A bacteriophage based virus-like particle vaccine induces cross-reactive neutralizing antibodies against porcine epidemic diarrhea viruses (PEDV)

A bacteriophage based virus-like particle vaccine induces cross-reactive neutralizing antibodies against porcine epidemic diarrhea viruses (PEDV) | 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 A bacteriophage based virus-like particle vaccine induces cross-reactive neutralizing antibodies against porcine epidemic diarrhea viruses (PEDV) Jixiang Gu, Xu Zheng, Chunhui Li, Shipeng Wang, Xiangyu Xie, Martin F. Bachmann, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6027078/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Veterinary Research → Version 1 posted You are reading this latest preprint version Abstract Although vaccines against porcine epidemic diarrhea viruses (PEDV) are available currently, PED outbreaks still occur in many countries due to emerging new variants. Therefore, more endeavors are required to develop efficient and broadly-protective vaccines. To this end, we here present a nanoparticles vaccine candidate AP205-S1 which effectively elicited antibody responses in mice and pigs. The vaccine was generated by coupling S1 protein of PEDV-KB2013, a G-II strain to bacterially expressed AP205-VLP via SpyCatcher/SpyTag. AP205-S1 demonstrated intact and homogenous viral particle structure and packed E. coli -derived ssRNA. Upon administration in mice, AP205-S1 induced high titers of S1-specific IgG antibodies in sera as well as in gastrointestinal tracts, especially after booster. Importantly, these antibodies were able to neutralize PEDV in vitro , indicating the vaccine is able to induce protective antibodies against PEDV infection. Of note, AP205-S1 elicited antibodies exhibited cross-neutralizing potential against a G-I strain, PEDV-AH2018-HF1, which was preserved in our lab. Last but not least, S1-specific IgG antibodies were stimulated in piglets after AP205-S1 immunization, which could neutralize PEDV in vitro . Most interestingly, AP205-S1 immunized piglets showed reduced viral loads compared to control piglets upon viral challenge. In conclusion, we generated a VLP-based vaccine candidate against PEDV demonstrating excellent immunogenicity in mice and piglets, which granted potential protection against viral infection. Our work provides an efficient option for prevention of future PEDV epidemics. PEDV AP205-VLP vaccine neutralizing antibody Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Since porcine epidemic diarrhea viruses (PEDVs) were firstly reported in 1970s, they have caused enormous losses to the pork industry over the world 1 , 2 . PEDV is a ssRNA virus and belongs to genus Alphacoronavirus of Coronaviridae family. The genome of PEDV frequently mutates so that new variants continuously emerge 3 , 4 . According to spike (S) protein sequence, PEDVs are designated to two groups: classic strains (G-I) and mutant strains (G-II), which have been overwhelmingly circulating among the fields in the past decade 5 . Of note, G-II strains are more pathogenic and contagious than G-I strains, leading to higher mortality and many endemic outbreaks in different countries 1 , 6 . Due to the significant genomic divergences between G-I and G-II strains, the immune responses elicited by G-I strains are poorly cross-reactive to G-II strains. Hence, the vaccines derived from G-I strains granted limited protection against G-II strains, hindering the eradication of the virus. The ideal approach is to develop a vaccine candidate that induce cross-protection against both G-I and G-II viruses. PEDV mainly infects gastrointestinal tracts, resulting in vomiting, watery feces, diarrhea, and dehydration in pigs 7 . After invading epithelial cells, the viruses trigger innate immune responses involving dendritic cells, natural killer cells and macrophages, which further activate adaptive immunity via cytokine secretion and antigen processing 8 , 9 . B cells in response to those innate signals as well as virus antigens would differentiate to plasma cells and secrete antigen-specific antibodies, rendering predominant defenses in hosts against PEDV invasion. IgG and IgA antibodies are the most abundant antibody classes in serum and mucosa respectively and play essential role in defending against viral infection 10 . It is believed that neutralizing IgG antibodies can effectively prevent PEDV attaching and internalizing to cells 11 . On the other hand, high level of IgA in serum has been shown to correlate with low PEDV viral shedding in stool. In addition, IgA antibodies in colostrum and milk are indispensable for enteric virus defenses of suckling piglets 12 . Recently, a correlation study demonstrated that the neutralizing titers in serum, feces and colostrum were highly correlated with S1 protein specific IgA titers 13 . Taken together, an ideal PEDV vaccine should be capable to induce high amounts of both IgG and IgA antibodies. Despite that porcine receptors of PEDV are not defined yet, S protein is critical for the viral infection and independent studies have confirmed that S protein is a superior target for subunit vaccine design 7 , 14 – 16 . The 3D-structure of PEDV S protein revealed by Cryo-EM at a resolution of 3.1 Å defines the putative receptor binding domain (RBD) and sialic acid binding regions in S1 protein 17 , which are essential for virus-cell attachment and contain neutralizing epitopes 18 , 19 . Bacteriophage AP205-derived VLP, an icosahedral particle made up with 180 identical capsid protein, has been applied in various vaccine candidates. We have previously fused the receptor binding motif (RBM) of SARS-CoV-2 to AP205, called AP205-RBM, which induced IgA production in mice after subcutaneous administration 20 . In addition to genetic fusion, chemical coupling via a linker is another well-defined way to connect antigen with AP205-VLP. For example, SMPH has been used to link RBD of SARS-CoV-2 to CuMV TT -VLP 21 – 23 . Furthermore, the covalent bond between SpyCatcher and SpyTag (both bacterially derived sequences known to form covalent bonds) successfully presented the surface protein of Plasmodium falciparum Pfs47 on AP205 VLP, which strongly induced specific IgG antibodies of high-affinity in immunized mice 24 . This specific SpyCatcher/SpyTag covalent binding is spontaneously formed between lysine and aspartic acid. SpyCatcher/SpyTag reaction occurs in moderate conditions and production costs may be lower than chemical coupling, which is advantageous in veterinary vaccines, in particular for the food-producing industry. In this study, we developed a PEDV vaccine candidate AP205-S1 derived from PEDV-KB2013, a G-II virus, by SpyCatcher/SpyTag linkage. AP205-S1 remained the intact viral particle shape and packaged E. coli derived RNA during bacterial expression. After immunizing mice with AP205-S1, systemic and mucosal IgG antibodies were stimulated, and the titers induced were very high especially after a booster. Furthermore, immunized mice sera showed PEDV-neutralizing efficacy in vitro via reduction of cytopathogenic effect (CPE) of PEDV-KB2013, a G-II strain. Indeed, the 100% neutralizing titers of some sera at day 49 were as high as 1: 160, indicating the AP205-S1 was potent at elicit neutralizing antibodies. Moreover, AP205-S1 elicited antibodies could recognize and cross-neutralize a G-I virus AH2018-HF1 as well. In summary, the presented AP205-S1 has great potential to protect pigs from PEDV infection and is worthwhile to scale up for industrial application in the future. Materials and Methods 1. Expression and purification of AP205-SpyCatcher The cDNA encoding SpyCatcher domain was synthesized in General Biol (Anhui) Co. Ltd, which was then ligated to pET28a-AP205 using LightNing DNA Assembly Mix Plus (BestEnzymes Biotech, Lianyungang, China). Then the construct AP205-SpyCatcher was transformed into BL21 competent cells. Subsequently, expression of AP205-SpyCatcher protein was induced by adding 0.5 mM IPTG to medium. After lysing bacteria with sonication, the protein was precipitated with ammonium sulfate. Finally, the purified AP205-SpyCatcher was dialyzed into 20 mM Tris-HCl and characterized by running SDS-PAGE gel, agarose gel, dynamic light scattering (DLS) and transmission electron microscope (TEM). 2. Production of S1-SpyTag Vero cells were firstly infected with PEDV-KB2013 strain, then whole RNA was extracted and reverse transcribed into cDNA. S1 protein-encoding sequence was amplified from cDNA using specific primers infused with SpyTag-encoding sequence, then assembled into pTWIST-CMV-BetaGlobin-WPRE-Neo vector (TWIST Bioscience, California, USA) under the control of CMV promoter. When HEK293F cells reached 1 × 10 6 /ml, the plasmid was incubated with PEI in a ratio of 1:3 at room temperature for 15 min to form complexes before adding into cell suspension. After 5 days, the cell supernatant containing S1-SpyTag protein was collected and the target protein was purified using Ni-NTA column (GenScript, Nanjing, China), followed by PBS dialysis to remove imidazole. The purified protein was concentrated and quantified using BCA assay (Beyotime Biotech Inc, Jiangsu, China) before further application. 3. Generation of AP205-S1 vaccine The purified AP205-SpyCatcher and S1-Spytag were connected by simply incubating at 4˚C for 16 hours at molar ratio of 1:1. Subsequently, the coupling product was loaded in SDS-PAGE gel and coupling efficiency was calculated based on densitometry analysis. The formed AP205-S1 was loaded to 1% agarose gel to examine the packed RNA and Western blot using AP205-specific antibody was performed to verify the covalent bond. 4. Transmission electron microscope (TEM) VLPs at 0.2 mg/ml were dropped on copper grid, after which VLPs were stained. Briefly, the grid was floated on a drop of VLP solution for 3 min and then dried with filter paper. Next, the dried grid was floated on phosphotungstic acid staining solution for 30 s, after which the extra solution was removed with filter paper. Then the dry grid was observed under HT7700 (Hitachi, Tokyo, Japan) microscope. 5. Mice immunization 7-week-old BALB/c female mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co. Ltd and kept at SPF animal facility in Anhui Agricultural University according to the guidelines and regulations by the Animal Care and Use Committee, Anhui Agricultural University (SYXK (Anhui) 2016-007). Mice were kept for 1 week to adapt to the new environment before experiment. All experimental procedures were carried out strictly adhere to the guidelines of Animal Care and Use Committee, Anhui Agricultural University. 50 µg AP205-S1 vaccine, S1 protein or AP205-SpyCatcher was administered by subcutaneous injection at lateral abdominal area on day 0 and day 28, during which blood and feces were collected weekly until day 49. Tail vein blood was collected without anesthesia and mice were euthanized under CO 2 atmosphere using a controlled chamber at day 49. Sera were separated from blood and feces were suspended in PBS at 0.1g per 1 ml PBS, after which sera and fecal supernatants were collected and stored at -20˚C. 6. IgG antibody measurement The IgG antibody levels in serum and feces were determined via ELISA (enzyme-linked immunosorbent assay). 96-well plates were coated with S1-SpyCatcher protein at 1 µg/ml in PBS overnight at 4˚C. After washing plates with PBS for 4 times, PBS-1% Casein was added to block plates for 2 h at room temperature. Then sera or feces supernatants were added to plates and serially diluted in PBS-1% Casein, which then incubated for 1 h. Next, plates were washed with PBS-0.1% Tween for 4 times and goat anti-mouse IgG-HRP (Makewonderbio, Beijing, China) or goat anti-pig IgG-HRP (Cellwaylab, Luoyang, China) was incubated for 1 h to detect bound IgG antibodies. Finally, TMB developing solution (Makewonderbio, Beijing, China) was added and same volume of 1 M H 2 SO 4 solution was added to stop the reaction. The plates were read under OD 450 nm in a micro plate spectrophotometer (Molecular Device, California, USA). Antibody titers were calculated as the dilution folds that reached half the OD max . 7. IgA antibody measurement IgG may compete with IgA antibodies for S1 binding sites, so as IgG should be removed in case underdetermining IgA responses. Serum or feces supernatant was firstly incubated with Protein A beads (Senhui Microsphere Tech, Jiangsu, China) for 30 min at room temperature, after which supernatant was collected for IgA detection. As for IgA ELISA, most procedures were as described above except that the samples were pretreated and detection antibody was goat anti-mouse IgA conjugated with HRP (Abcam in China, Shanghai, China). The binding curve was drawn using OD 450 nm and dilution folds. 8. PEDV amplification and titer determination PEDV-KB2013 viruses were amplified in Vero cells, which were cultured in DMEM medium. When cells reached 80% confluent on plates, virus was seeded and incubated for 1 h, which then was washed away with PBS for 3 times. After culturing for 48 hours, cytopathic changes were observed, such as rounding, forming cluster or syncytium and detaching from plates. Infected cells were collected after trypsinization and frozen in -80˚C. Viral titer was determined using plaque formation assay. Basically, single layer of Vero cells (100% confluent) was infected with thawed virus-containing supernatant, which was serially diluted. Similarly, the infection lasted for 1 hour and then cells were kept in 37˚C, 5% CO 2 atmosphere until visible plaques were observed. Titers were calculated and the viral titer was 5.75 × 10 5 PFU/ml. 9. Indirect immunofluorescence assay (IFA) In addition to ELISA, IFA was carried out to assess whether the immunized sera were able to recognize and bind to natural viruses. Generally, 200 TCID 50 virus was added to 70% confluent Vero cells in 24-well plate for 1 hour. 24 hours later, cell supernatants were removed and washed with PBS for 3 time. Then cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with FoxP3/Transcription Factor Staining Buffer Kit (MuitiScience Biotech Co., Ltd, Hangzhou, Zhejiang, China). Next, PBS-1% Casein was applied to block extra binding sites of plate for 2 h at 37˚C. Subsequently, diluted sera were added and incubated at 37˚C for 1 h. After washing with PBS for 3 times, detection antibody goat-anti mouse IgG-FITC (Biosharp, Anhui, China) was incubated with cells at 37˚C for 1 h. Lastly, cell nuclei were stained with DAPI at room temperature for 10 min. The images were captured under fluorescent microscope. 10. Neutralizing assay Cytopathic effects (CPE) caused by pretreated PEDV viruses were used to reflect viral neutralizing abilities of immunized sera. Simply, sera were firstly inactivated at 56˚C for 30 min and PEDV-KB2013 was incubated with inactivated serum for 1 h at 37˚C, which was 1:2 serially diluted from 1:20. The reaction mixtures were added to Vero cells (70% confluent) and incubated at 37˚C for 1 h, which were then removed and washed with PBS for 3 times. Then, cells were cultured for 48 h and CPE was observed under microscope, from which the neutralizing titers were noted as the dilution folds to completely neutralize viruses. To visualize the neutralizing effects of immunized sera, IFA was performed as well. Instead of PEDV virus alone, immunized sera pre-incubated sera were used to infect Vero cells. In addition, poly clonal antibodies derived from rabbit against PEDV-N protein (self-made) were added to recognize infection-competent viruses. Goat-anti rabbit IgG-FITC was used as detection antibody, which reflects the amounts of infected viruses. 11. ELISpot assay To determine the plasma cell counts in immunized mice, spleens were harvested at day 49 after mice were euthanized. In brief, single cell suspension was obtained by smashing spleen on a 70 µm strainer. Afterwards, erythrocytes were removed by suspending cells in ACK buffer for 5 min at room temperature, after which same volume of medium was added and cells were spined at 300 g for 5 min. Then cells were resuspended in RPMI 1640 medium supplemented with 10% FBS and certain counts of cells were seeded in 96-well Multiscreen plate (MilliporeSigma, Massachusetts, United States). Before seeding cells, the plate was coated with 10 µg/ml S1 protein overnight at 4˚C and blocked with PBS-1%Casein at 37˚C for 2 hours. After 5 hours incubation at 37˚C, 5% CO 2 atmosphere, cells were discarded and primary antibody Goat Anti-Mouse IgG (ThermoFisher, Massachusetts, United States) was incubated overnight at 4˚C. Next, secondary antibody Donkey Anti-Goat IgG-AP (Biosharp, Anhui, China) was incubated at room temperature for 2 hours. Finally, spots were visulaized using BCIP/NBT substrate kit (Biosharp, Anhui, China). 12. Piglets immunization and viral challenge Neonatal piglets used in this study were kept at experimental animal center of Northwest Agriculture & Forestry University. The experiment protocol was reviewed and approved by the Animal Welfare Committee of Northwest A&F University. All animals were monitored on a daily basis for any clinical signs during the whole experimentation. Piglets at 7-day-old were primed (day − 21) and boosted at day − 7 with 100 µg AP205-S1 or AP205-SpyCatcher in 200 µl MONTANIDE ISA 206 VG (Seppic Shanghai Chemical Specialities, Shanghai, China) by intramuscular injection at neck area. Animals were orally challenged with 1*10 5 TCID50 KB2013 at day 0. Sera and rectal swabs were collected at 0, 3, 5, 7, 10 and 14 days after challenge. Each group was assigned randomly with 4 individuals from the same litter. The severity of diarrhea was determined as follows: normal and soft feces were scored as 0, loose as 1 and watery as 2. All animals were euthanized at day 14 post challenge by bleeding out from axillary artery of forelimb under deep anasthesia with intramuscular injection of 5 mg/kg xylazine. Note: one animal in AP205-S1 group died from congenital hypoplasia at day 4. 13. RT-qPCR PEDV loads in serum and feces of challenged piglets were assessed by RT-qPCR. Briefly, total RNA was isolated from serum or rectal swabs using Viral Genome Extraction Kit (Qingdao Lijian Bio-Tech, Qingdao, China) according to manual. Then cDNA was obtained from RNA using HyperScript III 1st Strand cDNA Synthesis Kit with gDNA Remover (EnzyArtisan Biotech, Shanghai, China), which was used as qPCR template. The reaction system was as follows (20 µl): 10 µl SYBR Green, 8 µl ddH 2 O, 0.5 µl Primer-F (10 µM), 0.5 µl Primer-R (10 µM), 1 µl cDNA. The primer sequences were F: GAGGGTGTTTTCTG -GGTTG, R: CGTGAAGTAGGAGGTGTGTTAG. To set up standard curve, the target gene (N gene) was constructed on pET28a and the Ct = -3.225Log 10 (Copies) + 36.87. 14. Statistical analysis The significance analysis was performed in GraphPad Prism 9 (GraphPad Software, Inc. La Jolla, CA, USA). A P value from unpaired t-test was indicated as ≤ 0.05 (*), ≤ 0.01 (**), ≤ 0.001 (***), ≤ 0.0001 (****). All error bars were displayed as mean ± SEM. Results 1. SpyCatcher/Tag bond effectively conjugated AP205-VLP and S1 protein without compromising advantages of VLP. Chemical coupling was the first option tried to link purified AP205-VLP and S1 protein. However, this did not work efficiently, probably due to the relatively large molecular size of S1 protein (data not shown). We then attempted to produce AP205-S1 vaccine via SpyCatcher/Tag specific binding as illustrated in Fig. 1 A. Firstly, AP205-SpyCatcher was successfully expressed and purified, with a molecular weight of 29 kD per subunit as shown in Fig. 1 B. In addition, agarose gel showed that ssRNA was packaged in AP205-SpyCatcher (Fig. 1 C). Although the molecular size of SpyCatcher (15 kD) was equivalent to that of AP205 monomer (14 kD), AP205-SpyCatcher still kept viral particle (Fig. 1 H). After purifying S1-SpyTag expressed by HEK293 cells, AP205-S1 was generated by incubating AP205-SpyCatcher and S1-SpyTag. To find out the optimal reaction conditions, different molecular ratios between AP205-SpyCatcher and S1-SpyTag were tested. The band slightly lifted above S1-SpyTag in SDS-PAGE gel was AP205-S1, which were shown at all ratios (Fig. 1 D). Meanwhile, the AP205-SpyCatcher band faded when mixed with S1-SpyTag. When the ratio was 1:1, the coupling efficiency was highest (49%), at which ratio the AP205-S1 vaccine was generated for further characterization and immunization. Western blot result using AP205-specific monoclonal antibody confirmed that covalent bond was formed in Fig. 1 E. Rather than a clear RNA band of AP205-SpyCatcher, RNA in AP205-S1 was diffuse, but co-localized with the protein band after coomassie blue staining (Fig. 1 F). Furthermore, DLS showed that AP205-S1 particles were homogenous at 80 nm diameter (Fig. 1 G) and TEM revealed the vaccine was in viral particle structure (Fig. 1 H). Taken together, these results suggested that AP205-S1 connected by SpyCatcher/Tag bond was homogeneous, remained stable spheric shape and packaged with prokaryotic ssRNA. 2. AP205-S1 vaccine exhibited superb immunogenicity in mice. To examine the capabilities of AP205-S1 to induce immune responses in vivo , BALB/c mice were primed at day 0 and boosted at day 28 via subcutaneous injection (Fig. 2 A). IgG antibody levels in sera were tested using ELISA, which demonstrated that AP205-S1 immunized mice produced S1-specific IgG antibodies as early at day 7 (Fig. 2 B). The IgG levels increased with time and remarkably further increased after booster, which peaked at day 35 and kept high until day 49. On the other hand, S1 protein immunized mice showed decent antibody levels only after second injection. As expected, AP205-SpyCatcher could not induce S1-specific antibody responses after two injections. S1-specific IgG titers induced by AP205-S1 were significantly higher than S1 or AP205-SpyCatcher alone, demonstrating that displays of S1 on AP205 greatly increased immunogenicity (Fig. 2 C). Accordingly, AP205-S1 stimulated significantly more plasma cells that secrete S1-specific IgG antibodies than S1 and AP205-SpyCatcher at day 49 via ELISpot assay (Fig. 2 D). Furthermore, IFA was performed to determine whether the immunized sera could recognize and bind to PEDV-infected cells in vitro . Consistent with the ELISA results, AP205-SpyCatcher immunized sera at day 49 did not bind to PEDV-infected cells, as no FITC signals were observed (Fig. 3 A). As for the S1 immunization, day 28 sera did not bind to PEDV, while day 49 sera could bind to PEDV, reflecting the higher antibody titers after booster (Fig. 3 B and 3 C). In addition, AP205-S1 elicited IgG antibodies at day 28 were able to bind to PEDV viruses in vitro as illustrated in Fig. 3 D. These results not only suggested that S1 displayed on AP205-VLPs was in natural conformation, but also indicated that AP205-S1 stimulated strong immune responses in mice. 3. S1-specific IgG antibodies in AP205-S1 immunized mice were of high affinity. High binding strengths between antibody and virus were correlated with neutralizing potentials, therefore avidity ELISA was performed to assess the amounts of high-avidity antibodies. The essence of avidity ELISA is that those antibodies bound weakly to S1 protein are washed away by 7 M urea, whereas high-avidity antibodies were remained on plates. As expected, the high-avidity antibodies were significantly less than total IgG levels as shown in Fig. 4 A. Consistent to total IgG levels, high-avidity IgG antibodies were relatively low before booster (day 28). In contrast, the high-avidity IgG antibodies were significantly elevated after booster at day 49 (Fig. 4 B), indicating that the affinity maturation was enhanced after booster. There results suggested that AP205-S1 could induce high-avidity antibodies to potentially neutralize PEDV. 4. Systemic IgA antibodies against S1 protein were induced by AP205-S1. IgA antibodies are of great importance in controlling viruses in addition to IgG, especially in mucosa. After first immunization, S1-specific IgA antibodies were stimulated by AP205-S1 and S1 (Fig. 5 A). Similar to IgG levels, IgA antibody levels in sera were elevated after booster, and AP205-S1 demonstrated higher immunogenicity than S1 protein in terms of IgA antibodies (Fig. 5 B). AP205-SpyCatcher failed to induce S1-specific IgA antibodies in mice sera even after two immunizations. Although the IgA levels in sera reflect those in mucosa to certain extent, IgA antibodies were not detected in fecal supernatants of all immunized mice (data not shown), indicating the systemic immunization failed to induce mucosal IgA antibodies. Nevertheless, systemic IgA antibodies were shown to grant excellent viral neutralizing effects in PEDV infected pigs, especially S1-specific IgA. In conclusion, AP205-S1 was capable to induce systemic high amounts of IgA antibody in mice upon systemic administration, which is likely to neutralize PEDV. 5. AP205-S1 immunized mice sera exhibited premium PEDV neutralizing capabilities. Next, the abilities of AP205-S1 elicited antibodies to neutralize PEDV viruses were assessed. The viruses were firstly incubated with immunized sera, which were then added into Vero cells. IFA results demonstrated that AP205-SpyCatcher immunized sera failed to neutralize KB2013 virus, thus FITC signals were detected as shown in Fig. 6 A. In contrast, AP205-S1 immunized mice sera completely neutralized PEDV virus from infecting Vero cells (Fig. 6 A). It’s noteworthy that S1 elicited antibodies partly neutralized PEDV, indicating that S1 protein could induce neutralizing antibodies but with strongly delayed kinetics and lower levels. Additionally, CPE assays were carried out to determine the neutralizing titers of vaccinated sera. As expected, AP205-SpyCatcher immunized sera did not show any neutralizing effects. S1 immunized sera showed slight viral neutralizing capacity after booster, whereas AP205-S1 immunized sera exhibited viral neutralizing effects throughout all tested time points. In accordance with antibody titers, neutralization titers of AP205-S1 immunized sera were evidently elevated after booster, some reaching 1:160 at day 49 (Fig. 6 B). To summarize, both IFA and CPE proved that AP205-S1 induced neutralizing antibodies. 6. Neutralizing mucosal IgG antibodies were stimulated by AP205-S1 in mice. Because PEDV is an enteric virus, local gastrointestinal antibodies play vital roles in defending viral infection at first place. To find out if mucosal antibodies were induced after immunization, mice fecal supernatants were used for ELISA as well. The results showed that mucosal IgG antibodies were induced by both AP205-S1 and S1 protein after booster (Fig. 7 A). Again, AP205-S1 induced substantially more mucosal S1-specific IgG antibodies than S1 protein. In general, mucosal IgG levels of both AP205-S1 and S1 immunized mice were obviously lower than those in sera. Nevertheless, they specifically bound to natural PEDV viruses in vitro as shown in Fig. 7 B. Furthermore, AP205-S1 elicited mucosal antibodies completely hindered PEDV viruses from infecting Vero cells, while S1 elicited mucosal antibodies partially did so (Fig. 7 C). Collectively, these results revealed that systemic administration of AP205-S1 was capable to induce superior systemic as well as mucosal antibody responses, both of which effectively neutralized PEDV. 7. AP205-S1 induced systemic and mucosal antibodies were cross-reactive to AH2018-HF1 (G-I strain) One major obstacle for PEDV control is the immune escape of emerging variants, which could be overcome by vaccines that render broadly cross-protections. To investigate whether antibodies induced by AP205-S1 could recognize and neutralize other PEDV strain, AH2018-HF1 (G-I strain) was used, whose S1 protein showed 88.5% similarity with PEDV-KB2013 (Supplementary material). IFA results demonstrated that AP205-S1 immunized mice sera (day 49) could bind to AH2018-HF1 in vitro (Fig. 8 A), which could neutralize the virus as well (Fig. 8 B). Furthermore, the AH2018-HF1 neutralizing titers of immunized sera were significantly higher than those of AP205-SpyCatcher immunized sera (Fig. 8 C), meaning that AP205-S1 could induce cross-neutralizing antibodies against a G-I virus. Additionally, whether AP205-S1 elicited mucosal antibodies cross-reacted with AH2018-HF1 virus was assessed. IFA results suggested that they could recognize (Fig. 9 A) and neutralize (Fig. 9 B) AH2018-HF1 virus. In conclusion, the KB2013 derived vaccine AP205-S1 induced potent systemic and local antibody responses that not only neutralized KB2013 virus also cross-reacted to a G-I strain AH2018-HF1. 8. Immunization of piglets with AP205-S1 posed protection against PEDV infection With the clear results of mice that AP205-S1 elicited neutralizing antibodies in serum and mucosa, we wanted to determine the protective effects in piglets after PEDV challenge. Suckling piglets were immunized with AP205-S1 twice and then challenged with a lethal dose (in 7-day old piglets) of KB2013 virus (Fig. 10 A). High levels of S1-specific IgG antibodies were induced in sera after booster (Fig. 10 B), which could recognize PEDV in vitro (Fig. 10 D). Moreover, PEDV viruses were entirely neutralized by immunized sera as shown in Fig. 10 C, indicating the AP205-S1 demonstrated excellent immunogenicity in piglets as well. Because the piglets were 28-day-old at the day of challenge, all animals survived even those immunized with AP205-SpyCatcher (control). In general, AP205-S1 immunized piglets showed more active movements (data not shown). Additionally, less watery feces were observed in AP205-S1 immunized piglets on 5 to 7 days post challenge although the difference was not significant (Fig. 10 E). In addition, the viral loads in immunized pigs were lower than control pigs yet not significant (Fig. 10 F). This latter observation is similar to the experience with COVID-19 vaccines, where limited impact on viral spread was observed despite strongly reduced disease. Overall, AP205-S1 demonstrated modest prophylactic effects in piglets against PEDV infection, which needs further development for industrial application. Discussion PEDV has been an important pathogen in pork industry for decades, and vaccines are under intense development around the world. To accommodate with the sustained novel variants, vaccine candidates are required to update with time, particularly after 2010, when G-II strains started being a major concern. Typically, S1 protein of coronaviruses specifically binds to receptors and S2 mediates membrane fusion, assisting to virus internalization. Separate studies have shown that blocking both S1 and S2 activities could cease the virus invasion. Cocktail antibodies REGN-COV against receptor binding domain (RBD) in S1 region of SARS-CoV-2 could effectively neutralize and reduce the viral loads in patients 25 , 26 . Serum antibodies from convalescent patients were identified to recognize an epitope located at the site of the fusion peptide of S2 protein, which could be a neutralizing epitope 27 . Regardless that porcine aminopeptidase N (pAN), sialic acid, C-type lectin, and heparan sulfate were reported to be involved in viral entry in different models, the host receptors for PEDV are not defined definitively, which may require coordination of protein-protein binding and glycan-protein binding 28 , 29 . After unveiling the S protein structure of PEDV by Cyro-EM, NTD and CTD located in S1 protein were regarded as functional domain responsible for glycan and protein binding, respectively 17 . Hence, there is no doubt that S1 protein of PEDV plays the key part in binding cell receptor, which could be the highlight of vaccine targets. Based on these rationales, we here chose S1 protein as targeting antigen to design PEDV vaccine. However, free proteins usually stimulate insufficient antigen presentation, consequently induce limited immune responses. Displaying targeting proteins on a VLP scaffold could amplify the antigen presenting signals, owing to the following merits: i. Nanometer size of VLP makes it freely drain in lymphatic vessels, so that be accessible to present antigens; ii. Repetitive organization of antigens on VLPs magnifies antigen presenting signaling; iii. Distances between adjacent antigens on VLPs are ideal for BCR crosslinking; iv. Nucleic acids packaged inside of VLPs stimulate innate immune responses, facilitating specific adaptive responses 30 – 32 . The AP205-S1 vaccine in this study demonstrated particulate shape with diameter of around 80 nm and prokaryotic ssRNA was packed. Different from a focused RNA band packed in AP205-SpyCatcher, the RNA packed in AP205-S1 was diffuse probably owing to the disparate numbers of S1-SpyTag protein on each particle. Thus, the fact that AP205-S1 vaccine incited potent specific antibody responses is coincident with the previous studies that TLR signaling triggered by VLP-packaged ssRNA was critical for antibody affinity maturation and secondary plasma generation 33 , 34 . The PEDV structural proteins envelop (E) protein, membrane (M) protein and S protein have been shown to form VLPs after external expression in both baculovirus and mammalian cells. Immunizing mice with PEDV-VLPs composed of E, M and S protein could induce IgG and IgA production and T cell responses 35 . When pigs were vaccinated with PEDV-VLPs, both humoral and cellular immune responses were developed, posing some extent of protection against viral infection 36 . In addition to PEDV VLPs, a putative B cell epitope in S protein displayed on Hepatitis B Core Antigen (HBcAg) VLP was reported to prompt neutralizing antibody production in mice and gilts 37 . The major disadvantage of AP205-S1 may be that the S1 protein was produced in eukaryotic cells, which bears relatively high costs. Therefore, it might not be easily feasible for scale-up production and veterinary application. Nevertheless, we show here that S1 contained neutralizing epitopes that induced a PEDV neutralizing antibodies response. Either alternative cheaper expression systems (like yeast or insect cells) to produce S1 protein or truncated S1 protein (RBD), which could be expressed in E. coli , will be tested to generate cost-friendly PEDV vaccine candidates. Moreover, when it comes to large-scale production, the costs of mammalian cell expressing protein can be greatly reduced by optimizing the expression system or constructing stable-expression cell line. Indeed, W. Guo et al. has used an efficient expression system in HEK293F cells to produce S protein as well, which demonstrated excellent immunogenicity regarding humoral and cellular immune responses 38 . In addition, X. Song et al. immunized pigs with eukaryotic cell produced bivalant subunit vaccines, which provided protection against both G-IIa and G-IIb strains 39 . Meanwhile, SpyCatcher/Tag system still will be used to link AP205-VLP and antigens due to the relatively high efficiency and the reaction is accomplished by easily mixed and incubated at moderate condition. The mechanisms of VLPs to promote IgA generation were reported as well. Norovirus VLP recalled IgA responses in a humanized mouse model, which showed superior virus-neutralizing activities 40 . In VLP immunized mice, IgA antibodies were successfully induced in serum by both systemic (subcutaneous) and mucosal (intranasal) administration 41 . In addition, both systemic and mucosal IgA antibody responses were dependent on TLR7 signaling in B cells or lung DC and alveolar macrophages, respectively 42 , 43 . Consistently, AP205-S1 not only strongly prompted IgG responses, but also induced S1-specific IgA antibodies in serum. Whether the IgA levels could be increased by one more booster or intranasal immunization would require further investigation. On the other hand, IgA antibodies in intestinal mucosa were not detected in our study, inferring the subcutaneous immunization failed to induce mucosal IgA responses. This might be defeated by changing vaccination routes to intranasal or intrarectal application. Germinal center reactions, which are crucial for antibody affinity maturation, were demonstrated to be favored after VLP immunization. We previously found that GC reactions were accelerated when TLR7 signaling was activated during VLP immunization 33 , 44 . The downstream MyD88 signaling, especially B cell-intrinsic TLR7 signaling was involved in GC reactions 45 , 46 . In this study, AP205-S1 possessed TLR7 ligands and induced high affinity antibodies in mice, suggesting the vaccine effectively stimulated S1-specific GC reactions although they were not examined. It was claimed that high affinity antibodies were related with viral neutralizing capacities, exemplified by HIV-1 bearing patients. With “extreme mutation” on Ig variable regions, high-affinity antibodies could broadly neutralize HIV-1 strains 47 . Accordingly, repeated exposure to antigens increased antibody affinities in convalescent patients, accompanied with improved SARS-CoV-2 neutralizing abilities 48 . In this study, AP205-S1 was so efficient as to incite high affinity antibodies after only one booster immunization. It is worthwhile to find out whether one more booster would induce even more high-affinity antibodies and durable antibodies, which was not assessed in this study as well. Knowing that high-affinity antibodies were generated by the mice, their neutralization potentials were tested. Consistent with great amounts of high-affinity antibodies in sera at day 49, S1-specific antibodies exhibited predominant neutralization capacities. The PEDV-neutralizing abilities increased with the growing antibodies levels and dramatically elevated after booster. The CPE results reflected the overall neutralizing effects of all types of antibodies in samples. According to IFA results, IgG antibodies in both serum and mucosa executed viral neutralizing function. On the other hand, whether IgA antibodies in immunized sera neutralized PEDV was not determined in our study. Others have claimed that IgA antibodies were able to neutralize SARS-CoV-2 49 and the specific binding of high-avidity IgA antibodies to surface antigens prevented entero-pathogens division, termed as “enchained growth” 50 . Therefore, we extrapolate that the S1-specific IgA antibodies in immunized sera contributed to viral neutralization as well. Mucosal IgA plays a critical role for PEDV defenses, whereas we did not detect IgA antibodies in fecal supernatants, which was because the immunization route was systemic. It has been reported that intranasal administration of VLPs induced mucosal IgA responses while subcutaneous injection failed to do so 41 , which is consistent with our data. In addition, we are testing the mucosal IgA antibody levels via intranasal immunization as well, which is not shown in the current manuscript. The results showed that AP205-S1 could effectively induce both respiratory and intestinal IgA antibodies via intranasal immunization. The ideal PEDV vaccine candidates ought to induce broadly cross-reactive antibody responses so that different variants could be prevented with a single vaccination. Here, we demonstrated that AP205-S1 elicited antibodies not only neutralized the parental PEDV strain (KB2013), but also could neutralize a G-I strain AH2018-HF1. Surprisingly, the neutralizing titers against AH2018-HF1 were slightly higher than KB2013. The results suggested that the AP205-S1 was potential to induce cross-reactive neutralizing antibodies in mice. Lastly, AP205-S1 immunized piglets produced S1-specific IgG antibodies after two doses, and these antibodies could recognize and neutralize PEDV viruses in vitro . At this premise, the piglets were protected by less severe diarrhea, less viral shedding and more energetic activities. However, these findings were not statistically significant, probably due to the limited group size, which can be larger in the future to improve. Besides, there were no animals died because they were too old to die from PEDV challenge in our settings. In this case, the vaccine should be applied to pregnant sows to protect the newborns. In line with observations for SARS-CoV-2 infected individuals after vaccination, viral shedding was not strongly affected, perhaps due to limited presence of specific IgA antibodies, which may be a common problem for coronavirus vaccines. Rationally, the newborn piglets count on passive immunity from maternal antibodies rather than active immunization. Due to the low permeability of sow placenta, piglets are born agammaglobulinemic so that they could only rely on the lactogenic immunity. In this case, the maternal immunity means the lactogenic immunity. To our knowledge, there’s no research systemically studies the lactogenic immune responses after VLP vaccination in mice. However, the lactogenic immunogenicity of VLP vaccines in pigs were demonstrated previously by Y. Lu and colleagues, who showed that VLP vaccines incorporated B-cell epitope of PEDV offered lactogenic immunity to neonatal piglets 37 . The efficacy of PEDV vaccine should focus on the ability to induce maternal antibodies, which is our next plan to dig more in their maternal immunity including lactogenic immunogenicity. Alternatively, younger piglets with stronger signs of disease may be used for challenge. In summary, we here presented a PEDV vaccine candidate AP205-S1 based on VLP, which was formed by a specific covalent bond between SpyCatcher and SpyTag. AP205-S1 kept viral nanoparticle structure and packaged ssRNA, so that it effectively stimulated strong immune responses in mice after immunization. High titers of IgG antibodies were induced systemically, which could completely neutralize PEDV viruses and prevent them from infecting Vero cells in vitro . Importantly, these antibodies cross-neutralized other PEDV strains, suggesting that AP205-S1 caused cross-reactive antibody responses in mice. Furthermore, the vaccine elicited specific IgG responses in piglets, which completely neutralized PEDV in vitro . The clinical pathologic signs of immunized pigs were less although not significant. This vaccine has great potential to be developed further, offering an attractive alternative for PEDV prevention. Declarations Ethics approval and consent to participate All animal experiments were conducted following the Animal Ethics Procedures and Guidelines of the People’s Republic of China. Mice experiments protocols were approved by the Animal Care and Use Committee of Anhui Agricultural University (Approval No. SYXK (Anhui) 2016-007). Pig experiments protocols were approved by the Animal Welfare Committee of Northwest A&F University (Approval No. ICAUC-2024-CVM032). Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article. Competing interests Lisha Zha is involved in a pet vaccine company and owns shares. Other authors claim no interest conflict. Funding This work was funded by National Key Research and Development Plan (2022YDF1801800, to Lisha Zha), Anhui Provincial Natural Science Foundation (2408085QC074, to Xinyue Chang) and Starting Foundation of Anhui Agricultural University (to Xinyue Chang). Authors' contributions J. G and X. Z performed animal experiments and provided all figures; C. L and S. W helped prepare VLP material; X. X helped analyze data; M. F. B and Y. N reviewed and corrected the manuscript; L. L and P. S provided virus material; L. Z supervised the project; X. C wrote the main manuscript text. All authors reviewed the manuscript. Acknowledgements Not applicable. 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Affinity maturation of SARS-CoV-2 neutralizing antibodies confers potency, breadth, and resilience to viral escape mutations. Immunity 1853–1868 Wang Z et al (2021) Enhanced SARS-CoV-2 neutralization by dimeric IgA. Sci Transl Med 13:eabf1555 Lavelle EC, Ward RW (2022) Mucosal vaccines — fortifying the frontiers. Nat Rev Immunol 22:236–250 Supplementary Files sequencealignment.jpg Sequence alignment of S1 proteins of AH-2018-HF1 and PEDV-KB2013 strains. Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Veterinary Research → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6027078","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":444287337,"identity":"de0ba827-4348-4617-bacd-38ee679604bb","order_by":0,"name":"Jixiang Gu","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jixiang","middleName":"","lastName":"Gu","suffix":""},{"id":444287338,"identity":"2326abdb-e0df-4615-a214-6c20d3bf1d29","order_by":1,"name":"Xu Zheng","email":"","orcid":"","institution":"Northwest Agriculture and Forestry University: Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Zheng","suffix":""},{"id":444287339,"identity":"aab89dc7-8a68-48f6-a6b6-a4348f24d430","order_by":2,"name":"Chunhui Li","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chunhui","middleName":"","lastName":"Li","suffix":""},{"id":444287340,"identity":"41a57749-c675-4807-a9f7-cfd7f66cab68","order_by":3,"name":"Shipeng Wang","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shipeng","middleName":"","lastName":"Wang","suffix":""},{"id":444287341,"identity":"e625a532-a131-4e9e-b4f5-859380dc34be","order_by":4,"name":"Xiangyu Xie","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiangyu","middleName":"","lastName":"Xie","suffix":""},{"id":444287342,"identity":"646f38ec-5d79-46be-a818-e9241aa1d8b7","order_by":5,"name":"Martin F. Bachmann","email":"","orcid":"","institution":"University of Bern: Universitat Bern","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"F.","lastName":"Bachmann","suffix":""},{"id":444287343,"identity":"32862633-b475-4334-b4e3-fd46d47ad6c8","order_by":6,"name":"Yuchen Nan","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Yuchen","middleName":"","lastName":"Nan","suffix":""},{"id":444287344,"identity":"7cbb0288-b804-4c41-b277-c70130d3eeb2","order_by":7,"name":"Liang Li","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Li","suffix":""},{"id":444287345,"identity":"07a16349-71e7-40ed-8784-8b5d05cf05b6","order_by":8,"name":"Pei Sun","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Pei","middleName":"","lastName":"Sun","suffix":""},{"id":444287346,"identity":"51b1d73b-c46c-427f-9e11-f2aeefe1f1bd","order_by":9,"name":"Lisha Zha","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lisha","middleName":"","lastName":"Zha","suffix":""},{"id":444287347,"identity":"eea4d7b8-80b2-4806-bd55-992f41d8ef37","order_by":10,"name":"Xinyue Chang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYDACZgiVAMSMDxgbwBwDorUwGxCnhQGhhU2CKC0Gx3mPSfNU2OXxz26/VvFzh11iA3vzNgmGmjs4tUg286VJ85xJLpa4c6bsZu+Z5MQGnmNlEgzHnuHUws/MYybN23YgseFGTtoN3jbmxAaJHDOgCw/j1MIG1vLvQOJ8oJbCv231iQ3yb/BrgdjScCBxw430Y8y8bYeBtvDg1yLZzGNsOedYcuLGGznM0rJtx43beNKKLRKO4dZicP6M4Y03NXaJ826kP/z4tq1atp/98MYbH2pwawECFgkIzQOJDjYQkYBPAzDSP0Bo9gf41Y2CUTAKRsGIBQDSLVQF8ShA8wAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-7027-3889","institution":"Anhui Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Xinyue","middleName":"","lastName":"Chang","suffix":""}],"badges":[],"createdAt":"2025-02-14 03:54:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6027078/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6027078/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13567-025-01559-z","type":"published","date":"2025-07-01T15:58:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80897619,"identity":"5cb052d1-b428-4948-848b-84e287f73606","added_by":"auto","created_at":"2025-04-18 12:28:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1902988,"visible":true,"origin":"","legend":"\u003cp\u003eGeneration and characterization of AP205-S1 vaccine. A: Diagram of the way displaying S1 protein on surface of AP205-VLP via SpyTag and SpyCatcher specific bonds; B: SDS-PAGE image of purified AP205-SpyCatcher (lane 1); C: Agarose gel images of purified AP205-SpyCatcher (lane 1), left: UV light, right: coomassie blue staining; D: SDS-PAGE image of different molar ratios of AP205-SpyCatcher to S1-SpyTag, lane 1: AP205-SpyCatcher alone, lane 2: S1-SpyTag alone; lane 3: 1:2, lane 4: 1:1, lane 5: 2:1, lane 6: 3:1; E: Western blot image of generated AP205-S1 using monoclonal anti-AP205 antibody, lane 1: AP205-SpyCatcher alone, lane 2: S1-SpyTag alone; lane 3: AP205-S1 (1:1 reaction); F: Agarose gel images of formed AP205-S1 (lane 1), left: UV light, right: coomassie blue staining; G: Dynamic light scattering reports of AP205-SpyCatcher and formed AP205-S1; H: TEM images of AP205-SpyCatcher and AP205-S1.\u003c/p\u003e","description":"","filename":"Figure120250113.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6027078/v1/02eafe74875e861ab627abaa.jpg"},{"id":80898415,"identity":"c1d9cb65-1a39-4bd8-96f2-38936df67070","added_by":"auto","created_at":"2025-04-18 12:44:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1817051,"visible":true,"origin":"","legend":"\u003cp\u003eS1-specific IgG antibody responses in mice sera after immunization. A: Mice immunization regimen. Serum and feces samples were collected weekly. B: ELISA results of AP205-SpyCatcher, S1 or AP205-S1 immunized mice sera against S1 protein at different time points. Each point represented 5 mice and error bar was shown as ±SEM. C: S1-specific IgG antibody titers of immunized sera at different time points. Each point represented one mouse and error bar was shown as ±SEM. D: Plasma cell counts that secrete S1-specific IgG antibodies in immunized spleens at day 49. One well of spots was displayed in each group. P≤0.05 was shown *, p≤0.01 as **.\u003c/p\u003e","description":"","filename":"Figure220250114.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6027078/v1/738871eccb4e8865868976e7.jpg"},{"id":80897614,"identity":"926e4c72-7585-4a50-8517-547d15a7a6dd","added_by":"auto","created_at":"2025-04-18 12:28:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2484544,"visible":true,"origin":"","legend":"\u003cp\u003eIFA images of immunized mice sera to recognize PEDV KB2013 viruses \u003cem\u003ein vitro\u003c/em\u003e. A: Day 49 mouse serum immunized with AP205-SpyCatcher. B and C: mice sera immunized with S1 protein at day 28 (B) and day 49 (C). D: Mouse serum immunized with AP205-S1 (day 28). DAPI stained nucleus and FITC reflected mouse IgG antibodies bound to viruses. Serum samples at day 49 and day 28 were diluted 1:1000 or 1:100, respectively.\u003c/p\u003e","description":"","filename":"Figure320250118.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6027078/v1/df35ac5f461d1ae2dfb74b04.jpg"},{"id":80897618,"identity":"f53df0ea-66e7-4da4-9c90-964ddd317c6a","added_by":"auto","created_at":"2025-04-18 12:28:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":593796,"visible":true,"origin":"","legend":"\u003cp\u003eAvidity ELISA results of immunized mice sera against S1 protein. A: ELISA curves of OD\u003csub\u003e450nm\u003c/sub\u003e values with diluted sera washed with(red) or without (black) 7 M urea. B: Area under curve values of avidity ELISA curves. P≤0.05 was shown *, p≤0.001 as ***.\u003c/p\u003e","description":"","filename":"Figure420240618.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6027078/v1/465487df07b09934d7087fad.jpg"},{"id":80897847,"identity":"106e3b4c-1bdc-4aba-bbf7-3cdbff543977","added_by":"auto","created_at":"2025-04-18 12:36:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1251669,"visible":true,"origin":"","legend":"\u003cp\u003eS1-specific IgA antibody levels in immunized mice sera. A: ELISA curves of day 28 and 49 sera. Each point represented 5 mice and error bar was shown as ±SEM. B: S1-specific IgA titers of each group at day 28 and 49. P≤0.05 was shown *, p≤0.01 as **.\u003c/p\u003e","description":"","filename":"Fig5new20241225.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6027078/v1/c86d5a52fab4f092407bbb27.jpg"},{"id":80897845,"identity":"3d3c1b86-682e-42f5-a01e-9424400a3822","added_by":"auto","created_at":"2025-04-18 12:36:43","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2326829,"visible":true,"origin":"","legend":"\u003cp\u003eImmunized mice sera to neutralize PEDV (KB2013) viruses \u003cem\u003ein vitro\u003c/em\u003e. A: IFA images of day 49 sera immunized with AP205-SpyCatcher, S1 protein or AP205-S1 to block viruses from infecting Vero cells. DAPI stained nucleus and FITC reflected viral N protein-specific antibodies derived from rabbit, i.e. the infected viruses. AP205-SpyCatcher immunized sera were diluted 1:10, S1 protein and AP205-S1 immunized sera were diluted 1:100. B: Neutralization titers of immunized sera from day 21 to day 49. The dilution folds of sera to completely (100%) inhibit CPE in Vero cells were determined as titers. P≤0.05 was shown *, p≤0.01 as **.\u003c/p\u003e","description":"","filename":"Figure620241225.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6027078/v1/6ce6b757cd14c23f7ecaa4bf.jpg"},{"id":80897622,"identity":"1b21ae57-3f49-4fe9-b8d0-109d781c395e","added_by":"auto","created_at":"2025-04-18 12:28:43","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3487558,"visible":true,"origin":"","legend":"\u003cp\u003eAntibody responses in mice intestinal mucosa at day 42 after prime immunization. A: ELISA curves of S1-specific IgG antibodies in fecal supernatants. B: IFA images of mice fecal supernatants to bind to PEDV (KB2013) viruses \u003cem\u003ein vitro\u003c/em\u003e. DAPI stained nucleus and FITC reflected mouse IgG antibodies bound to viruses. C: IFA images of mice fecal supernatants to neutralize PEDV (KB2013) viruses from infecting Vero cells. DAPI stained nucleus and FITC reflected viral N protein-specific antibodies derived from rabbit, i.e. the infected viruses. Fecal supernatants were not diluted. P≤0.05 was shown *.\u003c/p\u003e","description":"","filename":"Figure720241226.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6027078/v1/5ebc28f7eb2176dd062bf76a.jpg"},{"id":80898416,"identity":"deee7535-2970-4b42-98be-ea5bd3a59a6a","added_by":"auto","created_at":"2025-04-18 12:44:43","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1977226,"visible":true,"origin":"","legend":"\u003cp\u003eRecognition and neutralization of immunized mice sera to PEDV AH2018-HF1 (G-I strain) virus. A: IFA images of mice sera at day 49 to bind to AH2018-HF1 viruses \u003cem\u003ein vitro\u003c/em\u003e. DAPI stained nucleus and FITC reflected mouse IgG antibodies bound to viruses. Serum samples were diluted 1:1000. B: IFA images of mice sera at day 49 to neutralize AH2018-HF1 viruses from infecting Vero cells. DAPI stained nucleus and FITC reflected viral N protein-specific antibodies derived from rabbit, i.e. the infected viruses. Serum samples were diluted 1:10. C: Neutralization titers of immunized sera at day 49. The dilution folds of sera to completely (100%) inhibit CPE in cells caused by AH2018-HF1 infection were determined as titers. P≤0.05 was shown *.\u003c/p\u003e","description":"","filename":"Figure820241017.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6027078/v1/87437f214dc0d4fd8d121af3.jpg"},{"id":80897625,"identity":"dff17583-bfee-47d8-b4ea-7a8230464704","added_by":"auto","created_at":"2025-04-18 12:28:43","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1552013,"visible":true,"origin":"","legend":"\u003cp\u003eMucosal antibodies from immunized mice to recognize and neutralize PEDV AH2018-HF1 (G-I strain) virus. A: IFA images of mice fecal supernatants at day 42 to bind to AH2018-HF1 viruses \u003cem\u003ein vitro\u003c/em\u003e. DAPI stained nucleus and FITC reflected mouse IgG antibodies bound to viruses. B: IFA images of mice fecal supernatants at day 42 to neutralize AH2018-HF1 viruses from infecting Vero cells. DAPI stained nucleus and FITC reflected viral N protein-specific antibodies derived from rabbit, i.e. the infected viruses. Fecal supernatants were not diluted.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6027078/v1/3c108d538a055464ee05b3fd.jpg"},{"id":80897635,"identity":"01f0c079-c788-4c85-b903-72938045a7f9","added_by":"auto","created_at":"2025-04-18 12:28:43","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2543938,"visible":true,"origin":"","legend":"\u003cp\u003eProtection efficacy of AP205-S1 in piglets against PEDV infection after AP205-S1 immunization. A: Immunization and challenge regimen of pig experiments. B: S1-specific IgG titers in pig sera after challenge. C: Neutralization titers of pig sera after challenge. D: IFA results of day 0 pig sera to bind to PEDV \u003cem\u003ein vitro\u003c/em\u003e. Sera were diluted 1:1000. DAPI stained nucleus and FITC reflected pig IgG antibodies bound to viruses. E: Diarrhea scores of piglets after challenge. F: PEDV viral loads in pig rectal swabs after challenge.\u003c/p\u003e","description":"","filename":"Figure1020250118.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6027078/v1/51312566bf9f25addd34315a.jpg"},{"id":86179679,"identity":"2577f0e4-9ec3-4166-961b-48f42b51bb33","added_by":"auto","created_at":"2025-07-07 16:18:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20623889,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6027078/v1/3b1748da-5d52-456d-908c-72e205d506d6.pdf"},{"id":80897843,"identity":"f7b67f34-49b2-41ac-a17e-afe99d197bd4","added_by":"auto","created_at":"2025-04-18 12:36:42","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":451378,"visible":true,"origin":"","legend":"\u003cp\u003eSequence alignment of S1 proteins of AH-2018-HF1 and PEDV-KB2013 strains.\u003c/p\u003e","description":"","filename":"sequencealignment.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6027078/v1/070e1084427a4be2c171e8d9.jpg"}],"financialInterests":"","formattedTitle":"A bacteriophage based virus-like particle vaccine induces cross-reactive neutralizing antibodies against porcine epidemic diarrhea viruses (PEDV)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSince porcine epidemic diarrhea viruses (PEDVs) were firstly reported in 1970s, they have caused enormous losses to the pork industry over the world\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. PEDV is a ssRNA virus and belongs to genus \u003cem\u003eAlphacoronavirus\u003c/em\u003e of \u003cem\u003eCoronaviridae\u003c/em\u003e family. The genome of PEDV frequently mutates so that new variants continuously emerge\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. According to spike (S) protein sequence, PEDVs are designated to two groups: classic strains (G-I) and mutant strains (G-II), which have been overwhelmingly circulating among the fields in the past decade\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Of note, G-II strains are more pathogenic and contagious than G-I strains, leading to higher mortality and many endemic outbreaks in different countries\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Due to the significant genomic divergences between G-I and G-II strains, the immune responses elicited by G-I strains are poorly cross-reactive to G-II strains. Hence, the vaccines derived from G-I strains granted limited protection against G-II strains, hindering the eradication of the virus. The ideal approach is to develop a vaccine candidate that induce cross-protection against both G-I and G-II viruses.\u003c/p\u003e \u003cp\u003ePEDV mainly infects gastrointestinal tracts, resulting in vomiting, watery feces, diarrhea, and dehydration in pigs\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. After invading epithelial cells, the viruses trigger innate immune responses involving dendritic cells, natural killer cells and macrophages, which further activate adaptive immunity via cytokine secretion and antigen processing\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. B cells in response to those innate signals as well as virus antigens would differentiate to plasma cells and secrete antigen-specific antibodies, rendering predominant defenses in hosts against PEDV invasion. IgG and IgA antibodies are the most abundant antibody classes in serum and mucosa respectively and play essential role in defending against viral infection\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. It is believed that neutralizing IgG antibodies can effectively prevent PEDV attaching and internalizing to cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. On the other hand, high level of IgA in serum has been shown to correlate with low PEDV viral shedding in stool. In addition, IgA antibodies in colostrum and milk are indispensable for enteric virus defenses of suckling piglets\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Recently, a correlation study demonstrated that the neutralizing titers in serum, feces and colostrum were highly correlated with S1 protein specific IgA titers\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Taken together, an ideal PEDV vaccine should be capable to induce high amounts of both IgG and IgA antibodies.\u003c/p\u003e \u003cp\u003eDespite that porcine receptors of PEDV are not defined yet, S protein is critical for the viral infection and independent studies have confirmed that S protein is a superior target for subunit vaccine design\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The 3D-structure of PEDV S protein revealed by Cryo-EM at a resolution of 3.1 \u0026Aring; defines the putative receptor binding domain (RBD) and sialic acid binding regions in S1 protein\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, which are essential for virus-cell attachment and contain neutralizing epitopes\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBacteriophage AP205-derived VLP, an icosahedral particle made up with 180 identical capsid protein, has been applied in various vaccine candidates. We have previously fused the receptor binding motif (RBM) of SARS-CoV-2 to AP205, called AP205-RBM, which induced IgA production in mice after subcutaneous administration\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In addition to genetic fusion, chemical coupling via a linker is another well-defined way to connect antigen with AP205-VLP. For example, SMPH has been used to link RBD of SARS-CoV-2 to CuMV\u003csub\u003eTT\u003c/sub\u003e-VLP\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Furthermore, the covalent bond between SpyCatcher and SpyTag (both bacterially derived sequences known to form covalent bonds) successfully presented the surface protein of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e Pfs47 on AP205 VLP, which strongly induced specific IgG antibodies of high-affinity in immunized mice\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. This specific SpyCatcher/SpyTag covalent binding is spontaneously formed between lysine and aspartic acid. SpyCatcher/SpyTag reaction occurs in moderate conditions and production costs may be lower than chemical coupling, which is advantageous in veterinary vaccines, in particular for the food-producing industry.\u003c/p\u003e \u003cp\u003eIn this study, we developed a PEDV vaccine candidate AP205-S1 derived from PEDV-KB2013, a G-II virus, by SpyCatcher/SpyTag linkage. AP205-S1 remained the intact viral particle shape and packaged \u003cem\u003eE. coli\u003c/em\u003e derived RNA during bacterial expression. After immunizing mice with AP205-S1, systemic and mucosal IgG antibodies were stimulated, and the titers induced were very high especially after a booster. Furthermore, immunized mice sera showed PEDV-neutralizing efficacy \u003cem\u003ein vitro\u003c/em\u003e via reduction of cytopathogenic effect (CPE) of PEDV-KB2013, a G-II strain. Indeed, the 100% neutralizing titers of some sera at day 49 were as high as 1: 160, indicating the AP205-S1 was potent at elicit neutralizing antibodies. Moreover, AP205-S1 elicited antibodies could recognize and cross-neutralize a G-I virus AH2018-HF1 as well. In summary, the presented AP205-S1 has great potential to protect pigs from PEDV infection and is worthwhile to scale up for industrial application in the future.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e1. Expression and purification of AP205-SpyCatcher\u003c/p\u003e \u003cp\u003eThe cDNA encoding SpyCatcher domain was synthesized in General Biol (Anhui) Co. Ltd, which was then ligated to pET28a-AP205 using LightNing DNA Assembly Mix Plus (BestEnzymes Biotech, Lianyungang, China). Then the construct AP205-SpyCatcher was transformed into BL21 competent cells. Subsequently, expression of AP205-SpyCatcher protein was induced by adding 0.5 mM IPTG to medium. After lysing bacteria with sonication, the protein was precipitated with ammonium sulfate. Finally, the purified AP205-SpyCatcher was dialyzed into 20 mM Tris-HCl and characterized by running SDS-PAGE gel, agarose gel, dynamic light scattering (DLS) and transmission electron microscope (TEM).\u003c/p\u003e \u003cp\u003e2. Production of S1-SpyTag\u003c/p\u003e \u003cp\u003eVero cells were firstly infected with PEDV-KB2013 strain, then whole RNA was extracted and reverse transcribed into cDNA. S1 protein-encoding sequence was amplified from cDNA using specific primers infused with SpyTag-encoding sequence, then assembled into pTWIST-CMV-BetaGlobin-WPRE-Neo vector (TWIST Bioscience, California, USA) under the control of CMV promoter. When HEK293F cells reached 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e/ml, the plasmid was incubated with PEI in a ratio of 1:3 at room temperature for 15 min to form complexes before adding into cell suspension. After 5 days, the cell supernatant containing S1-SpyTag protein was collected and the target protein was purified using Ni-NTA column (GenScript, Nanjing, China), followed by PBS dialysis to remove imidazole. The purified protein was concentrated and quantified using BCA assay (Beyotime Biotech Inc, Jiangsu, China) before further application.\u003c/p\u003e \u003cp\u003e3. Generation of AP205-S1 vaccine\u003c/p\u003e \u003cp\u003eThe purified AP205-SpyCatcher and S1-Spytag were connected by simply incubating at 4˚C for 16 hours at molar ratio of 1:1. Subsequently, the coupling product was loaded in SDS-PAGE gel and coupling efficiency was calculated based on densitometry analysis. The formed AP205-S1 was loaded to 1% agarose gel to examine the packed RNA and Western blot using AP205-specific antibody was performed to verify the covalent bond.\u003c/p\u003e \u003cp\u003e4. Transmission electron microscope (TEM)\u003c/p\u003e \u003cp\u003eVLPs at 0.2 mg/ml were dropped on copper grid, after which VLPs were stained. Briefly,\u003c/p\u003e \u003cp\u003ethe grid was floated on a drop of VLP solution for 3 min and then dried with filter paper. Next, the dried grid was floated on phosphotungstic acid staining solution for 30 s, after which the extra solution was removed with filter paper. Then the dry grid was observed under HT7700 (Hitachi, Tokyo, Japan) microscope.\u003c/p\u003e \u003cp\u003e5. Mice immunization\u003c/p\u003e \u003cp\u003e7-week-old \u003cem\u003eBALB/c\u003c/em\u003e female mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co. Ltd and kept at SPF animal facility in Anhui Agricultural University according to the guidelines and regulations by the Animal Care and Use Committee, Anhui Agricultural University (SYXK (Anhui) 2016-007). Mice were kept for 1 week to adapt to the new environment before experiment. All experimental procedures were carried out strictly adhere to the guidelines of Animal Care and Use Committee, Anhui Agricultural University. 50 \u0026micro;g AP205-S1 vaccine, S1 protein or AP205-SpyCatcher was administered by subcutaneous injection at lateral abdominal area on day 0 and day 28, during which blood and feces were collected weekly until day 49. Tail vein blood was collected without anesthesia and mice were euthanized under CO\u003csub\u003e2\u003c/sub\u003e atmosphere using a controlled chamber at day 49. Sera were separated from blood and feces were suspended in PBS at 0.1g per 1 ml PBS, after which sera and fecal supernatants were collected and stored at -20˚C.\u003c/p\u003e \u003cp\u003e6. IgG antibody measurement\u003c/p\u003e \u003cp\u003eThe IgG antibody levels in serum and feces were determined via ELISA (enzyme-linked immunosorbent assay). 96-well plates were coated with S1-SpyCatcher protein at 1 \u0026micro;g/ml in PBS overnight at 4˚C. After washing plates with PBS for 4 times, PBS-1% Casein was added to block plates for 2 h at room temperature. Then sera or feces supernatants were added to plates and serially diluted in PBS-1% Casein, which then incubated for 1 h. Next, plates were washed with PBS-0.1% Tween for 4 times and goat anti-mouse IgG-HRP (Makewonderbio, Beijing, China) or goat anti-pig IgG-HRP (Cellwaylab, Luoyang, China) was incubated for 1 h to detect bound IgG antibodies. Finally, TMB developing solution (Makewonderbio, Beijing, China) was added and same volume of 1 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution was added to stop the reaction. The plates were read under OD\u003csub\u003e450 nm\u003c/sub\u003e in a micro plate spectrophotometer (Molecular Device, California, USA). Antibody titers were calculated as the dilution folds that reached half the OD\u003csub\u003emax\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e7. IgA antibody measurement\u003c/p\u003e \u003cp\u003eIgG may compete with IgA antibodies for S1 binding sites, so as IgG should be removed in case underdetermining IgA responses. Serum or feces supernatant was firstly incubated with Protein A beads (Senhui Microsphere Tech, Jiangsu, China) for 30 min at room temperature, after which supernatant was collected for IgA detection. As for IgA ELISA, most procedures were as described above except that the samples were pretreated and detection antibody was goat anti-mouse IgA conjugated with HRP (Abcam in China, Shanghai, China). The binding curve was drawn using OD\u003csub\u003e450 nm\u003c/sub\u003e and dilution folds.\u003c/p\u003e \u003cp\u003e8. PEDV amplification and titer determination\u003c/p\u003e \u003cp\u003ePEDV-KB2013 viruses were amplified in Vero cells, which were cultured in DMEM medium. When cells reached 80% confluent on plates, virus was seeded and incubated for 1 h, which then was washed away with PBS for 3 times. After culturing for 48 hours, cytopathic changes were observed, such as rounding, forming cluster or syncytium and detaching from plates. Infected cells were collected after trypsinization and frozen in -80˚C.\u003c/p\u003e \u003cp\u003eViral titer was determined using plaque formation assay. Basically, single layer of Vero cells (100% confluent) was infected with thawed virus-containing supernatant, which was serially diluted. Similarly, the infection lasted for 1 hour and then cells were kept in 37˚C, 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere until visible plaques were observed. Titers were calculated and the viral titer was 5.75 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e PFU/ml.\u003c/p\u003e \u003cp\u003e9. Indirect immunofluorescence assay (IFA)\u003c/p\u003e \u003cp\u003eIn addition to ELISA, IFA was carried out to assess whether the immunized sera were able to recognize and bind to natural viruses. Generally, 200 TCID\u003csub\u003e50\u003c/sub\u003e virus was added to 70% confluent Vero cells in 24-well plate for 1 hour. 24 hours later, cell supernatants were removed and washed with PBS for 3 time. Then cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with FoxP3/Transcription Factor Staining Buffer Kit (MuitiScience Biotech Co., Ltd, Hangzhou, Zhejiang, China). Next, PBS-1% Casein was applied to block extra binding sites of plate for 2 h at 37˚C. Subsequently, diluted sera were added and incubated at 37˚C for 1 h. After washing with PBS for 3 times, detection antibody goat-anti mouse IgG-FITC (Biosharp, Anhui, China) was incubated with cells at 37˚C for 1 h. Lastly, cell nuclei were stained with DAPI at room temperature for 10 min. The images were captured under fluorescent microscope.\u003c/p\u003e \u003cp\u003e10. Neutralizing assay\u003c/p\u003e \u003cp\u003eCytopathic effects (CPE) caused by pretreated PEDV viruses were used to reflect viral neutralizing abilities of immunized sera. Simply, sera were firstly inactivated at 56˚C for 30 min and PEDV-KB2013 was incubated with inactivated serum for 1 h at 37˚C, which was 1:2 serially diluted from 1:20. The reaction mixtures were added to Vero cells (70% confluent) and incubated at 37˚C for 1 h, which were then removed and washed with PBS for 3 times. Then, cells were cultured for 48 h and CPE was observed under microscope, from which the neutralizing titers were noted as the dilution folds to completely neutralize viruses.\u003c/p\u003e \u003cp\u003eTo visualize the neutralizing effects of immunized sera, IFA was performed as well. Instead of PEDV virus alone, immunized sera pre-incubated sera were used to infect Vero cells. In addition, poly clonal antibodies derived from rabbit against PEDV-N protein (self-made) were added to recognize infection-competent viruses. Goat-anti rabbit IgG-FITC was used as detection antibody, which reflects the amounts of infected viruses.\u003c/p\u003e \u003cp\u003e11. ELISpot assay\u003c/p\u003e \u003cp\u003eTo determine the plasma cell counts in immunized mice, spleens were harvested at day 49 after mice were euthanized. In brief, single cell suspension was obtained by smashing spleen on a 70 \u0026micro;m strainer. Afterwards, erythrocytes were removed by suspending cells in ACK buffer for 5 min at room temperature, after which same volume of medium was added and cells were spined at 300 g for 5 min. Then cells were resuspended in RPMI 1640 medium supplemented with 10% FBS and certain counts of cells were seeded in 96-well Multiscreen plate (MilliporeSigma, Massachusetts, United States). Before seeding cells, the plate was coated with 10 \u0026micro;g/ml S1 protein overnight at 4˚C and blocked with PBS-1%Casein at 37˚C for 2 hours. After 5 hours incubation at 37˚C, 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere, cells were discarded and primary antibody Goat Anti-Mouse IgG (ThermoFisher, Massachusetts, United States) was incubated overnight at 4˚C. Next, secondary antibody Donkey Anti-Goat IgG-AP (Biosharp, Anhui, China) was incubated at room temperature for 2 hours. Finally, spots were visulaized using BCIP/NBT substrate kit (Biosharp, Anhui, China).\u003c/p\u003e \u003cp\u003e12. Piglets immunization and viral challenge\u003c/p\u003e \u003cp\u003eNeonatal piglets used in this study were kept at experimental animal center of Northwest Agriculture \u0026amp; Forestry University. The experiment protocol was reviewed and approved by the Animal Welfare Committee of Northwest A\u0026amp;F University. All animals were monitored on a daily basis for any clinical signs during the whole experimentation. Piglets at 7-day-old were primed (day \u0026minus;\u0026thinsp;21) and boosted at day \u0026minus;\u0026thinsp;7 with 100 \u0026micro;g AP205-S1 or AP205-SpyCatcher in 200 \u0026micro;l MONTANIDE ISA 206 VG (Seppic Shanghai Chemical Specialities, Shanghai, China) by intramuscular injection at neck area. Animals were orally challenged with 1*10\u003csup\u003e5\u003c/sup\u003e TCID50 KB2013 at day 0. Sera and rectal swabs were collected at 0, 3, 5, 7, 10 and 14 days after challenge. Each group was assigned randomly with 4 individuals from the same litter. The severity of diarrhea was determined as follows: normal and soft feces were scored as 0, loose as 1 and watery as 2. All animals were euthanized at day 14 post challenge by bleeding out from axillary artery of forelimb under deep anasthesia with intramuscular injection of 5 mg/kg xylazine. Note: one animal in AP205-S1 group died from congenital hypoplasia at day 4.\u003c/p\u003e \u003cp\u003e13. RT-qPCR\u003c/p\u003e \u003cp\u003ePEDV loads in serum and feces of challenged piglets were assessed by RT-qPCR. Briefly, total RNA was isolated from serum or rectal swabs using Viral Genome Extraction Kit (Qingdao Lijian Bio-Tech, Qingdao, China) according to manual. Then cDNA was obtained from RNA using HyperScript III 1st Strand cDNA Synthesis Kit with gDNA Remover (EnzyArtisan Biotech, Shanghai, China), which was used as qPCR template. The reaction system was as follows (20 \u0026micro;l): 10 \u0026micro;l SYBR Green, 8 \u0026micro;l ddH\u003csub\u003e2\u003c/sub\u003eO, 0.5 \u0026micro;l Primer-F (10 \u0026micro;M), 0.5 \u0026micro;l Primer-R (10 \u0026micro;M), 1 \u0026micro;l cDNA. The primer sequences were F: GAGGGTGTTTTCTG -GGTTG, R: CGTGAAGTAGGAGGTGTGTTAG. To set up standard curve, the target gene (N gene) was constructed on pET28a and the Ct = -3.225Log\u003csub\u003e10\u003c/sub\u003e (Copies)\u0026thinsp;+\u0026thinsp;36.87.\u003c/p\u003e \u003cp\u003e14. Statistical analysis\u003c/p\u003e \u003cp\u003eThe significance analysis was performed in GraphPad Prism 9 (GraphPad Software, Inc. La Jolla, CA, USA). A \u003cem\u003eP\u003c/em\u003e value from unpaired t-test was indicated as \u0026le;\u0026thinsp;0.05 (*), \u0026le;\u0026thinsp;0.01 (**), \u0026le;\u0026thinsp;0.001 (***), \u0026le;\u0026thinsp;0.0001 (****). All error bars were displayed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e1. SpyCatcher/Tag bond effectively conjugated AP205-VLP and S1 protein without compromising advantages of VLP.\u003c/p\u003e \u003cp\u003eChemical coupling was the first option tried to link purified AP205-VLP and S1 protein. However, this did not work efficiently, probably due to the relatively large molecular size of S1 protein (data not shown). We then attempted to produce AP205-S1 vaccine via SpyCatcher/Tag specific binding as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. Firstly, AP205-SpyCatcher was successfully expressed and purified, with a molecular weight of 29 kD per subunit as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. In addition, agarose gel showed that ssRNA was packaged in AP205-SpyCatcher (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Although the molecular size of SpyCatcher (15 kD) was equivalent to that of AP205 monomer (14 kD), AP205-SpyCatcher still kept viral particle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter purifying S1-SpyTag expressed by HEK293 cells, AP205-S1 was generated by incubating AP205-SpyCatcher and S1-SpyTag. To find out the optimal reaction conditions, different molecular ratios between AP205-SpyCatcher and S1-SpyTag were tested. The band slightly lifted above S1-SpyTag in SDS-PAGE gel was AP205-S1, which were shown at all ratios (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Meanwhile, the AP205-SpyCatcher band faded when mixed with S1-SpyTag. When the ratio was 1:1, the coupling efficiency was highest (49%), at which ratio the AP205-S1 vaccine was generated for further characterization and immunization. Western blot result using AP205-specific monoclonal antibody confirmed that covalent bond was formed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE. Rather than a clear RNA band of AP205-SpyCatcher, RNA in AP205-S1 was diffuse, but co-localized with the protein band after coomassie blue staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Furthermore, DLS showed that AP205-S1 particles were homogenous at 80 nm diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG) and TEM revealed the vaccine was in viral particle structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Taken together, these results suggested that AP205-S1 connected by SpyCatcher/Tag bond was homogeneous, remained stable spheric shape and packaged with prokaryotic ssRNA.\u003c/p\u003e \u003cp\u003e2. AP205-S1 vaccine exhibited superb immunogenicity in mice.\u003c/p\u003e \u003cp\u003eTo examine the capabilities of AP205-S1 to induce immune responses \u003cem\u003ein vivo\u003c/em\u003e, \u003cem\u003eBALB/c\u003c/em\u003e mice were primed at day 0 and boosted at day 28 via subcutaneous injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). IgG antibody levels in sera were tested using ELISA, which demonstrated that AP205-S1 immunized mice produced S1-specific IgG antibodies as early at day 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The IgG levels increased with time and remarkably further increased after booster, which peaked at day 35 and kept high until day 49. On the other hand, S1 protein immunized mice showed decent antibody levels only after second injection. As expected, AP205-SpyCatcher could not induce S1-specific antibody responses after two injections. S1-specific IgG titers induced by AP205-S1 were significantly higher than S1 or AP205-SpyCatcher alone, demonstrating that displays of S1 on AP205 greatly increased immunogenicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Accordingly, AP205-S1 stimulated significantly more plasma cells that secrete S1-specific IgG antibodies than S1 and AP205-SpyCatcher at day 49 via ELISpot assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, IFA was performed to determine whether the immunized sera could recognize and bind to PEDV-infected cells \u003cem\u003ein vitro\u003c/em\u003e. Consistent with the ELISA results, AP205-SpyCatcher immunized sera at day 49 did not bind to PEDV-infected cells, as no FITC signals were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). As for the S1 immunization, day 28 sera did not bind to PEDV, while day 49 sera could bind to PEDV, reflecting the higher antibody titers after booster (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In addition, AP205-S1 elicited IgG antibodies at day 28 were able to bind to PEDV viruses \u003cem\u003ein vitro\u003c/em\u003e as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD. These results not only suggested that S1 displayed on AP205-VLPs was in natural conformation, but also indicated that AP205-S1 stimulated strong immune responses in mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3. S1-specific IgG antibodies in AP205-S1 immunized mice were of high affinity.\u003c/p\u003e \u003cp\u003eHigh binding strengths between antibody and virus were correlated with neutralizing potentials, therefore avidity ELISA was performed to assess the amounts of high-avidity antibodies. The essence of avidity ELISA is that those antibodies bound weakly to S1 protein are washed away by 7 M urea, whereas high-avidity antibodies were remained on plates. As expected, the high-avidity antibodies were significantly less than total IgG levels as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. Consistent to total IgG levels, high-avidity IgG antibodies were relatively low before booster (day 28). In contrast, the high-avidity IgG antibodies were significantly elevated after booster at day 49 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), indicating that the affinity maturation was enhanced after booster. There results suggested that AP205-S1 could induce high-avidity antibodies to potentially neutralize PEDV.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e4. Systemic IgA antibodies against S1 protein were induced by AP205-S1.\u003c/p\u003e \u003cp\u003eIgA antibodies are of great importance in controlling viruses in addition to IgG, especially in mucosa. After first immunization, S1-specific IgA antibodies were stimulated by AP205-S1 and S1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Similar to IgG levels, IgA antibody levels in sera were elevated after booster, and AP205-S1 demonstrated higher immunogenicity than S1 protein in terms of IgA antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). AP205-SpyCatcher failed to induce S1-specific IgA antibodies in mice sera even after two immunizations. Although the IgA levels in sera reflect those in mucosa to certain extent, IgA antibodies were not detected in fecal supernatants of all immunized mice (data not shown), indicating the systemic immunization failed to induce mucosal IgA antibodies. Nevertheless, systemic IgA antibodies were shown to grant excellent viral neutralizing effects in PEDV infected pigs, especially S1-specific IgA. In conclusion, AP205-S1 was capable to induce systemic high amounts of IgA antibody in mice upon systemic administration, which is likely to neutralize PEDV.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e5. AP205-S1 immunized mice sera exhibited premium PEDV neutralizing capabilities.\u003c/p\u003e \u003cp\u003eNext, the abilities of AP205-S1 elicited antibodies to neutralize PEDV viruses were assessed. The viruses were firstly incubated with immunized sera, which were then added into Vero cells. IFA results demonstrated that AP205-SpyCatcher immunized sera failed to neutralize KB2013 virus, thus FITC signals were detected as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. In contrast, AP205-S1 immunized mice sera completely neutralized PEDV virus from infecting Vero cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). It\u0026rsquo;s noteworthy that S1 elicited antibodies partly neutralized PEDV, indicating that S1 protein could induce neutralizing antibodies but with strongly delayed kinetics and lower levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, CPE assays were carried out to determine the neutralizing titers of vaccinated sera. As expected, AP205-SpyCatcher immunized sera did not show any neutralizing effects. S1 immunized sera showed slight viral neutralizing capacity after booster, whereas AP205-S1 immunized sera exhibited viral neutralizing effects throughout all tested time points. In accordance with antibody titers, neutralization titers of AP205-S1 immunized sera were evidently elevated after booster, some reaching 1:160 at day 49 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). To summarize, both IFA and CPE proved that AP205-S1 induced neutralizing antibodies.\u003c/p\u003e \u003cp\u003e6. Neutralizing mucosal IgG antibodies were stimulated by AP205-S1 in mice.\u003c/p\u003e \u003cp\u003eBecause PEDV is an enteric virus, local gastrointestinal antibodies play vital roles in defending viral infection at first place. To find out if mucosal antibodies were induced after immunization, mice fecal supernatants were used for ELISA as well. The results showed that mucosal IgG antibodies were induced by both AP205-S1 and S1 protein after booster (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Again, AP205-S1 induced substantially more mucosal S1-specific IgG antibodies than S1 protein. In general, mucosal IgG levels of both AP205-S1 and S1 immunized mice were obviously lower than those in sera. Nevertheless, they specifically bound to natural PEDV viruses \u003cem\u003ein vitro\u003c/em\u003e as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB. Furthermore, AP205-S1 elicited mucosal antibodies completely hindered PEDV viruses from infecting Vero cells, while S1 elicited mucosal antibodies partially did so (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Collectively, these results revealed that systemic administration of AP205-S1 was capable to induce superior systemic as well as mucosal antibody responses, both of which effectively neutralized PEDV.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e7. AP205-S1 induced systemic and mucosal antibodies were cross-reactive to AH2018-HF1 (G-I strain)\u003c/p\u003e \u003cp\u003eOne major obstacle for PEDV control is the immune escape of emerging variants, which could be overcome by vaccines that render broadly cross-protections. To investigate whether antibodies induced by AP205-S1 could recognize and neutralize other PEDV strain, AH2018-HF1 (G-I strain) was used, whose S1 protein showed 88.5% similarity with PEDV-KB2013 (Supplementary material). IFA results demonstrated that AP205-S1 immunized mice sera (day 49) could bind to AH2018-HF1 \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA), which could neutralize the virus as well (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Furthermore, the AH2018-HF1 neutralizing titers of immunized sera were significantly higher than those of AP205-SpyCatcher immunized sera (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC), meaning that AP205-S1 could induce cross-neutralizing antibodies against a G-I virus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, whether AP205-S1 elicited mucosal antibodies cross-reacted with AH2018-HF1 virus was assessed. IFA results suggested that they could recognize (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA) and neutralize (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB) AH2018-HF1 virus. In conclusion, the KB2013 derived vaccine AP205-S1 induced potent systemic and local antibody responses that not only neutralized KB2013 virus also cross-reacted to a G-I strain AH2018-HF1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e8. Immunization of piglets with AP205-S1 posed protection against PEDV infection\u003c/p\u003e \u003cp\u003eWith the clear results of mice that AP205-S1 elicited neutralizing antibodies in serum and mucosa, we wanted to determine the protective effects in piglets after PEDV challenge. Suckling piglets were immunized with AP205-S1 twice and then challenged with a lethal dose (in 7-day old piglets) of KB2013 virus (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA). High levels of S1-specific IgG antibodies were induced in sera after booster (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB), which could recognize PEDV \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eD). Moreover, PEDV viruses were entirely neutralized by immunized sera as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC, indicating the AP205-S1 demonstrated excellent immunogenicity in piglets as well. Because the piglets were 28-day-old at the day of challenge, all animals survived even those immunized with AP205-SpyCatcher (control). In general, AP205-S1 immunized piglets showed more active movements (data not shown). Additionally, less watery feces were observed in AP205-S1 immunized piglets on 5 to 7 days post challenge although the difference was not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eE). In addition, the viral loads in immunized pigs were lower than control pigs yet not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eF). This latter observation is similar to the experience with COVID-19 vaccines, where limited impact on viral spread was observed despite strongly reduced disease. Overall, AP205-S1 demonstrated modest prophylactic effects in piglets against PEDV infection, which needs further development for industrial application.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePEDV has been an important pathogen in pork industry for decades, and vaccines are under intense development around the world. To accommodate with the sustained novel variants, vaccine candidates are required to update with time, particularly after 2010, when G-II strains started being a major concern. Typically, S1 protein of coronaviruses specifically binds to receptors and S2 mediates membrane fusion, assisting to virus internalization. Separate studies have shown that blocking both S1 and S2 activities could cease the virus invasion. Cocktail antibodies REGN-COV against receptor binding domain (RBD) in S1 region of SARS-CoV-2 could effectively neutralize and reduce the viral loads in patients\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Serum antibodies from convalescent patients were identified to recognize an epitope located at the site of the fusion peptide of S2 protein, which could be a neutralizing epitope\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Regardless that porcine aminopeptidase N (pAN), sialic acid, C-type lectin, and heparan sulfate were reported to be involved in viral entry in different models, the host receptors for PEDV are not defined definitively, which may require coordination of protein-protein binding and glycan-protein binding\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. After unveiling the S protein structure of PEDV by Cyro-EM, NTD and CTD located in S1 protein were regarded as functional domain responsible for glycan and protein binding, respectively\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Hence, there is no doubt that S1 protein of PEDV plays the key part in binding cell receptor, which could be the highlight of vaccine targets. Based on these rationales, we here chose S1 protein as targeting antigen to design PEDV vaccine.\u003c/p\u003e \u003cp\u003eHowever, free proteins usually stimulate insufficient antigen presentation, consequently induce limited immune responses. Displaying targeting proteins on a VLP scaffold could amplify the antigen presenting signals, owing to the following merits: i. Nanometer size of VLP makes it freely drain in lymphatic vessels, so that be accessible to present antigens; ii. Repetitive organization of antigens on VLPs magnifies antigen presenting signaling; iii. Distances between adjacent antigens on VLPs are ideal for BCR crosslinking; iv. Nucleic acids packaged inside of VLPs stimulate innate immune responses, facilitating specific adaptive responses\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The AP205-S1 vaccine in this study demonstrated particulate shape with diameter of around 80 nm and prokaryotic ssRNA was packed. Different from a focused RNA band packed in AP205-SpyCatcher, the RNA packed in AP205-S1 was diffuse probably owing to the disparate numbers of S1-SpyTag protein on each particle. Thus, the fact that AP205-S1 vaccine incited potent specific antibody responses is coincident with the previous studies that TLR signaling triggered by VLP-packaged ssRNA was critical for antibody affinity maturation and secondary plasma generation\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe PEDV structural proteins envelop (E) protein, membrane (M) protein and S protein have been shown to form VLPs after external expression in both baculovirus and mammalian cells. Immunizing mice with PEDV-VLPs composed of E, M and S protein could induce IgG and IgA production and T cell responses\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. When pigs were vaccinated with PEDV-VLPs, both humoral and cellular immune responses were developed, posing some extent of protection against viral infection\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In addition to PEDV VLPs, a putative B cell epitope in S protein displayed on Hepatitis B Core Antigen (HBcAg) VLP was reported to prompt neutralizing antibody production in mice and gilts\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe major disadvantage of AP205-S1 may be that the S1 protein was produced in eukaryotic cells, which bears relatively high costs. Therefore, it might not be easily feasible for scale-up production and veterinary application. Nevertheless, we show here that S1 contained neutralizing epitopes that induced a PEDV neutralizing antibodies response. Either alternative cheaper expression systems (like yeast or insect cells) to produce S1 protein or truncated S1 protein (RBD), which could be expressed in \u003cem\u003eE. coli\u003c/em\u003e, will be tested to generate cost-friendly PEDV vaccine candidates. Moreover, when it comes to large-scale production, the costs of mammalian cell expressing protein can be greatly reduced by optimizing the expression system or constructing stable-expression cell line. Indeed, W. Guo et al. has used an efficient expression system in HEK293F cells to produce S protein as well, which demonstrated excellent immunogenicity regarding humoral and cellular immune responses\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In addition, X. Song et al. immunized pigs with eukaryotic cell produced bivalant subunit vaccines, which provided protection against both G-IIa and G-IIb strains\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Meanwhile, SpyCatcher/Tag system still will be used to link AP205-VLP and antigens due to the relatively high efficiency and the reaction is accomplished by easily mixed and incubated at moderate condition.\u003c/p\u003e \u003cp\u003eThe mechanisms of VLPs to promote IgA generation were reported as well. Norovirus VLP recalled IgA responses in a humanized mouse model, which showed superior virus-neutralizing activities\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In VLP immunized mice, IgA antibodies were successfully induced in serum by both systemic (subcutaneous) and mucosal (intranasal) administration\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In addition, both systemic and mucosal IgA antibody responses were dependent on TLR7 signaling in B cells or lung DC and alveolar macrophages, respectively\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Consistently, AP205-S1 not only strongly prompted IgG responses, but also induced S1-specific IgA antibodies in serum. Whether the IgA levels could be increased by one more booster or intranasal immunization would require further investigation. On the other hand, IgA antibodies in intestinal mucosa were not detected in our study, inferring the subcutaneous immunization failed to induce mucosal IgA responses. This might be defeated by changing vaccination routes to intranasal or intrarectal application.\u003c/p\u003e \u003cp\u003eGerminal center reactions, which are crucial for antibody affinity maturation, were demonstrated to be favored after VLP immunization. We previously found that GC reactions were accelerated when TLR7 signaling was activated during VLP immunization\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The downstream MyD88 signaling, especially B cell-intrinsic TLR7 signaling was involved in GC reactions\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. In this study, AP205-S1 possessed TLR7 ligands and induced high affinity antibodies in mice, suggesting the vaccine effectively stimulated S1-specific GC reactions although they were not examined. It was claimed that high affinity antibodies were related with viral neutralizing capacities, exemplified by HIV-1 bearing patients. With \u0026ldquo;extreme mutation\u0026rdquo; on Ig variable regions, high-affinity antibodies could broadly neutralize HIV-1 strains\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Accordingly, repeated exposure to antigens increased antibody affinities in convalescent patients, accompanied with improved SARS-CoV-2 neutralizing abilities\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In this study, AP205-S1 was so efficient as to incite high affinity antibodies after only one booster immunization. It is worthwhile to find out whether one more booster would induce even more high-affinity antibodies and durable antibodies, which was not assessed in this study as well.\u003c/p\u003e \u003cp\u003eKnowing that high-affinity antibodies were generated by the mice, their neutralization potentials were tested. Consistent with great amounts of high-affinity antibodies in sera at day 49, S1-specific antibodies exhibited predominant neutralization capacities. The PEDV-neutralizing abilities increased with the growing antibodies levels and dramatically elevated after booster. The CPE results reflected the overall neutralizing effects of all types of antibodies in samples. According to IFA results, IgG antibodies in both serum and mucosa executed viral neutralizing function. On the other hand, whether IgA antibodies in immunized sera neutralized PEDV was not determined in our study. Others have claimed that IgA antibodies were able to neutralize SARS-CoV-2\u003csup\u003e49\u003c/sup\u003e and the specific binding of high-avidity IgA antibodies to surface antigens prevented entero-pathogens division, termed as \u0026ldquo;enchained growth\u0026rdquo;\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Therefore, we extrapolate that the S1-specific IgA antibodies in immunized sera contributed to viral neutralization as well.\u003c/p\u003e \u003cp\u003eMucosal IgA plays a critical role for PEDV defenses, whereas we did not detect IgA antibodies in fecal supernatants, which was because the immunization route was systemic. It has been reported that intranasal administration of VLPs induced mucosal IgA responses while subcutaneous injection failed to do so\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, which is consistent with our data. In addition, we are testing the mucosal IgA antibody levels via intranasal immunization as well, which is not shown in the current manuscript. The results showed that AP205-S1 could effectively induce both respiratory and intestinal IgA antibodies via intranasal immunization.\u003c/p\u003e \u003cp\u003eThe ideal PEDV vaccine candidates ought to induce broadly cross-reactive antibody responses so that different variants could be prevented with a single vaccination. Here, we demonstrated that AP205-S1 elicited antibodies not only neutralized the parental PEDV strain (KB2013), but also could neutralize a G-I strain AH2018-HF1. Surprisingly, the neutralizing titers against AH2018-HF1 were slightly higher than KB2013. The results suggested that the AP205-S1 was potential to induce cross-reactive neutralizing antibodies in mice.\u003c/p\u003e \u003cp\u003eLastly, AP205-S1 immunized piglets produced S1-specific IgG antibodies after two doses, and these antibodies could recognize and neutralize PEDV viruses \u003cem\u003ein vitro\u003c/em\u003e. At this premise, the piglets were protected by less severe diarrhea, less viral shedding and more energetic activities. However, these findings were not statistically significant, probably due to the limited group size, which can be larger in the future to improve. Besides, there were no animals died because they were too old to die from PEDV challenge in our settings. In this case, the vaccine should be applied to pregnant sows to protect the newborns. In line with observations for SARS-CoV-2 infected individuals after vaccination, viral shedding was not strongly affected, perhaps due to limited presence of specific IgA antibodies, which may be a common problem for coronavirus vaccines. Rationally, the newborn piglets count on passive immunity from maternal antibodies rather than active immunization. Due to the low permeability of sow placenta, piglets are born agammaglobulinemic so that they could only rely on the lactogenic immunity. In this case, the maternal immunity means the lactogenic immunity. To our knowledge, there\u0026rsquo;s no research systemically studies the lactogenic immune responses after VLP vaccination in mice. However, the lactogenic immunogenicity of VLP vaccines in pigs were demonstrated previously by Y. Lu and colleagues, who showed that VLP vaccines incorporated B-cell epitope of PEDV offered lactogenic immunity to neonatal piglets\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The efficacy of PEDV vaccine should focus on the ability to induce maternal antibodies, which is our next plan to dig more in their maternal immunity including lactogenic immunogenicity. Alternatively, younger piglets with stronger signs of disease may be used for challenge.\u003c/p\u003e \u003cp\u003eIn summary, we here presented a PEDV vaccine candidate AP205-S1 based on VLP, which was formed by a specific covalent bond between SpyCatcher and SpyTag. AP205-S1 kept viral nanoparticle structure and packaged ssRNA, so that it effectively stimulated strong immune responses in mice after immunization. High titers of IgG antibodies were induced systemically, which could completely neutralize PEDV viruses and prevent them from infecting Vero cells \u003cem\u003ein vitro\u003c/em\u003e. Importantly, these antibodies cross-neutralized other PEDV strains, suggesting that AP205-S1 caused cross-reactive antibody responses in mice. Furthermore, the vaccine elicited specific IgG responses in piglets, which completely neutralized PEDV \u003cem\u003ein vitro\u003c/em\u003e. The clinical pathologic signs of immunized pigs were less although not significant. This vaccine has great potential to be developed further, offering an attractive alternative for PEDV prevention.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted following the Animal Ethics Procedures and Guidelines of the People’s Republic of China. Mice experiments protocols were approved by the Animal Care and Use Committee of Anhui Agricultural University (Approval No. SYXK (Anhui) 2016-007). Pig experiments\u0026nbsp;protocols\u0026nbsp;were approved by the Animal Welfare Committee of Northwest A\u0026amp;F University (Approval No. ICAUC-2024-CVM032).\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\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLisha Zha is involved in a pet vaccine company and owns shares. Other authors claim no interest conflict.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by National Key Research and Development Plan (2022YDF1801800, to Lisha Zha), Anhui Provincial Natural Science Foundation (2408085QC074, to Xinyue Chang) and Starting Foundation of Anhui Agricultural University (to Xinyue Chang).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ. G and X. Z performed animal experiments and provided all figures; C. L and S. W helped prepare VLP material; X. X helped analyze data; M. F. B and Y. N reviewed and corrected the manuscript; L. L and P. S provided virus material; L. Z supervised the project; X. C wrote the main manuscript text. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJung K, Saif LJ, Wang Q (2020) Porcine epidemic diarrhea virus (PEDV): An update on etiology, transmission, pathogenesis, and prevention and control. Virus Res 286:198045\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi M, Pan Y, Xi Y, Wang M, Zeng Q (2023) Insights and progress on epidemic characteristics, genotyping, and preventive measures of PEDV in China: A review. Microb Pathog 181:106185\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Q, Vlasova AN, Kenney SP, Saif LJ (2019) Emerging and re-emerging coronaviruses in pigs. 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Nat Rev Immunol 22:236\u0026ndash;250\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PEDV, AP205-VLP, vaccine, neutralizing antibody","lastPublishedDoi":"10.21203/rs.3.rs-6027078/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6027078/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough vaccines against porcine epidemic diarrhea viruses (PEDV) are available currently, PED outbreaks still occur in many countries due to emerging new variants. Therefore, more endeavors are required to develop efficient and broadly-protective vaccines. To this end, we here present a nanoparticles vaccine candidate AP205-S1 which effectively elicited antibody responses in mice and pigs. The vaccine was generated by coupling S1 protein of PEDV-KB2013, a G-II strain to bacterially expressed AP205-VLP via SpyCatcher/SpyTag. AP205-S1 demonstrated intact and homogenous viral particle structure and packed \u003cem\u003eE. coli\u003c/em\u003e-derived ssRNA. Upon administration in mice, AP205-S1 induced high titers of S1-specific IgG antibodies in sera as well as in gastrointestinal tracts, especially after booster. Importantly, these antibodies were able to neutralize PEDV \u003cem\u003ein vitro\u003c/em\u003e, indicating the vaccine is able to induce protective antibodies against PEDV infection. Of note, AP205-S1 elicited antibodies exhibited cross-neutralizing potential against a G-I strain, PEDV-AH2018-HF1, which was preserved in our lab. Last but not least, S1-specific IgG antibodies were stimulated in piglets after AP205-S1 immunization, which could neutralize PEDV \u003cem\u003ein vitro\u003c/em\u003e. Most interestingly, AP205-S1 immunized piglets showed reduced viral loads compared to control piglets upon viral challenge. In conclusion, we generated a VLP-based vaccine candidate against PEDV demonstrating excellent immunogenicity in mice and piglets, which granted potential protection against viral infection. Our work provides an efficient option for prevention of future PEDV epidemics.\u003c/p\u003e","manuscriptTitle":"A bacteriophage based virus-like particle vaccine induces cross-reactive neutralizing antibodies against porcine epidemic diarrhea viruses (PEDV)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-18 12:28:38","doi":"10.21203/rs.3.rs-6027078/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0e0323bc-67bf-49ce-adaf-d7c62377d465","owner":[],"postedDate":"April 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:09:04+00:00","versionOfRecord":{"articleIdentity":"rs-6027078","link":"https://doi.org/10.1186/s13567-025-01559-z","journal":{"identity":"veterinary-research","isVorOnly":false,"title":"Veterinary Research"},"publishedOn":"2025-07-01 15:58:42","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-04-18 12:28:38","video":"","vorDoi":"10.1186/s13567-025-01559-z","vorDoiUrl":"https://doi.org/10.1186/s13567-025-01559-z","workflowStages":[]},"version":"v1","identity":"rs-6027078","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6027078","identity":"rs-6027078","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}