Multiscale dynamic immunomodulation by a nanoemulsified Trojan-TLR7/8 adjuvant for robust protection against heterologous pandemic and endemic viruses

Categories: Disease & Virus

June 25, 2025

Design and characterization of SE(Trojan-TLR7/8a)

SE(Trojan-TLR7/8a) was fabricated by emulsifying an aqueous phase with Tween 80 and an oil phase comprising a mixture of Trojan-TLR7/8a, squalene, and Span-85 (Fig. 1, Supplementary Fig. S1). Analysis of the physical properties of SE(Trojan-TLR7/8a), including size, shape, and zeta potential, via dynamic light scattering and transmission electron microscopy revealed that the size of SE(Trojan-TLR7/8a) remained stable at approximately 100 nm even after long-term storage (Fig. 2A–D, Supplementary Fig. S2, Supplementary Table S1). Additionally, SE(Trojan-TLR7/8a) can be freeze-dried, providing advantages in terms of storing and distributing heat-sensitive products where cold chain management is challenging (Fig. 2E) [19]. SE(Trojan-TLR7/8a) preserved its size and immunostimulatory function when redispersed immediately or after 30 days of storage at −20 °C following lyophilization (Fig. 2F–H, Supplementary Table S2). To investigate the intracellular mechanism of action of SE(Trojan-TLR7/8a), bone marrow-derived dendritic cells (BMDCs) were pretreated with the endocytosis inhibitor dynasore®. After treatment, SE(Trojan-TLR7/8a) was not detected in the intracellular compartments, and IL-12(p70) secretion was reduced, indicating that SE(Trojan-TLR7/8a) could be delivered into endosomes via endocytosis (Fig. 2I).

Moreover, significant BMDC activation was observed only in the groups treated with SE(Trojan-TLR7/8a) or a mixture of SE and R848 (SE + R848), both of which contained TLR7/8a (Supplementary Fig. S3). Notably, there was a significant increase in the levels of IL-12(p70), which is crucial for Th1 differentiation and initiates an adaptive immune response, and IL-6, which is essential for T_FH cell (CXCR5⁺ PD-1⁺ in CD3⁺CD4⁺) differentiation that supports GC B-cell functions, including class-switch recombination and somatic hypermutation (Fig. 2J) [20,21,22]. Consequently, naïve CD4⁺ T cells cocultured with DCs treated with SE(Trojan-TLR7/8a) biased toward Th1 cells through increased secretion of IFN-γ/IL-4 and increased differentiation into T_FH cells (Fig. 2K). Next, we assessed systemic toxicity by measuring the serum levels of IL-6, TNF-α, ALT, and AST. Compared with SE + R848, SE(Trojan-TLR7/8a) administration resulted in reduced systemic toxicity, as indicated by the lower serum levels of these markers (Supplementary Fig. S4). This reduction in toxicity is due to the cholesterol-blocking structure of Trojan-TLR7/8a, which maintains a stable particle form by preventing release and temporarily masks the active site, enabling selective activation within specific cells (Supplementary Figs. S1 and S2f). We further evaluated long-term drug toxicity via hematological and clinical biochemical analyses and detected no observable changes due to the administration of SE(Trojan-TLR7/8a) (Supplementary Tables S3–5).

Multiscale dynamic immunomodulation by SE(Trojan-TLR7/8a)

Macroscopic vaccine kinetics by SE

Upon intramuscular administration, SE is known to elicit danger-associated molecular pattern signals, attracting immune cells from the bloodstream to the injection site [23]. In vivo trafficking demonstrated that DiD-loaded SEs and SE(Trojan-TLR7/8a) presented sustained fluorescence intensity at the injection site compared with free DiD, with the signal lasting up to 7 days postimmunization (Fig. 3A). To confirm whether SE(Trojan-TLR7/8a) facilitates immune cell recruitment to muscle, muscle tissue was analyzed one day after intramuscular injection. Hematoxylin and eosin (H&E)-stained muscle tissue images demonstrated that SE(Trojan-TLR7/8a) significantly increased cell recruitment (Fig. 3B). Further cellular analysis revealed that the muscles from the groups containing SE had increased proportions of various immune cells, monocytes (Ly6C⁺ Ly6G⁻), neutrophils (Ly6C⁻ Ly6G⁺), CD11c⁺, CD11b⁺, NK cells (CD3⁻ NK1.1⁺), CD8⁺ T cells (CD3⁺ CD8⁺), and CD4⁺ T cells (CD3⁺ CD4⁺) (Fig. 3C, Supplementary Figs. S5 and 6).

Time-dependent cellular activation by Trojan-TLR7/8a

We hypothesized that once immune cells congregate in the muscles, they subsequently migrate to the LNs after the uptake of antigen and SE(Trojan-TLR7/8a) at the injection site. First, we aimed to evaluate the in vitro effects of SE(Trojan-TLR7/8a) on immune cells and determine its specific actions. The timely activation of SE(Trojan-TLR7/8a) resulted in prolonged and sustained activation of BMDCs, facilitated by the GILT-responsive linker designed for intracellular cleavage, as indicated by the continuous secretion of IL-12(p70) (Fig. 3D, Supplementary Fig. S3a). After 12 h of incubation followed by media replacement, SE + R848-treated DCs ceased IL-12(p70) secretion within 4 h (exhausted DCs), whereas SE(Trojan-TLR7/8a)-treated DCs continued to secrete cytokines for 24 h (nonexhausted DCs) (Fig. 3E). Moreover, SE(Trojan-TLR7/8a) increased CCR7 expression, and CCR7 signaling plays a key role in the migration of DCs from the injection site to the LNs (Fig. 3F). Transwell assays demonstrated that SE(Trojan-TLR7/8a)-treated DCs (nonexhausted DCs) exhibited greater migration toward chemoattractants (CCL21 and CCL19) than SE- or SE + R848-treated DCs did (Fig. 3G, Supplementary Fig. S7). On the basis of these in vitro experimental results, we hypothesized that SE(Trojan-TLR7/8a) induces semimature DCs that continuously secrete cytokines, thereby increasing DC migration and antigen delivery to LNs. We found that the total number of migratory DCs (CD103⁺) and resident DCs (CD8α⁺) increased in the SE(Trojan-TLR7/8a) group for up to 3 days (Fig. 3H, I; Supplementary Figs. S8, 9). To study in vivo antigen trafficking, each sample was mixed with FITC-labeled OVA (OVA(FITC)) and administered via intramuscular injection. Compared with that in the SE group, the fluorescence intensity of OVA(FITC) in the inguinal LNs (iLNs) in the SE(Trojan-TLR7/8a)-injected group was greater for up to 7 days (Fig. 3J, Supplementary Fig. S10).

Synergistic effects of SE and Trojan-TLR7/8a

Upon administration of SE(Trojan-TLR7/8a), increased DC migration from the muscles to the LNs was observed, indicating that antigen and nanoemulsion delivery was enhanced, thereby resulting in increased immune responses in the LNs. On the basis of these results, we hypothesized that SE(Trojan-TLR7/8a), which involves both the NLRP3-independent ASC activation pathway and the TLR-dependent MyD88 activation pathway, might induce both humoral and cellular immune responses (Fig. 3K) [24]. Compared with SE, SE(Trojan-TLR7/8a) upregulated the expression of cytokines and surface markers that are indicative of DC activation (Il6, CD86, and Il12a), cellular responses (Irf7, Ifna1, and Ifng) and humoral responses (IL18 and IL1β) (Fig. 3L, M) [24, 25]. To determine whether these increases enhance GC formation, which is crucial for the humoral response, we conducted immunofluorescence staining of mouse iLNs 7 days after a single immunization. We confirmed that GC B cells, identified as GL7⁺ cell clusters, represented bona fide GC structures on the basis of their location in the B-cell zone, which was distinct from that of the T-cell zone according to CD3 staining. SE(Trojan-TLR7/8a) markedly increased the number of GC B cells (Fig. 3L, N, Supplementary Fig. S11) [14, 26]. Next, to examine the enhanced activation of immune cells induced by SE(Trojan-TLR7/8a), we analyzed the dynamics of various immune cells associated with both humoral and cellular responses. In the kinetics of CD11b⁺ cells and CD11c⁺ cells, which are innate immune cells, SE(Trojan-TLR7/8a) peaked on day 7, whereas in the other groups, it peaked on day 3 and subsequently declined (Fig. 3O). Compared with those in the other groups, the number of T_FH cells, which are critical modulators of GC responses, increased approximately 7-fold on day 7 in the SE(Trojan-TLR7/8a) group. Moreover, the kinetics of GC B (CD38⁻ Fas⁺ in B220⁺) and CD8⁺ T cells also exhibited similar trends, peaking on day 7 before declining (Fig. 3P). We also analyzed the kinetics of neutrophils (Ly6C⁻ Ly6G⁺ in CD11b⁺ CD11c⁻), monocytes (Ly6C⁺ Ly6G⁻ in CD11b⁺ CD11c⁻), cDC2s (XCR1⁻ CD11b⁺ in MHCII⁺ CD11c⁺), and cDC1s (XCR1⁺ CD11b⁻ in MHCII⁺ CD11c⁺) and observed a greater magnitude in the SE(Trojan-TLR7/8a) group (Supplementary Figs. S12–14). Furthermore, the responses of antigen-specific CD8⁺ T cells that secrete IFN-γ, TNF-α and polyfunctional (IFN-γ⁺ TNF-α⁺) CD8⁺ T cells, which are vital for virus clearance, were significantly augmented (Fig. 3Q, Supplementary Figs. S15, 16) [27].

Increased local and systemic immunity after prime-boost immunization

To investigate whether booster immunization could potentiate immune responses, we administered intramuscular immunization twice at 3-week intervals, with analyses performed one week after booster immunization (Fig. 4A, Supplementary Figs. S17 and 18). SE(Trojan-TLR7/8a) significantly increased the percentages of GC B cells, T_FH cells, and memory B cells in the iLNs (Fig. 4B). In addition, compared with other treatments, SE(Trojan-TLR7/8a) immunization induced a high magnitude of immune cells involved in humoral immunity, including B cells (CD19⁺ B220⁺), follicular dendritic cells (FDCs) (CD21/35⁺), and plasma cells (B220⁻ CD138⁺) (Supplementary Fig. S19). Similarly, the percentage of antigen-specific CD8⁺ and CD4⁺ T cells secreting IFN-γ, granzyme B (GzB), or IL-4, which are involved in cellular immunity, was also significantly increased (Fig. 4C, Supplementary Fig. S20). By analyzing the spleen, lung, and serum, we further established that SE(Trojan-TLR7/8a) elicits not only local but also systemic immune responses (Fig. 4D). SE(Trojan-TLR7/8a) also had a notable effect on the spleen by enhancing GC B, T_FH, and antigen-specific cytokine-secreting T-cell responses and increasing plasma and memory B-cell responses (Fig. 4E, F; Supplementary Figs. S21 and 22). We analyzed effector memory (CD44⁺ CD62L⁻) T cells that were primed for an immediate response to pathogens only in the lungs and detected them at the highest abundance in the SE(Trojan-TLR7/8a)-vaccinated group (Fig. 4G, Supplementary Fig. S23). Additionally, the antigen-specific T-cell responses in the lungs paralleled those in the LNs and spleens (Fig. 4H, Supplementary Fig. S24). OVA-specific IgG antibody titers, including those of IgG1- and IgG2c, which are indicative of Th2- or Th1-biased responses, respectively, were also highest in the serum of the SE(Trojan-TLR7/8a)-vaccinated group (Fig. 4I, Supplementary Fig. S25). Taken together, these results demonstrated that prime-boost immunization with SE(Trojan-TLR7/8a) effectively induced robust localized and systemic humoral and cellular immune responses.

A comparative study of commercialized vaccine formulations

Comparison with vaccine adjuvants

To further evaluate the clinical translational potential of SE(Trojan-TLR7/8a), we compared it with that of the FDA-approved vaccine adjuvants Alum and AS03. C57BL/6 mice were immunized with alum, AS03, or SE(Trojan-TLR7/8a) mixed with OVA twice at 3-week intervals. The performance of SE(Trojan-TLR7/8a), a squalene-based nanoemulsion incorporating TLR7/8a, was compared with that of AS03, which enhances cellular immunity by adding α-tocopherol to squalene-based emulsions (Fig. 4J–L) [23, 24]. Compared with alum, which is known to induce a strong humoral response, SE(Trojan-TLR7/8a) increased the proportion of humoral immune cells (T_FH, GC B, and memory B cells) in the iLNs (Fig. 4J–L). SE(Trojan-TLR7/8a) also elicited higher levels of antigen-specific cytokine-secreting T cells and antigen-specific antibody responses, especially IgG2c, than did AS03, highlighting the potential benefit of TLR7/8a integration (Fig. 4M, N).

Comparison with the mRNA vaccine

Next, the immune response elicited by SE(Trojan-TLR7/8a) was compared with that elicited by an mRNA vaccine, which was recently clinically approved for use as a SARS-CoV-2 vaccine. C57BL/6 mice were immunized with SE(Trojan-TLR7/8a) mixed with OVA or LNP (OVA-mRNA) twice at 3-week intervals (Fig. 5A). SE(Trojan-TLR7/8a) also significantly enhanced the immune defense system by markedly increasing the proportion of immune cells involved in the humoral response (T_FH, memory B cells, GC B, B cells, and FDCs) (Fig. 5B, C). OVA-specific antibody titers (IgG, IgG2c, and IgG1) were also significantly greater with SE(Trojan-TLR7/8a) (Fig. 5D). Furthermore, SE(Trojan-TLR7/8a) substantially increased the numbers of antigen-specific CD8⁺ and CD4⁺ T cells secreting IFN-γ, granzyme B (GzB), or IL-4, indicating a significant improvement in immune effectiveness over the mRNA vaccine (Fig. 5E, F).

Heterologous vaccination, also known as “mix-and-match” vaccination, involves administering a series of vaccines from different platforms to an individual [28]. Recent studies suggest that combining an mRNA vaccine with a vaccine based on a different platform can increase immunogenicity, broaden the spectrum of immune responses, and possibly improve the durability of protection compared with homologous vaccination strategies. We compared the immunological effects of homologous LNP (OVA-mRNA) vaccination with those of heterologous vaccination with SE(Trojan-TLR7/8a) after LNP (OVA-mRNA) (Fig. 5G). Notably, heterologous vaccination significantly increased the numbers of GC B, T_FH, and memory B cells and FDCs for up to 6 weeks, whereas homologous vaccination peaked at 4 weeks (Fig. 5H). Additionally, the number of antigen-specific cytokine-secreting CD8⁺ T cells was significantly greater after heterologous vaccination (Fig. 5I). Compared with homologous vaccination, the heterologous vaccination approach with SE(Trojan-TLR7/8a) is a more promising strategy for enhancing long-term immunogenicity by improving the durability of the immune response. Nine weeks after the second immunization (Fig. 5J), significantly greater levels of key indicators of humoral and cellular responses, including GC B, T_FH, and memory B cells and FDCs (Fig. 5K–M), and antigen-specific CD8⁺ T-cell responses were observed in the SE(Trojan-TLR7/8a) group (Fig. 5N). This finding underscores the superior immunogenicity of SE(Trojan-TLR7/8a), highlighting its potential to induce robust and comprehensive immune responses (Supplementary Fig. S26).

Cross-protection against SARS-CoV-2 and influenza

To verify that the promising SE(Trojan-TLR7/8a)-adjuvanted vaccine can effectively generate neutralizing antibodies and provide protection against SARS-CoV-2, the levels of these antibodies were measured via a pseudovirus neutralization assay. A mixture of SE(Trojan-TLR7/8a) and the HexaPro antigen, which is a prefusion-stabilized spike ectodomain, displayed excellent neutralizing antibody titers against all the variants tested (Fig. 6A) [29]. For further evaluation, we administered spike-stabilized trimers from the BA.2 variant as antigens to BALB/c mice, either alone or combined with SE or SE(Trojan-TLR7/8a), via intramuscular injection at weeks 0 and 3. Following the last immunization, the mice from each group were challenged with 100 LD₅₀ of mouse-adapted SARS-CoV-2 (Wuhan strain) to assess immunogenicity and cross-protective responses (Fig. 6B). The groups receiving the spike protein with SE(Trojan-TLR7/8a) demonstrated complete protection against SARS-CoV-2 infection, as evidenced by the 100% survival rate and minimal body weight loss, in contrast to the spike protein-only group, which demonstrated 100% mortality within five days (Supplementary Fig. S27). By day 3 postchallenge, the SE(Trojan-TLR7/8a) group presented significantly reduced viral loads in the lungs, achieving complete viral clearance by day 5, in contrast to the persistent presence of viruses in the SE group (Fig. 6C). Additionally, the numbers of T_FH (PD-1⁺ IL-21⁺ in CD4⁺) and GC B cells (GL7⁺ AID⁺ in CD19⁺) were markedly greater in the SE(Trojan-TLR7/8a) group than in the other groups, indicating improved antibody production (Fig. 6D). Consequently, SE(Trojan-TLR7/8a) significantly increased neutralizing titers against both homologous (BA.2) and heterologous variants (Fig. 6E, F). Notably, the SE(Trojan-TLR7/8a) group exhibited superior CD8⁺ cytotoxic T lymphocyte (CTL) activity, which is essential for eradicating virus-infected cells, as evidenced by the increase in IFN-γ spot-forming units in response to homologous (BA.2) and heterologous (S peptide pool and inactivated SARS-CoV-2 variants) variants (Fig. 6G). Furthermore, the polyfunctional capacity of CTLs to clear the virus rapidly and provide long-term immunity was highest in the SE(Trojan-TLR7/8a) group (Fig. 6H, Supplementary Fig. S28). These findings underscore the powerful role of SE(Trojan-TLR7/8a) in amplifying both humoral and cellular immune defenses against SARS-CoV-2, allowing rapid clearance of viruses from the lung and acting as a significant enhancer of vaccine-induced protection against viruses.

Next, we evaluated the efficacy of the SE(Trojan-TLR7/8a) adjuvant against influenza through intramuscular immunization in combination with sM2HA2, a promising antigen that targets a broad range of influenza subtypes and is composed of influenza matrix protein 2 (sM2) and the stalk domain of hemagglutinin protein (HA2) [30]. The sM2HA2 used in this study comprises the conserved extracellular (residues 1–24) and cytoplasmic (residues 44–97) domains of influenza sM2 from H5N1, H1N1, and H9N2 subtypes, as well as the stalk domain (residues 15-137) of HA2 from the A/EM/Korea/W149/06 (H5N1) strain [31]. Immunization with SE(Trojan-TLR7/8a) has been found to elicit sM2HA2-specific humoral and cell-mediated immunity, both locally and systemically, indicating potential protection against lethal influenza infection (Supplementary Figs. S29–31). To assess the protective ability of SE(Trojan-TLR7/8a), immunized mice were challenged with a 10 LD₅₀ dose of various mouse-adapted influenza A virus subtypes (H1N1, H5N2, H7N3, H9N2, or H3N2) (Fig. 7A). Body weights and survival rates were then monitored until 13 days post infection (dpi) (Fig. 7B–F). All control mice died from infection between days 5 and 7, resulting in significant body weight loss of more than 20%. However, all the mice immunized with SE(Trojan-TLR7/8a) demonstrated complete protection against diverse influenza subtypes, with rapid reversal of body weight loss and notably, no weight loss upon challenge with H1N1. The virus titers in the lungs of the mice challenged with H1N1 or H5N2 were significantly lower in the SE(Trojan-TLR7/8a) group than in the control group within 5 dpi (Fig. 7G). Furthermore, in mice immunized with SE(Trojan-TLR7/8a), virus clearance from the lungs was correlated with histopathological analysis (Fig. 7H, Supplementary Fig. S32). Pathologic signs such as alveolar wall thickening and inflammatory cell infiltration (yellow arrows) were rarely observed in the SE(Trojan-TLR7/8a) group.

Long-term protection against influenza and SFTS viruses

To determine whether SE(Trojan-TLR7/8a) can sustain enhanced immune responses, we conducted mRNA sequencing to observe the upregulation of genes associated with long-term immunity, including genes related to the proliferation and differentiation of memory T cells (Tsc1), GC B cells (Bcl6 and Il21), and plasma cells (Prdm1, Lgals1, and Nfkbiz) (Fig. 8A, B) [32]. Given that GC B cells play a crucial role in generating sustained and high-quality antibody responses, we sought to assess whether SE(Trojan-TLR7/8a), which enhances GC B-cell responses, also facilitates differentiation into long-lived plasma cells (LLPCs) and influences sustained antigen-specific antibody secretion. Differentiated LLPCs migrate to the bone marrow, where they reside, produce antibodies, and maintain serum antibody levels against pathogens. At six weeks post-immunization, a significant increase in the LLPC population was observed in the bone marrow of the SE(Trojan-TLR7/8a)-immunized mice (Fig. 8A, C).

To assess the effectiveness of a vaccine, evaluating its ability to elicit durable immune responses and provide sustained protection against pathogens is crucial. BALB/c mice were immunized with SE(Trojan-TLR7/8a)-adjuvanted sM2HA2 at 3-week intervals (Fig. 8D). Twenty-four weeks after the initial immunization, the measured serum IgG levels and ELISPOT results for IFN-γ and IL-4 in splenocytes, all of which were specific to sM2HA2, sM2, or HA2, demonstrated that SE(Trojan-TLR7/8a) induces long-lasting humoral and cellular immune responses (Fig. 8E, F; Supplementary Figs. S33, 34). At that point, the mice were challenged with H1N1, after which the mice in the SE(Trojan-TLR7/8a) group fully recovered, with a 100% survival rate for 13 days, which was comparable to the survival rate immediately following immunization (Fig. 8G).

SFTSV is associated with increased morbidity and mortality in elderly patients [33]. Similarly, aged ferrets (≥3 years old) infected with SFTSV exhibit mortality with clinical symptoms resembling those observed in human cases [34, 35]. To evaluate the efficacy of the SE(Trojan-TLR7/8a) vaccine, aged ferrets (3 years old), an established immunocompetent model for SFTSV infection, were intramuscularly administered an inactivated SFTSV vaccine with or without SE(Trojan-TLR7/8a) twice at two-week intervals (Fig. 8H). Serum was collected two weeks after each immunization to assess neutralizing antibody (SN) production, with the highest SN titer observed with SE(Trojan-TLR7/8a) after boosting (Fig. 8I). At 30 weeks postpriming, the results of the IFN-γ ELISPOT assays revealed stronger SFTSV-specific T-cell responses in the SE(Trojan-TLR7/8a) and vaccine combination groups than in the vaccine alone group (Fig. 8J). To evaluate the protective efficacy of SE(Trojan-TLR7/8a), ferrets were challenged with a lethal dose (10⁶ TCID₅₀) of the CB1/2014 strain. Viral RNA was present in all unvaccinated ferret tissues at 4 and 6 dpi but was undetectable in all animals in the SE(Trojan-TLR7/8a) vaccine group, indicating a faster rate of virus clearance (Fig. 8K, Supplementary Fig. S35). Additionally, the ferrets were monitored for clinical signs of infection, changes in body weight and temperature, platelet count, and serum neutralization (SN) titers until 6 dpi (Supplementary Fig. S35). The unvaccinated group presented high fever, 20% body weight loss, and severe thrombocytopenia. In contrast, the SE(Trojan-TLR7/8a) group presented no clinical symptoms (Fig. 8L–N). Overall, the SE(Trojan-TLR7/8a) vaccine enhanced SFTSV-specific T-cell responses, providing sufficient protection against lethal challenges and enabling rapid virus clearance even after long-term vaccination.