Enhancing effect of targeted intestinal delivery of Alhagi honey Polysaccharide-Alum Pickering emulsion adjuvant on the immune response to the BVDV vaccine in cattle

Enhancing effect of targeted intestinal delivery of Alhagi honey Polysaccharide-Alum Pickering emulsion adjuvant on the immune response to the BVDV vaccine in cattle | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhancing effect of targeted intestinal delivery of Alhagi honey Polysaccharide-Alum Pickering emulsion adjuvant on the immune response to the BVDV vaccine in cattle Yan Wu, Jianmin Li, Guangyan He, Peng Huang, En Zhou, Alimu Aersilan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8142640/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Background The mucosa represents the first line of defense against pathogenic invasion, triggering strong mucosal immunity by vaccination for the prevention of infectious diarrheal diseases. Methods In this study, a new assembled Pickering emulsion (AHPPE) was created. This emulsion was constructed by employing an Alum adjuvant (Alum) loaded with Alhagi honey polysaccharide (AHP)-a known enhancer of intestinal mucosal immunity-as the shell, and squalene containing all-trans retinoic acid (RA), an agent targeting intestinal mucosa, as the core, utilizing ultrasonic emulsification techniques. Furthermore, the systemic and mucosal immune responses elicited by AHPPE as an adjuvant for bovine viral diarrhea virus (BVDV) vaccines, along with the associated mechanistic pathways, were investigated. Results The results showed that AHPPE had raspberry-like morphology with a mean particle size of around 2000 nm and a positive surface charge. The emulsion proved to have effective loading capacity for BVDV vaccine antigen and good stability for 30 days. When used as an adjuvant for BVDV vaccine, AHPPE greatly enhanced the titers of BVDV-specific IgG and IgA antibodies and the expression of cytokine interleukin-10 (IL-10), chemokine CCL28, and chemokine receptor CCR9 ( P < 0.05). Transcriptomic analysis revealed that AHPPE was highly phagocytosed by dendritic cells (DCs), which in turn upregulated the expression of Toll-like receptors and major histocompatibility complex class II (MHC II) molecules, thereby activating T and B lymphocytes to trigger strong systemic immune responses. At the same time, induction of chemokines enabled DCs to migrate specifically to the intestinal tract, triggering intestinal mucosal immune activation. The authenticity of the sequencing data was further validated using confocal microscopy and enzyme-linked immunosorbent assay (ELISA). Conclusion To conclude, the current study provides evidence that AHPPE is a viable vaccine adjuvant for BVDV, which not only generates a robust systemic immune response but also efficiently induces an intestine-targeted mucosal immune response through inducing extensive dendritic cell chemotaxis. Bovine viral diarrhea virus (BVDV) Dextran sulfate-Alum Pickering emulsion Vaccine adjuvant Intestinal mucosal immunity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. INTRODUCTION Bovine viral diarrhea virus (BVDV) is the causative agent of bovine viral diarrhea/mucosal disease (BVD/MD), responsible for significant economic losses in the world livestock industry [ 1 ]. BVDV infects the mucosal epithelium and related lymphoid tissues prior to being spread to other organ systems, resulting in abortion in mature cattle as well as acute gastrointestinal and respiratory diseases [ 2 ]. Immunization is the most efficient measure of preventing and controlling infectious diseases [ 3 ]. Nonetheless, the BVDV subunit vaccine is constrained in its poor immunogenicity, short duration of protection, and requirement for multiple doses [ 4 ]; thus, adjuvants must play a critical role in improving delivery efficiency and inducing a strong immune response [ 5 ]. Alum-based and oil-emulsion adjuvants are the most widely used in veterinary settings [ 6 ]. Alum adjuvants are one of the safest, they can stimulate an effective humoral immune response but will not trigger strong cellular or intestinal mucosal immunity. In contrast, oil emulsion adjuvants will often elicit strong humoral and cellular responses with less desirable safety profiles and also will not be able to trigger intestinal mucosal immunity. BVDV infection usually begins on the mucosal surface, and this mainly impacts intestinal mucosal tissues [ 7 ]; hence, an efficient mucosal immune response is important to contain BVDV. IgA antibodies, which are mainly secreted by gut-associated lymphoid tissue (GALT), are critical in eliciting the intestinal mucosal immune response[ 8 ]. They create a vital immune barrier that defends the host from invasion by pathogens [ 9 , 10 ] and guard intestinal homeostasis [ 8 ] by inhibiting attachment of pathogens, suppressing viral shedding, and inhibiting viral replication and spread through localized immunity responses [ 11 , 12 ]. Therefore, IgA-dependent immune response is just as important as systemic humoral and cellular immunity for BVDV infecting the intestinal mucosa. Hence, the creation of adjuvants capable of inducing strong systemic immunity and high intestinal IgA responses is essential for increasing the vaccine efficacy and duration of the BVDV subunit vaccine. Alhagi honey polysaccharide (AHP), is a water-soluble polysaccharide which induces the expression of CD4⁺ and CD8a⁺ T lymphocytes in the mesenteric lymph nodes, enhances intraepithelial lymphocytes in the small intestine, greatly increases the percentage of IgA-positive cells in the intestinal lamina propria, enhances secretory IgA production, and triggers cytokine secretion such as IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, and TNF-α [ 13 ]. AHP, however, does not have inherent intestinal targeting capacity, precluding its utility for evoking efficacious intestinal mucosal immune responses through parenteral immunization. All-trans retinoic acid (RA) is a hydrophobic small molecule that triggers the differentiation of non-adaptive regulatory T cells (nTregs) into an intestinal homing phenotype characterized by α4β7 and CCR9 expression, which allows DC migration to the lamina propria of the small intestine [ 14 ]. RA is also responsible for the preservation of immune homeostasis in the intestinal lamina propria and is an efficient tool for targeted regulation of mucosal immunity, significantly influencing the shaping of mucosal immune responses [ 15 , 16 ]. A Pickering emulsion (PE) with an Alum adjuvant is a capable delivery system that can encapsulate high payloads of both water-soluble and hydrophobic agents simultaneously. This PE possesses various benefits such as increased stability, controlled release, and low toxicity [ 17 – 19 ]. Additionally, the flexible core of the emulsion enhances the contact area between immune cells and antigens, stimulates cellular internalization of the Alum adjuvant, and enhances antibody production while sustaining high antibody titers over a more extended period. It also increases the induction of memory B and T cells, enhances cellular immune responses substantially, and increases the duration of immune protection [ 20 ]. Therefore, the co-loading of RA and AHP onto Alum-stabilized Pickering emulsions as adjuvants for BVDV vaccines is expected to take advantage of RA's intestinal mucosal targeting and AHP's DC-recruiting activity, and thus induce strong intestinal mucosal and systemic immune responses. The objective of this research is to prepare an intestine-targeted Pickering emulsion, known as the Alhagi honey polysaccharide-based Pickering emulsion (AHPPE). The emulsion adjuvant was prepared with Alum as the carrier by ultrasonic assembly, giving a structure in which the shell is composed of AHP-adsorbed Alum and the core is composed of squalene, dissolved RA being dissolved in it. The immunogenicity and safety of AHPPE as an adjuvant for a BVDV vaccine in cattle were assessed by quantifying serum BVDV-specific antibody titers, serum cytokine levels, and fecal concentrations of IgA. Transcriptomics was also conducted to decipher the molecular mechanisms of AHPPE in the intestinal mucosa. 2. MATERIALS AND METHODS 2.1 Preparation of Saccharomyces cerevisiae polysaccharide peeling emulsion (AHPPE) and AHPPE/BVDV AHPPE was prepared via ultrasonic emulsification. Alum (InvivoGen, USA) at a concentration of 6 mg/mL was subjected to ultrasonic treatment in an ice bath at 40% power. Each cycle consisted of 1 minute of sonication followed by a 4-second interval, repeated twice. The treated Alum was then uniformly mixed with AHP (concentration: 1.25 mg/mL) to form the aqueous phase (W) of AHPPE. RA (Shanghai Macklin Biochemical Technology Co., Ltd.) was dissolved in squalene (Shanghai Macklin Biochemical Technology Co., Ltd.) to prepare the oil phase (O) containing RA at 0.625 mg/mL. The aqueous and oil phases were thoroughly mixed at a 11:1 (W:O) volume ratio and then emulsified via ultrasonic treatment in an ice bath at 20% power, with an amplitude setting of 6. The emulsification process consisted of two cycles, each lasting 3 minutes with a 4-second interval between pulses, resulting in the final AHPPE formulation. The BVDV solution was added to the AHPPE solution at a 1:3 ratio (BVDV:AHPPE), and the mixture was shaken for 30 minutes to ensure complete adsorption of BVDV. The final concentration of BVDV was 20 µg/mL. 2.2 Optimization of AHPPE Preparation Conditions through Single-Factor Experiments In this experiment, three factors—Alum concentration, ultrasonic power, and oil-to-water ratio—were evaluated individually to investigate their effects on the BVDV loading efficiency of AHPPE, and the emulsification was performed under varying conditions. The AHPPE/BVDV samples prepared under different experimental conditions were centrifuged at 12,000 rpm for 30 minutes to separate the oil and aqueous phases, and the supernatant was collected. The BVDV loading efficiency in AHPPE/BVDV was determined using a BCA protein assay kit [21]. Not: M 1 represents the mass of BVDV added, M 2 denotes the mass of unbound BVDV, and M 3 refers to the total mass of the AHPPE/BVDV formulation. 2.3 Optimization of AHPPE Preparation Conditions Using Response Surface Methodology Based on the single-factor experiments, the optimal levels of each factor were determined by using BVDV loading efficiency as the response variable. A three-factor, three-level experimental design was conducted (Table 1). Response surface methodology was employed to investigate the effects of Alum concentration, ultrasonic power, and water-to-oil ratio on antigen encapsulation efficiency, thereby identifying the optimal preparation process. ------------------------------------- Insert Table 1 -------------------------------------- 2.4 Characterization of AHPPE AHPPE and AHPPE/BVDV were prepared under the optimized conditions. The morphologies of AHPPE and AHPPE/BVDV were examined using optical microscopy and transmission electron microscopy. The particle size was assessed by cryogenic scanning electron microscopy. Confocal laser scanning microscopy was employed to visualize the morphology of AHPPE. The aqueous phase was labeled with fluorescein isothiocyanate (FITC), and the oil phase was labeled with CY5.5 dye. The average hydrodynamic diameter, zeta potential, and polydispersity index (PDI) of AHPPE were measured using a Malvern particle size analyzer at 4 °C and 37 °C for 0, 3, 5, 14, 30, 56, and 120 days to evaluate its physical stability. 2.5 Experimental Animals and Group Allocation Twenty-four calves were randomly assigned to four groups: the control group (CON), the BVDV group, the Alum/BVDV group, and the AHPPE/BVDV group, with six animals in each group. The animals were maintained under standardized indoor housing conditions at Bachu Tianrun Animal Husbandry Co., Ltd. throughout the experimental period, with ad libitum access to hay and commercially available pelleted feed (Approval Number: 2022016). The CON group received an intramuscular injection of 1 mL of normal saline per calf, the BVDV group received 1 mL of BVDV antigen, the Alum/BVDV group received 1 mL of a mixture of BVDV antigen and a commercially available Alum adjuvant (V BVDV :V Alum =1:3), and the AHPPE/BVDV group received 1 mL of a mixture of BVDV antigen and AHPPE adjuvant (V BVDV : V AHPPE =1:3) during the experiment period. The final concentration of BVDV antigen was 50 μg/mL in all treatment groups. All animals were immunized once. 2.6 Determination of Antibody Levels and Cytokine Concentrations Blood and fecal samples were collected and processed on days 7, 14, 21, and 28 post-immunizations for subsequent antibody detection. According to the manufacturer's instructions, ELISA kits (Shanghai Kexing Trading Co., Ltd.) were used to measure specific IgG levels in serum and IgA levels in fecal samples from calves in each group during the immunization period. Additionally, the concentrations of IL-4, IL-10, IL-17, TNF-α, CCR6, CCR9, CCL20, and CCL28 in the serum were determined on day 14 post-immunization. 2.7 Cytotoxicity Evaluation of AHPPE Bovine blood was diluted 1:1 with PBS, then layered onto lymphocyte separation medium and centrifuged. The intermediate milky-white cell layer was collected and suspended in red blood cell lysis buffer to lyse erythrocytes. After that, it was centrifuged to remove the lysed cells. The resulting pellet, containing a mixture of lymphocytes and DCs, was suspended in complete culture medium supplemented with recombinant bovine GM-CSF and IL-4 proteins. Half-volume medium changes were performed every other day for three straight days, and the supernatant containing non-adherent lymphocytes was discarded. After 7 days of culture, mature bovine peripheral blood-derived dendritic cells were obtained. Cells were adjusted to a density of 2 × 10⁶ cells/mL and seeded into 96-well plates at 200 μL per well. After 24 hours of incubation, AHPPE was diluted in complete culture medium to various concentrations (1000, 500, 250, 125, 62.5, 31.3, and 15.6 μg/mL). Blank and control groups were included in the experimental design. After 24 hours of treatment, the supernatant was removed, and the cells were washed twice with PBS. Cell viability was assessed using the CCK-8 assay [22] to determine the safe concentration range of AHPPE for bovine dendritic cells. 2.8 Evaluation of DCs' Phagocytic Activity toward AHPPE FITC and AHPPE were uniformly mixed to prepare FITC-labeled AHPPE (AHPPE-FITC). The prepared AHPPE-FITC was co-cultured with DCs, and samples were collected at predetermined time points (1, 3, 6, 9, 12, and 24 hours). The intracellular FITC fluorescence intensity in DCs was measured by laser-scanning confocal microscopy to assess DC phagocytic capacity for AHPPE. 2.9 Transcriptomic Sequencing Analysis Cells were processed as described in Section 2.6, and the cell concentration was adjusted to 2 × 10⁶ cells/mL. After 7 days, AHPPE was added, and the cells were incubated for 12 or 36 hours. Subsequently, both the cells and the supernatants were collected. Trizol reagent was added to the cell samples, which were then immediately frozen in liquid nitrogen and sent to Majorbio Bio-Pharm Technology Co., Ltd. for transcriptomic sequencing analysis. 2.10 Distribution of AHPPE in DCs AHPPE-FITC was co-cultured with DCs in confocal dishes at 1, 3, 6, 9, 12, and 24 hours post-incubation. The dishes were then fixed with 4% paraformaldehyde and washed three times with PBS. The fluorescently labeled antibodies were added to stain the nuclei and lysosomes, respectively, according to the manufacturer’s instructions, while unbound dye was carefully removed by washing with PBS. Fluorescence images were acquired randomly across multiple fields of view using a laser scanning confocal microscope. 2.11 Evaluation of Protein Secretion by Dendritic Cells Following AHPPE Stimulation The supernatant from cells collected at 36 hours in Section 2.8 was used to measure the concentrations of IL-4, IL-10, IL-17, TNF-α, CCR6, CCR9, CCL20, and CCL28 using ELISA kits according to the manufacturer’s instructions. 2.12 Statistical Analysis All the experimental data are presented as mean ± standard deviation (X ± SD). P-value of less than 0.05 was considered statistically significant. *Denotes a significant difference compared with the control group at the corresponding time point ( P < 0.05), and ** denotes an extremely highly significant difference compared with the control group ( P < 0.01). Statistical analyses were performed using GraphPad Prism version 8.0. 3. RESULTS 3.1 Single-Factor Experimental Results Under fixed ultrasonic power (40%) and an oil-to-water ratio of 1:9, the effect of Alum concentration on the antigen loading rate of AHPPE showed an initial increase, followed by a decrease (Figure 1A). The antigen loading rate reached its peak at an Alum concentration of 6 mg/mL, which was significantly higher ( P < 0.05) than those observed at 1.5, 3, 4.5, and 7.5 mg/mL. This formulation also exhibited excellent emulsification efficiency, with no phase separation observed over 64 days64 days (Figure 1B). Under fixed conditions of Alum concentration (6 mg/mL) and ultrasonic power (40%), the effect of the oil-to-water phase ratio on the antigen loading rate of AHPPE showed an initial decrease, followed by an increase, and then a subsequent decrease (Figure 1C). The antigen loading rate reached its peak at an oil-to-water ratio of 1:11, which was significantly higher ( P < 0.05) than those observed at ratios of 1:5, 1:7, 1:9, and 1:13. This formulation also exhibited excellent emulsification efficiency, with no phase separation observed over 64 days64 days (Figure 1D). Under fixed conditions of Alum concentration (6 mg/mL) and an oil-to-water ratio of 1:11, the antigen loading rate of AHPPE peaked at an ultrasonic power of 20% (Figure 1E). This value was significantly higher than those observed at 10%, 30%, 40%, and 50% ( P < 0.05). This formulation also demonstrated excellent emulsification efficiency, with no phase separation observed over 64 days (Figure 1F). Based on the efficiency and emulsification stability of AHPPE, the optimal single-factor conditions for its preparation were determined as an Alum concentration of 6 mg/mL, an oil-to-water phase ratio of 1:11, and an ultrasonic power intensity of 20%. ------------------------------------- Insert Figure 1 -------------------------------------- 3.2 Results of the Response Surface Optimization Experiment The data in Table 2 were subjected to quadratic regression analysis using Design-Expert 13, with Alum concentration (A), oil-to-water ratio (B), and ultrasonic instrument power (C) as independent variables. The resulting regression equation is: Y = 88.56 - 1.2A + 0.5141B + 0.6096C + 0.2056AB + 0.8079AC - 0.3231BC - 4.16A² - 6.34B² - 8.21C². ------------------------------------- Insert Table 2 -------------------------------------- The regression model was evaluated, and the results are presented in Table 3. The model yielded P < 0.0001, indicating high statistical significance, with a lack-of-fit P value of 0.0507 (significant), a coefficient of determination ( R 2 ) of 0.9875, an Adjusted Coefficient of Determination ( AdjR 2 ) of 0.9714, close to unity, and a coefficient of variation ( CV% ) of 1.31%. These metrics suggest that the model strong goodness-of-fit and is reliable for predicting the actual preparation process. The order of influence of the independent variables on antigen loading efficiency is as follows: Alum concentration (A) > ultrasonic power (C) > oil-to-water ratio (B). The optimal preparation conditions for AHPPE were determined to be an Alum concentration of 6 mg/mL, an oil-to-water ratio of 1:11, and an ultrasonic power setting of 20%. The nature of interactions between variables was assessed based on the contour patterns in the response surface plots and the trends observed in the corresponding three-dimensional graphs (Figure S1 in the supplementary material). The results show that, as Alum concentration, ultrasonic power, and oil-to-water ratio increase, the initial increase is followed by a decrease. To validate the response surface model, the software-optimized parameters were refined to the following conditions: An Alum concentration of 6 mg/mL, an oil-to-water ratio of 1:11, and an ultrasonic power setting of 20%. Under these conditions, the predicted antigen loading efficiency was 88.555%. The experimentally obtained antigen loading efficiency was 89.657 ± 1.11%, which is in close agreement with the predicted value and shows a slight deviation. This consistency indicates that the optimized conditions are feasible and reliable. ------------------------------------- Insert Table 3 -------------------------------------- 3.3 Characterization and Analytical Results of AHPPE and AHPPE/BVDV Figure 2A and 2B display the appearance of AHPPE and AHPPE/BVDV at room temperature. Both exhibit a uniform milky white color. Under optical microscopy, as shown in Figures 2C and 2D, both AHPPE and AHPPE/BVDV display spherical morphology, uniform particle size, and no noticeable sedimentation. AHPPE exhibits a raspberry-like morphology, with Alum uniformly adsorbed on the surface of squalene, forming a surface-rough particle with an approximate size of 2000 nm (Figure 2E). As illustrated in Figure 2 F, Dextran-FITC (emitting green fluorescence) is conjugated to Alum, while Cy5.5 (emitting purple fluorescence) is associated with the oil phase. The particle size remains around 2000 nm. The localization of Alum at the oil–water interface confirms that AHPPE is a Pickering emulsion with the oil phase as the core and the aqueous phase as the outer shell. AHPPE remained stable at both 4 °C and 37 °C during a 120-day storage period (Figure 2G). No noticeable changes in appearance were observed, and no precipitation or phase stratification was seen at days 0, 3, 5, 14, 30, 56, and 120. These results indicate that the nanoparticles exhibit excellent stability. As shown in Figure 2 H–I, the particle size distribution of AHPPE and AHPPE/BVDV remained relatively uniform over 56 days at both 4 °C and 37 °C. The zeta potential of AHPPE was positive under both storage conditions (Figure 2J). The average PDI of AHPPE remained below 0.3 over 30 days (Figure 2J). These results indicate that AHPPE maintains good colloidal stability for at least 30 days at 4 °C and 37 °C. The results suggest that AHPPE maintains stability for at least 30 days at both 4 °C and 37 °C. ------------------------------------- Insert Figure 2 -------------------------------------- 3.4 Determination of Serum IgG-Cytokines and Fecal IgA The laboratory personnel administered immunizations to each group of calves at the cattle farm (Figure 3A). As illustrated in Figure 3B, IgG levels in all vaccinated groups peaked on day 28 after vaccination. On days 7, 14, 21, and 28 after immunization, serum BVDV-specific IgG levels in the AHPPE/BVDV, Alum/BVDV, and BVDV groups were significantly higher than those in the CON group ( P < 0.01). Furthermore, on day 28 after immunization, the BVDV-IgG level in the AHPPE/BVDV group was significantly higher than that in the Alum/BVDV group ( P < 0.01). ------------------------------------- Insert Figure 3 -------------------------------------- 3.5 Analysis of Serum Cytokine Levels The AHPPE/BVDV group exhibited significantly higher ( P < 0.05) expression levels of IL-10, IL-17, CCR6, CCR9, CCL20, and CCL28 compared with the CON group (Figure 4 A–H) after 28 days of immunization. When compared with the BVDV group, the AHPPE/BVDV group showed significantly increased expression levels of IL-10, IL-17, CCR6, CCR9, and CCL28 ( P < 0.05). Furthermore, the AHPPE/BVDV group showed significantly higher expression levels than the Alum/BVDV group. The AHPPE/BVDV group showed significantly elevated expression of IL-10, CCR9, and CCL28 ( P < 0.05). ------------------------------------- Insert Figure 4 -------------------------------------- 3.6 Cell Safety and the Phagocytic Capacity of DCs towards AHPPE The experimental results are presented in Figure 5A. When the concentration of AHPPE ranged from 15.6 to 1000 µg/mL, cell viability was non-significantly reduced compared with the cell control group and remained above 100% in all cases. These findings indicate that AHPPE exhibits no cytotoxicity to DCs at concentrations up to 1000 µg/mL and demonstrates favorable biocompatibility. As shown in Figure 5 B, after co-incubating DCs with AHPPE-FITC, green fluorescence was observed around the nuclei of DCs, indicating that AHPPE was taken up by antigen-presenting cells. When AHPPE-FITC was co-incubated with DCs for 1 hour, AHPPE was mainly located outside the cells. At 3 hours, most of the AHPPE had entered the cells. By 12 h and 24 h, all of the AHPPE-FITC had entered the DCs. ------------------------------------- Insert Figure 5 -------------------------------------- 3.7 Transcriptomic Profiling Results Across Time Points The results show that at 12 h and 36 h post-treatment, compared with the Control group, the AHPPE group up-regulated 3,843 and 1,716 genes, respectively, and down-regulated 3,372 and 2,355 genes, respectively (Figures 6A and 6C). To investigate the immunomodulatory pathways associated with AHPPE, a heat map was generated to visualize the changes in gene expression. Compared with the Control group, the AHPPE group showed upregulation of TLR2, TLR4, CCL20, CCL25, CCL28, CCR3, CCR10, CXCR6, CD28, and ICOS at 12 h (Figure 6B). As shown in Figure 6D, compared with the Control group, the expression of ICOS, CCL20, CCL25, CCL28, CCR3, and CD28 genes was also significantly increased at 36 h in the AHPPE group. KEGG pathway analysis results (Figure 6E) indicate that at 12 h post-treatment, the AHPPE group showed significant enrichment in immune-related pathways, including endocytosis. As shown, the AHPPE group showed pronounced activation of the B cell receptor signaling pathway, the Toll-elevated receptor signaling pathway, and the IL-17 signaling pathway at 12 h and 36 h (Figure 6E and 6F). Whereas GO analysis results presented in Figure 6G and 6H, at 12 h and 36 h post-treatment, showed that the AHPPE group exhibited significant enrichment in biological processes, including the chemokine signaling pathway, positive regulation of T cell activation, lysosomal activity, and the MHC class II antigen presentation pathway. The research suggests that within 12 hours, AHPPE is largely engulfed by DCs, which subsequently activate DCs to highly express Toll-like receptors and MHC II receptors, stimulating T cells and B cells to generate effective systemic immune responses. Meanwhile, DCs are targeted and chemotaxed by elevated chemokine expression in the intestinal tract, thereby inducing intestinal mucosal immune responses. ------------------------------------- Insert Figure 6 -------------------------------------- 3.8 Distribution Results of AHPPE in DCs As shown in Figures 7A and 7 B, the Pearson correlation coefficients between AHPPE (green fluorescence) and lysosomes (red fluorescence) following co-culture with DCs for 1 h, 9 h, and 12 h were 0.64 ± 0.02, 0.53 ± 0.01, and 0.42 ± 0.01, respectively, indicating predominant lysosomal uptake of the carriers during the early stages. Upon extending the incubation time to 24 h, the localization coefficient decreased significantly to 0.23 ± 0.01 ( P < 0.05), suggesting substantial lysosomal degradation of the carriers and the potential release of a fraction into the cytoplasm, which is indicative of successful endolysosomal escape. ------------------------------------- Insert Figure 7 -------------------------------------- 3.9 Cytokine and Chemokine Assay Immune-related cytokines play a critical role in activating various immune cells. As shown in Figure 8 A–D, the AHPPE/BVDV group exhibited significantly higher levels of IL-4, IL-10, and IL-17 compared with both the CON and Alum groups ( P < 0.05). Furthermore, Figures 8 E–H depicted that the AHPPE/BVDV group showed a markedly increased expression of CCR6, CCR9, CCL20, and CCL28 relative to the CON group ( P < 0.05), and significantly elevated expression of CCR9 and CCL28 compared with the Alum group ( P < 0.05). ------------------------------------- Insert Figure 8 -------------------------------------- 4. DISCUSSION Bovine viral diarrhea virus is an acute infectious agent that invades the intestinal mucosa, leading to severe diarrhea, infertility, abortion, congenital malformations, and persistent infection in cattle [ 23 ]. Traditional adjuvants, such as Alum and oil emulsions, have demonstrated limited efficacy in inducing robust intestinal mucosal immune responses [ 5 , 6 ]. In the present study, a targeted intestinal Pickering emulsion adjuvant was developed, termed AHPPE, through the precise alignment of functional components and optimization of the carrier structure. This novel approach overcomes the shortcomings in mucosal immunity elicited by the currently available BVDV vaccines. The preparation parameters of AHPPE were optimized using response surface methodology, followed by a complete examination of its physicochemical properties, antigen loading capacity, and stability. PDI, which reflects the molecular weight distribution of the polymer [ 24 ], was an important indicator of nanoparticle dispersion in the emulsion; the lower the value, the better the dispersion and higher stability [ 25 ]. Indeed, according to Fig. 2 J, the PDI of AHPPE remained less than 0.3 during the 30-day course, which pointed toward an excellent dispersion state of the particles within the emulsion. From scanning electron microscopy and particle size distribution analyses (Figs. 2 F and 2 H), it can be observed that AHPPE particles had an average diameter of approximately 2000 nm with a distinctive raspberry-like morphology and high antigen-loading capacity. The raspberry-like topography in the surface provides numerous binding sites for the attachment of antigens, a feature that greatly increases the antigen-loading efficiency of the emulsion system. Immunological evaluation focused on quantifying antigen-specific IgG and intestinal IgA antibodies. IgG is the predominant immunoglobulin in serum [ 26 ], which is a direct marker of adjuvant-enhanced humoral immune responses. As shown in Fig. 3 B, at day 28 post-immunization, the IgG antibody titers in the AHPPE/BVDV group were significantly higher than those in the CON and Alum/BVDV groups. IgA is the most abundant immunoglobulin on the intestinal mucosal surface-over 75% of total immunoglobulins-on the surface. It is produced via differentiation and proliferation of IgA-secreting plasma cells [ 27 ] and has an important role in maintaining intestinal mucosal immunity [ 28 ]. Figure 3 C shows that the IgA antibody levels in the AHPPE/BVDV group were significantly higher compared to the CON and Alum/BVDV groups. Collectively, these results further confirm that AHPPE effectively induces a strong systemic humoral immune response against BVDV, remarkably enhancing IgA antibody production, promoting mucosal immune activation, and contributing to preservation of intestinal mucosal immunity. IL-10 is a pleiotropic cytokine that acts not only as a pivotal mediator in facilitating Th2-type cellular immune responses, but also plays a direct role in antigen-specific IgA responses at mucosal effector sites. It has been identified as a major inducer of IgA production in the colonic mucosa [ 29 ]. IL-10 drives the differentiation of antigen-specific surface IgA + B cells into IgA-secreting plasma cells and modulates initiation and maintenance of mucosal immune responses [ 29 ]. Likewise, IL-17, another pleiotropic cytokine secreted by Th17 cells and innate immune cells, controls IgA secretion in mucosal tissues and promotes the activation and persistence of mucosal immune responses [ 30 ]. Figure 4 A–D shows that compared to the CON, the AHPPE/BVDV group had significantly enhanced secretion of IL-10 and IL-17. Therefore, these findings indicate that AHPPE triggers a strong BVDV-specific Th2-type cellular immune response, thereby facilitating humoral immunity and enhancing intestinal mucosal IgA responses. Chemokines and their respective receptors are crucial for the directional migration of immune cells [ 31 ]. Among these, CCR9 is an intestine-specific chemokine receptor primarily expressed on lymphocytes and dendritic cells. Following interaction with its ligand, CCR9 promotes T cell trafficking into the intestinal mucosa to facilitate the activation of mucosal immune responses in the gut [ 32 , 33 ]. CCL28, also named mucosal-associated epithelial chemokine (MEC), is primarily produced by intestinal epithelial cells (IECs) and acts as a fundamental regulator of immune cell trafficking into the intestinal mucosa through its receptors CCR10 (the primary receptor) and CCR3 [ 34 ]. This chemokine ensures the trafficking of IgA antibody-secreting cells (IgA-ASCs) into the intestinal mucosa, representing a source of antibody-secreting cells that is necessary for mucosal humoral immunity. Moreover, CCL28 contributes to the accumulation of T cells in the intestinal mucosa and therefore to cellular immune responses in this site. Collectively, these mechanisms form an essential regulatory system for intestinal mucosal immune defense and mucosal immune homeostasis [ 35 ]. As shown in Figs. 4 F and 4 H, CCR9 and CCL28 expressions are much more significantly augmented in the AHPPE/BVDV group than in the Alum/BVDV group. These results show that AHPPE is efficient to support the activation of mucosal IgA response by promoting the intestinal homing of immune cells. In fact, the phagocytic ability of DCs against vaccine adjuvants is associated with their immunostimulatory effectiveness. Therefore, enhanced phagocytosis of vaccine adjuvants by DCs would lead to more effective antigen loading and uptake, thus promoting a robust immune response. In Fig. 5 B, it can be observed that AHPPE is being internalized by DCs, with its complete uptake occurring within 24 hours. Correspondingly, comparing Figs. 6 E and 6 F shows that the endocytic pathway is significantly upregulated in the AHPPE-treated group at 12 hours but not at 36 hours. All these observations support that AHPPE is effectively phagocytosed by DCs within 12 hours, thereby initiating immune activation. Toll-like receptors (TLRs) play a crucial role in recognizing pathogen-associated molecular patterns and triggering an immune response against BVDV. Indeed, TLRs, through their activation, initiate signaling cascades that result in the production of inflammatory cytokines and chemokines [ 36 ]. These processes subsequently activate not just the immediate host defense mechanisms but also facilitate antigen-specific adaptive immunity [ 37 ]. Moreover, the regulation of chemokine and chemokine receptor expression is deeply associated with the maintenance of immune homeostasis [ 38 ]. For instance, CCL20 belongs to the CC motif chemokine ligand that binds exclusively to the chemokine receptor CCR6. It selectively recruits effector or memory CD4 + T cells and B cells [ 39 ]. This interaction, therefore, provides a critical cellular basis for the initiation, maintenance, and functional execution of intestinal mucosal immune responses [ 40 ]. Among key immunoregulatory molecules, the inducible co-stimulatory molecule (ICOS) is a type I transmembrane receptor homodimer that is structurally and functionally homologous to CD28 and expressed on DCs [ 41 ] while exhibiting immune activation capabilities analogous to CD28. ICOS enhances basic T cell responses to exogenous antigens, including proliferation, lymphokine secretion, and upregulation of intercellular adhesion molecules, besides providing support to B-cell-mediated production of antigen-specific antibodies [ 42 ]. Furthermore, it plays an essential role in inducing Th2-type immune responses [ 43 ] as it potently stimulates Th2 cells to produce the cytokine IL-10 [ 42 ]. This cytokine environment favors the differentiation of IgA-producing plasma cells along The results also showed that the expressions of TLR2, TLR4, ICOS, and CD28 genes are significantly increased in the AHPPE group as compared to the control group, as evidenced by gene expression heatmap analyses shown in Figs. 6 B and 6 D. Figures 6 E-H also present that the activation of several signaling pathways is enhanced in the AHPPE group at 12 and 36 hours' post-treatment, including Toll-like receptor signaling, T cell receptor signaling, B cell receptor signaling, IL-17 signaling, lysosomal enrichment, chemokine signaling, and MHC class II antigen presentation. It has already been established that exogenous antigens internalized by DCs traffic into lysosomes, where antigen processing occurs. These molecules, upon lysosomal digestion, will be degraded into small peptide fragments and then presented by MHC class II molecules, thus activating Th2 cells and eliciting humoral immune responses. These results, combined with heatmap analysis of gene expression, suggest that AHPPE elicits a Th2-type immune response mainly through DCs. Additionally, this preparation influences chemokine expression and pathways, thus orchestrating the migratory behavior of immune cells and contributing to chemotaxis in the intestinal mucosal immune response. To further validate the sequencing findings, the intracellular localization of AHPPE within lysosomes of cells and the protein secretion following AHPPE stimulation of DCs were assessed using techniques such as laser confocal microscopy and ELISA. Exogenous antigens are internalized by DCs and trafficked to lysosomes, where they are degraded into small peptide fragments. These peptides are subsequently presented by major histocompatibility complex class II (MHC II) molecules, ultimately activating T helper 2 (Th2) cells and initiating humoral immune responses. This mechanism aligns with the observed significant upregulation of MHC class II expression on DCs at the transcriptional level (Fig. 6 H), thus providing corroborative molecular and cellular evidence for AHPPE-mediated activation of downstream immune pathways. The data presented in Figs. 8 A-D reveal that, relative to the Alum and CON, AHPPE more effectively induces DCs to secrete interleukins IL-4, IL-10, and IL-17. These findings suggest that AHPPE is not only efficiently internalized and presented by DCs but also substantially promotes their maturation and activation. AHPPE lays a robust foundation for subsequent immune responses by stimulating the secretion of additional immunomodulatory cytokines. At the cellular level, the immunostimulatory capacity of AHPPE suggests a greater potential to elicit cellular immune responses. Moreover, the results demonstrate that, compared with the Alum group, AHPPE significantly enhances the expression of CCR9 and CCL28, which further substantiates its role in promoting the intestinal homing of immune cells and in effectively activating mucosal immune responses. This study showed that DCs internalized AHPPE effectively. The regulation of genes like TLR2/4, ICOS, and CD28, together with their signal pathways, promotes the activation of Th2-type immune responses via the lysosome-MHC class II antigen presentation pathway. It enhances the secretion of IL-10 and other immunomodulatory molecules, enhancing BVDV-specific IgG and intestinal IgA production. Moreover, AHPPE enhances key factors, such as CCR9 and CCL28, which increase the homing of immune cells to intestinal mucosa and guide the recruitment of immune cells to this site. Therefore, this mechanism effectively stimulates humoral, cellular, and mucosal immunity. These data provide substantial promise to overcome the major shortcomings of the currently available BVDV subunit vaccines, namely the transient immune responses and poor mucosal protection, and constitute strong support for AHPPE's use as a novel adjuvant in bovine BVDV vaccine formulations. 5. CONCLUSION In this work, an AHPPE emulsion exhibiting excellent intestinal targeting efficacy was successfully developed. Emulsion surface testing showed that the AHPPE emulsion has a strawberry-like surface, with strong intestinal targeting and sustained antigen release. DCs efficiently phagocytose the AHPPE emulsion and activate critical immune signaling pathways, including Toll-like receptor, chemokine, and MHC II pathways, which induce robust and sustained antigen-specific IgA and IgG responses. Therefore, all findings suggest that the AHPPE/BVDV formulation is a novel and effective adjuvant for bovine vaccines, inducing targeted immune protection against BVDV infection and related diseases and therefore representing a valuable strategy for the prevention and control of BVDV. Abbreviations AHP: Alhagi· honey polysaccharides; AHPPE: Alhagi Honey polysaccharide Pickering emulsion; RA: retinoic acid; Alum: Alum adjuvant; BVDV: Bovine viral diarrhea virus; BVD/MD: bovine viral diarrhea/mucosal disease; GALT: gut-associated lymphoid tissue; nTregs: non-adaptive regulatory T cells; PE: Pickering emulsion; FITC: fluorescein isothiocyanate; PDI: polydispersity index; DCs: dendritic cells; CON: control group; IL-10: interleukin-10; TCR: T-cell receptor; MEC: mucosal-associated epithelial chemokine; IgA-ASCs: IgA antibody-secreting cells; TLRs: Toll-like receptors; ICOS: inducible co-stimulatory molecule; ELISA: enzyme-linked immunosorbent assay MHC II: major histocompatibility complex class II. Declarations Ethics approval and consent to participate: The experimental animals were supplied by Bachu Tianrun Livestock Co., Ltd., which authorized their use for the study. All experimental procedures were reviewed and approved by the Animal Welfare and Ethics Committee of Xinjiang Agricultural University (Approval Number: 2022016). The research was conducted in full compliance with applicable local laws, regulations, and institutional guidelines. Consent for publication: Not applicable. Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests: The authors declare no conflict of interest. Funding: Xinjiang Uygur Autonomous Region Major Science and Technology Special Project (2023A02007-2). National Natural Science Foundation of China Youth Fund Project (32202855), Key Laboratory of New Drugs for Herbivores of Xinjiang Province (XJCDVMHDRC-T202301), Autonomous Region-level Entrepreneurship Training Project (S202510758007X). 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Tables Table 1: Factors and levels of the response surface experiment Level A： Concentration of Alum B： Oil-water phase ratio C： Power consumption -1 4.5 mg/mL 1:9 10 % 0 6 mg/mL 1:11 20 % 1 7.5 mg/mL 1:13 30 % Table 2: Arrangement and results of response surface experiment Test No. Factor Y: Rate of encapsulation A B C Measured value Predictive value 1 0 1 -1 74.5 74.23 2 0 0 0 88.37 88.56 3 0 -1 1 74.15 74.42 4 1 1 0 77.61 77.58 5 0 0 0 88.23 88.56 6 0 1 1 75.68 74.8 7 0 -1 -1 71.68 72.55 8 0 0 0 87.94 88.56 9 -1 -1 0 78.91 78.94 10 -1 1 0 78.38 79.56 11 -1 0 -1 78.5 77.59 12 1 0 -1 73.27 73.58 13 0 0 0 89.37 88.56 14 -1 0 1 77.5 77.19 15 1 0 1 75.5 76.41 16 0 0 0 88.87 88.56 17 -1 -1 0 77.32 76.14 Table 3: The results of the variance analysis for the quadratic regression model incorporating factors A, B, and C Source Sum of Squares df Mean Square F-Value p-value Prob > F significance Model 601.78 9 66.86 61.48 < 0.0001 ** A 11.46 1 11.46 10.54 0.0141 B 2.11 1 2.11 1.94 0.2059 C 2.97 1 2.97 2.73 0.1423 AB 0.1692 1 0.1692 0.1555 0.7050 AC 2.61 1 2.61 2.40 0.1652 BC 0.4177 1 0.4177 0.3841 0.5551 A 2 72.71 1 72.71 66.86 < 0.0001 ** B 2 169.47 1 169.47 155.82 < 0.0001 ** C 2 283.78 1 283.78 260.93 < 0.0001 ** Residual Error 7.61 7 1.09 Lack of fit 6.32 3 2.11 6.54 0.0507 Pare error 1.29 4 0.3224 Cor Toal 609.39 16 Note: **represented an exceptionally significant influence ( P <0.01) Additional Declarations No competing interests reported. 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Data are presented as mean ± standard deviation (n = 3).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8142640/v1/61a5bebf73c48d13f14d9aa7.jpg"},{"id":98321578,"identity":"78e5a24e-c014-44cd-bf95-70f5f54a4f22","added_by":"auto","created_at":"2025-12-16 14:06:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1851529,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe results of on-site immunization maps, serum IgG and fecal IgA tests.\u003c/strong\u003e (A) On-site picture of immunized calves, (B) Serum IgG level graph, (C) Fecal IgA level graph. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 and ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8142640/v1/b2698cad68cb74cf45debdc0.jpg"},{"id":98437988,"identity":"4364354e-7a86-41df-94a1-6a01ca9080e6","added_by":"auto","created_at":"2025-12-17 16:58:22","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":967783,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe detection results of various cytokines in the serum. \u003c/strong\u003e(A) IL-4 level graph, (B) IL-10 level graph, (C) IL-17 level graph, (D) TNF-α level graph, (E) CCR6 level graph, (F) CCR9 level graph, (G) CCL20 level graph, (H) CCL28 level graph. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 and **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8142640/v1/6d397494c71215ae11346ccf.jpg"},{"id":98437686,"identity":"336fb672-7193-4671-86cc-739b0e3857fd","added_by":"auto","created_at":"2025-12-17 16:57:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of cell safety and the phagocytic ability of AHPPE towards dendritic cells.\u003c/strong\u003e (A) Results of cell safety concentration, expressed as X ± S (n = 5), (B) Cell uptake diagram. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-8142640/v1/9c52a88710316d91a8d7beb7.png"},{"id":98435772,"identity":"9866d104-4745-4dfb-b831-4f74bb86218b","added_by":"auto","created_at":"2025-12-17 16:54:24","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2582329,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome detection results at different time periods. \u003c/strong\u003eTranscriptomic analysis of AHPPE VS CON (12 h and 36 h). (A, C) volcano plots, (B, D) heat maps, (E, F) KEGG plots, and (G, H) GO plots\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8142640/v1/6144e501c502d0f6240ae05a.jpg"},{"id":98436117,"identity":"80ba7f77-61ff-4054-8403-1e85d0bec9d8","added_by":"auto","created_at":"2025-12-17 16:54:57","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":946050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe distribution of AHPPE in the DCs. \u003c/strong\u003e(A) Confocal laser scanning microscopy images illustrating lysosomal escape; (B) plots of Pearson correlation coefficients. Data are presented as mean ± standard deviation (n = 3). ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8142640/v1/d1f77c2227e533a385c25c32.jpg"},{"id":98435681,"identity":"499bb7d0-59bf-4934-8965-5311e6d85ed1","added_by":"auto","created_at":"2025-12-17 16:54:12","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":742975,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe determination results of various cytokines and chemokines. \u003c/strong\u003e(A) IL-4 level graph, (B) IL-10 level graph, (C) IL-17 level graph, (D) TNF-α level graph, (E) CCR6 level graph, (F) CCR9 level graph, (G) CCL20 level graph, (H) CCL28 level graph. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 and ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8142640/v1/89e7f2c6ad182be90ca489ba.jpg"},{"id":98445692,"identity":"be45ce0a-4b8b-4cf2-9052-4d5d0bb62e64","added_by":"auto","created_at":"2025-12-17 17:20:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12954468,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8142640/v1/b952d216-0fb6-43b0-b40f-3ba81670113a.pdf"},{"id":98438014,"identity":"690f66ec-d952-43e8-8856-15dd49820987","added_by":"auto","created_at":"2025-12-17 16:58:25","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1556630,"visible":true,"origin":"","legend":"","description":"","filename":"attachmentFigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8142640/v1/c2cca3bb9c8ab62d9df38597.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancing effect of targeted intestinal delivery of Alhagi honey Polysaccharide-Alum Pickering emulsion adjuvant on the immune response to the BVDV vaccine in cattle","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eBovine viral diarrhea virus (BVDV) is the causative agent of bovine viral diarrhea/mucosal disease (BVD/MD), responsible for significant economic losses in the world livestock industry [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. BVDV infects the mucosal epithelium and related lymphoid tissues prior to being spread to other organ systems, resulting in abortion in mature cattle as well as acute gastrointestinal and respiratory diseases [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Immunization is the most efficient measure of preventing and controlling infectious diseases [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Nonetheless, the BVDV subunit vaccine is constrained in its poor immunogenicity, short duration of protection, and requirement for multiple doses [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]; thus, adjuvants must play a critical role in improving delivery efficiency and inducing a strong immune response [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Alum-based and oil-emulsion adjuvants are the most widely used in veterinary settings [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Alum adjuvants are one of the safest, they can stimulate an effective humoral immune response but will not trigger strong cellular or intestinal mucosal immunity. In contrast, oil emulsion adjuvants will often elicit strong humoral and cellular responses with less desirable safety profiles and also will not be able to trigger intestinal mucosal immunity. BVDV infection usually begins on the mucosal surface, and this mainly impacts intestinal mucosal tissues [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]; hence, an efficient mucosal immune response is important to contain BVDV. IgA antibodies, which are mainly secreted by gut-associated lymphoid tissue (GALT), are critical in eliciting the intestinal mucosal immune response[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. They create a vital immune barrier that defends the host from invasion by pathogens [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and guard intestinal homeostasis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] by inhibiting attachment of pathogens, suppressing viral shedding, and inhibiting viral replication and spread through localized immunity responses [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, IgA-dependent immune response is just as important as systemic humoral and cellular immunity for BVDV infecting the intestinal mucosa. Hence, the creation of adjuvants capable of inducing strong systemic immunity and high intestinal IgA responses is essential for increasing the vaccine efficacy and duration of the BVDV subunit vaccine. Alhagi honey polysaccharide (AHP), is a water-soluble polysaccharide which induces the expression of CD4⁺ and CD8a⁺ T lymphocytes in the mesenteric lymph nodes, enhances intraepithelial lymphocytes in the small intestine, greatly increases the percentage of IgA-positive cells in the intestinal lamina propria, enhances secretory IgA production, and triggers cytokine secretion such as IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, and TNF-α [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. AHP, however, does not have inherent intestinal targeting capacity, precluding its utility for evoking efficacious intestinal mucosal immune responses through parenteral immunization.\u003c/p\u003e\u003cp\u003eAll-trans retinoic acid (RA) is a hydrophobic small molecule that triggers the differentiation of non-adaptive regulatory T cells (nTregs) into an intestinal homing phenotype characterized by α4β7 and CCR9 expression, which allows DC migration to the lamina propria of the small intestine [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. RA is also responsible for the preservation of immune homeostasis in the intestinal lamina propria and is an efficient tool for targeted regulation of mucosal immunity, significantly influencing the shaping of mucosal immune responses [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A Pickering emulsion (PE) with an Alum adjuvant is a capable delivery system that can encapsulate high payloads of both water-soluble and hydrophobic agents simultaneously. This PE possesses various benefits such as increased stability, controlled release, and low toxicity [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Additionally, the flexible core of the emulsion enhances the contact area between immune cells and antigens, stimulates cellular internalization of the Alum adjuvant, and enhances antibody production while sustaining high antibody titers over a more extended period. It also increases the induction of memory B and T cells, enhances cellular immune responses substantially, and increases the duration of immune protection [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, the co-loading of RA and AHP onto Alum-stabilized Pickering emulsions as adjuvants for BVDV vaccines is expected to take advantage of RA's intestinal mucosal targeting and AHP's DC-recruiting activity, and thus induce strong intestinal mucosal and systemic immune responses. The objective of this research is to prepare an intestine-targeted Pickering emulsion, known as the Alhagi honey polysaccharide-based Pickering emulsion (AHPPE). The emulsion adjuvant was prepared with Alum as the carrier by ultrasonic assembly, giving a structure in which the shell is composed of AHP-adsorbed Alum and the core is composed of squalene, dissolved RA being dissolved in it.\u003c/p\u003e\u003cp\u003eThe immunogenicity and safety of AHPPE as an adjuvant for a BVDV vaccine in cattle were assessed by quantifying serum BVDV-specific antibody titers, serum cytokine levels, and fecal concentrations of IgA. Transcriptomics was also conducted to decipher the molecular mechanisms of AHPPE in the intestinal mucosa.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e2.1 Preparation of Saccharomyces cerevisiae polysaccharide peeling emulsion (AHPPE) and AHPPE/BVDV\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAHPPE was prepared via ultrasonic emulsification. Alum (InvivoGen, USA) at a concentration of 6 mg/mL was subjected to ultrasonic treatment in an ice bath at 40% power. Each cycle consisted of 1 minute of sonication followed by a 4-second interval, repeated twice. The treated Alum was then uniformly mixed with AHP (concentration: 1.25 mg/mL) to form the aqueous phase (W) of AHPPE. RA (Shanghai Macklin Biochemical Technology Co., Ltd.) was dissolved in squalene (Shanghai Macklin Biochemical Technology Co., Ltd.) to prepare the oil phase (O) containing RA at 0.625 mg/mL. The aqueous and oil phases were thoroughly mixed at a 11:1 (W:O) volume ratio and then emulsified via ultrasonic treatment in an ice bath at 20% power, with an amplitude setting of 6. The emulsification process consisted of two cycles, each lasting 3 minutes with a 4-second interval between pulses, resulting in the final AHPPE formulation. The BVDV solution was added to the AHPPE solution at a 1:3 ratio (BVDV:AHPPE), and the mixture was shaken for 30 minutes to ensure complete adsorption of BVDV. The final concentration of BVDV was 20 \u0026micro;g/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Optimization of AHPPE Preparation Conditions through Single-Factor Experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this experiment, three factors\u0026mdash;Alum concentration, ultrasonic power, and oil-to-water ratio\u0026mdash;were evaluated individually to investigate their effects on the BVDV loading efficiency of AHPPE, and the emulsification was performed under varying conditions. The AHPPE/BVDV samples prepared under different experimental conditions were centrifuged at 12,000 rpm for 30 minutes to separate the oil and aqueous phases, and the supernatant was collected. The BVDV loading efficiency in AHPPE/BVDV was determined using a BCA protein assay kit [21].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cimg 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width=\"241\" height=\"55\"\u003e\u003c/p\u003e\n\u003cp\u003eNot: M\u003csub\u003e1\u003c/sub\u003e represents the mass of BVDV added, M\u003csub\u003e2\u003c/sub\u003e denotes the mass of unbound BVDV, and M\u003csub\u003e3\u003c/sub\u003e refers to the total mass of the AHPPE/BVDV formulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Optimization of AHPPE Preparation Conditions Using Response Surface Methodology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the single-factor experiments, the optimal levels of each factor were determined by using BVDV loading efficiency as the response variable. A three-factor, three-level experimental design was conducted (Table 1). Response surface methodology was employed to investigate the effects of Alum concentration, ultrasonic power, and water-to-oil ratio on antigen encapsulation efficiency, thereby identifying the optimal preparation process.\u003c/p\u003e\n\u003cp\u003e-------------------------------------\u003c/p\u003e\n\u003cp\u003eInsert Table 1\u003c/p\u003e\n\u003cp\u003e--------------------------------------\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Characterization of AHPPE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAHPPE and AHPPE/BVDV were prepared under the optimized conditions. The morphologies of AHPPE and AHPPE/BVDV were examined using optical microscopy and transmission electron microscopy. The particle size was assessed by cryogenic scanning electron microscopy. Confocal laser scanning microscopy was employed to visualize the morphology of AHPPE. The aqueous phase was labeled with fluorescein isothiocyanate (FITC), and the oil phase was labeled with CY5.5 dye. The average hydrodynamic diameter, zeta potential, and polydispersity index (PDI) of AHPPE were measured using a Malvern particle size analyzer at 4 \u0026deg;C and 37 \u0026deg;C for 0, 3, 5, 14, 30, 56, and 120 days to evaluate its physical stability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Experimental Animals and Group Allocation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty-four calves were randomly assigned to four groups: the control group (CON), the BVDV group, the Alum/BVDV group, and the AHPPE/BVDV group, with six animals in each group. The animals were maintained under standardized indoor housing conditions at Bachu Tianrun Animal Husbandry Co., Ltd. throughout the experimental period, with ad libitum access to hay and commercially available pelleted feed (Approval Number: 2022016). The CON group received an intramuscular injection of 1 mL of normal saline per calf, the BVDV group received 1 mL of BVDV antigen, the Alum/BVDV group received 1 mL of a mixture of BVDV antigen and a commercially available Alum adjuvant (V\u003csub\u003eBVDV\u003c/sub\u003e:V\u003csub\u003eAlum\u003c/sub\u003e=1:3), and the AHPPE/BVDV group received 1 mL of a mixture of BVDV antigen and AHPPE adjuvant (V\u003csub\u003eBVDV\u003c/sub\u003e: V\u003csub\u003eAHPPE\u003c/sub\u003e=1:3) during the experiment period. The final concentration of BVDV antigen was 50 \u0026mu;g/mL in all treatment groups. All animals were immunized once.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Determination of Antibody Levels and Cytokine Concentrations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood and fecal samples were collected and processed on days 7, 14, 21, and 28 post-immunizations for subsequent antibody detection. According to the manufacturer\u0026apos;s instructions, ELISA kits (Shanghai Kexing Trading Co., Ltd.) were used to measure specific IgG levels in serum and IgA levels in fecal samples from calves in each group during the immunization period. Additionally, the concentrations of IL-4, IL-10, IL-17, TNF-\u0026alpha;, CCR6, CCR9, CCL20, and CCL28 in the serum were determined on day 14 post-immunization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Cytotoxicity Evaluation of AHPPE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBovine blood was diluted 1:1 with PBS, then layered onto lymphocyte separation medium and centrifuged. The intermediate milky-white cell layer was collected and suspended in red blood cell lysis buffer to lyse erythrocytes. After that, it was centrifuged to remove the lysed cells. The resulting pellet, containing a mixture of lymphocytes and DCs, was suspended in complete culture medium supplemented with recombinant bovine GM-CSF and IL-4 proteins. Half-volume medium changes were performed every other day for three straight days, and the supernatant containing non-adherent lymphocytes was discarded. After 7 days of culture, mature bovine peripheral blood-derived dendritic cells were obtained.\u003c/p\u003e\n\u003cp\u003eCells were adjusted to a density of 2 \u0026times; 10⁶ cells/mL and seeded into 96-well plates at 200 \u0026mu;L per well. After 24 hours of incubation, AHPPE was diluted in complete culture medium to various concentrations (1000, 500, 250, 125, 62.5, 31.3, and 15.6 \u0026mu;g/mL). Blank and control groups were included in the experimental design. After 24 hours of treatment, the supernatant was removed, and the cells were washed twice with PBS. Cell viability was assessed using the CCK-8 assay [22] to determine the safe concentration range of AHPPE for bovine dendritic cells.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"609\" height=\"63\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Evaluation of DCs\u0026apos; Phagocytic Activity toward AHPPE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFITC and AHPPE were uniformly mixed to prepare FITC-labeled AHPPE (AHPPE-FITC). The prepared AHPPE-FITC was co-cultured with DCs, and samples were collected at predetermined time points (1, 3, 6, 9, 12, and 24 hours). The intracellular FITC fluorescence intensity in DCs was measured by laser-scanning confocal microscopy to assess DC phagocytic capacity for AHPPE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Transcriptomic Sequencing Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were processed as described in Section 2.6, and the cell concentration was adjusted to 2 \u0026times; 10⁶ cells/mL. After 7 days, AHPPE was added, and the cells were incubated for 12 or 36 hours. Subsequently, both the cells and the supernatants were collected. Trizol reagent was added to the cell samples, which were then immediately frozen in liquid nitrogen and sent to Majorbio Bio-Pharm Technology Co., Ltd. for transcriptomic sequencing analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 Distribution of AHPPE in DCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAHPPE-FITC was co-cultured with DCs in confocal dishes at 1, 3, 6, 9, 12, and 24 hours post-incubation. The dishes were then fixed with 4% paraformaldehyde and washed three times with PBS. The fluorescently labeled antibodies were added to stain the nuclei and lysosomes, respectively, according to the manufacturer\u0026rsquo;s instructions, while unbound dye was carefully removed by washing with PBS. Fluorescence images were acquired randomly across multiple fields of view using a laser scanning confocal microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11 Evaluation of Protein Secretion by Dendritic Cells Following AHPPE Stimulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supernatant from cells collected at 36 hours in Section 2.8 was used to measure the concentrations of IL-4, IL-10, IL-17, TNF-\u0026alpha;, CCR6, CCR9, CCL20, and CCL28 using ELISA kits according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12 Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the experimental data are presented as mean \u0026plusmn; standard deviation (X \u0026plusmn; SD). P-value of less than 0.05 was considered statistically significant. *Denotes a significant difference compared with the control group at the corresponding time point (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and ** denotes an extremely highly significant difference compared with the control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). Statistical analyses were performed using GraphPad Prism version 8.0.\u003c/p\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003e\u003cstrong\u003e3.1 Single-Factor Experimental Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder fixed ultrasonic power (40%) and an oil-to-water ratio of 1:9, the effect of Alum concentration on the antigen loading rate of AHPPE showed an initial increase, followed by a decrease (Figure 1A). The antigen loading rate reached its peak at an Alum concentration of 6 mg/mL, which was significantly higher (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) than those observed at 1.5, 3, 4.5, and 7.5 mg/mL. This formulation also exhibited excellent emulsification efficiency, with no phase separation observed over 64 days64 days (Figure 1B).\u003c/p\u003e\n\u003cp\u003eUnder fixed conditions of Alum concentration (6 mg/mL) and ultrasonic power (40%), the effect of the oil-to-water phase ratio on the antigen loading rate of AHPPE showed an initial decrease, followed by an increase, and then a subsequent decrease (Figure 1C). The antigen loading rate reached its peak at an oil-to-water ratio of 1:11, which was significantly higher (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) than those observed at ratios of 1:5, 1:7, 1:9, and 1:13. This formulation also exhibited excellent emulsification efficiency, with no phase separation observed over 64 days64 days (Figure 1D).\u003c/p\u003e\n\u003cp\u003eUnder fixed conditions of Alum concentration (6 mg/mL) and an oil-to-water ratio of 1:11, the antigen loading rate of AHPPE peaked at an ultrasonic power of 20% (Figure 1E). This value was significantly higher than those observed at 10%, 30%, 40%, and 50% (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). This formulation also demonstrated excellent emulsification efficiency, with no phase separation observed over 64 days (Figure 1F).\u003c/p\u003e\n\u003cp\u003eBased on the efficiency and emulsification stability of AHPPE, the optimal single-factor conditions for its preparation were determined as an Alum concentration of 6 mg/mL, an oil-to-water phase ratio of 1:11, and an ultrasonic power intensity of 20%.\u003c/p\u003e\n\u003cp\u003e-------------------------------------\u003c/p\u003e\n\u003cp\u003eInsert Figure 1\u003c/p\u003e\n\u003cp\u003e--------------------------------------\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Results of the Response Surface Optimization Experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data in Table 2 were subjected to quadratic regression analysis using Design-Expert 13, with Alum concentration (A), oil-to-water ratio (B), and ultrasonic instrument power (C) as independent variables. The resulting regression equation is: Y = 88.56 - 1.2A + 0.5141B + 0.6096C + 0.2056AB + 0.8079AC - 0.3231BC - 4.16A² - 6.34B² - 8.21C².\u003c/p\u003e\n\u003cp\u003e-------------------------------------\u003c/p\u003e\n\u003cp\u003eInsert Table 2\u003c/p\u003e\n\u003cp\u003e--------------------------------------\u003c/p\u003e\n\u003cp\u003eThe regression model was evaluated, and the results are presented in Table 3. The model yielded P \u0026lt; 0.0001, indicating high statistical significance, with a lack-of-fit P value of 0.0507 (significant), a coefficient of determination (\u003cem\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/em\u003e) of 0.9875, an Adjusted Coefficient of Determination (\u003cem\u003eAdjR\u003csup\u003e2\u003c/sup\u003e\u003c/em\u003e) of 0.9714, close to unity, and a coefficient of variation (\u003cem\u003eCV%\u003c/em\u003e) of 1.31%. These metrics suggest that the model strong goodness-of-fit and is reliable for predicting the actual preparation process. The order of influence of the independent variables on antigen loading efficiency is as follows: Alum concentration (A) \u0026gt; ultrasonic power (C) \u0026gt; oil-to-water ratio (B). The optimal preparation conditions for AHPPE were determined to be an Alum concentration of 6 mg/mL, an oil-to-water ratio of 1:11, and an ultrasonic power setting of 20%. The nature of interactions between variables was assessed based on the contour patterns in the response surface plots and the trends observed in the corresponding three-dimensional graphs (Figure S1 in the supplementary material). The results show that, as Alum concentration, ultrasonic power, and oil-to-water ratio increase, the initial increase is followed by a decrease.\u003c/p\u003e\n\u003cp\u003eTo validate the response surface model, the software-optimized parameters were refined to the following conditions: An Alum concentration of 6 mg/mL, an oil-to-water ratio of 1:11, and an ultrasonic power setting of 20%. Under these conditions, the predicted antigen loading efficiency was 88.555%. The experimentally obtained antigen loading efficiency was 89.657 ± 1.11%, which is in close agreement with the predicted value and shows a slight deviation. This consistency indicates that the optimized conditions are feasible and reliable.\u003c/p\u003e\n\u003cp\u003e-------------------------------------\u003c/p\u003e\n\u003cp\u003eInsert Table 3\u003c/p\u003e\n\u003cp\u003e--------------------------------------\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Characterization and Analytical Results of AHPPE and AHPPE/BVDV\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 2A and 2B display the appearance of AHPPE and AHPPE/BVDV at room temperature. Both exhibit a uniform milky white color. Under optical microscopy, as shown in Figures 2C and 2D, both AHPPE and AHPPE/BVDV display spherical morphology, uniform particle size, and no noticeable sedimentation. AHPPE exhibits a raspberry-like morphology, with Alum uniformly adsorbed on the surface of squalene, forming a surface-rough particle with an approximate size of 2000 nm (Figure 2E). As illustrated in Figure 2 F, Dextran-FITC (emitting green fluorescence) is conjugated to Alum, while Cy5.5 (emitting purple fluorescence) is associated with the oil phase. The particle size remains around 2000 nm. The localization of Alum at the oil–water interface confirms that AHPPE is a Pickering emulsion with the oil phase as the core and the aqueous phase as the outer shell. AHPPE remained stable at both 4 °C and 37 °C during a 120-day storage period (Figure 2G). No noticeable changes in appearance were observed, and no precipitation or phase stratification was seen at days 0, 3, 5, 14, 30, 56, and 120. These results indicate that the nanoparticles exhibit excellent stability. As shown in Figure 2 H–I, the particle size distribution of AHPPE and AHPPE/BVDV remained relatively uniform over 56 days at both 4 °C and 37 °C. The zeta potential of AHPPE was positive under both storage conditions (Figure 2J). The average PDI of AHPPE remained below 0.3 over 30 days (Figure 2J). These results indicate that AHPPE maintains good colloidal stability for at least 30 days at 4 °C and 37 °C. The results suggest that AHPPE maintains stability for at least 30 days at both 4 °C and 37 °C.\u003c/p\u003e\n\u003cp\u003e-------------------------------------\u003c/p\u003e\n\u003cp\u003eInsert Figure 2\u003c/p\u003e\n\u003cp\u003e--------------------------------------\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Determination of Serum IgG-Cytokines and Fecal IgA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe laboratory personnel administered immunizations to each group of calves at the cattle farm (Figure 3A). As illustrated in Figure 3B, IgG levels in all vaccinated groups peaked on day 28 after vaccination. On days 7, 14, 21, and 28 after immunization, serum BVDV-specific IgG levels in the AHPPE/BVDV, Alum/BVDV, and BVDV groups were significantly higher than those in the CON group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). Furthermore, on day 28 after immunization, the BVDV-IgG level in the AHPPE/BVDV group was significantly higher than that in the Alum/BVDV group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003e-------------------------------------\u003c/p\u003e\n\u003cp\u003eInsert Figure 3\u003c/p\u003e\n\u003cp\u003e--------------------------------------\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Analysis of Serum Cytokine Levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe AHPPE/BVDV group exhibited significantly higher (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) expression levels of IL-10, IL-17, CCR6, CCR9, CCL20, and CCL28 compared with the CON group (Figure 4 A–H) after 28 days of immunization. When compared with the BVDV group, the AHPPE/BVDV group showed significantly increased expression levels of IL-10, IL-17, CCR6, CCR9, and CCL28 (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Furthermore, the AHPPE/BVDV group showed significantly higher expression levels than the Alum/BVDV group. The AHPPE/BVDV group showed significantly elevated expression of IL-10, CCR9, and CCL28 (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e-------------------------------------\u003c/p\u003e\n\u003cp\u003eInsert Figure 4\u003c/p\u003e\n\u003cp\u003e--------------------------------------\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Cell Safety and the Phagocytic Capacity of DCs towards AHPPE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental results are presented in Figure 5A. When the concentration of AHPPE ranged from 15.6 to 1000 µg/mL, cell viability was non-significantly reduced compared with the cell control group and remained above 100% in all cases. These findings indicate that AHPPE exhibits no cytotoxicity to DCs at concentrations up to 1000 µg/mL and demonstrates favorable biocompatibility.\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 5 B, after co-incubating DCs with AHPPE-FITC, green fluorescence was observed around the nuclei of DCs, indicating that AHPPE was taken up by antigen-presenting cells. When AHPPE-FITC was co-incubated with DCs for 1 hour, AHPPE was mainly located outside the cells. At 3 hours, most of the AHPPE had entered the cells. By 12 h and 24 h, all of the AHPPE-FITC had entered the DCs.\u003c/p\u003e\n\u003cp\u003e-------------------------------------\u003c/p\u003e\n\u003cp\u003eInsert Figure 5\u003c/p\u003e\n\u003cp\u003e--------------------------------------\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 Transcriptomic Profiling Results Across Time Points\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results show that at 12 h and 36 h post-treatment, compared with the Control group, the AHPPE group up-regulated 3,843 and 1,716 genes, respectively, and down-regulated 3,372 and 2,355 genes, respectively (Figures 6A and 6C). To investigate the immunomodulatory pathways associated with AHPPE, a heat map was generated to visualize the changes in gene expression. Compared with the Control group, the AHPPE group showed upregulation of TLR2, TLR4, CCL20, CCL25, CCL28, CCR3, CCR10, CXCR6, CD28, and ICOS at 12 h (Figure 6B). As shown in Figure 6D, compared with the Control group, the expression of ICOS, CCL20, CCL25, CCL28, CCR3, and CD28 genes was also significantly increased at 36 h in the AHPPE group. KEGG pathway analysis results (Figure 6E) indicate that at 12 h post-treatment, the AHPPE group showed significant enrichment in immune-related pathways, including endocytosis. As shown, the AHPPE group showed pronounced activation of the B cell receptor signaling pathway, the Toll-elevated receptor signaling pathway, and the IL-17 signaling pathway at 12 h and 36 h (Figure 6E and 6F). Whereas GO analysis results presented in Figure 6G and 6H, at 12 h and 36 h post-treatment, showed that the AHPPE group exhibited significant enrichment in biological processes, including the chemokine signaling pathway, positive regulation of T cell activation, lysosomal activity, and the MHC class II antigen presentation pathway. The research suggests that within 12 hours, AHPPE is largely engulfed by DCs, which subsequently activate DCs to highly express Toll-like receptors and MHC II receptors, stimulating T cells and B cells to generate effective systemic immune responses. Meanwhile, DCs are targeted and chemotaxed by elevated chemokine expression in the intestinal tract, thereby inducing intestinal mucosal immune responses.\u003c/p\u003e\n\u003cp\u003e-------------------------------------\u003c/p\u003e\n\u003cp\u003eInsert Figure 6\u003c/p\u003e\n\u003cp\u003e--------------------------------------\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8 Distribution Results of AHPPE in DCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figures 7A and 7 B, the Pearson correlation coefficients between AHPPE (green fluorescence) and lysosomes (red fluorescence) following co-culture with DCs for 1 h, 9 h, and 12 h were 0.64 ± 0.02, 0.53 ± 0.01, and 0.42 ± 0.01, respectively, indicating predominant lysosomal uptake of the carriers during the early stages. Upon extending the incubation time to 24 h, the localization coefficient decreased significantly to 0.23 ± 0.01 (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), suggesting substantial lysosomal degradation of the carriers and the potential release of a fraction into the cytoplasm, which is indicative of successful endolysosomal escape.\u003c/p\u003e\n\u003cp\u003e-------------------------------------\u003c/p\u003e\n\u003cp\u003eInsert Figure 7\u003c/p\u003e\n\u003cp\u003e--------------------------------------\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.9 Cytokine and Chemokine Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmune-related cytokines play a critical role in activating various immune cells. As shown in Figure 8 A–D, the AHPPE/BVDV group exhibited significantly higher levels of IL-4, IL-10, and IL-17 compared with both the CON and Alum groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Furthermore, Figures 8 E–H depicted that the AHPPE/BVDV group showed a markedly increased expression of CCR6, CCR9, CCL20, and CCL28 relative to the CON group (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05), and significantly elevated expression of CCR9 and CCL28 compared with the Alum group (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e-------------------------------------\u003c/p\u003e\n\u003cp\u003eInsert Figure 8\u003c/p\u003e\n\u003cp\u003e--------------------------------------\u003c/p\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eBovine viral diarrhea virus is an acute infectious agent that invades the intestinal mucosa, leading to severe diarrhea, infertility, abortion, congenital malformations, and persistent infection in cattle [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Traditional adjuvants, such as Alum and oil emulsions, have demonstrated limited efficacy in inducing robust intestinal mucosal immune responses [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In the present study, a targeted intestinal Pickering emulsion adjuvant was developed, termed AHPPE, through the precise alignment of functional components and optimization of the carrier structure. This novel approach overcomes the shortcomings in mucosal immunity elicited by the currently available BVDV vaccines.\u003c/p\u003e\u003cp\u003eThe preparation parameters of AHPPE were optimized using response surface methodology, followed by a complete examination of its physicochemical properties, antigen loading capacity, and stability. PDI, which reflects the molecular weight distribution of the polymer [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], was an important indicator of nanoparticle dispersion in the emulsion; the lower the value, the better the dispersion and higher stability [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Indeed, according to Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ, the PDI of AHPPE remained less than 0.3 during the 30-day course, which pointed toward an excellent dispersion state of the particles within the emulsion. From scanning electron microscopy and particle size distribution analyses (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH), it can be observed that AHPPE particles had an average diameter of approximately 2000 nm with a distinctive raspberry-like morphology and high antigen-loading capacity. The raspberry-like topography in the surface provides numerous binding sites for the attachment of antigens, a feature that greatly increases the antigen-loading efficiency of the emulsion system.\u003c/p\u003e\u003cp\u003eImmunological evaluation focused on quantifying antigen-specific IgG and intestinal IgA antibodies. IgG is the predominant immunoglobulin in serum [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], which is a direct marker of adjuvant-enhanced humoral immune responses. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, at day 28 post-immunization, the IgG antibody titers in the AHPPE/BVDV group were significantly higher than those in the CON and Alum/BVDV groups. IgA is the most abundant immunoglobulin on the intestinal mucosal surface-over 75% of total immunoglobulins-on the surface. It is produced via differentiation and proliferation of IgA-secreting plasma cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and has an important role in maintaining intestinal mucosal immunity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC shows that the IgA antibody levels in the AHPPE/BVDV group were significantly higher compared to the CON and Alum/BVDV groups. Collectively, these results further confirm that AHPPE effectively induces a strong systemic humoral immune response against BVDV, remarkably enhancing IgA antibody production, promoting mucosal immune activation, and contributing to preservation of intestinal mucosal immunity.\u003c/p\u003e\u003cp\u003eIL-10 is a pleiotropic cytokine that acts not only as a pivotal mediator in facilitating Th2-type cellular immune responses, but also plays a direct role in antigen-specific IgA responses at mucosal effector sites. It has been identified as a major inducer of IgA production in the colonic mucosa [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. IL-10 drives the differentiation of antigen-specific surface IgA\u0026thinsp;+\u0026thinsp;B cells into IgA-secreting plasma cells and modulates initiation and maintenance of mucosal immune responses [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Likewise, IL-17, another pleiotropic cytokine secreted by Th17 cells and innate immune cells, controls IgA secretion in mucosal tissues and promotes the activation and persistence of mucosal immune responses [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;D shows that compared to the CON, the AHPPE/BVDV group had significantly enhanced secretion of IL-10 and IL-17. Therefore, these findings indicate that AHPPE triggers a strong BVDV-specific Th2-type cellular immune response, thereby facilitating humoral immunity and enhancing intestinal mucosal IgA responses.\u003c/p\u003e\u003cp\u003eChemokines and their respective receptors are crucial for the directional migration of immune cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Among these, CCR9 is an intestine-specific chemokine receptor primarily expressed on lymphocytes and dendritic cells. Following interaction with its ligand, CCR9 promotes T cell trafficking into the intestinal mucosa to facilitate the activation of mucosal immune responses in the gut [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. CCL28, also named mucosal-associated epithelial chemokine (MEC), is primarily produced by intestinal epithelial cells (IECs) and acts as a fundamental regulator of immune cell trafficking into the intestinal mucosa through its receptors CCR10 (the primary receptor) and CCR3 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This chemokine ensures the trafficking of IgA antibody-secreting cells (IgA-ASCs) into the intestinal mucosa, representing a source of antibody-secreting cells that is necessary for mucosal humoral immunity. Moreover, CCL28 contributes to the accumulation of T cells in the intestinal mucosa and therefore to cellular immune responses in this site. Collectively, these mechanisms form an essential regulatory system for intestinal mucosal immune defense and mucosal immune homeostasis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, CCR9 and CCL28 expressions are much more significantly augmented in the AHPPE/BVDV group than in the Alum/BVDV group. These results show that AHPPE is efficient to support the activation of mucosal IgA response by promoting the intestinal homing of immune cells.\u003c/p\u003e\u003cp\u003eIn fact, the phagocytic ability of DCs against vaccine adjuvants is associated with their immunostimulatory effectiveness. Therefore, enhanced phagocytosis of vaccine adjuvants by DCs would lead to more effective antigen loading and uptake, thus promoting a robust immune response. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, it can be observed that AHPPE is being internalized by DCs, with its complete uptake occurring within 24 hours. Correspondingly, comparing Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF shows that the endocytic pathway is significantly upregulated in the AHPPE-treated group at 12 hours but not at 36 hours. All these observations support that AHPPE is effectively phagocytosed by DCs within 12 hours, thereby initiating immune activation. Toll-like receptors (TLRs) play a crucial role in recognizing pathogen-associated molecular patterns and triggering an immune response against BVDV. Indeed, TLRs, through their activation, initiate signaling cascades that result in the production of inflammatory cytokines and chemokines [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These processes subsequently activate not just the immediate host defense mechanisms but also facilitate antigen-specific adaptive immunity [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Moreover, the regulation of chemokine and chemokine receptor expression is deeply associated with the maintenance of immune homeostasis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. For instance, CCL20 belongs to the CC motif chemokine ligand that binds exclusively to the chemokine receptor CCR6. It selectively recruits effector or memory CD4\u0026thinsp;+\u0026thinsp;T cells and B cells [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This interaction, therefore, provides a critical cellular basis for the initiation, maintenance, and functional execution of intestinal mucosal immune responses [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Among key immunoregulatory molecules, the inducible co-stimulatory molecule (ICOS) is a type I transmembrane receptor homodimer that is structurally and functionally homologous to CD28 and expressed on DCs [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] while exhibiting immune activation capabilities analogous to CD28. ICOS enhances basic T cell responses to exogenous antigens, including proliferation, lymphokine secretion, and upregulation of intercellular adhesion molecules, besides providing support to B-cell-mediated production of antigen-specific antibodies [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Furthermore, it plays an essential role in inducing Th2-type immune responses [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] as it potently stimulates Th2 cells to produce the cytokine IL-10 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This cytokine environment favors the differentiation of IgA-producing plasma cells along The results also showed that the expressions of TLR2, TLR4, ICOS, and CD28 genes are significantly increased in the AHPPE group as compared to the control group, as evidenced by gene expression heatmap analyses shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD. Figures\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-H also present that the activation of several signaling pathways is enhanced in the AHPPE group at 12 and 36 hours' post-treatment, including Toll-like receptor signaling, T cell receptor signaling, B cell receptor signaling, IL-17 signaling, lysosomal enrichment, chemokine signaling, and MHC class II antigen presentation. It has already been established that exogenous antigens internalized by DCs traffic into lysosomes, where antigen processing occurs. These molecules, upon lysosomal digestion, will be degraded into small peptide fragments and then presented by MHC class II molecules, thus activating Th2 cells and eliciting humoral immune responses. These results, combined with heatmap analysis of gene expression, suggest that AHPPE elicits a Th2-type immune response mainly through DCs. Additionally, this preparation influences chemokine expression and pathways, thus orchestrating the migratory behavior of immune cells and contributing to chemotaxis in the intestinal mucosal immune response.\u003c/p\u003e\u003cp\u003eTo further validate the sequencing findings, the intracellular localization of AHPPE within lysosomes of cells and the protein secretion following AHPPE stimulation of DCs were assessed using techniques such as laser confocal microscopy and ELISA. Exogenous antigens are internalized by DCs and trafficked to lysosomes, where they are degraded into small peptide fragments. These peptides are subsequently presented by major histocompatibility complex class II (MHC II) molecules, ultimately activating T helper 2 (Th2) cells and initiating humoral immune responses. This mechanism aligns with the observed significant upregulation of MHC class II expression on DCs at the transcriptional level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH), thus providing corroborative molecular and cellular evidence for AHPPE-mediated activation of downstream immune pathways. The data presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-D reveal that, relative to the Alum and CON, AHPPE more effectively induces DCs to secrete interleukins IL-4, IL-10, and IL-17. These findings suggest that AHPPE is not only efficiently internalized and presented by DCs but also substantially promotes their maturation and activation. AHPPE lays a robust foundation for subsequent immune responses by stimulating the secretion of additional immunomodulatory cytokines. At the cellular level, the immunostimulatory capacity of AHPPE suggests a greater potential to elicit cellular immune responses. Moreover, the results demonstrate that, compared with the Alum group, AHPPE significantly enhances the expression of CCR9 and CCL28, which further substantiates its role in promoting the intestinal homing of immune cells and in effectively activating mucosal immune responses.\u003c/p\u003e\u003cp\u003eThis study showed that DCs internalized AHPPE effectively. The regulation of genes like TLR2/4, ICOS, and CD28, together with their signal pathways, promotes the activation of Th2-type immune responses via the lysosome-MHC class II antigen presentation pathway. It enhances the secretion of IL-10 and other immunomodulatory molecules, enhancing BVDV-specific IgG and intestinal IgA production. Moreover, AHPPE enhances key factors, such as CCR9 and CCL28, which increase the homing of immune cells to intestinal mucosa and guide the recruitment of immune cells to this site. Therefore, this mechanism effectively stimulates humoral, cellular, and mucosal immunity. These data provide substantial promise to overcome the major shortcomings of the currently available BVDV subunit vaccines, namely the transient immune responses and poor mucosal protection, and constitute strong support for AHPPE's use as a novel adjuvant in bovine BVDV vaccine formulations.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eIn this work, an AHPPE emulsion exhibiting excellent intestinal targeting efficacy was successfully developed. Emulsion surface testing showed that the AHPPE emulsion has a strawberry-like surface, with strong intestinal targeting and sustained antigen release. DCs efficiently phagocytose the AHPPE emulsion and activate critical immune signaling pathways, including Toll-like receptor, chemokine, and MHC II pathways, which induce robust and sustained antigen-specific IgA and IgG responses. Therefore, all findings suggest that the AHPPE/BVDV formulation is a novel and effective adjuvant for bovine vaccines, inducing targeted immune protection against BVDV infection and related diseases and therefore representing a valuable strategy for the prevention and control of BVDV.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAHP: Alhagi\u0026middot; honey polysaccharides; AHPPE: Alhagi Honey polysaccharide Pickering emulsion; RA: retinoic acid; Alum: Alum adjuvant; BVDV: Bovine viral diarrhea virus; BVD/MD: bovine viral diarrhea/mucosal disease; GALT: gut-associated lymphoid tissue; nTregs: non-adaptive regulatory T cells; PE: Pickering emulsion; FITC: fluorescein isothiocyanate; PDI: polydispersity index; DCs: dendritic cells; CON: control group; IL-10: interleukin-10; TCR: T-cell receptor; MEC: mucosal-associated epithelial chemokine; IgA-ASCs: IgA antibody-secreting cells; TLRs: Toll-like receptors; ICOS: inducible co-stimulatory molecule; ELISA: enzyme-linked immunosorbent assay MHC II: major histocompatibility complex class II.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental animals were supplied by Bachu Tianrun Livestock Co., Ltd., which authorized their use for the study. All experimental procedures were reviewed and approved by the Animal Welfare and Ethics Committee of Xinjiang Agricultural University (Approval Number: 2022016). The research was conducted in full compliance with applicable local laws, regulations, and institutional guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXinjiang Uygur Autonomous Region Major Science and Technology Special Project (2023A02007-2). National Natural Science Foundation of China Youth Fund Project (32202855), Key Laboratory of New Drugs for Herbivores of Xinjiang Province (XJCDVMHDRC-T202301), Autonomous Region-level Entrepreneurship Training Project (S202510758007X).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Data curation, Methodology, Validation, Writing-original draft, Investigation, YW; Conceptualization, Data curation, Writing-original draft, Investigation, Methodology, JL, GH, PH; Conceptualization, Data curation, Investigation, Methodology, EZ, AA; Formal Analysis, Methodology, DH, LD, SW; Data curation, Investigation, Methodology, Supervision, Validation, Writing-review, editing, AW.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTechnical supports from all members of the Chinese Veterinary Medicines Lab in animal handling, sampling, and lab analysis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYang NN, Zhang JW, Xu MG, Yi JH, Wang Z, Wang Y, et al. 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BMC Veterinary Research. 2025;21(1):315-315. http://doi.org/10.1186/S12917-025-04761-5.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1: Factors and levels of the response surface experiment\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLevel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eA：\u0026nbsp;Concentration of Alum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eB：\u0026nbsp;Oil-water phase ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eC：\u0026nbsp;Power consumption\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.5 mg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1:9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10 %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6 mg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1:11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e20 %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.5 mg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1:13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e30 %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Arrangement and results of response surface experiment\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 16px;\"\u003e\n \u003cp\u003eTest No.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 20px;\"\u003e\n \u003cp\u003eFactor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 62px;\"\u003e\n \u003cp\u003eY: Rate of encapsulation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003eMeasured value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003ePredictive value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e74.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e74.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e88.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e88.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e74.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e74.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e77.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e77.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e88.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e88.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e75.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e74.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e71.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e72.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e87.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e88.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e78.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e78.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e78.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e79.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e78.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e77.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e73.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e73.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e89.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e88.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e77.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e77.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e75.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e76.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e88.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e88.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e77.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e76.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: The results of the variance analysis for the quadratic regression model incorporating factors A, B, and C\u003c/strong\u003e\u003c/p\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eSum of Squares\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cem\u003edf\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003cp\u003eSquare\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eF-Value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cem\u003ep-value\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eProb \u0026gt; F\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003esignificance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e601.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e66.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e61.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e11.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e11.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e10.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e0.0141\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e1.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e0.2059\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e2.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e0.1423\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eAB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e0.1692\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e0.1692\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e0.1555\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e0.7050\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e2.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e0.1652\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e0.4177\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e0.4177\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e0.3841\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e0.5551\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eA\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e72.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e72.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e66.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eB\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e169.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e169.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e155.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eC\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e283.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e283.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e260.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eResidual\u003c/p\u003e\n \u003cp\u003eError\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e7.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eLack of fit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e6.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e6.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e0.0507\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003ePare error\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e0.3224\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eCor Toal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e609.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNote: **represented an exceptionally significant influence (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01)\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bovine viral diarrhea virus (BVDV), Dextran sulfate-Alum Pickering emulsion, Vaccine adjuvant, Intestinal mucosal immunity","lastPublishedDoi":"10.21203/rs.3.rs-8142640/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8142640/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe mucosa represents the first line of defense against pathogenic invasion, triggering strong mucosal immunity by vaccination for the prevention of infectious diarrheal diseases.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eIn this study, a new assembled Pickering emulsion (AHPPE) was created. This emulsion was constructed by employing an Alum adjuvant (Alum) loaded with Alhagi honey polysaccharide (AHP)-a known enhancer of intestinal mucosal immunity-as the shell, and squalene containing all-trans retinoic acid (RA), an agent targeting intestinal mucosa, as the core, utilizing ultrasonic emulsification techniques. Furthermore, the systemic and mucosal immune responses elicited by AHPPE as an adjuvant for bovine viral diarrhea virus (BVDV) vaccines, along with the associated mechanistic pathways, were investigated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe results showed that AHPPE had raspberry-like morphology with a mean particle size of around 2000 nm and a positive surface charge. The emulsion proved to have effective loading capacity for BVDV vaccine antigen and good stability for 30 days. When used as an adjuvant for BVDV vaccine, AHPPE greatly enhanced the titers of BVDV-specific IgG and IgA antibodies and the expression of cytokine interleukin-10 (IL-10), chemokine CCL28, and chemokine receptor CCR9 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Transcriptomic analysis revealed that AHPPE was highly phagocytosed by dendritic cells (DCs), which in turn upregulated the expression of Toll-like receptors and major histocompatibility complex class II (MHC II) molecules, thereby activating T and B lymphocytes to trigger strong systemic immune responses. At the same time, induction of chemokines enabled DCs to migrate specifically to the intestinal tract, triggering intestinal mucosal immune activation. The authenticity of the sequencing data was further validated using confocal microscopy and enzyme-linked immunosorbent assay (ELISA).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eTo conclude, the current study provides evidence that AHPPE is a viable vaccine adjuvant for BVDV, which not only generates a robust systemic immune response but also efficiently induces an intestine-targeted mucosal immune response through inducing extensive dendritic cell chemotaxis.\u003c/p\u003e","manuscriptTitle":"Enhancing effect of targeted intestinal delivery of Alhagi honey Polysaccharide-Alum Pickering emulsion adjuvant on the immune response to the BVDV vaccine in cattle","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-16 14:06:37","doi":"10.21203/rs.3.rs-8142640/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-27T08:27:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-24T20:46:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254175003640326535706297239171903466658","date":"2026-01-08T11:32:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-06T00:16:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-24T09:57:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16376507712797969202947104026493917485","date":"2025-12-16T23:04:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53951347958886751595182366237612177790","date":"2025-12-11T02:38:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-09T11:36:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-09T11:27:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-05T14:54:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-05T03:58:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2025-12-05T03:51:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"83068545-1c45-40f1-91c7-8b963bb1638e","owner":[],"postedDate":"December 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T18:23:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-16 14:06:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8142640","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8142640","identity":"rs-8142640","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}