Ethics statement

This study was performed in light of the National Guidelines for Experimental Animal Welfare (Minister of Science and Technology, People’s Republic of China, 2006). All animal experiments in this study were approved by the institutional Life Science Ethics Committee of Zhengzhou University (No. ZZUIRB GZR 2023–1397).

Trichinella species and experimental animal

Trichinella spiralis (ISS534) was collected from a naturally infected swine in Henan province of China, and maintained by serial passage in BALB/c mice in our department [21]. The female BALB/c mice with 6–8 weeks old were purchased from the Experimental Animal Center of Zhengzhou University.

Preparation of rTsSPc protein

The full-length TsSPc cDNA sequence was cloned into pQE-80L to construct recombinant expression plasmid pQE-80L/TsSPc [16]. The pQE-80L/TsSPc was expressed in E. coli BL21 (DE3) (Novagen, USA) under induction of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 6 h at 37 °C, and purified by a Ni-NTA-Sefinose resin containing His tag (Sangon Biotech, China). Finally, rTsSPc was refolded by dialysis and renatured methods [22].

Subcutaneous immunization of mice with rTsSPc and sample collection

A total of 150 mice were randomly divided into three groups (50 animals per group). The immunization group was subcutaneously vaccinated with 20 µg rTsSPc protein emulsified in ISA 201 adjuvant (SEPPIC, France). Two boosting injections were administered at 2-week intervals using the same dose of rTsSPc combined with ISA 201 adjuvant. Two control groups received either PBS or ISA 201 adjuvant alone. At two weeks following the final vaccination, all mice were orally challenged with 300 T. spiralis ML. Serum samples were collected from ten mice per group at weeks 0, 2, 4, 6, 7, 9 and 11 after vaccination. Tail blood (100 µl) was obtained and centrifuged to isolate sera, which were stored at −80 °C until use. To assess intestinal mucosal immune responses, five mice per group were euthanized, intestinal tissues, spleens, mesenteric lymph nodes (MLNs) and Peyer’s patches (PPs) were collected at weeks 0, 6, 7 and 11 after vaccination, their cells were isolated and cultivated to measure the production of cytokines (IFN-γ and IL-4) [23]

To evaluate the protective efficacy of rTsSPc vaccination, an additional ten mice of each group were sacrificed respectively at 7 and 11 weeks after vaccination, i.e., 1 and 5 weeks after infection [7 days post-infection (dpi) and 35 dpi]. Adult worm burden, female reproductive capacity (the in vitro production of NBL deposited by each female for 72 h), and ML burden were assessed as previously described [13]. Furthermore, to investigate intestinal barrier integrity and inflammation reaction, intestinal permeability, goblet cells and expression levels of gut epithelial TJs and mucins, additional five unvaccinated and uninfected mice were also used [19,24]. Moreover, to evaluate the macrophage polarization after rTsSPc immunization and infection, peritoneal macrophages were isolated from ten mice of each group to ascertain macrophage’s surface markers (CD86, CD206), intracellular enzymes (iNOS, Arg1) and cytokine profiles (IL-6, TNF-α, IL-10 and TGF-β). The vaccination schedule is summarized in Fig 1A.

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Fig 1. The designed vaccination scheme and anti-rTsSPc antibody detection.

A: The designed vaccination scheme and detection protocol. Mice were subcutaneously vaccinated with 20 µg rTsSPc protein emulsified in ISA 201 adjuvant three times at weeks 0, 2, and 4. At two weeks following the final vaccination, the vaccinated mice were orally challenged with 300 T. spiralis ML. Anti-rTsSPc antibodies (IgG, IgG1/IgG2a, and IgA) were measured by rTsSPc-ELISA at weeks 0, 2, 4, and 6 post-immunization, respectively, and at weeks 7, 9 and 11 post-immunization (e.g., 1, 3, and 5 weeks after infection). Five mice per group were euthanized at 6 weeks after immunization, 1 and 5 weeks after infection to assess levels of sIgA and cytokines (IFN-γ and IL-4). At 1 and 5 weeks after infection, ten mice per group were sacrificed to determine intestinal adult burden, female fecundity, and muscle larval burden (larvae per gram of skeletal muscles, LPG). Histopathological examination of intestines and muscles from infected mice was performed. Furthermore, peritoneal macrophages were isolated from ten mice per group at 2 weeks after final immunization and 1 week after infection to evaluate the macrophage polarization. B: Serum anti-rTsSPc IgG titers. Anti-rTsSPc IgG levels were assayed in serum sample from vaccinated mice two weeks after the final vaccination. All serum samples were tested in duplicate. The data represent the OD values of anti-rTsSPc IgG levels derived from 10 vaccinated mice. Additionally, 45 serum samples from normal mice diluted at 1:100 were measured as negative controls. The ELISA cut-off values were calculated based on 2.1-fold of the mean OD value of negative control sera from normal mice. Serum samples with OD values equal to or greater than the cut-off were defined as positive. The cut-off value (0.17) is indicated by a dotted line. C: Serum anti-rTsSPc antibody kinetics at various times after immunization. The figure illustrates the total IgG response at various time points post vaccination, as well as the specific IgG1/IgG2a subclass responses and IgA levels in vaccinated mice. All serum samples were tested in duplicate, and the OD values are presented as mean ± standard deviation (SD) (n = 10). D: Total intestinal sIgA and rTsSPc-specific sIgA levels in enteral washing fluid of vaccinated mice. All enteral fluid samples were assayed in duplicate. No significant sIgA response was detected in the ISA201 or PBS group. The data are presented as mean OD values ± SD (n = 5). Arrows (↑) indicate vaccination times, triangles (▲) denote challenge time. *P < 0.05 compared to the PBS group.

https://doi.org/10.1371/journal.pntd.0014161.g001

ELISA determination of serum antibody

Serum specific antibodies (IgG, IgG1/IgG2a and IgA) were measured using an indirect ELISA with rTsSPc as coating antigen [14]. Briefly, 96-well microtiter plates were coated with 100 µl of 1.5 µg/ml rTsSPc and incubated at 4 °C overnight. After washing three times with phosphate-buffered saline containing 0.05% Tween 20 (PBST), the plates were blocked with 5% skim milk in PBST at 37 °C for 2 h. Following being washed again, the plates were incubated at 37 °C for 1 h with primary antibody (immune serum), then with HRP-conjugated goat anti-mouse IgG, IgG1/IgG2a, and IgA (1:10000; Sigma, USA) at 37 °C for 1 h. Finally, the plates were colored using the substrate o-phenylenediamine dihydrochloride (OPD; Sigma) plus 0.15% H₂O₂, and reaction was stopped by using 2 M H₂SO₄. Optical density (OD) values at 492 nm were measured using a microplate reader [6,25]. Positive results were defined as a ratio of sample OD value to mean OD value of negative serum control ≥ 2.1.

Sandwich ELISA determining total and specific sIgA in intestinal fluid

The levels of gut secretory IgA (sIgA) in immunized mice were determined using a sandwich ELISA [26]. Briefly, a 20-cm small intestine was cut, and intestinal content was washed three times with 1 ml of cold PBS containing 1% protease inhibitor (Sangon Biotech, Shanghai, China). The washing enteral fluid was collected and centrifuged at 12000 g for 5 min at 4 °C, and then supernatants were collected. Total enteral sIgA was measured by a sandwich ELISA, and rTsSPc-specific sIgA was assayed by an indirect ELISA. Color development was performed with OPD substrate, and the OD values at 492 nm were measured using a microplate reader as previously described [26].

ELISA measurement of cytokines

To ascertain cellular immune response to the rTsSPc immunization, five mice of each group were scarified at 0 and 6 weeks after immunization, and at 1 and 5 weeks after infection. The spleen, MLNs and PPs were recovered from all immunized mice, homogenized in complete DMEM medium (Gibco, Auckland, New Zealand). The cells were collected following centrifugation at 1000 g for 5 min, and isolated as reported before [6]. The cell density was adjusted to 2 × 10⁶ cells/ml in DMEM medium containing 5% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin, stimulated with 10 μg/ml rTsSPc at 37˚C and 5% CO₂ for 72 h [27]. The culture supernatant was obtained and two cytokines (IFN-γ and IL-4) were assayed using a sandwich ELISA (BD Biosciences Pharmingen, USA). The cytokine level was showed as pictograms per milliliter (pg/ml). Additionally, the contents of inflammatory cytokines (TNF-α, IL-6, TGF-β and IL-10) in intestinal fluids were also assayed by ELISA as described before [28].

Challenge infection and evaluation of immune protection

To evaluate the immune protection induced by rTsSPc vaccination, all immunized mice were orally challenged with 300 T. spiralis ML at two weeks after the final immunization. At 7 dpi, AWs were collected from the intestines of ten mice per group. The remaining ten mice from each group were euthanized at 35 dpi. Murine carcasses were weighed, skeletal muscle were artificially digested, and muscle larvae were collected and numbered as previously described [29,30]. The protective efficacy of the rTsSPc vaccination was assessed based on the reduction of intestinal worm burden and larvae per gram (LPG) of skeletal muscles compared to the PBS control group. Additionally, female fecundity was determined as reported before [23,31].

qPCR and Western blot assay of gut mucosal TJs, mucins and inflammatory cytokines from infected mice

To assess the expression levels of gut mucosal TJs, mucin and inflammatory cytokines of infected mice, mouse intestinal tissues were collected at 7 dpi, qPCR and Western blot assay were performed [19]. Briefly, total RNA was extracted using TRIzol reagent (Takara) and reverse-transcribed into cDNA using a cDNA synthesis kit. Subsequently, qPCR amplification was performed on an ABI Prism 7500 Fast Sequence Detection System (Applied Biosystems, USA). The mRNA levels were normalized to β-actin as an internal reference gene, no significant differences in β-actin expression were observed among diverse groups. PBS negative controls were set for each assay. Relative expression changes of the murine genes were calculated by comparing Ct values using the 2^−ΔΔCt method [32]. All experiments were repeated three times. The primers used for qPCR in this study are detailed in Table 1 [33,34]

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Table 1. Primer sequences of murine TJs, mucin and cytokines for qPCR assays.

https://doi.org/10.1371/journal.pntd.0014161.t001

For Western blot analysis, mouse enteral tissues collected at 7 dpi were lysed in RIPA buffer and ground on ice for 30 min, followed by centrifugation at 12,000 g for 15 min at 4 °C to remove cell debris. Extracted enteral tissue proteins were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, USA). The membranes were blocked with 5% skim milk in TBST at 37 °C for 2 h, then cut into strips and incubated with primary antibodies against Occludin (1.5 µg/ml), Claudin-1 (1.5 µg/ml), and E-cad (1: 40,000, Abcam, USA), as well as anti-β-actin antibody (1:1000, Servicebio, Wuhan, China) at 4 °C overnight [35,36]. After washing with TBST, the strips were incubated with the secondary antibodies (1:5000 dilutions of HRP-anti-mouse IgG conjugate or HRP-conjugated anti-rabbit IgG; Upingbio, Shenzhen, China) at room temperature for 1 h. Finally, the bands were visualized using an enhanced chemiluminescence kit (ECL; Epizyme, Shanghai, China), and the relative intensity of the bands was analyzed using Image J software (National Institutes of Health, USA) [28].

Immunofluorescent assay (IFA) and Western blotting of expression of RACK1 and ERK1/2 pathway

To investigate whether rTsSPc immunization prevents the secretion of parasite-derived TsSPc into the mouse intestinal epithelium and blocks its interaction with gut receptor RACK1, small intestines from the rTsSPc, ISA201 and PBS groups were fixed, embedded and sectioned at 7 dpi [16]. The IFA was performed with primary antibodies including anti-rTsSPc serum and anti-RACK1 antibody, followed by secondary antibodies (Alexa Fluor 488 conjugated-goat anti-mouse IgG and Cy3 conjugated-goat anti-rabbit IgG). Nuclei were counterstained red with DAPI [37]. Moreover, mRNA and protein expression of RACK1 and ERK1/2 pathway was also ascertained by qPCR and Western blot as reported before [17].

Histopathological examination of small intestine and masseter muscles

At 7 and 35 dpi, small intestine and masseter muscles were collected from infected mice and blank control mice. Tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin wax, and cut into 3-µm-thick tissue cross-sections. Deparaffinized sections were stained using hematoxylin and eosin (HE) stain and periodic acid Schiff reagent (PAS; Baso, Zhuhai, China) [14,26]. Gut mucosal morphology was examined under light microscopy, the width of enteral villi and the number of goblet cells per high-power field (400×) were quantified. On muscle sections, the number of encapsulated larvae per low-power field (100×) and inflammatory cell infiltration (eosinophils, neutrophils, and lymphocytes) per high-power field (400×) were counted as previously described [38,39].

Measurement of intestinal permeability

Intestinal TJs protein expression levels are directly correlated with intestinal permeability, which is typically determined in vivo using 4 kDa FITC-dextran (FD-4) [18,20]. To evaluate the intestinal permeability changes following subcutaneous immunization with rTsSPc, the mice were fasted overnight at 7 dpi. A 50 mg/ml FD-4 solution was prepared by dissolving 4 kDa FD-4 powders in sterile PBS, and 100 µl FD-4 was intragastrically administered to each mouse, then water access was restored. Four hours later, blood was collected from the orbital sinus, and plasma was separated by centrifugation at 4 °C and 1000 g for 10 min. The collected plasma was diluted with PBS (1:100) and added to a 96-well ELISA plate. Fluorescence intensity was measured using a microplate reader at excitation and emission wavelengths of 485 nm and 520 nm, respectively [17,40].

Flow cytometry of murine peritoneal macrophages

Five mice of each group were euthanized at 2 weeks after final immunization and 1 week post infection, subsequently immersed in 75% alcohol for 2 min [41]. A total of 10 ml sterile PBS was injected intraperitoneally, followed by gentle pressure on peritoneal wall for 5 min before slowly aspirating the fluid into 15 ml centrifuge tubes [42,43]. Peritoneal cells were centrifuged at 500 g for 10 min at 4°C, and the supernatant was discarded. The cell pellet was resuspended in complete DMEM culture medium and seeded at a density of 5 × 10⁶ cells per well in 6-well plates. After incubation for 4 h, non-adherent cells were removed by medium replacement, and cells were washed three times with PBS. The resulting adherent cells were defined as peritoneal macrophages and continued to be cultured in complete DMEM. To analyze macrophage polarization states, cells were also treated with LPS (M1) or IL-4 (M2). Macrophage surface markers were analyzed via flow cytometry using FITC-F4/80, PerCP-Cyanine5.5-CD11b and PE-CD86, while intracellular expression of APC-CD206 was detected after permeabilization [44,45].

qPCR and ELISA measurement of macrophage M1/M2 factors

Peritoneal macrophages were collected from three groups of mice at 2 weeks post immunization and 1 week after infection. Total RNA was extracted, and cDNA was synthesized via reverse transcription. qPCR was performed using the synthesized cDNA as a template to assess transcription levels of M1 (CD86, iNOS, TNF-α and IL-6) and M2-related factors (CD206, Arg1, TGF-β and IL-10). LPS (200ng/ml) served as the M1 control, while IL-4 (20ng/ml) served as the M2 control. DMEM served as the negative control. β-actin was used as the internal reference gene. Gene transcription levels were analyzed by comparing Ct values using the 2^−ΔΔCt method, and all assays were performed with triplicate [32], and qPCR primers used in this study are listed in Table 1. Additionally, macrophage culture supernatant was centrifuged at 1000 g, 4 °C for 10 min, and macrophage-secreted inflammatory cytokines (TNF-α, IL-6, TGF-β and IL-10) were assayed by a sandwich ELISA kit (Biolegend, USA [26,38]. Cytokine concentration was showed as pictograms per milliliter (pg/ml).

Western blot analysis was performed to measure the expression levels of M1 (iNOS) and M2-related factors (Arg1). Primary antibodies consisted of rabbit anti-iNOS (1:1000, Abcam) and mouse anti-Arg1 (1:5000, Proteintech), which were incubated at 4°C overnight [46]. Secondary antibodies included goat anti-rabbit IgG (1:5000, Upingbio) and goat anti-mouse IgG (1:5000, Upingbio), incubated at 37°C for 1 h. β-actin served as the internal reference, with three biological replicates per group [47]. Protein band intensities were analyzed using ImageJ software, and statistical analysis was conducted based on the ratio of target protein gray values to β-actin gray values.

Antibody-dependent cell-mediated cytotoxicity (ADCC) assay

Specific antibody-mediated cytotoxicity killing T. spiralis NBL was assessed as previously described [45]. Briefly, female adult worms were collected at 6 dpi, cultivated in DMEM containing 10%FBS (Gibco) at 37 °C under 5% CO₂ for 24 h, and NBL were recovered. A total of 100 NBL were co-cultured with 3 × 10⁵ murine peritoneal exudate cells (PECs) in a 48-well plate. The cultures were then incubated with serial dilutions (1:50–1:800) of immune sera from rTsSPc, ISA 201 and PBS groups, T. spiralis infection sera were used as a positive control. After incubation at 37 °C for 72 h, NBL viability was determined based on larval morphology and motility [48]. Live larvae exhibited active movement, whereas dead larvae appeared rigid and immobile. Cytotoxicity was defined as the percentage of dead larvae relative to the total number of larvae observed in each well [49].

Statistical analysis

Data were analyzed using GraphPad Prism 9.5.0 software. Results are expressed as the mean ± standard deviation (SD). The normality and homogeneity of the datum variance were evaluated by using the Shapiro-Wilk test and Levene’s test, respectively. Statistical analysis was performed using one-way analysis of variance (ANOVA) and t-tests. A P value less than 0.05 was considered statistically significant.

Anti-rTsSPc antibodies in immunized mice

Serum anti-rTsSPc IgG titers were determined by ELISA at two weeks after the final vaccination. The results demonstrated that anti-rTsSPc IgG levels in vaccinated mice were significantly elevated compared to the pre-immunization levels (F = 17.22, P < 0.0001). The specific IgG titer reached 1:10⁵ at two weeks following final immunization (Fig 1B), indicating that rTsSPc had good immunogenicity. However, no anti-rTsSPc IgG responses were detected in mice immunized with ISA201 and PBS alone.

The kinetics of serum anti-rTsSPc antibodies were assayed by ELISA, the results showed that compared with the PBS group, the levels of anti-rTsSPc IgG in the rTsSPc-immunized group were significantly elevated at weeks 2, 4, 6, 7, 9 and 11 post immunization (F_2W = 47.13, F_4W = 4775, F_6W = 596.3, F_7W = 396.4, F_9W = 169, F_11W = 317; P < 0.0001). Furthermore, the IgG1 levels were significantly higher than IgG2a levels at various times after immunization (t_2W = 85.1, t_4W = 9.741, t_6W = 27.94; t_7W = 37.28, t_9W = 11.87, t_11W = 8.479; P < 0.0001), indicating that rTsSPc vaccination induced a mixed Th1/Th2 immune response with a Th2 dominance. Additionally, anti-rTsSPc IgA in the sera of immunized mice was also measured, and the results demonstrated that IgA levels were significantly increased at weeks 2, 4, 6, 7, 9 and 11 compared to the PBS control group (F_2W = 1768, F_4W = 287.4, F_6W = 136.7, F_7W = 256.8, F_9W = 97.95, F_11W = 61.58; P < 0.0001) (Fig 1C). After challenge infection, both ISA201 and PBS groups exhibited significantly elevated anti-rTsSPc IgG and IgA levels compared to pre-infection levels. These findings showed that specific IgG, IgG1/IgG2a and IgA levels in the rTsSPc group were gradually increased after immunization, and further elevated following challenge, indicated that rTsSPc vaccination elicited an evident antibody response.

Intestinal mucosal total and rTsSPc-specific sIgA responses were measured by sandwich and indirect ELISA. The results showed that there were no significant differences of total and specific sIgA levels among the rTsSPc, ISA201 and PBS groups before immunization (P > 0.05). But at two weeks after the final immunization, total sIgA levels in intestinal fluid of rTsSPc-immunized mice were significantly elevated compared to the PBS group (F = 60.58, P < 0.0001); specific sIgA levels in the rTsSPc group were also significantly higher than the PBS group (F = 48.04, P < 0.0001) (Fig 1D). No specific sIgA responses were detected in only ISA201 or PBS alone group. Moreover, at 7 and 11 weeks after immunization (e.g., 1 and 5 weeks after infection), both ISA201 and PBS groups exhibited significantly increased total and rTsSPc-specific sIgA levels compared to pre-infection levels (1 week after infection: total sIgA: t_ISA201 = 6.207; P = 0.0003; t_PBS = 8.453; P < 0.0001; specific sIgA: t_ISA201 = 3.180; P = 0.013; t_PBS = 4.858; P = 0.0013; 5 weeks after infection: total sIgA: t_ISA201 = 5.746; P = 0.0004; t_PBS = 10.07; P < 0.0001; specific sIgA: t_ISA201 = 13.96; P < 0.0001; t_PBS = 30.05; P < 0.0001). These results indicated that rTsSPc immunization induced a strong specific gut mucosal sIgA response.

Analysis of cellular immune response type (Th1/Th2) of immunized mice

To investigate the cellular immune response type (Th1/Th2) induced by rTsSPc vaccination, spleen, MLN and Peyer’s patch were isolated from immunized BALB/c mice. Their cells were stimulated with rTsSPc, and the cytokine secretion levels in culture supernatants were detected by ELISA. The results showed that the levels of Th1 (IFN-γ) and Th2 cytokine (IL-4) of three groups had no significant differences prior to immunization (P > 0.05). At 6 weeks after immunization, the levels of IFN-γ and IL-4 secreted by spleen cells in the rTsSPc group were significantly elevated compared to the PBS group (F_IFN-γ = 233.5, F_IL-4 = 89.09, P < 0.0001). Furthermore, the expression levels of IFN-γ and IL-4 were further increased at 7 weeks after immunization (1 week after challenge; F_IFN-γ = 247.5, F_IL-4 = 41.85, P < 0.0001). And the levels of IFN-γ and IL-4 of spleen cells were further elevated at 11 weeks after immunization (5 weeks after challenge; F_IFN-γ = 351.5, F_IL-4 = 27.17, P < 0.0001). The levels of IFN-γ and IL-4 secreted by MLN cells in the rTsSPc group were also significantly higher than the PBS group (6 weeks: F_IFN-γ = 167.7, F_IL-4 = 252.8, P < 0.0001; 11 weeks: F_IFN-γ = 360.8, F_IL-4 = 25.05, P < 0.0001). Similarly, the levels of IFN-γ and IL-4 secreted by PP cells in the rTsSPc group were significantly higher than the PBS group at 6 weeks post immunization (F_IFN-γ = 54.52, F_IL-4 = 220.9, P < 0.0001); but at 11 weeks post immunization, only IFN-γ levels secreted by PP cells in the rTsSPc group was higher the PBS group(F_IFN-γ = 479.8, P < 0.0001; F_IL-4 = 2.926, P > 0.05) (Fig 2). These findings indicate that subcutaneous immunization with rTsSPc not only induced systemic (spleen) cellular immunity, but also elicited gut local (MLN and PP) mucosal cellular immunity, resulting in a mixed Th1/Th2 immune response.

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Fig 2. Cytokine secretion profiles from spleen, mesenteric lymph nodes (MLN) and Peyer’s patches (PP) of rTsSPc-immunized mice at different times after vaccination.

Concentrations of IFN-γ and IL-4 were quantified in culture supernatants following stimulation with 10 µg/ml rTsSPc at 37 °C and 5% CO₂ for 72 h. Data represent the mean picograms per milliliter (pg/ml) ± SD (n = 5). All samples were assayed in duplicate. *P < 0.05 compared to the PBS control group.

https://doi.org/10.1371/journal.pntd.0014161.g002

rTsSPc immunization increased TJs expression in infected mice

qPCR results showed that at 7 dpi, the transcript levels of E-cad, Occludin and Claudin-1 in three group of rTsSPc, ISA 201 and PBS groups after infection were obviously declined compared to the uninfected PBS group (F_E-cad = 71.39, F_Occludin = 128.7, F_Claudin-1 = 29.74, P < 0.001), but their transcript levels in the rTsSPc group were significantly higher than the infected PBS group (t_E-cad = 6.410, P = 0.003; t_Occludin = 5.562, P = 0.0051; t_Claudin-1 = 4.041, P = 0.0156) (Fig 3A). Western blot results revealed that at 7 dpi, the protein expression levels of E-cad, Occludin and Claudin-1 in the three group were evidently lower than the uninfected PBS group (F_E-cad = 29.06, F_Occludin = 31.13, F_Claudin-1 = 11.52, P < 0.01), but their protein levels in the rTsSPc group were significantly higher than the infected PBS group (t_E-cad = 8.741, P = 0.0009; t_Occludin = 5.660, P = 0.0048; t_Claudin-1 = 2.882, P = 0.0449) (Fig 3B). These findings indicated that rTsSPc immunization counteracted and restored the TJs protein downregulation caused by T. spiralis infection, suggesting that rTsSPc vaccination enhanced murine intestinal epithelial integrity and barrier function after infection.

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Fig 3. qPCR, Western blot and ELISA determination of mRNA and protein expression of gut TJs and cytokines in immunized mice at 7 days post infection.

A: qPCR analysis of mRNA expression levels of TJs (E-cad, Occludin and Claudin-1) in intestines of immunized mice (n = 3). B: Western blot analysis of TJs protein expression levels in intestines of immunized mice (n = 3). C: qPCR analysis of mRNA levels of TNF-α, IL-6, TGF-β and IL-10 in intestinal tissues (n = 3). β-actin was used as an internal control. Each experiment was repeated three times. D: ELISA analysis of protein expression levels of TNF-α, IL-6, TGF-β and IL-10 in intestinal lavage fluids (n = 5). *P < 0.05 compared with the uninfected PBS group; ^# P < 0.05 compared with the infected PBS group.

https://doi.org/10.1371/journal.pntd.0014161.g003

Cytokine transcription and expression levels in intestinal mucosa of infected mice

qPCR results showed that compared with the infected PBS group, the transcription levels of pro-inflammatory cytokines (TNF-α and IL-6) in murine intestinal mucosa of the rTsSPc group were significantly decreased (t_TNF-α = 9.553, P = 0.0007; t_IL-6 = 5.755, P = 0.0045), while the transcription levels of anti-inflammatory cytokines (TGF-β and IL-10) were significantly increased (t_TGF-β = 17.87, P < 0.0001; t_IL-10 = 4.882, P = 0.0081) (Fig 3C). ELISA results revealed that the expression levels of pro-inflammatory cytokines (TNF-α and IL-6) in intestinal lavage fluids of the rTsSPc-immunized mice were significantly lower than the infected PBS group (t_TNF-α = 16.70, t_IL-6 = 8.820, P < 0.0001), whereas the expression levels of anti-inflammatory cytokines (TGF-β and IL-10) were significantly higher than the infected PBS group (t_TGF-β = 10.19, t_IL-10 = 9.253, P < 0.0001) (Fig 3D). These results indicated that rTsSPc immunization significantly reduced the transcription and secretion levels of pro-inflammatory cytokines (TNF-α and IL-6), but increased expression of anti-inflammatory cytokines (TGF-β and IL-10), suggesting that rTsSPc immunization alleviated intestinal inflammation.

rTsSPc immunizations blocked the binding between parasite-derived TsSPc and gut epithelial RACK1 receptor

IFA results showed that at 7 dpi, TsSPc-specific green fluorescence was not detected in intestinal epithelium of the rTsSPc group. But green fluorescence was observed in both the ISA201 and PBS groups. Anti-RACK1 antibody identified the natural RACK1 receptor in intestinal epithelium as red fluorescence. After being merged, compared with the infected PBS group, the intensity of yellow fluorescence resulting from co-localization between rTsSPc and RACK1 in intestinal epithelium was significantly reduced in the rTsSPc group. Pearson correlation coefficients for the rTsSPc, ISA201, infected PBS and uninfected PBS groups were 0.357, 0.638, 0.734, and 0.227, respectively. Pearson correlation coefficient of the rTsSPc group was obviously lower than the infected PBS group (F = 33.17, P < 0.0001) (Fig 4A).

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Fig 4. rTsSPc immunizations blocked the binding of parasite-derived TsSPc and gut RACK1 receptor after infection.

A: IFA co-localization of TsSPc and RACK1 on gut epithelium. Small intestinal sections were incubated with anti-rTsSPc serum, followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. Intestinal sections were incubated with anti-RACK1 antibody and stained with Cy3-conjugated goat anti-rabbit IgG for detecting gut epithelial RACK1 receptors. Cellular nuclei were counterstained blue with DAPI. Scale bar: 100 μm. B: qPCR analysis of mRNA expression levels of RACK1. C: Western blot analysis of protein expression levels of RACK1 and p-ERK1/2/ERK1/2. *P < 0.05 compared to the uninfected PBS group; ^#P < 0.05 compared with the infected PBS group.

https://doi.org/10.1371/journal.pntd.0014161.g004

qPCR results showed that at 7 dpi, RACK1 transcription level in the rTsSPc group was obviously lower than the infected PBS group (t = 7.925, P < 0.0001), it was not statistically different from the uninfected PBS group (P > 0.05) (Fig 4B). Western blot analysis revealed that at 7 dpi, protein expression levels of RACK1 and phosphorylated ERK1/2 (p-ERK1/2) in the rTsSPc group were significantly lower than the infected PBS group (t_RACK1 = 3.897, P = 0.0046; t_{p-ERK1/2/ERK1/2} = 2.737, P = 0.0256), but they had no statistical difference from the uninfected PBS group (P > 0.05). Moreover, expression levels of RACK1 and p-ERK1/2 in ISA201 and infected PBS group were markedly elevated compared with the uninfected PBS group (F_RACK1 = 11.74, P = 0.0003; F_{p-ERK1/2/ERK1/2} = 12.01, P = 0.0002) (Fig 4C).

These findings suggested that anti-rTsSPc antibodies produced by rTsSPc immunization might neutralize the parasite-derived TsSPc and block its binding to gut epithelial RACK1 receptor, subsequently prevent the activation of MAPK/ERK1/2 pathway and destruction of gut epithelial integrity, and ultimately impede the parasite invasion.

Intestinal and muscle histopathological change in infected mice

Intestinal sections HE staining revealed that both ISA201 and infected PBS groups exhibited severe damage to intestinal villi with significant edema at 7 dpi. In contrast, the rTsSPc-immunized mice only showed mild inflammatory responses, with slight villous edema and relatively intact villous architecture. Compared to the infected PBS group, the villus width of the rTsSPc group was significantly reduced (F = 45.16, P < 0.0001) (Fig 5A). PAS staining showed that the number of goblet cells was distinctly increased and their volume became larger in the ISA201 and PBS groups, whereas the rTsSPc group exhibited a clear decrease of goblet cell numbers and smaller cell size. Compared to the infected PBS group, the number of goblet cells in the rTsSPc group was significantly reduced (F = 211.3, P < 0.0001) (Fig 5B). Furthermore, qPCR results showed that transcription levels of Muc2 and Muc5ac of the rTsSPc group were significantly lower than the infected PBS group (F_Muc2 = 259.2, F_Muc5ac = 58.07, P < 0.0001) (Fig 5C, 5D). These findings suggested that rTsSPc immunization effectively inhibited the T. spiralis larva invasion of intestinal mucosa, alleviated intestinal inflammation, and decreased mucin expression in intestinal mucosa.

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Fig 5. Histopathological changes in murine intestinal tissues at 7 days post infection.

A: HE staining of duodenum section under a microscope. The pathological alterations were significantly alleviated in the rTsSPc group compared to ISA201 and PBS control groups. In contrast, severe inflammation was observed in the intestinal mucosa of the ISA201 and PBS groups, as characterized by obvious villus shortening and edema. Intestinal villus width of each group was measured (n = 3). B: PAS staining of duodenum tissue sections under a microscope. Compared to the ISA201 and PBS control groups, the rTsSPc-immunized group exhibited a significant reduction of goblet cell number. C: Relative expression levels of Muc2 mRNA. D: Relative expression levels of Muc5ac mRNA. Scale bar = 200 μm. *P < 0.05 compared to the uninfected PBS group; ^#P < 0.05 compared with the infected PBS group.

https://doi.org/10.1371/journal.pntd.0014161.g005

At 35 dpi, muscle sectional HE staining revealed obvious inflammation around the encapsulated larvae in the diaphragm of the PBS and ISA201 groups, characterized by massive infiltration of inflammatory cells. However, inflammatory infiltration around the encapsulated larvae in the rTsSPc group was relieved (Fig 6A). Compared to the PBS group, the number of the larvae within collagen capsules of the rTsSPc group was significantly reduced (F = 23.11, P = 0.0015). The inflammatory infiltration surrounding collagen capsules in the rTsSPc group was also significantly lower than the PBS groups (F = 129.8, P < 0.0001).

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Fig 6. Histopathology of murine diaphragm, gut permeability and worm burdens in infected mice.

A: HE staining of diaphragm muscle sections under a microscope. The rTsSPc-immunized group exhibited mild inflammatory reactions and fewer larvae compared to the ISA201 and PBS control groups. Scale bar = 200 μm. Quantification of encapsulated muscle larvae (ML) and inflammatory infiltration around larval capsules was performed in various groups. B: gut permeability of various groups of mice after infection. The passage of FD-4 through the gut was significantly reduced in rTsSPc group compared to the infected PBS group following T. spiralis infection (n = 5). C: Intestinal adult worm burden (n = 10). D: Newborn larvae (NBL) production per female worm (n = 30). E: Muscle larval burden (LPG, n = 10). Data are expressed as mean ± SD. *P < 0.05 compared to the PBS group. ^# P < 0.05 compared to infected PBS group.

https://doi.org/10.1371/journal.pntd.0014161.g006

rTsSPc immunization decreased gut permeability and reduced worm burdens

To evaluate intestinal permeability following rTsSPc immunization, mice were challenged with 300 T. spiralis ML two weeks after the final immunization. At 7 dpi, mice were orally administered with FD-4, and plasma was collected to be ascertained 4 hours later. The results showed that compared to the infected PBS group, the FD-4 content in the rTsSPc group was significantly reduced by 40.43% (F = 15.66, P < 0.0001) (Fig 6B). These findings indicated that rTsSPc immunization significantly declined the gut permeability increased from T. spiralis infection, thereby enhancing intestinal mucosal epithelial integrity. This protective effect may be associated with the rTsSPc immunization to prevent larval invasion and reduce intestinal inflammation.

At 7 dpi, AWs were collected from the intestines of ten mice per group and enumerated. The results showed that compared to the PBS control group, intestinal adult burden in the rTsSPc group was significantly reduced, with a 65.7% reduction of adult burden (F = 46.24, P < 0.0001) (Fig 6C). To assess female fecundity, three female worms from each group were randomly selected and cultured at 37 °C and 5% CO₂ for 72 h, and the NBL were collected. The results revealed that the adult female reproductive capacity of the rTsSPc group was significantly lower than the PBS group, with a 61.13% reduction of female fecundity (F = 16.32, P < 0.0001) (Fig 6D). Furthermore, at 35 dpi, the muscle larval burden in the rTsSPc group was decreased with a reduction of 58.10% (F = 17.80, P < 0.0001) (Fig 6E). These findings demonstrated that rTsSPc immunization significantly reduced adult burden, hindered enteral worm growth and decreased female fecundity, thereby reduced muscle larval burden, indicating that rTsSPc vaccination elicited an obvious immune protection against T. spiralis infection.

Polarization of peritoneal macrophages in rTsSPc-immunized mice

Flow cytometry was performed to evaluate macrophage polarization of immunized murine. Peritoneal macrophages were identified based on F4/80⁺/CD11b⁺ expression, with CD86⁺ cells defined as the M1 phenotype and CD206⁺ cells defined as the M2 phenotype. The results showed that compared to the PBS group, the expression levels of both CD86⁺ and CD206⁺ in peritoneal macrophages from the rTsSPc group were significantly increased at 2 weeks post-immunization (F_CD86+ = 2581, F_CD206+ = 45.65, P < 0.001) (Fig 7A). However, compared to the PBS group, CD86⁺ expression of the rTsSPc group at 1 week post-infection was significantly decreased(F = 566.5; P < 0.0001), but CD206⁺ levels were obviously increased (F = 1260, P < 0.0001) (Fig 7B). The data indicated that peritoneal macrophages of the rTsSPc group exhibited a mixed M1/M2 polarization pattern at 2 weeks after final immunization, whereas the macrophages displayed a M2 polarization at 1 week after infection.

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Fig 7. Flow cytometry of murine peritoneal macrophages after rTsSPc immunization and larval infection.

A: Assessment of CD86 (M1) expression levels (n = 3). LPS (200 ng/ml) served as the positive control for M1 polarization. Macrophages were identified using F4/80⁺/CD11b⁺ markers. B: Assessment of CD206 (M2) expression levels. IL-4 (20 ng/ml) served as the positive control for M2 polarization. *P < 0.05 compared to the PBS group.

https://doi.org/10.1371/journal.pntd.0014161.g007

qPCR, ELISA and Western blotting of mRNA and protein expression level of M1/M2 factors in peritoneal macrophages

qPCR results revealed that compared to the PBS group, peritoneal macrophages of the rTsSPc group exhibited significantly increased mRNA expression levels of both M1- and M2-associated genes at two weeks post last immunization (M1: F_CD86 = 13.45, F_iNOS = 57.75, F_IL-6 = 75.18, F_TNF-α = 60.88, P < 0.01; M2: F_CD206 = 150.0, F_Arg1 = 152.2, F_IL-10 = 54.87, F_TGF-β = 36.66, P < 0.001). However, at one week post infection, the mRNA levels of M1-associated factors have a significant decrease compared to the PBS group (F_CD86 = 43.77, P = 0.0003; F_iNOS = 25.44, P = 0.0012; F_IL-6 = 11.17, P = 0.0095; F_TNF-α = 24.75, P = 0.0013), while the mRNA levels of M2-associated factors showed an obvious elevation (F_CD206 = 311.6, F_Arg1 = 420.7, F_IL-10 = 113.3, F_TGF-β = 549.2, P < 0.0001) (Fig 8A).

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Fig 8. qPCR, ELISA and Western blotting of mRNA and protein expression levels of M1/M2-related factors in murine peritoneal macrophages.

A: qPCR analysis of mRNA levels of M1 (CD86, iNOS, TNF-α and IL-6) and M2-related factors (CD206, Arg1, TGF-β and IL-10) in murine peritoneal macrophages (n = 3). B: ELISA measurement of macrophages-secreted cytokines (TNF-α, IL-6, TGF-β and IL-10) (n = 3). C: Western blotting of iNOS (M1) and Arg1 (M2) expression in murine peritoneal macrophages. iNOS and Arg1 protein expression levels of murine peritoneal macrophages were assayed at 2 weeks post immunization and 1 week after infection (n = 3). LPS (200 ng/ml) served as the M1 positive control, while IL-4 (20 ng/ml) served as the M2 positive control. *P < 0.05 compared to the PBS group or DMEM group.

https://doi.org/10.1371/journal.pntd.0014161.g008

ELISA results showed that compared with the PBS group, rTsSPc group exhibited significantly increased expression level of macrophage pro-inflammatory cytokines at 2 weeks after final immunization (F_IL-6 = 3565, F_TNF-α = 40.88, P < 0.0001); simultaneously anti-inflammatory cytokine expression level was also significantly increased (F_IL-10 = 409.1, F_TGF-β = 39.11, P < 0.0001). However, at 1 week post-infection, pro-inflammatory cytokine level of rTsSPc group was obviously declined compared to the PBS group (F_IL-6 = 255.6, F_TNF-α = 37.86, P < 0.0001), but anti-inflammatory cytokine levels were evidently elevated (F_IL-10 = 17.99, F_TGF-β = 51.86, P < 0.001) (Fig 8B).

Western blot results showed that compared to the PBS group, the expression levels of both iNOS and Arg1 in peritoneal macrophages from the rTsSPc group were significantly upregulated at 2 weeks post-immunization (F_iNOS = 19.97, P < 0.01; F_Arg1 = 6.094, P = 0.0359). However, at 1 week post-infection, iNOS expression was obviously decreased (F_iNOS = 7.661, P = 0.0223), Arg1 expression was significantly increased (F_Arg1 = 34.05, P < 0.001) (Fig 8C). No significant differences of iNOS and Arg1 expression between the PBS and ISA201 groups were observed at 2 weeks post-immunization and 1 week post-infection (P > 0.05).

These findings further confirmed that peritoneal macrophages from rTsSPc-immunized mice exhibited a mixed M1/M2 polarization phenotype at 2 weeks after immunization, but the macrophages displayed a M2 polarization at 1 week after infection.

ADCC killing NBL

Anti-rTsSPc antibodies mediated the adhesion of PECs to the NBL and induced larval damage and death (Fig 9A). When 1:100 dilutions of anti-rTsSPc sera were co-cultured with normal murine PECs and NBL for 72 h, ADCC efficiently killed the NBL with a 81.68% cytotoxicity, which was significantly higher than the sera from the PBS group (F = 258.7, P < 0.0001) (Fig 9B). This cytotoxic effect exhibited a dose-dependent on immune serum dilution (r = 0.964, P < 0.0001). Furthermore, the cytotoxicity was increased gradually with prolonged culture time (F_24h = 13.03, P = 0.0019; F_48h = 65.26, F_72h = 302.8, P < 0.0001) (Fig 9C).

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Fig 9. Anti-rTsSPc antibodies mediated ADCC of various macrophages killing NBL.

A: Anti-rTsSPc serum mediated PECs adhesion to NBL and subsequent destruction of NBL. One hundred NBL were co-cultured for 72 h with normal murine PECs (3 × 10⁵) and different dilutions of anti-rTsSPc serum. Infection serum served as the positive control, while sera from ISA201 and PBS groups served as negative controls. B: Cytotoxicity was dependent on dose of anti-rTsSPc antibodies. C: Cytotoxicity was dependent on the culture time. D: Cytotoxicity of various groups of murine macrophage killing NBL via ADCC. One hundred newborn larvae were co-cultured for 48 h with peritoneal macrophages and immune sera collected from different groups of mice at 2 weeks post vaccination. When anti-rTsSPc serum or infection serum was used, macrophage cytotoxicity of the rTsSPc group was also significant higher than the PBS and ISA 201 groups. Scale bar = 50 μm. *P < 0.05 compared to the PBS group.

https://doi.org/10.1371/journal.pntd.0014161.g009

When various macrophages from three groups of mice at 2 weeks post immunization were co-cultured with the NBL and 1:100 dilutions of anti-rTsSPc sera for 48 h, the macrophages’ cytotoxicity of the rTsSPc, ISA201 and PBS was 64.67, 40.00 and 41.33%, respectively (F = 18.69, P = 0.0026); the cytotoxicity of macrophages from the rTsSPc group was obviously higher than the PBS group (t= 4.709, P = 0.0092) (Fig 9D). When the macrophages from three groups of mice were co-cultured with NBL and 1:100 dilutions of infection serum, the cytotoxicity were 54.67, 39.33 and 43.0%, respectively (F = 10.07, P = 0.0121). The infection serum-mediated cytotoxicity of macrophages from the rTsSPc group was also significant higher than the PBS group (t = 3.127, P = 0.0353). The results indicated that rTsSPc immunization obviously enhanced murine macrophages’ ADCC killing newborn larvae.

Additionally, the raw data of Figs 1-9 in this study were shown in S1 Table.