Abstract

Background

   Flavivirus is a highly prevalent and outbreak-prone disease, affecting
   billions of individuals annually and posing substantial public health
   challenges. Vaccination is critical to reducing the global impact of
   flavivirus infections, making the development of a safe and effective
   vaccine a top priority. The self-assembled pan-epitope vaccine presents
   key advantages for improving immunogenicity and safety without relying
   on external vectors or adding immunomodulatory elements, both of which
   are essential for successful vaccine development.

Results

   In this study, the pan-epitope peptide TBT was combined with adjuvant
   CpG to form the TBT-CpG nanovaccine (TBT-CpG NaVs), which was found to
   be spherical, uniform in shape, and demonstrated strong serum
   stability. In vitro studies showed that the TBT-CpG NaVs were
   efficiently taken up and internalized by bone marrow-derived dendritic
   cells (BMDCs). Flow cytometry and transcriptomic analysis indicated
   that the antigens were effectively presented to antigen-presenting
   cells (APCs) via the MHC II pathway, which facilitated BMDCs maturation
   and promoted the release of pro-inflammatory cytokines IL-1β, TNF-α,
   and IL-6. In vivo studies confirmed that TBT-CpG NaVs enhanced
   antigen-specific IgG levels, significantly increased IFN-γ and IL-4
   expression in spleen cells, and offered protective effects against
   Dengue virus (DENV) and Zika virus (ZIKV) infections. Safety
   evaluations revealed no hepatotoxicity and no significant organ damage
   in immunized mice.

Conclusion

   The self-assembled candidate nanovaccine TBT-CpG NaVs effectively
   activates BMDCs and triggers a targeted immune response, providing
   antiviral effects against DENV and ZIKV. This vaccine demonstrates good
   immunogenicity and safety, establishing a promising foundation and a
   new strategy for the development of safe and effective vaccines.

Graphical Abstract

   [46]graphic file with name 12951_2024_3031_Figa_HTML.jpg

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12951-024-03031-0.

   Keywords: Flavivirus, Pan-epitope peptide, CpG, Nanovaccine

Introduction

   Flaviviruses, a group of viruses transmitted by arthropods, include
   several significant human pathogens, such as Dengue virus (DENV), Zika
   virus (ZIKV), Yellow fever virus (YFV), and West Nile virus (WNV)
   [[47]1, [48]2]. These viruses threaten billions of people worldwide,
   representing an increasing global health risk [[49]3, [50]4]. DENV,
   with its four serotypes (DENV1-4), is the most widespread
   mosquito-borne virus, while ZIKV, known for its severe teratogenic and
   neurotoxic effects, garners particular attention due to their
   substantial impact on public health [[51]5]. Vaccination remains a
   critical strategy for controlling infectious diseases, with ample
   evidence of its effectiveness. For instance, the success of COVID-19
   vaccines in reducing disease severity and incidence underscores the
   essential role of vaccines in mitigating severe outcomes and
   hospitalizations. Globally, vaccines continue to play an irreplaceable
   role in controlling communicable diseases.

   Currently, no vaccines for ZIKV have received approval, and only two
   live-attenuated dengue vaccines—Dengvaxia (CYD-TDV) and DENVax
   (TAK003)—are clinically approved [[52]6]. Although live-attenuated
   vaccines retain a nearly complete virus structure to trigger mucosal,
   humoral, and cellular immune responses [[53]7, [54]8], their protective
   efficacy against the four DENV serotypes remains inconsistent.
   Additionally, the use of live pathogens carries a risk of pathogenicity
   recovery [[55]9, [56]10]. In contrast, a new class of epitope-based
   subunit vaccines, which contain only specific pathogen antigen
   fragments, offers enhanced safety and scalability for large-scale
   production [[57]11]. Epitope-based peptide vaccines target specific
   regions of a pathogen’s proteins, known as epitopes, ensuring a focused
   immune response against critical areas of the pathogen, thereby
   improving efficacy and reducing the risk of off-target effects [[58]12,
   [59]13]. The design and synthesis of peptide vaccines can be rapidly
   accomplished once a pathogen’s genetic information is available, a
   crucial advantage during outbreaks of new infectious diseases.

   The primary limitation of epitope-based subunit vaccine is their low
   immunogenicity, due to the absence of a complete organism [[60]14].
   This limitation can be addressed by combining subunit vaccines with
   adjuvants and enhancing the capacity of dendritic cells (DCs) to uptake
   and present antigen epitopes, both of which are effective for boosting
   antiviral immune responses and achieving long-lasting protective
   efficacy [[61]15–[62]17]. CpG, a widely used adjuvant, act as a TLR9
   agonist that promotes antigen endocytosis to enhance the immune
   response, with a safety profile similar to conventional vaccines
   [[63]18, [64]19]. Among CpG options, ODN 1826 (5’-TCC ATG ACG TTC CTG
   ACG TT-3’) is a well-known immunological adjuvant, activating the
   innate immune response and improving the function of professional
   antigen-presenting cells (APCs) with proven safety [[65]20].
   Furthermore, the programmability and chemical functionalization of DNA
   sequences allow for the formation of diverse nanostructures [[66]21,
   [67]22]. Co-delivering antigens with CpG ODN in nanocarriers promotes a
   robust immune response and enhances antitumor effects, both within and
   outside these carriers [[68]23]. As professional APCs, DCs play a
   central role in immune induction by capturing and processing antigens.
   Immature DCs, in particular, exhibit high migratory abilities and
   preferentially uptake virus-sized particles (20–200 nm)
   [[69]24–[70]26]. Linking target peptide epitopes to nanoparticles aids
   in the maturation of bone marrow-derived dendritic cells (BMDCs) and
   facilitates the organized presentation of multivalent epitopes
   [[71]27]. This approach enhances antigen bioavailability and
   effectively activates both innate and adaptive immune responses, which
   is critical for developing an effective candidate vaccine [[72]28,
   [73]29].

   Self-assembled nanovaccines, which combine antigens with adjuvants,
   have proven to be an excellent vaccine platform, offering several
   advantages: (1) They avoid the inclusion of unnecessary components,
   such as delivery carriers, thereby reducing the risk of carrier-related
   toxicity thanks to their simple structure [[74]30]; (2) They ensure the
   co-delivery of antigens and adjuvants directly to APCs, inducing
   targeted immune responses and minimizing the risk of immune tolerance
   [[75]21, [76]31, [77]32]; and (3) They enhance antigen stability,
   prolong antigen release, and support sustained immune responses over
   time [[78]28]. Various types of nanovaccine have shown promise in
   antiviral and antitumor studies, with self-assembled peptide
   nanoparticles due to their high antigen load and lack of external
   carriers [[79]33–[80]35]. However, no research has yet explored a
   self-assembled co-delivery system specifically based on flavivirus
   epitope antigens and adjuvants.

   In this study, we developed a self-assembled nanovaccine to combat
   flavivirus, using a conjugate of the pan-epitope peptide TBT with the
   adjuvant CpG. The hydrophobic TBT was conjugated with the hydrophilic
   CpG using DTDP and the reductant TCEP, resulting in self-assembly
   through hydrophobic and electrostatic interactions to form the TBT-CpG
   nanovaccines (TBT-CpG NaVs). This nanovaccine co-delivers antigens and
   adjuvants to dendritic cells (DCs), promoting the maturation of bone
   marrow-derived dendritic cells (BMDCs) and stimulating cytokine
   secretion. TBT-CpG NaVs effectively activated both cellular and humoral
   immunity against the flavivirus with excellent biosafety, establishing
   a promising foundation for the next generation of flavivirus vaccine
   candidates.

Materials and methods

Preparation of TBT-CpG NaVs

   The synthesis of self-assembled nanovaccine was carried out as follows:
   CpG ODN (Sangon Biotech, China) was mixed with Tris (2-carboxyethyl)
   phosphine (TCEP) solution at a molar ratio of 1:100 for 2 h at 25 °C.
   This mixture was then combined with 4,4’-Dithiodipyridine (DTDP)
   solution at a molar ratio of 1:1000 for 30 min at 25 °C. The resulting
   product was analyzed with mass spectrometer (Thermo, USA) and purified
   using an NAP-5 nucleic acid purification column (Cytiva, USA) to remove
   excess TCEP and DTDP. The pan-epitope peptide TBT
   (KYVKQNTLKLAT-GG-VDRGWGNGCGLFGKG-LL-LEYIPEITLPVIAALSIAES, Chinese
   Peptide, China) was then mixed with purified products at molar ratios
   of 1:1, 2:1, 3:1, 4:1, 5:1 and 6:1 for 30 min at 25 °C to produce
   self-assembled nanovaccine TBT-CpG NaVs with varying loading degrees.

Gel electrophoresis experiment

   The conjugated products of TBT-CpG and free CpG were loaded into a 1%
   agarose gel and electrophoresed for 80 min at 60 V. Free CpG, TBT, and
   TBT-CpG were also loaded into a 15% native polyacrylamide gel
   electrophoresis (PAGE) and run for 2 h at 80 V. Following
   electrophoresis, the gel was stained with a 3× Gel Red staining
   solution in the dark for 30 min at 25 °C. Visualization and analysis
   were conducted using a Fusion SoloS gel imager.

Characterization of TBT-CpG NaVs

   To characterize the TBT-CpG NaVs, 20 µL of the conjugated products were
   diluted in PBS at a volume ratio of 1:50. The average particle size and
   Zeta potential of the sample were measured at 25 °C using a Zetasizer
   Nano-ZS90 (Malvern Instruments Ltd., UK). For morphological analysis,
   self-assembled TBT-CpG NaVs were prepared by negative staining
   transmission electron microscopy (TEM). Briefly, 10 µL of the sample
   was dropped onto a copper grid and absorbed for 10 min. After removing
   excess liquid, the sample was stained with 10 µL of uranyl acetate for
   1 min. Once dried, the sample’s dispersion and morphology were observed
   with JEM 1200EX transmission electron microscope.

In vitro pan-epitope peptide TBT Release Study

   The release profile of pan-epitope peptide TBT from TBT-CpG NaVs was
   evaluated through dialysis, with TBT labled by Cy5. Briefly, TBT-CpG
   NaVs were placed in dialysis bags (Viskase, USA), which were securely
   sealed. The dialysis bags were immersed in 50 mL of release medium,
   consisting of phosphate-buffered saline solution buffer with 1mM
   glutathione (GSH), and incubated at 37 °C with continuous shaking at
   100 rpm. 0.5 mL of dialysate was collected and replaced with an equal
   volume of fresh release medium to maintain sink conditions. The
   fluorescence intensity of TBT was measured using a SYNERGY H1
   fluorescence spectrophotometer (BioTek, USA), with a series of diluted
   Cy5-labeled TBT used to establish standard curves.

Serum stability study of TBT-CpG NaVs

   To assess serum stability, TBT-CpG NaVs and a control group of free CpG
   were treated with DMEM medium containing 10% FBS for 24 h, 48 h, and
   72 h. After each time point, samples were loaded into a 1% agarose gel
   and electrophoresed for 80 min at 60 V to evaluate their stability.

Isolation and cytotoxicity analysis of mouse bone marrow-derived dendritic
cells (BMDCs)

   BMDCs were isolated under sterile conditions from the femurs and tibias
   of mouse and cultured in RPMI1640 medium with 10% FBS, 1%
   penicillin/streptomycin, 20 ng/mL mGM-CSF and 10 ng/mL mIL-4
   (Novoprotein, China) at 37 °C in a cell incubator. Half of the medium
   was replaced on the 2nd and 4th days, and immature cells were collected
   for cytotoxicity analysis on the 6th day. BMDCs were seeded in a
   96-well plate at a density of 1 × 10^4 cells/well and incubated
   overnight. TBT, TBT + CpG, and TBT-CpG NaVs were then added at final
   concentrations of 50, 100, 200, 400, and 600 nM, with PBS as a control.
   Cell viability was determined using the Cell Counting Kit-8 (Beyotime,
   China) after 24 h at 37 °C. Absorbance at 450 nm was measured using a
   microplate reader. Cell viability (%) was calculated using the formula:
   (Atreat/Acontrol) × 100%, where Atreat represented the absorbance of
   TBT/TBT + CpG/TBT-CpG NaVs treated cells, and Acontrol represented the
   absorbance of the control group cells.

In Vitro uptake and activation of BMDCs

   BMDCs were seeded at in a 24-well culture plate at a density of
   1 × 10^5 cells/well, and incubated overnight. Cy5-labeled TBT,
   TBT + CpG, and TBT-CpG NaVs were co-incubated with BMDCs for 2–24 h.
   After 2 h of incubation, the samples were fixed with 4%
   paraformaldehyde for 15 min and stained with DAPI for 5 min. The
   fluorescence signal was visualized under a confocal microscopy (ZEISS,
   LSM880, Germany). The percentage of Cy5-positive cell was analyzed via
   flow cytometer (BD FACS Celesta, USA). After 24 h of incubation, the
   culture supernatant was collected to measure inflammatory cytokines
   (TNF-α, IL-1β, and IL-6) using ELISA kits (Thermo Fisher, USA). Cells
   were then stained with antibodies CD11c-APC, CD80-PE, CD86-FITC, and
   MHC-II-PE/Cyanine7 (BioLegend, USA). The expression of surface markers
   CD11C, co-stimulatory molecules CD80 and CD86, and MHC II on BMDCs was
   detected by flow cytometry, with BMDCs cultured alone serving as a
   negative control.

RNA-Seq and data analysis

   Sample RNA was reverse transcribed using the Illumina NovaSeq Reagent
   Kit to construct a cDNA library, followed by high-throughput sequencing
   on the Illumina NovaSeq 6000 platform. Raw sequencing data were
   processed with fastp software to obtain high-quality clean reads for
   further analysis. These clean reads were aligned to the mouse genome
   (Mus_musculus. GRCm39) using Hisat2. Gene and transcript levels were
   quantified with RSEM software. Differential gene expression analysis
   was conducted using DESeq2, identifying genes as differentially
   expressed if they met both criteria (p < 0.05 & |log2FC| ≥ 1). GO
   enrichment analysis was performed using Goatools, and KEGG pathway
   enrichment analysis was conducted with KOBAS.

Animal immunization

   Female Balb/c mice aged 6–8 weeks were purchased from the Army Medical
   University Laboratory Animal Center. All animal procedures were
   approved by the Army Medical University Animal Welfare and Ethics
   Committee (AMUWEC20240036). After a 7-day acclimation period, the mice
   were randomly assigned to four groups (PBS, TBT, TBT + CpG, and TBT-CpG
   NaVs), with six mice per group. Vaccination were administered via
   subcutaneous injection on days 0, 14, and 28, with each dose containing
   30 µg TBT in a volume of 200 µL. Serum samples were collected on days
   35 and 56 post-initial immunization for IgG antibody analysis. Seven
   days after the final immunization, organs were aseptically collected
   for Enzyme-linked Immunospot Assay (ELISPOT) and safety evaluation.

IgG antibody titer detection

   TBT was dissolved in coating buffer at a final concentration of
   5 µg/mL, with 100 µL of antigen solution per well in a 96-well plate,
   and incubated overnight at 4 °C. The plate was washed five times with
   wash buffer containing 0.1% Tween 20 to remove unbound antigen. Wells
   were blocked with 200 µL of blocking solution (carbonate buffer
   containing 5% bovine serum albumin) and incubated at 37 °C for 2 h to
   prevent non-specific binding. After washing five times, diluted serum
   samples were incubated at 37 °C for 2 h. Following additional washing,
   100 µL of horseradish peroxidase-labeled anti-mouse IgG secondary
   antibody at a dilution of 1:5000 was added and incubated at 37 °C for
   1 h. The reaction was developed with TMB substrate solution for 10 min,
   stopped with 100 µL of 2M H[2]SO[4], and absorbance was measured at
   450 nm using a microplate reader Synergy H1.

Enzyme-linked immunospot assay (ELISPOT)

   Seven days after the final immunization, mouse spleen was aseptically
   harvested to prepare single-cell suspension. The cell suspension was
   adjusted to a concentration of 5 × 10^6 cells/mL with RPMI1640 medium,
   and 100 µL per well was added to pre-coated 96-well plates. Peptide TBT
   was used as a stimulant, and cells were incubated at 37 °C for 36 h.
   Cytokines IFN-γ and IL-4 were detected using ELISPOT kits (Dakewei,
   China), and spots were analyzed with an immune spot microscope.

In vivo viral challenge protection experiment

   On day 35 after the initial immunization, blood samples were collected
   from each group and centrifuged at 1000 g for 10 min to isolate serum.
   The serum was then inactivated at 56 °C for 30 min, diluted with PBS,
   and mixed with 10 µL of DENV-2, DENV-4, or ZIKV at 37 °C for 1 h. The
   virus-serum mixture was injected intracranially into suckling mice
   using a microsyringe, and survival was monitored for 15 days
   post-infection.

Bio-safety analysis

   On day 35 post-immunization, mice were euthanized to collect blood and
   major organs (heart, liver, spleen, lungs, kidneys, brain. Serum levels
   of ALT and AST were measured using biochemical assay kits (Solarbio,
   China) following the manufacturer’s instructions. Organs were preserved
   in 4% paraformaldehyde and prepared for hematoxylin-eosin staining.

Statistical analysis

   All data are presented as mean ± SD. Statistical analyses were
   performed using GraphPad Prism 9.0 software, employing Student’s t-test
   or one-way analysis of variance (ANOVA). A P-value < 0.05 is considered
   statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****
   P < 0.0001).

Results

Preparation and characterization of TBT-CpG nanovaccines (NaVs)

   The thiolated CpG ODN (CpG-SH) was coupled with DTDP to creat a new
   product CpG-DTDP, with its molecular weight displayed in Fig. [81]S1.
   The purified CpG-DTDP was then conjugated with the flavivirus
   pan-epitope peptide TBT via disulfide bonds, resulting in the TBT-CpG
   product. To quantify the binding capacity between the antigenic peptide
   TBT to the adjuvant CpG ODN, TBT was mixed with CpG ODN at different
   molar ratios, and unbound CpG ODN was monitored via agarose gel
   electrophoresis. Uptake of the peptide by DC2.4 cells was visualized
   using the fluorescence microscopy, revealing a marked increase in
   cellular uptake for TBT when coupled with CpG ODN compared to free TBT
   (Fig. [82]S2). Full binding of CpG ODN to TBT occurred at a molar ratio
   exceeding 3:1 (Fig. [83]1A).

Fig. 1.

   [84]Fig. 1
   [85]Open in a new tab

   Preparation and characterization of TBT-CpG NaVs. A Agarose gel
   electrophoresis showing the conjugation reaction between TBT and CpG
   ODN at varying molar ratios. Lane 1: Free CpG ODN; Lanes 2–7:
   Conjugation reactions with TBT and CpG ODN at different molar ratios. B
   and C Dynamic Light Scattering (DLS) analysis for particle size (B) and
   zeta potential (C) of TBT-CpG NaVs at different TBT-to-CpG ODN ratios.
   D Transmission Electron Microscopy (TEM) image displaying the
   morphology of TBT-CpG NaVs. E Native polyacrylamide gel electrophoresis
   (PAGE) with Gel Red staining, comparing TBT-CpG NaVs at a 4:1 molar
   ratio with a physical mixture of TBT and CpG ODN at the same ratio
   (TBT + CpG). F Agarose gel electrophoresis results for serum stability
   testing of TBT-CpG NaVs and the physically mixed TBT + CpG at 24, 48,
   and 72 h. Data are represented as mean ± SD (n = 3). Statistical
   analysis was performed using Student’s t-test (B, C). ns, p > 0.05;
   ***p < 0.001

   To identify the optimal TBT to CpG ODN binding ratio, the conjugated
   products at various ratios were analyzed using the Dynamic Light
   Scattering (DLS) for particle size and zeta potential. TBT-CpG NaVs
   formed at a 4:1 molar ratio exhibited a smaller diameter of
   153.8 ± 1.7 nm with a PDI of 0.287 ± 0.023 (Fig. [86]1B and Fig.
   [87]S3) and the highest zeta potential at 28.1 ± 1.7 mV (Fig. [88]1C),
   which is favorable for cellular uptake.

   Following optimizing the nanovaccine synthesis, the self-assembled
   nanovaccine TBT-CpG NaVs were further characterized. Transmission
   electron microscopy revealed that TBT-CpG NaVs were spherical and
   uniformly distributed (Fig. [89]1D). Compared with free CpG ODN,
   TBT-CpG NaVs showed a reduced migration rate with no detectable
   residual CpG, indicating the adjuvant CpG was bound successfully to TBT
   via disulfide bonds, resulting in a high loading rate (Fig. [90]1E).
   The cumulative release of TBT was 57.2 ± 1.7% over 48 h under 1mM GSH
   (Fig. [91]S4A). These results confirmed the successful development of a
   self-assembled spherical nanovaccine with high encapsulation efficiency
   and slow antigen peptide TBT released.

   For practical application, TBT-CpG NaVs, as a novel vaccine delivery
   system, should be physiological stability. Serum stability was assessed
   through agarose gel electrophoresis, the result revealed that the
   self-assembled TBT-CpG NaVs have significantly enhanced stability in
   DMEM medium with 10% FBS compared to the physically mixed TBT + CpG
   group (Fig. [92]1F). Stability was also maintained in DMEM medium
   containing 30% and 50% FBS (Fig. [93]S4B).

TBT-CpG NaVs facilitate rapid uptake and internalization by BMDCs

   Flow cytometry analysis revealed that TBT-CpG NaVs exhibited a
   significantly higher antigen uptake capacity compared to the TBT and
   TBT + CpG groups (Fig. [94]2A). To further investigate the
   immunological mechanism of the TBT-CpG NaVs vaccine, Cy5-labeled TBT
   was employed to track the internalization of TBT-CpG NaVs
   (Fig. [95]2B). After co-culturing 200 nM Cy5-TBT with BMDCs for 2 h,
   TBT-CpG NaVs demonstrated markedly increased red fluorescence,
   primarily localized in the cytoplasm, compared to the free TBT and
   TBT + CpG groups. These findings suggested that TBT-CpG NaVs are
   rapidly taken up and internalized by BMDCs, potentially enhancing the
   effectiveness of immune response activation.

Fig. 2.

   [96]Fig. 2
   [97]Open in a new tab

   Uptake and internalization of TBT-CpG NaVs by BMDCs. A Flow cytometry
   anslysis to evaluate the uptake efficiency of different nanoparticles
   in BMDCs after 2 h at a concentration of 200 nM. B Confocal laser
   scanning microscopy images showing the internalization of different
   nanoparticles in BMDCs at a concentration of 200 nM after 2 h. TBT is
   labeled with Cy5 (red), and cell nuclei are stained with DAPI (blue).
   Scale bars represent 20 μm. Data are shown as mean ± SD (n = 3).
   Statistical analysis was performed using one-way ANOVA. ****p < 0.0001

TBT-CpG NaVs induce maturation of BMDCs and promote cytokine secretion

   To evaluate the effect of TBT-CpG NaVs on the activation and maturation
   of BMDCs, BMDCs from C57BL/6 mouse were cultured with mGM-CSF and mIL-4
   for 6 days before being treated with 200 nM TBT-CpG NaVs for 24 h. Flow
   cytometry was employed to measure the expression levels of cell surface
   molecules MHC II and co-stimulatory molecules CD80 and CD86. CD11C, a
   marker commonly used for dendritic cell identification, was expressed
   over 95% of cells in all groups, confirming the successful isolation of
   high purity BMDCs (Fig. [98]S5A).

   The percentage of CD80^+CD86^+BMDCs was significantly higher in the
   TBT-CpG NaVs group than in the TBT + CpG group (Fig. [99]3A and Fig.
   [100]S5B). Additionally, TBT-CpG NaVs treatment led to increased MHC II
   expression following antigen stimulation (Fig. [101]3B and Fig.
   [102]S5C). ELISA analysis of the cell culture supernatant revealed that
   TBT-CpG NaVs significantly upregulated the levels of pro-inflammatory
   factors IL-1β, TNF-α, and IL-6 (Fig. [103]3C-E). These results
   demonstrated that TBT-CpG NaVs enhanced antigen presentation through
   the MHC II pathway, thereby stimulating the maturation of dendritic
   cells.

Fig. 3.

   [104]Fig. 3
   [105]Open in a new tab

   TBT-CpG NaVs induce maturation of BMDCs and promote cytokine secretion.
   Flow cytometry was used to measure the expression of CD80^+CD86^+ (A)
   and MHC II^+ (B) in BMDCs, after co-culturing with different stimuli
   for 24 h. The bar graph of (A) represented the statistical analysis of
   CD80^+CD86^+. ELISA was used to assess cytokine levels in the
   supernatant of cells stimulated by different treatments: IL-1β (C),
   TNF-α (D), and IL-6 (E). Data are presented as mean ± SD (n = 3).
   Statistical analysis was conducted using one-way ANOVA. ns, p > 0.05;
   *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Transcriptomic analysis of BMDCs stimulated by TBT-CpG NaVs

   To explore the underlying mechanisms by which TBT-CpG NaVs promote BMDC
   maturation, transcriptomic analysis was performed on BMDCs treated with
   either TBT alone or TBT-CpG NaVs. Differentially expressed gene (DEG)
   analysis revealed substantial gene expression changes with TBT-CpG NaVs
   compared to free TBT, with 394 genes upregulated and 58 genes
   downregulated, showing a predominance of upregulated genes
   (Fig. [106]4A).

Fig. 4.

   [107]Fig. 4
   [108]Open in a new tab

   Transcriptome analysis of BMDCs stimulated by TBT-CpG NaVs. A Volcano
   plot showing differentially expressed genes, with upregulated genes
   marked in red and downregulated genes in blue. B GO enrichment analysis
   of upregulated genes, where the vertical axis represented the GO term
   and the horizontal axis represented the Rich factor. Dot size indicates
   the number of genes in each GO term, and dot color corresponded to
   different range of adjust p-values. C KEGG enrichment analysis of the
   upregulated genes. The right side shows pathways significantly enriched
   in differential genes, while the left side lists genes in each pathway,
   ordered by log2 Fold Change (log2FC). A higher log2FC indicates a
   greater fold increase in gene expression. D Reactome enrichment
   analysis of upregulated genes. The vertical axis represents Reactome
   pathways. The upper horizontal axis shows the number of genes mapped to
   each pathway, while the lower axis represents the significance level of
   enrichment

   Gene Ontology (GO) enrichment analysis indicated that these
   differential genes were primarily involved in myeloid dendritic cell
   activation, antigen processing and presentation of exogenous peptide
   antigen via MHC class II, and the regulation of T cell proliferation
   and differentiation (Fig. [109]4B). These findings align with previous
   results, further supporting that TBT-CpG NaVs could enhance antigen
   presentation through the MHC II pathway, thereby promoting DCs
   maturation to initiate an immune response.

   KEGG and Reactome analyses were also conducted to investigate the
   signaling pathways modulated by TBT-CpG NaVs in BMDCs. KEGG enrichment
   analysis showed that upregulated genes were predominantly associated
   with the regulation of cytokine-cytokine receptor interactions,
   NF-kappa B signaling pathway. Key genes such as CD40, LTa, TNF, CXCL1,
   CXCL2, and CXCL2 were involved in both signaling pathways, underscoring
   their importance in inducing an immune response (Fig. [110]4C).
   Reactome analysis highlighted that these differential genes were
   primarily involved in the regulation of the immune system
   (Fig. [111]4D). Overall, these results suggested that TBT-CpG NaVs
   induce significantly changes in the cellular mRNA transcriptome,
   playing a crucial role in modulating the immune system and immune
   response.

TBT-CpG NaVs induce specific immune responses and immunoprotection

   To evaluate the immune response induced by TBT-CpG NaVs, mice were
   vaccinated with a dose of 30 µg per injection. Control groups included
   PBS, free TBT, and TBT + CpG, and immune responses along with
   protective efficacy were assessed after three immunizations
   (Fig. [112]5A). Results showed that the specific antibody titer induced
   by the nanoparticle vaccine TBT-CpG NaVs was significantly higher than
   that of the other groups on day 35 (Fig. [113]5B) and remained elevated
   on day 56 (Fig. [114]S6). ELISPOT analysis of IFN-γ (Fig. [115]5C) and
   IL-4 (Fig. [116]5D) secretion revealed a significantly stronger
   cellular immune response in the TBT-CpG NaVs group compared to PBS,
   TBT, and TBT + CpG groups.

Fig. 5.

   [117]Fig. 5
   [118]Open in a new tab

   TBT-CpG NaVs induce specific immune responses. A Schematic diagram
   illustrating the immunization procedure for nanovaccine administration
   in BALB/c mice and suckling mice. B Measurement of serum-specific IgG
   antibody titers on day 35 post-initial immunization, using a serial
   dilution method. C-D ELISPOT assay to detect the number of IFN-γ (C)
   and IL-4 (D) spot-forming cells following 48-hour in vitro stimulation
   with peptide TBT, including statistical analysis. Images were obtained
   and quantified using an ELISPOT reader. E-F Survival rates of 3-day-old
   BALB/c suckling mice infected with DENV-2 or DENV-4 (E) and 1-day-old
   newborn mice infected with ZIKV (F) over a 15-day observation period,
   with survival rate expressed as the percentage of survivors. Data are
   shown as mean ± SD (n = 3). Statistical analysis was performed using
   one-way ANOVA. **p < 0.01; ***p < 0.001

   To assess protective efficacy, serum from immunized mice was mixed with
   DENV-2 or DENV-4 and used to infect 3-day-old Balb/c suckling mouse.
   Similarly, serum mixed with ZIKV was used to infect 1-day-old C57BL/6J
   newborn mice, with survival monitored over 15 days. The survival rate
   of the TBT-CpG NaVs-immunized group was significantly higher than that
   of the control group when infected with DENV (Fig. [119]5E) or ZIKV
   (Fig. [120]5F). These findings suggested that, unlike the free TBT
   peptide vaccine, TBT-CpG NaVs offer enhanced protective efficacy
   against DENV and ZIKV infections, demonstrating significant antiviral
   potential.

Safety assessment of TBT-CpG NaVs

   Ensuring biosecurity is essential for clinical applications. To assess
   the safety of the self-assembled nanovaccine, both in vitro and in vivo
   toxicity evaluations were conducted. The CCK-8 assay indicated a slight
   dose-dependent effect, with cell viability remaining above 85% even at
   a TBT concentration of 600 nM, suggesting that TBT-CpG NaVs exhibit no
   significant cytotoxicity to BMDCs and demonstrate good safety and
   biocompatibility (Fig. [121]6A). In immunized mice, the biochemical
   indicators alanine aminotransferase (ALT) and aspartate
   aminotransferase (AST) remained within normal ranges (Fig. [122]6B).
   Furthermore, histological analysis revealed no significant damage or
   inflammatory response in major organs (heart, liver, spleen, lung,
   kidney) in mice treated with the nanovaccine (Fig. [123]6C). These
   results indicated that our self-assembled TBT-CpG NaVs possess a high
   level of safety and potential suitability for clinical research.

Fig. 6.

   [124]Fig. 6
   [125]Open in a new tab

   Safety evaluation of TBT-CpG NaVs. A Cell viability of BMDCs incubated
   with nanoparticles at various concentrations for 24 h. B Biochemical
   analysis of AST and ALT levels on day 7 following the final
   immunization. C Hematoxylin and Eosin (H&E) staining to assess
   histological changes in the main organs of mice on day 7 post-final
   immunization. Data are presented as mean ± SD (n = 3). Statistical
   analysis was performed using one-way ANOVA. ns, p > 0.05

Discussion

   Flaviviruses are becoming more prevalent globally due to uncontrolled
   urbanization and globalization, leading to a rise in cases and
   presenting new challenges for disease prevention and control
   [[126]36–[127]38]. This underscores the urgent need for a safe and
   effective vaccine to curb the spread of flaviviruses. In recent years,
   self-assembled nanoparticle platforms have garnered attention due to
   their biocompatibility, biodegradability, ease of production [[128]39],
   and ability to be effectively recognized by antigen-presenting cells
   (APCs) [[129]15]. These platforms have been widely used for anti-tumor
   and antiviral therapy, positioning them as a promising direction for
   next-generation subunit vaccine development [[130]35, [131]40,
   [132]41]. The use of self-assembled nanoparticle platforms combining
   adjuvants with epitope peptide antigens specifically for flaviviruses
   remains underexplored. In this study, we developed a multi-epitope
   peptide nanovaccine delivery system conjugated with the adjuvant CpG
   ODN.

   Our findings demonstrate that CpG ODN, serving as an adjuvant, can be
   covalently linked to an immunodominant epitope peptide to form a
   positively charged spherical nanovaccine. This spherical nanovaccine
   mimics the structure of flaviviruses, facilitating uptake by BMDCs. The
   electrostatic attraction between the positively charged nanoparticles,
   which have high antigen density, and the negatively charged cell
   surfaces promotes effective binding and internalization of the antigen
   [[133]42, [134]43]. Efficient delivery of peptide epitope antigens is
   essential to boost vaccine immunogenicity. Once internalized, these
   peptide epitopes stimulate the maturation of DCs and enable antigen
   presentation to CD4^+ and CD8^+ T cells via MHC molecules, inducing a
   highly targeted immune response [[135]34, [136]44].

   Extensive research has also shown that nanovaccines can deliver
   antigens to specific tissues, improving the bioavailability of
   antigens, activating the body’s humoral and cellular immunity, and
   inducing a robust immune response [[137]45, [138]46]. Notably, our
   bioinformatics analysis revealed that BMDCs presented epitope peptides
   via the MHC II pathway and identified that TBT-CpG NaVs regulated
   pathways related to BMDC activation and T cell proliferation, which are
   crucial for mounting an effective immune response. The self-assembled
   nanovaccine TBT-CpG NaVs effectively activated the immune system in
   mice, significantly elevated serum IgG levels, and stimulated the
   spleen to secrete IFN-γ and IL-4, thereby enhancing antiviral
   capability.

   Epitope-based subunit vaccines are known for their specificity, safety,
   and reproducibility [[139]47]. Selecting appropriate antigenic epitopes
   is essential for optimizing the immunogenicity of these vaccines, with
   conserved regions within viral protein sequences serving as primary
   targets [[140]15]. B cell epitopes bind to B cell receptors, triggering
   the humoral immune response and producing specific antibodies that help
   resist viral invasion [[141]48]. In flaviviruses, the envelope (E)
   protein is the main target for neutralizing antibodies, providing
   long-term immunity and protection following natural infection or
   vaccination [[142]49]. Thus, selecting B cell epitopes derived from the
   E protein of flaviviruses can enhance humoral immune activation. The B
   cell epitope VDRGWGNGCGLFGKG, conserved across DENV1-4, ZIKV, and other
   flaviviruses, effectively produces neutralizing antibodies and induces
   broad-spectrum immunity [[143]5, [144]50]. Our neutralization test
   results indicated that immune serum neutralized both ZIKV and DENV,
   improving the survival rate of suckling mice and enhancing resistance
   to viral infection. The selected B cell epitopes demonstrated efficacy
   in producing neutralizing antibodies with broad-spectrum immune
   effects.

   It is worth noting that to further enhance the immunogenicity and
   stability of the vaccine, we added T-helper epitopes on both sides of
   the B-cell epitope with flexible peptides glycine G and leucine L. The
   addition of T1 helper epitope (KYVKQNTLKLAT) and T2 helper epitope
   (LEYIPEITLPVIAALSIAES) not only extended the length of the peptide to
   reduce the risk of degradation but also banded with MHC II molecules.
   This configuration facilitated antibody-mediated humoral immunity and
   effectively activated cellular immunity [[145]51].

   An ideal vaccine should induce a robust immune response while
   maintaining safety and non-toxicity [[146]45]. Self-assembled
   nanovaccines, which do not rely on external carriers, reduced potential
   systemic toxicity and simplified preparation, opening up a new field
   for vaccine delivery systems [[147]30]. In our study, both in vitro and
   in vivo safety assessments showed no significant cytotoxic effects or
   inflammatory responses, confirming the favorable safety profile of
   TBT-CpG NaVs—an essential factor for clinical application. This
   nano-delivery system proved to be stable and effectively activated both
   cellular and humoral immune responses to exert antiviral effects. Our
   nanovaccine demonstrated potent immune responses against Dengue and
   Zika viruses; however, further studies are necessary to evaluate its
   efficacy against other flaviviruses.

   In conclusion, this study highlighted the potential of TBT-CpG NaVs in
   inducing balanced humoral and cellular immune responses, offering a
   promising strategy for flavivirus vaccine development. Future research
   should focus on optimizing the vaccine formulation, evaluating its
   efficacy across a broader range of flaviviruses, and conducting
   clinical trials to establish its safety and effectiveness in humans.

Conclusion

   In summary, our study showed that the self-assembled nanovaccine
   TBT-CpG NaVs, possessed excellent biocompatibility and a favorable
   safety profile. TBT-CpG NaVs effectively stimulated the maturation of
   dendritic cells, promoting antigen uptake and presentation, and induced
   robust immune responses with high antibody titers and strong cellular
   immunity. These responses included the production of effective
   neutralizing antibodies, conferring a broad-spectrum antiviral effect
   against flaviviruses. Our results indicate that this self-assembled
   nanovaccine offers significant advantages in immunogenicity and safety,
   holding promising potential for combating flavivirus infections.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [148]Supplementary Material 1^ (1.8MB, docx)

Acknowledgements