The recombinant plasmids containing P29 used in this study were stored in our laboratory. The expression and purification of rEg.P29 were performed according to our previously published method (16). Briefly, the strains were inoculated in LB liquid medium containing 0.1 mM isopropyl β-d-thiogalactoside (IPTG, Invitrogen, Waltham, USA) and incubated at 37°C for 10h. Then, rEg.P29 purification was performed according to the manufacturer’s instructions of the histidine-tagged protein purification kit (Merck, Kenilworth, USA). Endotoxin was removed from the antigen using an Endotoxin Removal Kit (Genscript, Nanjing, China). The purified rEg.P29 with endotoxin removed was identified by SDS-PAGE and the antigen concentration was determined using a BCA kit (KeyGenBiotech, Nanjing, China).

Thirty-six 4–6-month-old female Chinese Yan chi Tan sheep were randomly selected, and individual information is presented in Supplementary Table 1 . Sheep were randomly divided into four groups of nine animals each, and each group was immunized according to three subcutaneous points ( Supplementary Figure 1 ): the PBS control group was injected with 1 mL sterile PBS, the Quil A adjuvant control group was injected with 1 mg Quil A adjuvant solution, the rEg.P29 immunization group was injected with 50 μg rEg.P29, and the rEg.P29+Quil A immunization group was injected 50 μg rEg.P29 + 1 mg Quil A adjuvant. We conducted primary and booster immunizations at weeks 0 and 4, respectively.

Peripheral blood was collected via the jugular vein at weeks 0, 4, 6, 8, 12 and 20, respectively. Sheep were euthanized at week 20 and spleens and mesenteric lymph nodes were collected for testing. Serum and peripheral blood mononuclear cells (PBMCs) were separated according to our previously published method (17). Briefly, anticoagulated peripheral blood was obtained by centrifugation at 1000 g for 10 min followed by aspiration of the upper layer. PBMCs were obtained by density gradient centrifugation, but without the buffy-coat stage. Whole blood was diluted 1:1 with PBS, mixed, and spread over an equal volume of lymphocyte isolate (Lymphoprep 1.087 g/L, TBD Science, Tianjin, China), centrifuged at 1130 g for 30 min at 22°C without brakes, and the white PBMCs layer was collected and washed twice. Sheep were euthanized by intravenous injection of 100 mg/kg potassium chloride under sedation with 20 mg/kg propofol intravenously. The spleen and mesenteric lymph nodes were harvested after euthanasia of the sheep, and the corresponding lymphocytes were isolated and obtained separately. The tissue was ground using a syringe piston and filtered through a 70-micron filter. The subsequent separation of lymphocytes was performed in the same way as for PBMCs. Cells were counted and then adjusted to 1×10⁶/mL using RPMI-1640 medium (GIBCO, Grand Island, USA) containing 10% fetal bovine serum (FBS), 2mM l-glutamine, 50 µM beta-mercaptoethanol 100 IU/mL penicillin, and 50 μg/mL streptomycin (Sigma-Aldrich, Saint Louis, USA) (18).

To determine the cellular phenotypes of PBMCs, cells were directly stained with mouse anti-sheep CD4 (Clone: 44.38) and mouse anti-sheep CD8 (Clone: 38.65) antibodies (Bio-Rad AbD Serotec, Munich, USA), and incubated at 4°C for 30 min in the dark, followed by two washes with washing buffer. For intracellular cytokine assays, cells were incubated with or without rEg.P29 (10 μg/mL), and both had anti-CD28 (1 μg/mL) added at 37°C and 5% CO₂ for 24 h. BFA (10 μg/mL) was added 6 h before the end of incubation. The cells were collected and washed twice with PBS containing 0.1% bovine serum albumin (BSA), blocked with 20% normal mouse serum, and stained with mouse anti-sheep CD4 (Clone: 44.38) and mouse anti-sheep CD8 (Clone: 38.65), and incubated at 4°C for 30 min in the dark, followed by two washes. Cells were fixed with 4% paraformaldehyde, washed twice, and permeabilized with PBS containing 0.1% BSA and 0.1% saponin overnight at 4°C. Cells were stained with mouse anti-bovine IFN-γ (Clone : CC302; Bio-Rad AbD Serotec, Munich, USA), mouse anti-bovine IL-4 (Clone : CC303; Bio-Rad AbD Serotec, Munich, USA), and mouse anti-human IL-17A(Clone : MT504; Mabtech, Nacka, Sweden) (three antibodies cross-reacted with sheep) at 4°C for 30 min in the dark. After cell washing, data were acquired using a BD FACSCelesta flow cytometer (Becton Dickinson, USA), and data analysis was performed using FlowJo software (Becton Dickinson, USA). Data were analyzed using fluorescence minus one (FMO) control for the gating strategy.

Cells were adjusted to 1×10⁶/mL using RPMI-1640 medium. We used a bovine IFN-γ/IL-4/IL-17A ELISPOTPLUS kit (Mabtech, Nacka, Sweden) for detecting intracellular factor production according to the manufacturer’s instructions. ELISPOT plates were closed with RPMI-1640 medium containing 10% FBS, and 200 µL of cell suspension was added to each well with or without rEg.P29 (10 μg/mL) in the presence of anti-CD28 (1 μg/mL), then plates were incubated at 37°C with 5% CO₂ for 24 h. After discarding the cell suspension, the plates were washed with PBS and the biotinylated detection antibody was added followed by Streptavidin-HRP. Finally, the plates were developed using TMB substrate and stopped using deionized water. The number of spots was read on an AID ELISPOT Reader Classic (Autoimmun Diagnostika Gmbh, Straßberg, Germany). Each group was set up in triplicate wells and the results are shown as their mean values.

Cells were adjusted to 2×10⁶/mL using RPMI-1640 medium, and 200 µL of cell suspension was added to each well of a 96-well round bottom plate with or without rEg.P29 (10 μg/mL) in the presence of anti-CD28 (1 μg/mL) (19, 20). Plates were incubated at 37°C with 5% CO₂ for 96 h, and the cytokine in the cell culture supernatant was quantified using the Bovine IFN-γ/IL-4 ELISA kit and Sheep IL-17A ELISA kit (Mabtech, Nacka, Sweden) according to the manufacturer’s instructions. The optical density of the plates was read at 450 nm using a Multiskan SkyHigh Microporous plate spectrophotometer (Thermo Fisher, Waltham, USA), and standard curves were generated to calculate cytokine concentrations.

The carboxyfluorescein diacetate succinimidyl ester (CFSE; Thermo Fisher, Waltham, USA) method was used to evaluate cell proliferation. CFSE was prepared using room temperature PBS to a concentration of 5μM, and PBMCs (1×10⁷/mL) were labeled with CFSE and incubated at 37°C with 5% CO₂ for 15 min. The reaction was terminated by adding pre-cooled medium containing 10% FBS at 4°C for 5 min. Cells were washed with cold PBS and incubated with or without rEg.P29 (10 μg/mL) at 37°C with 5% CO₂ for 96 h. Cells were collected and labeled with antibodies, and data were collected using a BD FACSCelesta flow cytometer and analyzed using FlowJo software.

All results were analyzed using SPSS 22.0 (GraphPad Software Inc., San Diego, USA) and GraphPad Prism 8.0 (IBM Corp., Armonk, NY, USA). For comparing two groups, Mann-Whitney test was used, and one-way ANOVA was used for comparing three or more groups. Data are shown as mean or mean ± standard deviation (SD). Differences were considered statistically significant when P < 0.05.

Peripheral blood samples were collected from each immunization group at different immunization times, and CD4⁺ and CD8⁺ T cells in PBMCs were analyzed using flow cytometry. Consistent with other animals, CD4⁺ and CD8⁺ T cells were relatively stable, and the percentage of CD4⁺ T cells was significantly higher than that of CD8⁺ T cells ( Figure 1A ). After immunization with rEg.P29 supplemented with adjuvant, there was a tendency for CD4⁺ T cells and CD4⁺/CD8⁺ T cells to increase compared with other immunization groups, especially CD4⁺ T cells, but none of these results were statistically significant ( Figures 1B, D, E ). rEg.P29 had no effect on CD4⁺ and CD8⁺ T cells in PBMCs, but the trend indicates that it has the potential to enhance immunity.

ELISPOT assays can sensitively detect cells expressing specific cytokines, and to observe vaccine-induced specific cytokines, we first measured cells secreting specific cytokines IFN-γ, IL-4, and IL-17A under stimulation with rEg.P29, and the results were shown as spot-forming cells (SFC). The number of cells secreting IFN-γ was significantly higher in the rEg.P29+Quil A group compared to the other immunization groups, and we found that the rEg.P29 immunization group also produced a number of IFN-γ-secreting cells, but without statistical differences ( Figures 2A, B ). In the IL-17A ELISPOT assay, the number of IL-17A-secreting cells in the rEg.P29+Quil A group was also significantly higher than in the other groups ( Figures 2C, D ). The IL-4 ELISPOT assay showed that IL-4-secreting cells were not found in any group (Data not presented).

We also measured the levels of these three antigen-specific cytokines in the cell culture supernatants of each group at different immunization times using an ELISA. No IFN-γ was produced in the PBS, Quil A, or rEg.P29 groups, while a high level of IFN-γ was produced in the cell supernatant of the rEg.P29+Quil A group, which was significantly higher than the other three immunization groups ( Figure 3A ). The time-varying results of specific IFN-γ showed that IFN-γ gradually increased in the rEg.P29+QuilA group, peaking at week 8, and then gradually decreased, but remained significantly higher than other groups, where specific IFN-γ production was consistently not detected ( Figure 3B ). The ELISA results and temporal patterns of the specific cytokine IL-17A in culture supernatants were similar to those of IFN-γ. Relatively high levels of IL-17A were produced only in the rEg.P29+QuilA group, but not in the other groups ( Figures 3C, D ). The specific cytokine IL-4 was consistently not detected in the cell culture supernatant of all groups (Data not presented).

Sheep were euthanized and the results of the three cytokine levels in the culture supernatants of lymphocytes isolated from the spleen and mesenteric lymph nodes were consistent with the ELISA results of PBMCs, with both indicating that rEg.P29 induced the production of IFN-γ and IL-17A ( Figure 4 ), but not IL-4 (Data not presented). The results of ELISPOT and ELISA showed that rEg.P29 induced the production of the antigen-specific cytokines IFN-γ and IL-17A in sheep to protect against E.granulosus infection.

ELISPOTs and ELISAs are capable of highly sensitive, quantitative detection of cytokines; however, they cannot determine which population of cells produced the cytokines. To further identify which group of cells produced IFN-γ and IL-17A, cells were stimulated in vitro by antigen, and the cytokine expression in CD4⁺ and CD8⁺ T cells was detected using flow cytometry. The expression of IFN-γ in both CD4⁺ and CD8⁺ T cells was significantly higher in PBMCs of the rEg.P29+QuilA group after antigen stimulation than in the other groups, and the level of IFN-γ in CD4⁺ T cells was higher than that in CD8⁺ T cells ( Figures 5A, B, D ). Moreover, the cytokine IFN-γ in CD4⁺ and CD8⁺ T cells gradually increased over time and peaked at week 8, after which it gradually decreased but remained at a high level, always significantly higher than the other groups ( Figures 5C, E ). High levels of antigen-specific IL-17A expression were also detected in CD4⁺ T cells, but IL-17A was not produced in CD8⁺ T cells ( Figures 5F, G, I, J ). In addition, the level of IL-17A in CD4⁺ T cells changed over time in a manner consistent with that of IFN-γ in CD4⁺ T cells ( Figure 5H ).

We also examined cytokine expression in CD4⁺ and CD8⁺ T cells after in vitro culturing of lymphocytes from the spleen and mesenteric lymph nodes stimulated with rEg.P29. The findings were consistent with those of PBMCs which expressed the specific cytokines IFN-γ and IL-17A in CD4⁺T cells, and IFN-γ in CD8⁺T cells ( Figures 6A–C , 7A–C , 6D–F , 7D–F ). For the IL-4-expressing cell population, none were detected in lymphocytes from PBMCs, the spleen, or the mesenteric lymph nodes, which is consistent with the ELISPOT and ELISA results (Data not presented). These results strongly suggest that rEg.P29 supplemented with adjuvant-induced sustained Th1, Tc1, and Th17 cellular immune responses in sheep.

PBMCs from each group were labeled with CFSE, stimulated in vitro with or without rEg.P29, and CD4⁺ and CD8⁺ T cell proliferation was assessed by detecting the decrease in CFSE fluorescence intensity of labeled cells using flow cytometry. The CFSE intensity of lymphocytes, CD4⁺ and CD8⁺ T cells in the rEg.P29+QuilA group decreased in the presence of rEg.P29, indicating that significant cell proliferation occurred; however, no cell proliferation occurred in the absence of rEg.P29 ( Figure 8 ). Moreover, the proliferation of CD8⁺ T cells stimulated by rEg.P29 was higher than that of CD4⁺ T cells ( Figures 8C, D ).