Female BALB/c mice, 6–8 week-old and free of specific pathogens, were obtained from Laboratory Animal Services Center of Capital Medical University (Beijing, China). All mice were maintained under specific pathogen-free conditions at an appropriate temperature and humidity. Experimental procedures were consistent in strict with the NIH Guide for the Care and Use of Laboratory Animals, that were carried out in accordance with the protocols approved by the Capital Medical University Animal Care and Use Committee (Permission No: 2012-X-108).

The DNA encoding full-length TsPmy (GenBank ID: EF429310) was cloned into the eukaryotic expression vector pVAX1, and the recombinant pVAX1-TsPmy plasmid DNA was transformed into an attenuated S. typhimurium (SL7207) which was aroA mutant strain with 2337–2365 derivative hisG46 DEL407 (aroA::Tn10 {Tc-s}) by electroporation (2.5 kV, 25 μF, and 200 Ω) as an oral DNA vaccine (SL7207/pVAX1-TsPmy), the transformants were selected on plates with 50 μg/ml kanamycin and identified by PCR amplification and restriction enzyme digestion. The PCR products were then confirmed by DNA sequencing as previous described (Wang et al., 2016).

DNA encoding TsPmy without signal peptide was cloned into the expression vector pET-28a (+). The recombinant TsPmy protein (rTsPmy) was expressed and identified in an Escherichia coli (BL21) expression system in our laboratory. The rTsPmy with a His-tag at the C terminus was expressed in E. coli under 1 mM IPTG induction and purified with Ni-affinity chromatography (Qiagen, United States) as previously described (Yang et al., 2008).

Trichinella spiralis (ISS 533) parasites used in this study were originally isolated from an infected pig in Heilongjiang, China and maintained by serial passage in female ICR mice in our laboratory (Cui and Wang, 2001). The adult worms from the small intestines of infected mice were isolated 5 days post-infection (dpi). T. spiralis muscle larvae (ML) from infected mice were recovered at 42 dpi via a previously described pepsin-hydrochloric acid digestion method (Gamble et al., 2000).

BALB/c mice were randomly divided into six groups and each group was composed of 40 animals. For the heterologous prime-boost group (DNA+Protein), each mouse was inoculated orally with 1 × 10⁸ attenuated S. typhimurium SL7207 containing pVAX1-TsPmy (SL7207/pVAX1-TsPmy) as previously described (Wang et al., 2016) and then boosted intramuscularly twice with 25 μg rTsPmy emulsified with an equal volume of the water-in-oil adjuvant ISA 50V2 (SEPPIC, France) at 2-weeks interval. For DNA or recombinant protein vaccination groups, mice were immunized either orally with 1 × 10⁸ bacteria of SL7207/pVAX1-TsPmy (DNA) or intramuscularly with 25 μg of rTsPmy formulated with ISA 50V2 (Protein) for three times at 2-weeks interval. The other three control groups were given with either the same number bacteria with empty vector of SL7207/pVAX1 orally (Vector), or PBS alone (emulsified with ISA 50V) intramuscularly for three times, or first oral vector followed with two PBS intramuscularly to mimic heterologous prime-boost regimen (Vector+PBS) as shown in Table 1.

Before immunization and 1 week after each immunization, five mice from each group were sacrificed; the lavage fluid of intestines, serum, mesenteric lymph nodes (MLNs), and spleen were collected from each mouse to examine the levels of mucosal, humoral and cellular immune responses, as previously described (Wang et al., 2016). All animals were daily monitored for general appearance, abnormal locomotion, eye abnormality, labored breathing, head tilt, hyperactivity, lethargy, paresis, lameness, paralysis, ruffled fur, and tremors. Mice would be euthanized immediately by CO₂ inhalation if there was any severe sign of abnormality including bleeding diarrhea, leg injuries or in moribund condition.

The levels of TsPmy-specific total IgG, IgG1, and IgG2a antibodies in the sera of the immunized mice were detected via a modified ELISA using rTsPmy coated on plates as described previously (Yang et al., 2010). Briefly, microtiter plates were coated with rTsPmy (1 μg/ml) at 4°C overnight. Then, 100 μl of sera samples diluted in PBS at 1:1000 was added to each well. HRP-conjugated goat-anti-mouse IgG, IgG1, or IgG2a antibodies were added as secondary antibodies. 3,3′,5,5′-tetramethylbenzidine was used as a substrate and the reactions were stopped with 50 μl per-well of 2 M H₂SO₄. Absorbance at 450 nm was measured with a microplate reader. All samples were run in triplicate.

To evaluate the secreted IgA (sIgA) level, the 10 cm interior small intestine from each sacrificed mouse was washed twice with 2 ml of cold PBS. After centrifugation at 800 × g for 10 min, the supernatants were harvested. Intestinal total sIgA was quantified with sandwich-type ELISA using rat anti-mouse IgA antibody as the capture antibody. The specific TsPmy-specific sIgA in intestinal lavage fluid of each mouse was measured by standard ELISA using rTsPmy as antigen coated on plates, respectively, as described previously (Yang et al., 2010; Wang et al., 2016).

To examine the specific cellular immune responses against rTsPmy, the cytokine profiles in splenocytes and MLN cells from immunized mice were analyzed upon re-stimulation with the rTsPmy antigen in vitro using a modified ELISPOT assay (BD Bioscience, United States). In brief, spleen and the MLN cells were isolated aseptically from mice 7 days after each immunization and the single lymphocyte suspensions were prepared in RPMI-1640. 1 × 10⁶ lymphocytes were added to each well of plates pre-coated with the capture antibody (anti-mouse IFN-γ, IL-2, IL-4, IL-6, and IL-10) at 1: 200 dilutions in PBS and incubated at 4°C overnight. After stimulation with 1 μg/ml of rTsPmy for 48 h, the biotinylated secondary antibodies were added for 2 h, then incubated with streptavidin-HRP for 1 h and developed with 100 μL of 3-amino-9-ethylcarbazole substrate solution (BD Biosciences, United States) for 30 s–5 min. The spots secreted with IFN-γ, IL-2, IL-4, IL-6, and IL-10 were counted as spot forming units (SFUs) with a CTL ELISPOT reader and analyzed using ImmunoSpot image analyzer software v4.0.

Two weeks after the final boost, the remaining 20 mice in each group were orally challenged with 500 T. spiralis ML per mouse. Ten mice from each group were euthanized 5 days after challenge and the adult worms were collected and counted. The left 10 mice were euthanized 42 days after challenge infection and the muscle larva were collected and counted. The adult worm and muscle larvae reduction in immunized mouse groups was calculated compared with those from the PBS control mice.

One-way ANOVA was used for data comparison among different groups with SPSS version 17.0 software and expressed as the means ± standard deviation (SD), p < 0.05 was regarded as statistically significant.

Mouse sera collected 1 week after each immunization were used to determine the antibody levels of anti-TsPmy IgG and its subtype (IgG1 and IgG2a). The anti-TsPmy specific IgG was detected in sera of mice immunized with DNA+Protein, DNA, or Protein alone after first immunization compare to the three empty control groups. The anti-TsPmy IgG reached the highest titers after three immunizations. Specifically, mice immunized with protein prime-boost (Protein) produced significant high titers of anti-TsPmy IgG after the first immunization and remained the plateau after two boosts. The anti-TsPmy IgG level in mice of DNA+Protein prime-boost group was significantly increased after first boost with rTsPmy and reached to even higher titer than mice in Protein prime-boost group after three immunizations. The mice immunized with DNA prime-boost produced lower anti-TsPmy IgG titer than other two vaccine groups after three immunizations. However, none of the mice in control groups received Vector+PBS, Vector or PBS alone showed any detectable TsPmy specific IgG response (Figure 1A). Similarly, after three immunizations, mice immunized with DNA+Protein produced highest anti-TsPmy IgG1 and IgG2a compared to DNA or Protein homologous prime-boost groups and the IgG1 and IgG2a level was proximately equal, indicating balanced Th1/Th2 immune responses were induced in heterologous prime-boost immunization. Mice immunized with Protein prime-boost produced higher IgG1 than IgG2a, indicating predominant Th2 responses. Mice immunized with DNA alone produced lowest anti-TsPmy IgG1 and IgG2a compared to other two vaccine groups, with IgG2a predominant (Figure 1B).

The intestinal total sIgA were measured by a sandwich ELISA and anti-TsPmy specific sIgA were evaluated using an rTsPmy coated plate in intestinal mucosal washings. Intestinal total sIgA level was significantly increased in the intestinal mucosa of mice immunized with DNA+Protein, DNA, and Vector alone compared to other groups after second immunization. Mice immunized with DNA+Protein (only one immunization of oral DNA) produced lower total sIgA compared to mice immunized with three immunizations of DNA or Vector alone, indicating that the total sIgA was induced mostly by attenuated Salmonella bacteria (Figure 2A). The anti-TsPmy specific sIgA was detected only in mice immunized with DNA and DNA+Protein. Mice immunized with once oral DNA followed by two boosts of protein (DNA+Protein) elicited the similar level of antigen-specific sIgA as mice immunized with oral DNA for three times (DNA) without significant difference. Mice immunized with protein (intramuscular) or empty vector did not induce any anti-TsPmy sIgA response (Figure 2B).

The cytokine responses including IFN-γ, IL-2, IL-4, IL-5, IL-6, and IL-10 in splenocytes (Figure 3) and MLN cells (Figure 4) were measured by ELISPOT 1 week after each immunization. All of the cytokines were significantly increased after the first immunization and reached the highest after three immunizations in mice immunized with DNA+Protein, DNA, or Protein alone in both splenocytes and MLN cells upon re-stimulation of rTsPmy. In the systematic splenocyte responses, mice immunized with DNA+Protein produced significant higher Th1 cytokines including IFN-γ and IL-2 than mice immunized with DNA or Protein alone, similar levels of Th2 cytokines (IL-4, IL-5, IL-6, and IL-10) to the mice immunized with recombinant protein (Protein). Even though mice immunized with DNA prime-boost produced significant level of Th1 cytokines (IFN-γ and IL-2), the level of Th2 cytokines (IL-4, IL-5, IL-6, and IL-10) were much lower than groups immunized with DNA+Protein or Protein alone (Figure 3). The similar pattern of cytokine profile was observed in the local MLN cell responses to the re-stimulation of rTsPmy, with high Th1 cytokine responses (IFN-γ and IL-2) in mice immunized with DNA prime-boost, high Th2 cytokine (IL-4, IL-5, IL-6, and IL-10) responses in mice immunized with Protein prime-boost, and with highest Th1/Th2 cytokine responses for mice immunized with DNA+Protein prime-boost (Figure 4). There was no significant cytokine response in both splenocytes and MLN cells from empty control mice. These results suggest that mice immunized with TsPmy using heterologous prime-boost immunization regimen induced highest Th1 and Th2 cytokine responses in both systemic lymphocytes (spleen) and local lymphocytes around the intestine (MLNs).

Two weeks after final immunization with TsPmy in different regimens, six groups of mice were challenged orally with 500 ML per mouse. The worm burden results reveal that mice immunized with DNA+Protein prime-boost, DNA alone and Protein alone prime-boost produced 41.8, 44.8, and 10.1%, respectively, adult worm reduction compared with mice received PBS control (Figure 5A). Mice immunized with DNA+Protein and DNA alone prime-boost induced significant higher adult worm reduction than mice immunized with Protein alone prime-boost. Muscle larvae collection 42 days after challenge demonstrated that mice immunized with DNA+Protein prime-boost, DNA and Protein alone prime-boost produced 55.4, 46.6, or 36.6% muscle larvae reduction, respectively, compared to mice received PBS control (p < 0.01) (Figure 5B). The number of adult worms and ML collected from each group of mice was shown in Supplementary Table 1. The statistical analysis shows that mice immunized with TsPmy in DNA/protein heterologous prime-boost regimen produced significant higher larvae reduction than mice in groups of DNA prime-boost (p < 0.05) and protein prime-boost (p < 0.05), mice immunized with TsPmy in DNA prime-boost regimen produced higher larvae reduction than mice in protein prime-boost (p < 0.05). These adult and muscle larvae reduction results indicate that TsPmy immunization in DNA/protein heterologous prime-boost regimen induced significantly higher protective immunity than the DNA or protein homologous prime-boost regimens against T. spiralis infection in BALB/c mice, especially at the larvae reduction level.