy, and the images showed that the uptake level gradually increased over time, almost reaching a plateau at 9 h (Figure S7). Considering that Toll‐like receptor 9, predominantly localized on the endolysosomal membrane, is specifically activated by CpG motifs to initiate innate immune signaling cascades, we investigated the intracellular trafficking and lysosomal localization of MnCA in DC2.4 cells. Confocal microscopy analysis revealed that SYBR Green II‐labeled MnCA nanoparticles efficiently accumulated within lysosomal compartments as early as 4 h post‐incubation, demonstrating significant colocalization with LysoTracker Red‐stained lysosomes. The colocalization area persisted with prolonged uptake time, suggesting efficient endo/lysosomal delivery of the nanoadjuvant (Figure S8). The dissolution of the nanoadjuvant structure under acidic condition suggested that the lysosomal environment promoted the hydrolysis of the inorganic pyrophosphate mineralization layer on its surface (Figure S9), thereby accelerating the release of Mn²⁺. Simultaneously, the widely present pyrophosphatase in cells further catalyzed this hydrolysis process, synergistically enhancing the degradation of the nanoadjuvant. Surprisingly, despite the degradation of the surface inorganic salt, the red fluorescence of lysosomes still overlapped with the green fluorescence of the stained DNA long‐chain even after 24 h incubation. This observation revealed the long‐term retention within lysosome, which facilitated sufficient binding of CpG to TLR9 (Figure 2A,B).

FIGURE 2.

In vitro immune activation capacity of MnCA + RBD. (A) Confocal fluorescence images of DC2.4 after being incubated with MnCA for 24 h. (B) Colocalization analysis of MnCA and lysosomes in DC2.4. The MnCA and lysosomes were stained with SYBR Green II and Lyso–Tracker Red, respectively. (C, D) Cytokine secretion level of IL‐6 (C) and IFN‐β (D) from BMDCs following treatment with PBS, MnCA_nA/nC + RBD, MnCA_nA/C + RBD, MnCA, MnCA + RBD, MgCA + RBD and LPS for 12 h. The data are shown as the mean ± SEM, n = 3. Statistical significance was tested with one‐way ANOVA followed by Tukey's multiple comparisons test (ns, not significant, ****p < 0.0001). (E) Schematic diagram of Mn²⁺ activation of cGAS‐STING pathway. (F) Western blot analyzing the cGAS‐STING pathway‐associated proteins in DC2.4 after treating with PBS, MnCA + RBD and MgCA + RBD for 12 h.

To evaluate the activation of the MnCA+RBD on DCs, we conducted quantitative analysis of immunostimulatory cytokine secretion profiles (IL‐6 and IFN‐β) from bone marrow‐derived dendritic cells (BMDCs) following 12 h incubation with various experimental groups, PBS, MnCA_nA/nC + RBD, MnCA_nA/C + RBD, MnCA, MnCA + RBD, and MgCA + RBD [29]. The MnCA_nA/nC represented nanoparticles lacking both Apt and CpG sequences, while MnCA_nA/C represented nanoparticles containing CpG but not Apt sequences. Meanwhile, Mn²⁺ was substituted for Mg²⁺ in the Mn₂PPi‐mineralized particles to construct Mg‐based nanoadjuvant named MgCA, which also possessed a flower‐like morphology with a size of around 700 nm (Figure S10). The quantitative analysis of cytokine secretion profiles revealed that all treatment groups exhibited significantly elevated cytokine expression compared to PBS‐treated controls (Figure 2C,D and Figure S11). However, statistical analysis indicated minimal differences among MnCA_nA/nC + RBD, MnCA_nA/C + RBD, and MnCA + RBD groups, suggesting that variations in DNA sequence composition had limited impact on cytokine induction in vitro. This observation led to two key hypotheses: (1) the incorporation of RBD antigen did not significantly enhance cytokine secretion, as evidenced by comparable results between MnCA and MnCA+RBD groups, potentially diminishing the apparent efficacy of aptamer‐mediated targeting; (2) MnCA + RBD exhibited significantly enhanced activation of IL‐6 secretion (∼2‐fold increase) relative to MgCA + RBD, indicating more efficient promotion of dendritic cell maturation and subsequent cellular immune responses. Specifically, MnCA + RBD demonstrated remarkable induction of IFN‐β production (∼20‐fold increase compared to MgCA + RBD). This indicated that MnCA + RBD was inclined to stimulate the expression of type I interferon, which was beneficial for coordinating innate immune response against viral infections [16]. Furthermore, CpG potently induced the production of IFN‐β through the activation of TLR‐9 signaling pathway, while demonstrating a favorable immunomodulatory profile characterized by minimal induction of pro‐inflammatory cytokines, including IL‐6.

To further elucidate the molecular mechanisms underlying immune activation by MnCA + RBD, we performed western blot analysis to investigate the activation status of key components in the cGAS‐STING signaling pathway (Figure 2E). The results demonstrated that although the expression levels of cGAS, STING, TBK1 showed no significant differences [30, 31, 32], the phosphorylation of downstream proteins, including P‐TBK1, P‐STING, and P‐IRF3, was markedly upregulated in the MnCA + RBD‐treated group compared to controls, providing molecular‐level evidence for the activation of cGAS‐STING pathway (Figure 2F and Figure S12). This phosphorylation cascade ultimately led to the nuclear translocation of IRF3 and subsequent induction of type I interferons, thereby establishing a robust antiviral state and potentiating both cellular and humoral immune responses [33].

2.3. In Vivo Immune Activity of Mn‐Based DNA Nanovaccine

The in vivo immune activity of Mn‐based DNA nanovaccine was first evaluated by RBD antigen‐specific antibody titers measurement. The dose of RBD antigen was fixed as 0.5 µg, and the mass ratio of Mn/Mg‐based DNA nanovaccines to RBD was selected as 100:1 to ensure sufficient adsorption of RBD. Different formulations of Mn/Mg‐based DNA nanovaccines, namely MnCA_nA/nC + RBD, MnCA_nA/C + RBD, MnCA, MnCA + RBD, and MgCA + RBD, were administered into mice, and the serum samples from the eye sockets of mice were analyzed for antibody titers following the second and third immunization to investigate the in vivo immune response (Figure 3A). The antibody titer analysis demonstrated that all vaccine formulations, except PBS and MnCA controls, effectively induced RBD‐specific IgG antibody production (Figure 3B–I). While the Mn‐based nanovaccines generated slightly lower IgG1 titers compared to conventional Al(OH)₃ + RBD formulation, they exhibited remarkable enhancement in IgG2a production, achieving approximately 1–2 orders of magnitude higher titers than the Al(OH)₃ + RBD group. This substantial increase in IgG2a levels resulted in significantly elevated IgG2a/IgG1 ratios, indicating that Mn‐based nanoadjuvants possessed superior Th1‐polarizing capacity compared to traditional alum‐based formulations. Interestingly, the sequence variations among different Mn‐based nanovaccines showed minimal impact on antibody production, which could be attributed to two primary factors: (1) The inherent porous structure of the nanoadjuvants facilitated non‐specific antigen adsorption even in the absence of aptamer sequences; (2) The relatively low CpG content in the formulations may limit its immunostimulatory potential. Furthermore, the comparable IgG2a/IgG1 ratios between MnCA + RBD and MgCA + RBD groups following tertiary immunization (Figure 3I) suggested that the current Mn²⁺ concentration in the nanovaccine formulation might be suboptimal for achieving maximal enhancement of Th1‐type immune response. This observation highlighted the need for further optimization of Mn²⁺ loading to fully exploit its immunopotentiating capabilities.

FIGURE 3.

In vivo antibody response induced by Mn‐based DNA nanovaccine (MnCA + RBD). (A) Procedure of mice immunization and serum collection for analysis. Female BALB/c mice were immunized with three doses of different formulations of nanovaccines via intramuscular injection, and serum was collected at 35 and 56 days after initial vaccination (n = 8). (B‐E) SARS‐CoV‐2‐specific IgG (B), IgG1 (C), IgG2a (D) antibody titers, and IgG2a/lgG1 ratio (E) determined by ELISA at 35 days after initial vaccination. (F‐I) SARS‐CoV‐2‐specific IgG (F), IgG1 (G), IgG2a (H) antibody titer, and lgG2a/IgG ratio (I) determined by ELISA at 56 days after initial vaccination. The data are shown as the mean ± SEM, n = 8. Statistical significance was tested with one‐way ANOVA followed by Tukey's multiple comparisons test (ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001).

2.4. Immune Activity of Mn‐Based DNA Nanovaccine With Increased Dosage of Mn and CpG

Building upon these promising findings, we implemented strategic modifications to optimize the nanovaccine formulation: (1) increasing Mn²⁺ concentration to 6–10 mM to enhance cGAS‐STING pathway activation, (2) tripling the CpG content to amplify TLR9‐mediated immune stimulation, and (3) eliminating the aptamer sequence to streamline the design (designated as MnCA_3CpG, Figure 4A and Table S1), and the immune effects of this optimized formulation were re‐evaluated.

FIGURE 4.

Preparation, characterization and immune response effect of MnCA_3CpG nanovaccine containing three segments of CpG. (A) Schematic illustration of the construction of MnCA_3CpG. (B) TEM images of MnCA_3CpG at different concentrations of MnCl₂. (C) Western blot analyzing the expression level of cGAS‐STING pathway‐associated proteins in DC2.4 with treatment of PBS, MnCA_3CpG + RBD and MgCA_3CpG + RBD for 12 h. The results within the dotted box are from the same experiment. (D) Schematic diagram of MnCA_3CpG nanovaccine stimulating BMDCs maturation and cytokine secretion. (E, F) ELISA assay for detecting cytokine secretion level of IFN‐β (E) and TNF‐α (F) by BMDCs, which were treated with PBS, MnCA_CpG + RBD, MnCA_3CpG, MnCA_3CpG + RBD, MgCA_3CpG + RBD and LPS for 12 h. (G) Flow cytometry analysis of the percentage of CD80⁺CD86⁺ in CD11c from BMDCs treated with different nanovaccines for 12 h. The data are shown as the mean ± SEM, n = 3. (H) Procedure of mice immunization with MnCA_3CpG nanovaccine and serum collection. (I) SARS‐CoV‐2‐specific antibody titers (IgG, IgG1, IgG2a) from the serum of mice after two immunizations, which were intramuscularly injected with Al(OH)₃ + RBD, MnCA_CpG + RBD, MnCA_3CpG + RBD and MgCA_3CpG + RBD. The data are shown as the mean ± SEM, n = 5. Statistical significance was tested with one‐way ANOVA followed by Tukey's multiple comparisons test (ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

MnCA_3CpG with diverse Mn²⁺ concentrations were synthesized via RCA and characterized using TEM (Figure 4B). Structural characterization revealed a concentration‐dependent morphological transition, with the porous framework and particle size becoming progressively less defined as Mn²⁺ concentration increased. The size variation likely influenced the enrichment efficiency of the nanoadjuvants in lymph nodes [34]. Based on this observation, we established 8 mM as the optimal Mn²⁺ concentration for MnCA_3CpG synthesis, effectively balancing structural integrity with immunological potency. Complementary cytotoxicity assessment demonstrated excellent biocompatibility, with cell viability remaining unaffected even at the maximum tested Mn²⁺ concentration of 10 mM, confirming the safety profile of the optimized formulation (Figure S13). Meanwhile, Mg‐based nanoadjuvant (MgCA_3CpG) containing three fragments of CpG was successfully synthesized as a control (Figure S14). To confirm the cGAS‐STING pathway activated by MnCA_3CpG, western blot was conducted and the results showed that the MnCA_3CpG remarkably elevated the expression of cGAS‐STING pathway‐related proteins, P‐TBK1/NAK and P‐STING, while also reducing the expression levels of cGAS and STING (Figure 4C and Figure S15). Furthermore, the ability of MnCA_3CpG + RBD to induce the maturation of BMDCs was investigated by evaluating the cytokine secretion levels of IFN‐β and TNF‐α, as well as CD80/CD86 phenotype on BMDCs (Figure 4D). As illustrated in Figure 4E,F and Figure S16, MnCA_3CpG + RBD triggered BMDCs to secrete significantly higher concentrations of IFN‐β in comparison of MnCA_CpG + RBD and MgCA_3CpG + RBD groups, which indicated that Mn/CpG biadjuvant played a positive role in activating type I IFN‐dependent innate immunity via STING‐mediated pathways and TLR‐9 pathways, as well as promoting Th1 response and IFN‐induced antiviral activity [35, 36, 37]. Interestingly, the MnCA_3CpG + RBD formulation demonstrated limited efficacy in enhancing TNF‐α expression, with its performance being inferior to that of MnCA_CpG + RBD. At the same time, the expression levels of CD80 and CD86 in BMDCs were similar in BMDCs treated with the MnCA_3CpG + RBD and MnCA_CpG + RBD (Figure 4G and Figure S17). This phenomenon might be attributed to potential dose‐dependent immunomodulatory effects, where elevated CpG concentrations could paradoxically induce certain tolerogenic pathways, thereby attenuating pro‐inflammatory cytokine production [38]. Additionally, the expression of these two co‐stimulatory molecules in BMDCs treated with Mn^{2 +}‐containing nanovaccine was higher than those treated with MgCA_3CpG+RBD, highlighting the potent immune effects of Mn^{2 +} in stimulating BMDCs maturation and further regulating T cell activation (Figure 4G).

To comprehensively evaluate the in vivo immunogenicity, we administered BALB/c mice with Al(OH)₃ + RBD, MnCA_CpG + RBD, MnCA_3CpG + RBD, and MgCA_3CpG + RBD formulations at 3‐week intervals (Figure 4H). Post‐secondary immunization analysis revealed that Mn‐based nanovaccines generated IgG and IgG1 titers comparable to conventional Alum adjuvants, while demonstrating slight enhancement in IgG2a production and IgG2a/IgG1 ratios compared to both Alum and Mg‐based formulations (Figure 4I). This distinct antibody profile underscored the superior Th1‐polarizing capacity of Mn‐containing adjuvants, highlighting their potential advantage in eliciting balanced humoral and cellular immune responses. Notably, the MnCA_3CpG formulation containing elevated CpG concentrations failed to demonstrate enhanced immunogenicity compared to its lower‐CpG counterpart (MnCA_CpG), potentially due to dose‐dependent immunosuppressive effects associated with high‐density CpG motifs. This observation aligned with our previous in vitro findings, suggesting a potential threshold for CpG‐mediated immune activation beyond which additional adjuvant incorporation may not proportionally enhance immunostimulatory effects and could potentially induce counterproductive immune modulation [39].

2.5. Immune Activity and Long‐Term Immune Activity Evaluation of MnCA_CpG

Based on these findings, we optimized the CpG content in the Mn‐based nanovaccine by reverting to a single CpG segment in the DNA template while maintaining the optimal Mn²⁺ concentration at 8 mM (Figure 5A). The resulting Mn‐based nanoadjuvant, referred to as MnCA_CpG, also exhibited a porous flower‐like morphology and demonstrated excellent RBD adsorption (Figure S18). At an MnCA_CpG‐to‐RBD mass ratio of 13:1, the adsorption efficiency exceeded 90%, which was primarily attributed to the confinement effect of the porous structure. Next, the in vivo immune activity of the MnCA_CpG was evaluated based on RBD‐specific antibody levels, neutralizing antibody (NAb) titers, and phenotypic differentiation of T‐cell populations. The immunization schedule, blood collection, and analytical timelines for diverse indicators were shown in Figure 5B. The antibody titer analysis confirmed that the modified formulation outperformed both alum and Mg‐based adjuvants in terms of IgG2a/IgG1 ratios following secondary immunizations and reached a comparable level to alum and Mg‐based adjuvants after the third immunization (Figure 5C and Figure S19). This persistent Th1‐biased immune profile, characterized by elevated IgG2a/IgG1 ratios, provided compelling evidence for the intrinsic Th1‐polarizing capacity of Mn‐containing nanoadjuvant and its potential advantage in eliciting cellular immune responses. Additionally, in terms of NAb titers, MnCA_CpG achieved levels comparable to those induced by alum at 14 days, 1 month, and even 3 months after the third immunization, suggesting its potential for long‐term protection against viral pathogens (Figure 5D and Figure S20).

FIGURE 5.

Immune response and long‐term immune efficacy of MnCA_CpG nanovaccine. (A) Structural diagram of MnCA_CpG nanoadjuvant. (B) Procedure of mice immunization with MnCA_CpG nanovaccine and immune response evaluation. (C) SARS‐CoV‐2‐specific antibody titers (IgG, IgG1, IgG2a) from the serum of mice after two immunizations, which were intramuscularly injected with PBS, Al(OH)₃ + RBD, MnCA_CpG + RBD, MgCA_CpG + RBD. The data are shown as the mean ± SEM, n = 8. (D) Neutralizing antibody (NAb) titers from the serum of vaccinated mice with different formulations. The data are shown as the geometric mean ± geometric SD, n = 30. (E, F) Flow cytometry analysis for the percentage of IFN‐γ⁺ and TNF‐α⁺ in CD4⁺ Th1 cells (E) and CD8⁺ CTL cells (F) from the splenocytes of vaccinated mice. The data are shown as the mean ± SEM, n = 6 except for outliers. (G) FluoroSpot assay for detecting IFN‐γ secretion level in splenocytes. The data are shown as the mean ± SEM, n = 6. (H) Schematic illustration of in vivo immune system interaction after intramuscular injection of MnCA_CpG nanovaccine. (I,J) Percentage of naïve CD4⁺, CD4⁺ TCM, and CD4⁺ TEM (I), as well as CD8⁺, CD8⁺ TCM, and CD8⁺ TEM (J) from splenocytes of vaccinated mice with different formulations, PBS, Al(OH)₃ + RBD, MnCA_CpG + RBD, MgCA_CpG + RBD, at 14 days after three immunizations. The data are shown as the mean ± SEM, n = 6. Statistical significance was tested with one‐way ANOVA followed by Tukey's multiple comparisons test (ns, not significant; *p < 0.1, **p < 0.01, ***p < 0.001).

To further evaluate the Th1‐polarizing capacity of Mn‐based nanoadjuvant, we analyzed the proportions of CD4⁺ Th1 and CD8⁺ cytotoxic T lymphocyte (CTL) populations secreting IFN‐γ and TNF‐α using flow cytometry and FluoroSpot assays at 14 days after three immunizations. The analysis of T‐cell immune responses revealed that MnCA_CpG showed a relative advantage over both PBS and Mg‐based nanoadjuvant in elevating the percentage of CD4⁺ Th1 and CD8⁺ CTL secreting IFN‐γ and TNF‐α (Figure 5E–G), indicating a robust Th1‐biased cellular immunity of MnCA_CpG for the clearance of intracellular pathogens. In addition, we selected CD134, a critical co‐stimulatory molecule involved in promoting the proliferation and survival of activated CD4⁺ and CD8⁺ T cells, as a phenotypic marker for T cell response evaluation. Flow cytometric analysis of T cell populations revealed that MnCA_CpG + RBD specifically enhanced CD134 expression in CD8⁺ T cells compared with MgCA_CpG + RBD, while showing minimal effect on CD4⁺ T cell populations. This selective activation profile suggested that the Mn‐based nanovaccine preferentially stimulated cytotoxic T lymphocyte responses, potentially enhancing its antiviral efficacy through targeted cellular immunity (Figure S21). These findings suggested that Mn‐containing nanovaccines preferentially elicited Th1/CTL immune responses. In detail, the nanoscale characteristics of Mn/DNA nanovaccine formulations facilitated their efficient drainage within the lymph node and subsequent uptake by DCs, stimulating their activation and maturation processes. Furthermore, the activated DCs initiated robust antigen processing and presentation cascades through MHC, leading to the binding and activation of T lymphocyte receptors. This process preferentially promoted the differentiation of Th1 CD4⁺ cells and CD8⁺ CTLs, thereby enhancing the secretion of related cytokines (such as IFN‐γ and TNF‐α). As a result, the formulation demonstrated a pronounced advantage in driving IgG2a antibody production, while its capacity to promote Th2‐type cellular differentiation remained relatively modest (Figure 5H).

Furthermore, to evaluate the long‐term immunity of Mn‐based nanoadjuvant, we measured the proportions of memory CD4⁺ and CD8⁺ T cell subsets—including central memory T cells (TCM, CD44^hiCD62L^hi) and effector memory T cells (TEM, CD44^hiCD62L^lo)—in splenocytes at 14 days and 3 months after three‐dose vaccination (Figure 5I,J). The statistical analysis revealed that, compared with PBS and Al(OH)₃ + RBD control groups, the Mn‐based DNA nanovaccine group showed a significant reduction in naïve CD4⁺ T cells and a marked increase in both CD4⁺ and CD8⁺ TEM cells at early time points. However, by the 3‐month time point, the proportions of TEM and TCM subsets in all groups showed no statistically significant differences (Figure S22). In parallel, we evaluated memory B cell populations and observed no enhancement in their proportions among different formulations at either 1 month or 3 months post‐vaccination (Figure S23). These results suggested that the Mn‐based nanoadjuvant primarily promoted an early effector memory T‐cell response, which facilitated rapid antigen clearance upon re‐exposure. The convergence of TEM and TCM levels across groups at later stages, along with the absence of sustained memory B cell expansion, could be attributed to the transient antigen presentation of this nanoadjuvant platform and its limited capacity to maintain prolonged germinal center activity. Collectively, our results demonstrated that Mn‐based nanovaccines effectively induced robust immune activation, with particular efficacy in promoting Th1‐type cellular immunity and cellular effector memory. Furthermore, the distinct immunological profiles observed between Mn‐ and Mg‐based formulations indicated fundamentally different immune activation mechanisms, potentially mediated through unique signaling pathways that warrant further investigation.

2.6. RNA Transcriptome Analysis Comparing Mn‐ and Mg‐Based Nanovaccines

To compare the immunomodulatory effects of Mn‐ versus Mg‐based nanovaccines, we performed RNA‐seq analysis of BMDCs treated with MnCA_CpG + RBD, MgCA_CpG + RBD, or PBS. Differential expression of cytokines and immune pathway genes associated with the induction of T‐cell polarization (including TNF, TLR9, and DNA sensing pathways) was quantified by FPKM and visualized via hierarchical clustering (Figure 6A) [40]. The mRNA sequencing analysis revealed distinct immunostimulatory properties between the nanovaccine formulations. Notably, MnCA_CpG + RBD demonstrated superior efficacy in upregulating the transcriptional expression of multiple cytokines, including Tnf, Il12rb1/2, Il1a, Il1b, Il6, Il17ra, and Il23a, compared to MgCA_CpG + RBD. Comprehensive analysis of the cytokine profile indicated that the Mn‐based nanovaccine preferentially induced a myeloid cell polarization conducive to Th1‐ and Th17‐type immune responses, while also exhibiting moderate activation of gene programs associated with Th2‐skewing microenvironments. Furthermore, transcriptomic profiling demonstrated that MnCA_CpG + RBD significantly enhanced the activation of TNF signaling pathway, as evidenced by the upregulation of key mediators including Cxcl3, Cxcl2, Il6, Cxcl1, and Edn1, suggesting its potent capacity to augment immune cell functionality and overall immune response. The incorporation of CpG sequences in both MgCA_CpG + RBD and MnCA_CpG + RBD formulations conferred the capacity to activate the TLR9 signaling pathway, which orchestrated innate immune responses through downstream signaling cascades to combat pathogenic invasion. However, quantitative analysis revealed that the expression levels of certain pathway components were comparable to those in the PBS control group, potentially attributable to suboptimal CpG‐TLR9 binding efficiency and limited CpG accessibility within the nanovaccine architecture. Notably, MnCA_CpG + RBD formulation demonstrated significant activation of cytoplasmic DNA sensing pathway. Comprehensive mechanistic investigations revealed that Mn²⁺ mediated this immunostimulatory effect through two distinct molecular mechanisms: (1) direct activation of the cGAS‐STING signaling cascade in a dsDNA‐independent manner, and (2) enhancement of cGAS protein sensitivity toward both endogenous cytosolic double‐stranded DNA and dsDNA fragments generated through the degradation of Mn‐based nanoadjuvant. This dual activation mechanism resulted in substantial upregulation of type I interferon expression, ultimately conferring potent antiviral activity.

FIGURE 6.

RNA sequencing analysis of BMDCs treated with Mn/Mg‐based nanovaccine. (A) RNA‐seq analyzing the cytokine activation associated with BMDCs. The transcriptomic profile reflected the intrinsic gene expression of myeloid cells, which established the cytokine milieu for subsequent T‐cell differentiation. (B) Gene ontology enrichment analysis illustrating the pathways with the smallest p‐values for differential genes in comparison of Mn‐ versus Mg‐based nanovaccines, based on the biological process category. (C) GO enrichment analysis of different pathway‐expressed genes in Mn‐ versus Mg‐based nanovaccine.

Subsequently, we performed comprehensive Gene Ontology (GO) enrichment analysis focusing on biological processes to systematically compare the immunomodulatory effects between Mn‐ and Mg‐based nanovaccines. The top ten significantly enriched pathways were selected for comparative analysis based on their statistical significance. The results indicated that MnCA_CpG+RBD was effective in activating immune‐related pathways, mainly reflected in innate and adaptive immune responses, immune system process, inflammatory response and immune responses (Figure 6B). Additionally, RNA‐seq‐based GO enrichment analysis was conducted on the differentially expressed genes related to MnCA_CpG+RBD and MgCA_CpG+RBD (Figure 6C). Complementary in vivo experiments—including flow cytometry and FluoroSpot assays—were conducted to evaluate the proportions of regulatory CD4⁺ T cells and Th17 CD4⁺ T cells, as well as the expression of their relevant signature cytokines. Compared with the PBS group, neither MnCA_CpG + RBD nor MgCA_CpG + RBD altered the proportion or frequency of CD4⁺ Foxp3⁺ regulatory T cells. However, in the FluoroSpot assay, IL‐10 secretion was relatively reduced in the MnCA_CpG + RBD group compared with the MgCA_CpG + RBD group. It was proposed that MnCA_CpG may impair the immunosuppressive function of regulatory T cells without affecting their differentiation (Figure S24). This attenuation of immunosuppressive capacity contributed to maintaining Mn nanovaccine‐induced Th1‐type protective immunity. Regarding Th17 differentiation, a discrepancy was observed between the transcriptomic data at the cellular level and the phenotypic data from animal experiments. This inconsistency might be attributed to the relatively weak intrinsic capacity of the Mn nanovaccine to promote Th17 polarization, as well as the multiple physiological barriers in vivo that could attenuate the regulatory effects of the nanovaccine. Notably, excessive Th17 responses are undesirable, as they might lead to the overactivation of autoimmune pathways and compromise nanovaccine safety. Overall, the Mn‐based nanoadjuvant demonstrated enhanced capacity in promoting DCs differentiation and maturation compared to their Mg‐based counterparts, and induced significantly higher IgG2a antibody titers, indicative of a Th1‐biased cellular immune response. Moreover, analysis of T cell subset differentiation and cytokine profiles revealed its modulatory effect on Treg responses and the establishment of an immune microenvironment conducive to a balanced T‐cell response [41, 42].

2.7. Pharmacokinetics, Biodistribution, and Biosafety Assessment of Mn‐Based Nanovaccine

Finally, we conducted comprehensive pharmacokinetics, biodistribution, and biosafety evaluation of the Mn‐based DNA nanovaccines. The pharmacokinetic profile of MnCA_CpG in blood was evaluated following intramuscular administration. Blood samples were collected at 1, 3, 6, and 12 h post‐injection, and the content of Mn was quantified using inductively coupled plasma‐mass spectrometry (ICP‐MS). The results showed that the concentration of Mn in blood decreased progressively over time and rapidly returned to the normal baseline range (< 50 µg/L) within 1 h [43, 44], indicating rapid systemic clearance of the administered formulation and low risk of long‐term accumulation (Figure S25). In the biodistribution study, SYBR Green II‐stained MnCA was administered into mice via intramuscular injection, and the major organs were harvested 24 h post‐administration. The imaging data revealed distinct tissue tropism, with the majority of Mn‐based nanovaccine accumulation observed in hepatic tissues, while renal tissues showed minimal but detectable levels (Figure S26A). This distribution pattern suggested that the nanovaccine clearance predominantly occurred through hepatic metabolism and renal excretion pathways, consistent with typical nanoparticle pharmacokinetic profiles.

In addition, systemic biosafety analyses were performed on blood, serum, and major organ samples collected from mice administered with MnCA_CpG + RBD, MnCA + RBD nanovaccines, and PBS control group following a three‐dose immunization protocol. A variety of serum biochemical indicators, including liver function‐related alanine transaminase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP); kidney function‐related urea (UREA); heart function‐related lactate hydrogenase (LDH); glycosylated serum protein (GSP), as well as hematological parameters, including white blood cell count (WBC), lymphocyte count (Lymp), red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT), and hematocrit (HCT), were used to assess the organ functions and blood status (Tables S4 and S5). There were negligible differences between PBS and nanovaccine‐treated groups, and these values were within the normal range (Figure S26B,C and Figure S27A,B). Histopathological evaluation through hematoxylin and eosin (H&E) staining further corroborated the biosafety profile of Mn‐based nanovaccines. Comparative analysis of tissue morphology in major organs (including cardiac, hepatic, splenic, pulmonary, and renal tissues) revealed no significant histological alterations between the nanovaccine‐treated groups and the PBS control group (Figure S26D and Figure S27C).

To further assess long‐term biosafety, particularly in view of the potential risk of Mn‐induced neurotoxicity associated with cerebral accumulation, we monitored mouse behavior and brain Mn levels during repeated administration of MnCA_CpG at 3‐week intervals over a period of nearly 3 months (Figure S28A). The gait analysis, performed using ink‐based tracking, revealed negligible differences between the Mn nanoadjuvant‐treated and PBS control groups (Figure S28B,C). Moreover, brain Mn concentrations remained comparable to those of the control group throughout the study (Figure S28D). Collectively, these findings provided compelling evidence for the favorable biosafety characteristics of Mn‐based nanovaccines in the context of antiviral immunity development.

3. Conclusion

In summary, we have successfully engineered a novel manganese‐based DNA nanocomposite adjuvant system, integrating CpG ODN and Mn²⁺ components, which demonstrated enhanced cellular immunogenicity to address the limitations of conventional alum adjuvants in antiviral applications. The simple and efficient synthesis of MnCA was dependent on the synergistic synchronization of nucleic acid rolling circle amplification and Mn²⁺ inorganic salt‐mediated mineralization, allowing for the co‐delivery of multiple adjuvants. The nanoscale architecture and pH‐responsive degradation characteristics of MnCA facilitated its efficient cellular uptake by DCs, enabling controlled release and intracellular exposure of CpG and Mn dual adjuvants. The liberated components orchestrated a coordinated immune activation cascade: On the one hand, the elongated CpG‐containing DNA fragments initiated TLR‐9 signaling cascades to potentiate innate and adaptive immunity through the direct stimulation of pDCs and B lymphocytes, complemented by indirect activation via pattern recognition receptor (PRR)‐mediated detection of pathogen‐associated molecular patterns (PAMPs). On the other hand, Mn²⁺ released from the mineral matrix permeated into the cytosol and effectively enhanced type I interferon production and Th1‐polarized cellular immune responses via the cGAS‐STING signaling axis. This dual‐adjuvant synergy promoted efficient antigen presentation by DCs to T lymphocytes, inducing robust adaptive immunity characterized by both potent cellular and humoral responses.

The in vivo experiments showed that the Mn‐based DNA nanovaccine exhibited a distinct Th1‐polarizing capacity, as evidenced by enhanced IgG2a isotype switching and increased secretion of Th1‐associated cytokines (IFN‐γ, IL‐12, and TNF‐α). Comparative analysis with Mg‐based nanovaccine revealed that MnCA preferentially induced the differentiation of CD4⁺ T cells into Th1, Th17, and regulatory T cell subsets, consequently establishing robust cellular immunity that is particularly crucial for antiviral protection.

Compared to currently mature commercial adjuvants and Mn‐based adjuvant systems reported in the literature, the Mn/DNA nanovaccine in this work exhibited several advantages: (1) This preparation method was conducted via a one‐step reaction under ambient temperature, atmospheric pressure, and physiological pH conditions, which streamlined the manufacturing process relative to most reported Mn‐based systems (requiring high temperature, alkaline environment, or dropwise loading procedures) [45]. (2) The combination of multiple adjuvants facilitated synergistic activation of multiple immune pathways, offering an advantage over single Mn‐based adjuvant systems [46, 47]. (3) Compared to adjuvants that predominantly induced Th2‐driven humoral immunity (e.g., MF59) or those with limited CD8⁺ T cell‐inducing capacity (e.g., AS04), this nanovaccine was crucial for clearing virus‐infected cells. Furthermore, the in situ mineralized porous delivery system of the Mn adjuvant helped to stabilize and protect antigens, which was superior to the simple physical mixture of commercial adjuvants and antigens.

Overall, the Th1‐biased immunostimulation exhibited by Mn‐based DNA nanoadjuvants effectively addressed the inherent limitations of conventional alum adjuvants in mounting adequate Th1‐type immune responses [47]. The modular design of MnCA established a versatile nanoplatform that enabled programmable delivery of diverse DNA sequences, allowing for customizable immune modulation and significantly broadening the potential applications of this manganese‐based adjuvant system in vaccinology and immunotherapy.

Author Contributions

Shuangbo Dang: methodology, investigation, data curation, writing – original draft. Yun Sun: methodology, data curation, investigation, writing – original draft. Ling Chen: methodology, data curation, investigation, writing – original draft. Yuhang Dong: conceptualization, data curation, investigation, writing – original draft, funding acquisition, project administration. Zibo Han: methodology, data curation. Junwei Hou: methodology, data curation. Xiaoshuang Wang: methodology, data curation. Ziyao Kang: methodology, data curation. Zhaoming Liu: methodology, data curation. Zhuo Wang: methodology, data curation, investigation, project administration, writing – review and editing. Qiming Li: conceptualization, resources, project administration, writing – review and editing. Jin Ren: conceptualization, data curation, investigation, writing – review and editing, project administration, resources. Feng Li: conceptualization, data curation, investigation, writing – review and editing, project administration, funding acquisition, resources, methodology.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: anie72948‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.