The PEDV S1 gene was amplified from the genome of PEDV QY-2016 strain (GenBank ID: MH244927) preserved in our laboratory by PCR using primers S1-F and S1-R ( Table 1 ). The DNA sequence of the PA gene based on the peptidoglycan hydrolase AcmA (GenBank ID: U17696) was synthesized by Sangon Biotech (Shanghai, China). The S1-PA fusion protein gene S1-PA was obtained using overlap extension PCR (OE-PCR) by splicing the segments of S1 gene and PA gene through a flexible linker (GGSG). All the primers in this study were synthesized by Sangon Biotech (Shanghai, China). To obtain the recombinant expression plasmid pCold1-S1 or pCold1-S1-PA, the gene S1 and the fusion protein gene S1-PA were digested with EcoR I and Sal I, respectively, and then cloned into the pCold 1 vector carrying a 6×his tag (Takara Bio, #3360) also digested with the above restriction endonucleases using T4 ligase. Finally, the single clone carrying S1 or S1-PA gene was verified by PCR and double digestion with EcoR I and Sal I, and then was sequenced by Sangon Biotech (Shanghai).

The recombinant plasmids pCold1-S1 and pCold1-S1-PA were transformed into competent E. coli BL21 (DE3) cells (Tiangen Biotech, #CB105) using the heat shock method. The transformed E. coli cells were inoculated on Luria Broth (Amp⁺/LB) Agar plates containing 50 μg/mL ampicillin and cultured at 37°C for 18 h. The single colony containing the target gene was selected and inoculated into Amp⁺/LB broth for culture at 37°C. When the OD_{600 nm} value of the bacterial culture reached 0.6-0.8, the culture was moved to 4°C for 1 h. After adding 0.5 mmol/L isopropyl-β-d-thiogalactoside (IPTG), the bacteria were cultured at 16°C for 24 h to induce the expression of recombinant proteins. The bacterial cells were harvested and re-suspended in PBS (pH 7.4), followed by sonication in an ice bath, the supernatant was collected for SDS-PAGE analysis after centrifugation at 10,000 r/min for 10 min. The expressed proteins were purified using the HisTrap™ HP (GE company, #17524801) on the AKTA protein purification system (GE company, USA), and confirmed by Western blotting. The protein concentration was measured using the BCA protein assay kit (Takara Bio, #T9300A).

SDS-PAGE was used to analyze the expression and purification of S1 and S1-PA proteins, as well as the proteins bound to BLPs. S1 protein, S1-PA and BLPs-bound S1 protein were identified by Western blotting. Briefly, the recombinant protein was transferred to a polyvinylidene difluoride (PVDF) membrane following SDS-PAGE, and blocked overnight at 4°C. The membrane was incubated with anti-His-tagged mouse monoclonal antibody (1:5000; Cowin Biotech, #CW0285) or Rabbit anti-S1 protein polyclonal antibody (1:500; prepared and stored in our lab) at room temperature (RT) for 1 h. After washing, the membrane was incubated with 1:5000 diluted HRP-conjugated goat anti-mouse/-rabbit antibody (Solarbio Tech, #SE131, #SE134) at RT for 1 h. After washing, the membrane was incubated in the DAB chromogenic solution for color development, and the results were observed directly.

Lactococcus (L.) lactis MG1363 was cultured in GM17 broth (Hope Bio, #HB0391) at 30°C for 24 h with shaking. Bacterial cells were harvested by centrifugation at 8,000 r/min for 15 min, washed twice with sterile PBS, re-suspended in 25 mM sulfuric acid, and heated at 100°C for 30 min. The treated L. lactis (GEM) particles were centrifuged, and the pellet was washed three times with PBS. Finally, the GEM particles (GEMs) were re-suspended in PBS at a concentration of 2.5×10⁹ particles/mL, which was referred to as 1 U (28). The GEM particles were directly used for the preparation of S1-BLPs or stored at -20°C.

To prepare S1-BLPs, the GEM particles (2.5×10⁹ particles/mL) were mixed with 6.25 mg of the fusion protein S1-PA followed by incubation at RT for 30 min with shaking. The GEM particles bound to S1 protein via PA, designated S1-BLPs, were collected by centrifugation at 6,000 r/min for 5 min, washed 3 times with PBS, and re-suspended in PBS. To confirm the binding of the fusion protein S1-PA to the GEM particles, S1-BLPs was treated with 10% SDS at 100°C for 10 min to observe whether S1-PA was dissociated from the GEM particles. GEM particles control was set up at the same time. After centrifugation at 10,000 r/min for 2 min, the supernatant was collected and analyzed by SDS-PAGE to confirm the presence of S1-PA. Meanwhile, BCA protein assay kit was used to determine the total S1-PA protein concentration in the supernatant, and using the following formula to calculate the amount of protein bound by GEMs per unit (2.5×10⁹ particles/mL). Amounts of GEMs binding S1-PA protein per unit (μg) = (Total protein amounts in per unit of S1-BLP supernatant) - (Total protein amounts in per unit of GEM supernatant).

The samples were dropped onto the copper grid, negatively stained with 2% phosphotungstic acid, and vacuum dried. The samples were examined on a transmission electron microscope (TEM) (JEM1400, JEOL, Tokyo, Japan), which was operated at 80 kV and equipped with An AMT Camera. The particle sizes were measured using Image J.

S1-BLPs was evenly spread on the polylysine coated slides, air-dried, and blocked with 3% BSA in PBS at RT for 30 min. After washing twice with PBS, the slides were incubated with anti-PEDV S1 protein rabbit polyclonal antibody (1:100) at RT for 60 min. Meanwhile, the anti-S1 protein antibody negative rabbit serum control was set up. After washing 3 times with PBS, the slides were incubated with FITC-labeled goat anti-rabbit IgG (1:200, Solarbio Tech, # SF134) for 60 min at RT in the dark. The slides were washed three times with PBS, and the fluorescence was observed under an Axio Observer D1 fluorescence microscope (ZEISS, Gottingen, Germany).

The S1 protein or S1-BLPs were mixed with an equal volume of Montanide™ IMS1313N VG (IMS1313 for short) water adjuvant (Seppic, Paris, France). Forty specific-pathogen-free (SPF) female BALB/c mice (Changsheng Biotech, Liaoning, China) aged 6-8 weeks were randomly divided into 5 groups (S1/IM; S1-BLPs/IM, S1/IN, S1-BLPs/IN, and Blank group) with 8 mice in each group. Each mouse in S1/IM and S1/IN groups was immunized three times by intramuscular (IM) and intranasal inoculation (IN) with a dose 80 μL contained 40 μg of S1 protein at 2-week intervals, respectively. And each one in BLPs IM and BLPs IN groups was immunized with S1-BLPs containing 40 μg S1 protein by the same routes as above, respectively. In the blank group, mice were not immunization. Blood samples were collected from the tail vein of mice weekly before and after immunization, and serum collection was conducted and stored at -20°C for subsequent tests. At 14 days after the second immunization and 21 days after the third immunization, 4 mice were randomly selected from each group and anesthetized by intraperitoneal injection of 10% chloral hydrate (0.1 mL/10g body weight), and then intestine and lung airway lavage fluid were collected for specific secretory IgA detection.

PEDV S1-specific IgG, IgA, IgG1, IgG2a and IgG2b antibodies in serum were measured by ELISA, and IgG titer was determined on day 21 after the third immunization. Moreover, the ratio of IgG2a/IgG1 in serum of S1/IM and S1-BLPs/IM groups was analyzed. Briefly, 96-well ELISA plates (Biofil, #FEP101896) were coated with PEDV S1 protein (2 μg/well) diluted with Coating buffer (0.1 M carbonate buffer, pH 9.0) at 37°C for 1 h and overnight at 4°C. The plates were blocked with Blocking buffer (5% skim milk powder in PBST) at 37°C for 1 h. After washing 3 times with PBST (0.05% Tween-20 in phosphate-buffered saline (PBS), pH 7.4), 100 μL of samples (serum, or intestine or lung airway lavage fluid), or 4-fold serially diluted (1:100 to 1:204 800) serum were added in the wells of the plate to incubate at 37°C for 60 min. At the same time, positive, negative, and blank controls were set up. After washing 3 times, 100 μL of HRP-conjugated goat anti-mouse IgG (1:15000, Biodragon Tech, #BF03001) or IgA/IgG1/IgG2a/IgG2b antibodies (1:1000, Biodragon Tech, #BF03007, #BF03050, #BF03051, #BF03052) were added at 37°C for 60 min. After washing 3 times, TMB single-component substrate solution (Solarbio Tech, #PR1200) was added, 100 μL/well, to incubate in the dark at RT for 15 min. Finally, 50 μL stop solution was added to each well to terminate the reaction, and the OD₄₅₀ nm value was measured using a Multimode Microplate reader (Biotek Synergy HTX, USA). Three parallel repeated tests were performed for each sample.

The levels of IFN-γ, IL-2 and IL-4 in serum were detected using mouse cytokine ELISA kits (Shanghai Enzyme-linked Biotech, #m1002277, #ml063136, #ml063156) on day 21 after the third immunization. Briefly, 50 μL of serum diluted 1:3 and 100 μL of HRP-labeled specific antibody were added into each well followed by incubation at 37°C for 60 min. After washing 3 times with PBST, the substrate solution was added into each well to develop in the dark at 37°C for 15 min. The reaction was stopped immediately with the stop solution, 50 μL/well, and the OD values were measured at 450 nm using a Multimode Microplate reader (Biotek Synergy HTX, USA). Three parallel repeated tests were performed for each sample.

All data presented in this study were expressed as mean ± standard deviation (SD) and analyzed using GraphPad Prism 8.0. Statistical analysis was conducted using One-way ANOVA, followed by Duncan’s multiple comparisons. Differences among different groups were considered significant when P < 0.05 (*) or P < 0.01 (*), and the comparison without a statistics bar and asterisk were found to be non-significant (P > 0.05).

The constructed recombinant expression plasmids, pCold 1-S1 and pCold 1-S1-PA, were identified by PCR, the restriction endonuclease (EcoR I and Sal I) digestion, and sequencing and analysis, respectively. After agarose gel electrophoresis, target bands of the same size, 2244 bp and 2943 bp, as the expected S1 and the S1-PA recombinant protein was observed, respectively ( Supplementary Figure 1 ). The sequencing results were consistent with the reference sequences.

SDS-PAGE analysis and Western blotting showed the recombinant proteins of S1 and S1-PA were successfully expressed in the E. coli BL21 (DE3) cells by IPTG-induced 24 h at 16°C ( Figures 2A–C ). The expressed proteins existed mainly in the supernatant of the bacterial lysate at 80 kDa and 104 kDa, respectively. Recombinant S1 protein was purified by metal affinity chromatography, and after desalination and concentration, the protein concentration was 2.4-3.2 mg/mL. S1-PA recombinant protein was directly used for the subsequent preparation of S1-BLPs.

After incubation of GEM particles with the S1-PA protein, S1 antigen could be displayed on GEMs surface by anchoring protein PA. When S1-BLPs was subjected to SDS-PAGE electrophoresis, S1-PA recombinant protein bands were seen in the corresponding lanes ( Figure 2D ). BCA protein assay showed that each unit (2.5×10⁹ particles/mL) of the GEMs could bind to 371-410 μg of S1-PA protein. Furthermore, the electron microscopy revealed a ring of flocculation around the GEMs in S1-BLPs with a diameter of about 800-1000 nm, but not around the GEMs that did not bind the S1 protein ( Figure 2E ). These results showed that S1 protein was conjugated with GEMs and displayed on the surface of GEMs.

The antigens in S1-BLPs were detected by Western blotting and immunofluorescence assay. As shown in Figure 2F , a clear brown band of around 104 kDa appeared on the PVDF membrane in the lane of S1-BLPs following Western blotting. As expected, S1-BLPs glowed green fluorescence after indirect immunofluorescence staining while GEMs control not ( Figure 2G ). The results demonstrated that S1 protein was conjugated with GEMs by the protein anchor.

In order to analyze the influences of immunization routes on immune responses and compare systematic specific IgG antibody induced by S1-BLPs and S1 protein, female BALB/c mice were immunized with S1-BLPs or S1 protein through the intramuscular and intranasal route, respectively, and serum was collected weekly ( Figure 3A ). After immunization, the specific IgG antibody levels in two intramuscular immunization groups (S1/IM and S1-BLPs/IM) were significantly higher than those in two intranasal inoculation groups (S1/IN and S1-BLPs/IN) (P<0.05) ( Figure 3B ). Although the IgG antibody in S1-BLPs intranasal immunization (S1-BLPs/IN) group remained at a relatively low level, it higher than that in S1/IN group. At 14 days after the primary immunization, the IgG level in S1-BLPs/IM group was significantly higher than that in S1/IM group (P<0.05), but there were no significant differences after the second and third immunizations (P>0.05). No specific IgG antibody was found in S1/IN and the blank group. The antibody titers in groups S1/IM and S1-BLPs/IM reached 1:102400 and 1:51200, respectively on day 21 after the third immunization, which were significantly higher than 1:1600 in the intranasal S1-BLPs group ( Figure 3C ), indicating that the intranasal immunization of S1-BLPs has limited ability to stimulate systemic specific IgG antibodies.

The levels of specific IgG subclasses in serum were also tested by ELISA. Due to the low total IgG levels in the intranasal immunization groups, only IgG subclasses of the intramuscular immunization groups were measured. As shown in Figure 3D , the IgG1 antibody levels in the S1/IM group was slightly higher than that in the S1-BLPs/IM group at 14 and 28 days after the third immunization, but there was no significant difference between them (P>0.05). S1/IM group and S1-BLPs/IM group had similar IgG2a levels (P>0.05), and the former exhibited a higher IgG2b antibody level on day 14 after the second immunization (P<0.05) ( Figures 3E, F ). Moreover, S1-BLPs/IM group had a higher ratio of IgG2a/IgG1compared to the S1/IM group (P<0.05) ( Figure 3G ), indicating that the immune response induced by S1-BLPs was characterized by T helper (Th) cell type 1 (Th1) immunity.

To evaluate the effects of immunization routes on the specific IgA and sIgA responses, we compared the levels of IgA in serum and sIgA in the intestine and bronchoalveolar lavage fluid. From 7 days after immunization until the end of the experiment, only seroconversion of IgA presented in S1-BLPs/IN group (P<0.05). There was no significant difference between groups S1/IM, S1-BLPs/IM or S1/IN and the blank group (P>0.05) ( Figure 4A ). At 14 days after the second immunization and 21 days after the third immunization, the OD values of specific sIgA in intestine lavage fluid were 0.246 and 0.330 in S1/IN group, and 0.660 and 0.796 in S1-BLPs/IN group, respectively ( Figure 4B ). There was a significant difference between the two groups (P<0.01). No specific sIgA was found in the other groups ( Figure 4B ). In bronchoalveolar lavage fluid of all groups, specific sIgA was not detected ( Figure 4C ). The result showed that S1-BLPs could induce systemic and intestinal mucosal specific IgA responses by intranasal immunization.

To further evaluate cytokine responses induced by S1-BLPs, the levels of IFN-γ, IL-2 and IL-4 in serum were analyzed by ELISA, and the results were shown in Figure 5 . In addition to group S1/IN, serum IFN-γ levels in groups S1/IM, S1-BLPs/IM and S1-BLPs/IN were higher than that in the blank group. Furthermore, there was a stronger IFN-γ response in group S1-BLPs/IN compared to group S1-BLPs/IM and S1/IM (P<0.05), but there was no significant difference between groups S1/IN and S1/IM ( Figure 5A ). IL-2 levels were similar between the immunized groups and between the immunized and the blank group ( Figure 5B ). The level of IL-4 in group S1-BLPs/IN was higher than that in S1/IN and the blank group (P<0.05) ( Figure 5C ). The results indicated that S1-BLPs promoted IFN-γ response, and intranasal inoculation could significantly increase IFN-γ levels. Moreover, intranasal inoculation of S1-BLPs significantly induced IL-4 production.