Development of a Recombinant Adeno-Associated Virus Vaccine Expressing PEDV S1 Protein: Enhanced Humoral and Cellular Immunity with Superior Neonatal Protection

Development of a Recombinant Adeno-Associated Virus Vaccine Expressing PEDV S1 Protein: Enhanced Humoral and Cellular Immunity with Superior Neonatal Protection | 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 Article Development of a Recombinant Adeno-Associated Virus Vaccine Expressing PEDV S1 Protein: Enhanced Humoral and Cellular Immunity with Superior Neonatal Protection Maonan Pang, Meishen Ren, Dike Jiang, Teng Tu, You Zhou, Yin Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6224876/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Nov, 2025 Read the published version in npj Vaccines → Version 1 posted 9 You are reading this latest preprint version Abstract The continuous spread of porcine epidemic diarrhea virus (PEDV) has highlighted the importance and necessity of developing accessible, safe, effective, and versatile vaccine platforms. While approved inactivated and attenuated vaccines have been instrumental in reducing the burdens of prevention and control and economic impacts, the induction of antibody level remains an unmet need. Here, we constructed a recombinant adeno-associated virus (rAAV-CMV-PEDV S1) expressing regions of the PEDV S1 subunit based on Adeno-associated virus serotype 2 (AAV-2) vector). We assessed its immunogenicity, durability, and protective efficacy. In mice, rAAV-CMV-PEDV S1 induced significantly more remarkable and persistent serum-specific IgG compared with the inactivated vaccine and accompanied by robust CD3 + CD8 + T cells activation within 4 weeks post-immunization. In pregnant sows, compared with commercially available inactivated and attenuated vaccines, the rAAV-CMV-PEDV S1 induced significantly higher serum and colostral IgG as well as neutralizing antibody titers, which provided piglets with abundant maternal antibodies. Crucially, challenge experiments demonstrated that rAAV-CMV-PEDV S1 conferred 80% clinical protective efficacy for piglets, accompanied by significantly reduced viral shedding loads, surpassing other vaccine groups These findings suggest that rAAV-CMV-PEDV S1 candidate could be a promising PEDV vaccine for enhancing antibody levels and could strengthen the protection of piglets against PEDV infection. Biological sciences/Immunology Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Porcine epidemic diarrhea (PED), a highly detrimental intestinal infectious disease, has inflicted heavy losses on the global swine industry. The spread of its causative agent, the porcine epidemic diarrhea virus (PEDV), poses a severe threat to the health of pig populations and the sustainable development of the swine- farming sector [ 1 – 2 ]. PEDV belongs to the genus Alphacoronavirus within the Coronaviridae family. It is an enveloped, single -stranded positive-sense RNA virus with a diameter of approximately 95–190 nm [ 3 ]. Its genome, around 28 kb in length (excluding the poly A tail), encodes multiple proteins, including four structural proteins (S, M, E, and N), sixteen non-structural proteins (nsp1 - nsp16), and an accessory protein ORF3 [ 4 – 6 ]. Among these, the S glycoprotein is a crucial structural protein on the viral surface. It can be further divided into two subunits, S1 and S2[ 7 – 8 ]. The S1 subunit plays a crucial role in binding to the surface receptors of host cells, thereby initiating the viral infection process [ 9 ]. Moreover, it encompasses multiple neutralizing epitopes. Notably, through cryo-electron microscopy (cryo-EM) structural analysis, it has been found that the S1 trimer contains 4 conserved neutralizing epitopes [ 10 ]. This characteristic makes it an ideal antigenic target for the development of vaccines. The S2 subunit plays a vital role in the fusion of the virus with cells and cell-to-cell membranes, facilitating the virus's entry into host cells and subsequent replication [ 11 ]. PED was first identified in the United Kingdom in 1977. Subsequently, it spread rapidly across Europe, causing substantial damage to the local swine industry [ 12 ]. With the increasing frequency of global swine trade, PED gradually disseminated to numerous Asian countries, such as Japan, South Korea, Pakistan, Malaysia, and China [ 13 ]. Since 2011, PED has been prevalent on a large scale in China for an extended period, bringing about a huge economic impact on the domestic swine-farming industry [ 14 ]. In 2013, PED erupted in the United States, resulting in a large number of piglet deaths, a sharp decline in pig inventories, and a severe blow to the entire swine-farming industry chain, and caused an estimated $ 1.6 billion loss in the US swine industry during 2013–2014[ 15 ]. PEDV has a broad host range and can infect pigs of all ages through direct or indirect fecal-oral routes[ 16 ]. It is particularly devastating to suckling piglets within one week of age, with a mortality rate that can reach as high as 80–90% [ 17 ]. This not only leads to a significant reduction in the number of piglets and an increase in breeding costs but also severely affects the production efficiency and economic benefits of pig farms [ 18 ]. Currently, PEDV vaccines available on the market, such as traditional inactivated and attenuated vaccines, have several limitations in practical applications. Inactivated vaccines, although having a relatively short production cycle and high safety, are weak in stimulating the body to produce cellular and mucosal immunity, making it difficult to comprehensively and effectively resist virus invasion[ 19 , 20 ]. Attenuated vaccines can trigger a relatively strong immune response, but due to the instability of the PEDV genome, gene mutations and recombinations are likely to occur during the passage process. This may lead to the reversion of the virulence of attenuated strains, potentially causing harm to the pig herd. Additionally, the adaptability issues of PEDV variant strains in cell culture limit the large-scale production of traditional inactivated and attenuated vaccines. Adeno-associated virus (AAV), a single-stranded DNA virus lacking self-replication ability, possesses unique advantages that render it a mature and widely used gene delivery vector [ 21 , 22 ]. AAV can infect various cell types and stably express foreign proteins over an extended period. It also has low immunogenicity and causes no obvious side effects on the body [ 23 , 24 ]. In recent years, AAV has made remarkable progress in the field of gene therapy. In 2012, the European Medicines Agency (EMA) approved the AAV-based gene therapy drug "Glybera" for the treatment of inherited lipoprotein lipase deficiency (LPLD) [ 25 ]. In 2017, the US Food and Drug Administration (FDA) approved Spark Therapeutics' LUXTURNA, a recombinant adeno- associated virus serotype 2 (rAAV2-hRPE65) expressing human retinal pigment epithelium 65 kDa protein, for the treatment of patients with confirmed biallelic RPE65 mutation-associated retinal dystrophy [ 26 ]. AAV vectors are also extensively applied in the development of vaccines against various viral infectious diseases, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and influenza A virus subtype (H1N1) [ 27 – 30 ]. In addition, studies have shown that AAV2 demonstrated superior intestinal tropism (3.8-fold higher than AAV8 in porcine enteroids, p < 0.01), which makes it more suitable for the prevention and treatment of diseases caused by intestinal-related viruses[ 31 ]. Given the current severe threat of PEDV to the swine industry and the inadequacies of existing vaccines, the development of novel, efficient, and safe PEDV vaccines is of utmost urgency. In this study, we constructed a recombinant adeno-associated virus serotype 2 vector vaccine (rAAV-CMV-PEDV S1) expressing the PEDV S1 protein by cytomegalovirus (CMV) promoter. Our objectives were to comprehensively analyze its biological characteristics, safety, and immunogenicity, evaluate the immune responses it induced in mouse and sow models, and assess its protective efficacy in neonatal piglets. This research aims to provide a theoretical basis for the development of PEDV vaccines and hopes to open up new avenues for the prevention and control of PED and the healthy development of the swine industry. If successful, this AAV-based platform could be rapidly adapted to combat emerging coronaviruses in livestock, leveraging its modular design for antigen swapping. RESULT Construction, Characterization, and Genome-Based Quantification of rAAV-CMV-PEDV S1 Virions We obtained the two genome of the recombinant adeno-associated virus (rAAV-CMV-PEDV S1/rAAV-CMV-EGFP) by individually cloning the optimized porcine epidemic diarrhea virus (PEDV) S1 gene and enhanced green fluorescent protein (EGFP) gene into the AAV backbone plasmid and incorporating the CMV promoter (Figure.1a) . After separately transfecting the two backbone plasmids into HEK293T cells, successful expression of EGFP in HEK293T cells was observed in the pAAV-CMV-EGFP group, indicating the successful construction of the backbone plasmids (Figure.1b) . We obtained the fully packaged recombinant adeno-associated viruses (rAAV-CMV-PEDV S1 virus and rAAV-CMV-EGFP virus), after co-transfecting the two backbone plasmids with the helper plasmid respectively into HEK293T cells and culturing the cells for another 6 days. Subsequently, we verified through indirect immunofluorescence assay (IFA) and Western blot (WB) tests that both the rAAV-CMV-PEDV S1 virus and the rAAV-CMV-EGFP virus can infect cells and express either EGFP or S1 protein (Figure.1c,d) . Transmission electron microscopy (TEM) analysis revealed that both rAAV-CMV-PEDV S1 and rAAV-CMV-EGFP virions exhibited characteristic spherical morphology, with a uniform diameter of approximately 22 nm, consistent with the expected size of adeno-associated virus particles (Figure.1e) . Since rAAV-CMV-PEDV S1 infection does not induce cytopathic effects (CPE), rendering the TCID 50 (50% tissue culture infectious dose) assay inapplicable for viral titer determination, we developed an absolute quantitative qPCR standard curve targeting the viral genome. The equation Y= -3.221log 10 N + 36.441 (E = 104.4%, R 2 = 0.999) was established to quantify genome copies in viral preparations, providing a robust and reproducible metric for viral stock characterization. Following purification and removal of residual free DNA, the titer of rAAV-CMV-PEDV S1 viral particles was quantified as 1.86×10 10 vg/mL using an absolute qPCR standard curve, ensuring accurate measurement of encapsidated genomes. In summary, The recombinant adeno-associated virus expressing the PEDV S1 protein (rAAV-CMV-PEDV S1) was successfully constructed. To quantify antigen content in inactivated and live-attenuated PEDV vaccines and assess viral shedding loads during challenge protection experiments, we developed an absolute quantitative qPCR standard curve targeting the PEDV M gene. The regression equation Y= -3.337log 10 N + 38.601 demonstrated high amplification efficiency (E = 99.4%) and robust linearity (R 2 = 0.999), establishing a validated method for precise PEDV genome quantification across vaccine formulations and in vivo challenge models. The standard curves figures for absolute quantification of rAAV-CMV-PEDV S1 and PEDV genomes, including their respective regression equations (Y= -3.221log 10 N + 36.441 and Y= -3.337log 10 N + 38.601), are provided in Supplementary Fig. 1, 2 , demonstrating their high linearity (R 2 = 0.999 for both) and amplification efficiencies (E = 104.4% and 99.4%, respectively). rAAV-CMV-PEDV S1 vaccine elicits robust and sustained humoral-cellular immunity with superior CD8 + T-cell activation over inactivated vaccines in a murine model To evaluate the immunogenicity of the rAAV-CMV-PEDV S1 vaccine, we compared its efficacy in inducing serum anti-PEDV S1 IgG and activating CD3 + CD8 + and CD3 + CD4 + T-cells with that of a conventional inactivated PEDV vaccine in a murine model (Figure.2a) . The rAAV-CMV-PEDV S1 vaccine induced significantly higher ( P < 0.01) and more durable serum anti-PEDV S1 IgG titers than the inactivated vaccine, with elevated antibody levels persisting for at least 20 weeks ( Fig. 2 b ) . Furthermore, mice immunized with rAAV-CMV-PEDV S1 exhibited a marked increase in CD3 + CD8 + T-cells frequencies compared to both the inactivated vaccine and control groups at 4 weeks p.i. (1.39-fold increase vs. inactivated vaccine, P 0.05) ( Figures. 2 c,d ) . These findings demonstrate that the rAAV-CMV-PEDV S1 vaccine elicits potent and sustained humoral immunity (high IgG titers) and enhances cellular immunity (CD3 + CD8 + T-cell activation), outperforming traditional inactivated vaccines in inducing a balanced adaptive immune response. Safety and Tolerability of rAAV-CMV-PEDV S1 Vaccine in Pregnant Sows To assess the safety profile of the rAAV-CMV-PEDV S1 vaccine, we conducted a comparative study in pregnant sows, evaluating its immunogenicity alongside inactivated and attenuated PEDV vaccines (Figure. 3a) . Safety metrics included rectal temperature fluctuations and clinical symptom scores in sows monitored daily for 14 days post-immunization, as well as neonatal piglet viability (health proportion) and average birth weight (Figure.3d,e) . Notably, sows immunized with rAAV-CMV-PEDV S1 exhibited no significant differences in body temperature trends ( P > 0.05) or comprehensive score ( P > 0.05) compared to the inactivated vaccine, attenuated vaccine, and DMEM control groups ( Figures. 3 b,c ) . Similarly, neonatal piglets from the rAAV-CMV-PEDV S1 group showed comparable health proportions and average birth weights to those from other groups ( P > 0.05), with no adverse effects attributable to the vaccine ( Figures. 3 d,e ) . These data demonstrate that the rAAV-CMV-PEDV S1 vaccine is well-tolerated in pregnant sows and poses no detectable risks to neonatal piglets, supporting its potential for safe use in maternal immunization strategies against PEDV. rAAV-CMV-PEDV S1 vaccine enhances humoral immunity in sows and confers natural passive immunity to neonatal piglets To comprehensively explore the impact of the rAAV-CMV-PEDV S1 vaccine impact on humoral immunity, a series of experiments were conducted. Specifically, the serum anti-PEDV S IgG titers, colostrum anti-PEDV S IgG and IgA titers, as well as the colostric neutralizing antibody titers were detected through ELISA (Enzyme-Linked Immunosorbent Assay) and the virus neutralization test. The arrangement for collecting serum and colostrum is depicted in Fig. 3 a. The results revealed that the rAAV-CMV-PEDV S1 vaccine could prompt the body to generate higher and more stable titers of serum anti-PEDV S IgG compared to both the attenuated vaccine and the inactivated vaccine at 27 days post-immunization (d.p.i) and 60 d.p.i, ( P > 0.05 stability) (Figure.4a) . Moreover, when contrasted with the attenuated vaccine and the inactivated vaccine, this particular vaccine was able to increase the titer of colostric anti-PEDV S IgG ( P < 0.01 vs. attenuated vaccine; P 0.05 vs. attenuated vaccine and inactivated vaccine) (Fig. 4 c). Through the virus neutralization test, it was further demonstrated that the rAAV-CMV-PEDV S1 vaccine could enhance the neutralizing valence in colostrum ( log 2 7.08 vs. 6.06 and 5.48 in commercial vaccines P < 0.0001) (Fig. 4 d). To evaluate the natural passive immunity, the serum anti-PEDV S IgG titer of neonatal piglets was detected by ELISA at 5 days after birth. Intriguingly, compared with the other three groups, the rAAV-CMV-PEDV S1 vaccine could significantly increase the serum anti-PEDV S IgG titer of neonatal piglets, thereby enhancing the natural passive immunity ( P < 0.001 vs. attenuated vaccine; P < 0.0001 vs. inactivated vaccine) ( Fig. 4 e ) . In conclusion, based on all these findings, it can be firmly stated that the rAAV-CMV-PEDV S1 vaccine indeed plays a crucial role in strengthening the humoral immunity of sows and enhancing maternal passive immunity of piglets. rAAV-CMV-PEDV S1 vaccine could improve the cellular immunity and humoral immunity of sows in a certain sense. To assess the effects of the rAAV-CMV-PEDV S1 vaccine on cellular and humoral immunity in sows, serum concentrations of Th1-associated cytokines (IL-2, IFN-γ, TNF-β) and Th2-associated cytokine (IL-4) were analyzed across immunization groups ( Fig. 5 a-d ) . Compared to the attenuated vaccine group, the rAAV-CMV-PEDV S1 vaccine induced significantly higher levels of IL-2 ( P 0.05) ( Fig. 5 a-d ) . In contrast, when compared to the inactivated vaccine group, the rAAV vaccine elicited markedly elevated concentrations of all cytokines, including IL-4 ( P < 0.01), IL-2 ( P < 0.001), IFN-γ ( P < 0.05), and TNF-β ( P < 0.01) ( Figure.5a-d ). These results, to some extent, indicate that the rAAV-CMV-PEDV S1 vaccine robustly enhances both Th1-mediated cellular immunity (via IL-2, IFN-γ, and TNF-β) and Th2-driven humoral immunity (via IL-4), demonstrating its dual immunomodulatory efficacy in sows. rAAV-CMV-PEDV S1 vaccine confers superior protection in suckling piglets via passive immunity, enhancing survival and preserving intestinal integrity against PEDV challenge To evaluate the protective efficacy of various vaccines against PEDV infection, we conducted a comprehensive assessment in suckling piglets subjected to oral PEDV challenge, measuring four critical parameters: feed intake scores, diarrhea severity, viral shedding titers, and survival rates. Comparative analysis revealed that the rAAV-CMV-PEDV S1 vaccine demonstrated superior protective efficacy over conventional attenuated and inactivated vaccines, as evidenced by significantly improved feed intake scores, diarrhea symptom score, and markedly reduced viral shedding titers in challenged suckling piglets (Figure. 6a, b,c) . Post-challenge survival analysis demonstrated that piglets immunized with the rAAV-CMV-PEDV S1 vaccine achieved an 80% survival rate, significantly surpassing conventional attenuated (60%) and inactivated (40%) vaccines (Fig. 6 d). Histopathological evaluation demonstrated marked preservation of intestinal integrity in the piglets of rAAV-CMV-PEDV S1 group, with neither gross pathological alterations (e.g., distension or hemorrhage) nor microscopically detectable mucosal damage ( Fig. 7 ) . In stark contrast, The piglets in the other three group exhibited progressive intestinal pathology characterized by (1) macroscopic manifestations including severe intestinal distension, hemorrhagic foci, and pronounced wall thinning; (2) microscopic evidence of mucosal necrosis, epithelial desquamation, laminar propria vascular congestion, and muscular layer disintegration ( Fig. 7 ) . The above results confirmed that the rAAV-CMV-PEDV S1 candidate vaccine can provide highly effective protection against the infection of wild-type PEDV strains for suckling piglets through a passive immune mechanism. Moreover, its comprehensive protective efficacy is significantly superior to that of traditional attenuated vaccines and inactivated vaccines. Materials and methods Materials Plasmids, viruses, cells and antibodies The plasmids T-vector containing the EGFP gene, pAAV-CMV, pAAV-Helper, and pAAV-RC were preserved at Sichuan Agricultural University. HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Australia) at 37℃ with 5% CO 2 . The PEDV SNJ-P (GenBank No. GQ918139.1) and PEDV AJ1102-F12 (GenBank No. MK584552.1) was also preserved at Sichuan Agricultural University. Commercial reagents included Lipo293™ Transfection Reagent, RIPA Lysis Buffer, 4% PFA Fix Solution, Triton X-100, Trypsin solution, and Cy3-labeled Goat Anti-Mouse IgG (H + L) (Beyotime Biotech, China); DNAzyme I (Thermo Fisher Scientific, USA); PMSF (Solarbio, China); IL-2, IL-4, IFN-γ, and TNF-β cytokine detection kits (Zhuocai Biotechnology, China); PEDV IgA/IgG antibody test kits (IDEXX Laboratories, China; Kangbaote Biotechnology, China); APC Hamster Anti-Mouse CD3ε, FITC anti-mouse CD4, and PE-Cy™ anti-mouse CD8α antibodies (Biolegend, China); and PEDV inactivated/attenuated vaccines (Tianjin Ruipu Biotechnology; Zhongmu Industry, China). Mouse anti-PEDV S1 polyclonal antibody was prepared in-house. Animals Six-week-old female RIC mice ( n = 120) were obtained from Chengdu Dashuo Experimental Animal Co., Ltd. Twenty-five primiparous sows and and their neonatal piglets were purchased from Chengdu Wangjiang Agricultural and Animal Husbandry Technology Co., Ltd. (Chengdu, China). Prior to immunization, rectal swabs from the experimental sows tested negative for PEDV RNA by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Mice and piglets were housed in the specific pathogen-free (SPF) laboratory animal facility at Sichuan Agricultural University (Chengdu, China), which underwent rigorous disinfection and sterilization prior to the trial. Sows were raised in a PEDV-negative swine farm. All animals were provided with ad libitum access to water and feed. Sows were fed commercial swine diets, while piglets received maternal milk within the first 5 days of life and were manually fed animal milk replacer during the challenge protection trial. Mice were maintained on SPF-certified feed. Throughout the experiment, animals remained conscious, and no non-experimental procedures were conducted. Piglets and mice were humanely euthanized by CO₂ (≥ 70% (v/v), 5 minutes for mice; 10 minutes for piglets.) inhalation. This study was approved by the Sichuan Provincial Experimental Animal Management Committee (License No. SYXK (Chuan) 2019 − 187) and conducted in compliance with the Regulations on the Administration of Laboratory Animals (Ministry of Science and Technology, Beijing, China) and the Animal Research: Reporting of In Vivo Experiments (ARRIVE guidelines). Design and construction of rAAV-CMV-PEDV S1 virus and rAAV-CMV-EGFP virus The codon-optimized PEDV S1 gene (SNJ-P strain) and EGFP gene were cloned into the pAAV-CMV plasmid via AsiSI and MluI sites, generating pAAV-CMV-PEDV S1 and pAAV-CMV-EGFP backbone plasmid. HEK293T cells were co-transfected with plasmids pAAV-CMV-PEDV S1 (or pAAV-CMV-EGFP), pAAV-Helper, and pAAV-RC using Lipo293™ Transfection Reagent (Beyotime Biotech, China) in Opti-MEM® Medium (Gibco, USA). The plasmid structures of pAAV-Helper and pAAV-RC are shown in Fig. 1 a. Cells were monitored for EGFP expression by fluorescence microscopy at 24 h post-transfection. Recombinant viruses were harvested at 6 days, purified via cesium chloride ultracentrifugation, and treated with DNAzyme I to remove external DNA. Viral morphology and size were confirmed by transmission electron microscopy (TEM). Note: EGFP was inserted into the backbone plasmid as a control since PEDV plasmids could not accommodate it. Indirect immunofluorescence (IFA) and Western blot The HEK293T cells in a 6-well plate were infected with rAAV-CMV-PEDV S1 virus (100 µL) and rAAV-CMV-EGFP virus (100 µL) respectively, and cells inoculated with DMEM were used as mock. As for IFA, the cells were fixed with 4% Paraformaldehyde for 15 min after being cultured for 72 h at 37°C, and then stained with primary antibody of mouse anti-PEDV S1 polyclonal antibody and second antibody of Cy3-labeled Goat Anti-Mouse IgG (H + L) (Beyotime, China). The fluorescent signals were observed under an inverted fluorescence microscope (Olympus, IX71, Japan). As for western blot, the HEK293T cells infected with rAAV-CMV-PEDV S1 virus, rAAV-CMV-EGFP virus or mock respectively were lysed by 200 µL RIPA (Solarbio, China) with 0.5 mM PMSF(Solarbio, China). The lysed cells were centrifuged in 8000 g, 10 min, and supernatants were separated by a 12% SDS-PAGE and then transferred onto PVDF membrane (Millipore, USA). That was incubated with primary antibody of mouse anti-PEDV S1 polyclonal antibody and mouse anti-GAPDH monoclonal antibody(Abclonal, China), and secondary antibody of Goat Anti-Mouse IgG (HRP) (Abcam, UK), Ultimately, which visualized by the enhanced chemiluminescent (ECL) HRP substrate (Beyotime, China). Establishment of the qPCR standard curve and determination of the rAAV-CMV-PEDV S1 Titer. The plasmid copy numbers for the rAAV-CMV-PEDV S1 backbone plasmid and PEDV M gene T-vector were calculated using the formula: Plasmid copy number (copies/µL)= ( Cp ×10 − 9 × N A )/( Np ×648) ( Cp : Plasmid concentration (ng/µL); N A : Avogadro's constant (6.022×10 23 molecules/mol); Np : plasmid size (bp); 648: Average molecular weight of a dsDNA base pair (g/mol))[ 32 ]. Initial concentrations were determined as 1.84×10 11 copies/µL (rAAV-CMV-PEDV S1 plasmid) and 2.21×10 10 copies/µL (PEDV M gene plasmid). Both plasmids were serially diluted to 1.0×10 10 copies/µL using nuclease-free dH 2 O (Thermo Fisher Scientific, USA), followed by eight 10-fold serial dilutions to generate standards ranging from 1.0×10 9 to1.0×10 2 copies/µL. Quantitative PCR (qPCR) was performed using optimized cycling conditions (denaturation: 95°C for 30 s; 40 cycles of 95°C for 5 s, 60°C for 30 s). The threshold cycle (Cq) values were plotted against log 10 -transformed plasmid copy numbers (x-axis) to generate standard curves. A curve was deemed valid if it met the following criteria: amplification efficiency (E) = 95–105%, coefficient of determination (R 2 ) ≥ 0.998, and slope between − 3.58 and − 3.10. The viral nucleic acid was extracted using a DNA extraction kit(Tiangen China). qPCR amplification was performed according to the optimized amplification conditions described above. The Cq value obtained by qPCR was brought into the standard equation, which obtain the copy number of the virus genome per milliliter of virus liquid (vg/mL). Mouse Immunization Test Six-week-old female mice were divided into 3 groups ( n = 40/group). rAAV-CMV-PEDV S1 group and inactivated vaccine group were respectively injected 100 µL (1.00×10 7 vg/100 µL ) rAAV-CMV-PEDV S1 virus and 100 µL (1.00×10 7 vg/100 µL) inactivated vaccine, the negative control group was injected 100 µL DMEM. All the mice received one inoculations and no adverse events after vaccination. The blood samples of mine were collected every two weeks from orbit at 0 to 20 weeks.p.i. (Figure. 2a) , and the serum was harvested after centrifugation at 8000 g 5min. Serum anti-PEDV S1 IgG OD 450 value were determined with enzyme-linked immunosorbent assays (ELISA). Splenocytes Isolation and Flow Cytometry The spleens were dissected from the immunized mice and homogenized through a 70 µm strainer at 4 weeks.p.i. For analysis of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells, splenocytes were stained at 4 ℃ for 30 min with APC Hamster Anti-Mouse CD3ε, FITC anti-mouse CD4, and PE-Cy™ anti-mouse CD8α antibodies. The flow cytometry procedure was performed with a Beckman cytoflex, and the data were analyzed using CytExpert software. Immunity studies in sows Twenty-five primiparous sows were divided into 4 groups ( n = 5/group). rAAV-CMV-PEDV S1 virus group, attenuated vaccine group and inactivated vaccine group were respectively inoculated 1 mL (1.00×10 9 vg/mL) rAAV-CMV-PEDV S1 virus, 1.78 mL (1.00×10 9 vg ) attenuated vaccine and 2.11 mL (1.00×10 9 vg ) inactivated vaccine, the negative control group was injected 1ml DMEM in 34 days before delivery. Inactivated vaccine group was boosted once in 20 days before delivery. The immune procedure is shown in Figure.3a . From 0 to 14 days after immunization the body temperature and the comprehensive score ( the feeding, drinking water, mental condition and fecal morphology ) were monitored and observed, the scoring criteria are shown in Sumplmentary Table 1 . The health proportion and the body weight of neonatal piglets were counted after delivery. The blood of sows was collected in 0,27,60 days.p.i, and harvested after centrifugation at 8000× g 5min. Serum anti-PEDV S IgG S/P value was detected according to the ELISA kit instructions. The colostrum was collected after delivery, and harvested after centrifugation at 8000× g 5min. Anti-PEDV S IgG and IgA S/P value in milk serum was detected according to the ELISA kit instructions. Three 7-day-old neonatal piglets were randomly selected from each sow, and the serum anti-PEDV S IgG S/P value in piglets were detected according to the above method. The IL-2, IL-4, IFN-γ and TNF-β concentrations in serum of sows were measured at 27 days.p.i according to the instructions of cytokine ELISA detection kit. PEDV neutralization assay The colostrum was collected after delivery to determine PEDV virus neutralizing antibody (VNA) levels using end-point neutralization test. Colostrous supernatant is separated from colostrum and were inactivated at 56°C for 30 min. Colostrous supernatant were separated and serially diluted two-fold with DMEM from 2 − 1 to 2 − 8 and incubated with 100 TCID 50 of the PEDV AJ1102-F12 strain for 1 h at 37°C in a 5% CO 2 incubator. When the vero cells in the 96-well plate were 90% confluent, the above mixture of serum and virus or DMEM as negative control was added, and the cells were cultured for 2 h at 37°C. Next, DMEM containing trypsin (5 mg/mL), was added to each well, and the plate was incubated for 3 to 5 days at 37°C. Neutralizing antibody titers were expressed as the log 2 transformation of the reciprocal highest colostrous supernatant dilution that completely inhibited cytopathic effects (CPEs). Each sample was run in triplicate. Challenge protection in piglets Twenty 5-day-old neonatal piglets from each group (n = 5/group), and was orally infected with 1 mL(10 5 TCID 50 /mL) PEDV AJ1102-F12. The feed intake and diarrhea symptoms in infected neonatal piglets were observed and scored every 12 hours after challenge, the scoring criteria are shown in the Supplementary Table 2, 3 . The higher the score, the stronger the feeding ability and the lighter the diarrhea symptoms of suckling piglets. After PEDV challenge, anal swabs of infected neonatal piglets were collected every 12 hours, the amount of virus shedding was monitored by RT-qPCR, and the survival rate of infected neonatal piglets was monitored and counted every day. Five days post-challenge, surviving piglets were euthanized for necropsy to examine intestinal lesions. Jejunal segments were collected to prepare histopathological sections for observing microscopic pathological changes. Statistics and reproducibility GraphPad Prism 8 statistical software (GraphPad Software, San Diego, CA, USA) was used to analyze the data and draw graphs The data are presented as mean ± standard deviation (SD). Statistical differences among the groups were identified through one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. A threshold of P < 0.05 was considered statistically significant, with the following notation: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P 0.05. DISCUSSION This study successfully constructed a recombinant adeno-associated virus (rAAV-CMV-PEDV S1) expressing the S1 subunit of PEDV and systematically evaluated its immunogenicity and protective efficacy. The results demonstrate that the rAAV-based vaccine outperforms traditional inactivated and attenuated vaccines by inducing robust, durable humoral and cellular immune responses in mice, enhancing maternal antibody transfer in sows, and conferring significant protection to piglets against lethal PEDV challenge. These findings highlight the potential of AAV vectors as a novel platform for developing vaccines against enteric coronaviruses in livestock. The rAAV-CMV-PEDV S1 vaccine induced significantly higher and longer-lasting serum IgG titers in mice compared to inactivated vaccines ( Figure.2b ), likely due to sustained antigen expression mediated by the AAV vector. Unlike inactivated vaccines, which primarily rely on exogenous antigen presentation via MHC-II pathways, AAV-delivered antigens are synthesized intracellularly, enabling MHC-I presentation and subsequent activation of CD8 + T cells. This mechanism aligns with the observed increase in CD3 + CD8 + T-cell frequencies (Figures. 2 e-f), suggesting enhanced cytotoxic T lymphocyte (CTL) activity critical for eliminating virus-infected cells[ 33 ]. Such dual humoral-cellular immunity is a distinct advantage over traditional vaccines, which often fail to elicit robust CTL responses. In pregnant sows, the rAAV vaccine generated elevated serum and colostral IgG and neutralizing antibody titers (Figures. 4 a-d), translating to 80% survival rates in challenged piglets ( Figure.6d ). The high IgA levels in colostrum further suggest mucosal immune priming, which is vital for blocking PEDV entry at intestinal epithelial surfaces[ 34 ]. By contrast, commercial attenuated vaccines carry risks of virulence reversion due to PEDV’s genetic instability, while inactivated vaccines lack the durability and mucosal immune induction observed here[ 35 ]. The modular design of AAV vectors also allows rapid antigen updates to match emerging variants, a feature critical for addressing PEDV’s high mutation rate. The rAAV-CMV-PEDV S1 vaccine exhibited an excellent safety profile in sows, with no significant differences in body temperature, clinical scores, or neonatal piglet viability compared to controls ( Figure.3b-e ). This aligns with the inherent safety of AAV vectors, which lack pathogenicity and exhibit low immunogenicity[ 21 ]. Furthermore, histopathological analysis revealed preserved intestinal integrity in vaccinated piglets ( Figure.7 ), underscoring the vaccine’s ability to mitigate PEDV-induced enteric damage through passive immunity. These results position the rAAV platform as a safer alternative to live-attenuated vaccines, particularly in sensitive populations such as pregnant sows. While promising, this study has limitations. First, the experiments were conducted under controlled laboratory conditions; field trials are necessary to evaluate efficacy in diverse farming environments. Second, long-term safety assessments are needed to rule out potential risks of vector genome integration or immune tolerance after prolonged antigen exposure[ 36 ]. Third, the vaccine’s cross-protective efficacy against emerging PEDV variants (e.g., GIIb strains) remains untested. Future studies should explore multivalent AAV designs incorporating conserved epitopes from divergent strains to broaden protection. Additionally, optimizing production scalability and cost-effectiveness will be crucial for real-world application. The rAAV-CMV-PEDV S1 vaccine represents a significant advancement in PEDV prophylaxis, combining the safety of non-replicating vectors with the immunogenicity of live vaccines. Its ability to induce durable humoral immunity, activate CTLs, and confer passive protection to piglets addresses critical gaps in current PEDV vaccine strategies. As a modular platform, this approach could be rapidly adapted for other coronaviruses, offering a versatile tool to combat emerging zoonotic threats. Further translational research, including large-scale efficacy trials and cost-benefit analyses, will accelerate its transition from bench to farm. Declarations Data availability statement The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author/s. Authors’ contributions Methodology, M.P.; software,M.R.; validation, D.J.; data curation, M.P. and Y.Z; writing-original draft preparation, M.P.; writing-review and editing, T.T and Y.W; project administration, M.P. and M.R. All authors have read and agreed to the published version of the manuscript. Conflflict of interest The authors declare that they have no competing interest. Data availability statement The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author/s. Funding This project was supported by the Sichuan Province Science and Technology Planning Program (2021ZDZX0010), the Natural Science Foundation of Sichuan Province (2024NSFSC0374), and the Sichuan Innovation Team Project of National Modern Agricultural Industry Technology System (SCCXTD-2024-26) Acknowledgments We thank Prof. Wang for his insightful comments on the design of the study. Supplementary material The Supplementary Material for this article can be found online at: https: References Zhang H, Zou C, Peng O, Ashraf U, Xu Q, Gong L, Fan B, Zhang Y, Xu Z, Xue C, Wei X, Zhou Q, Tian X, Shen H, Li B, Zhang X, Cao Y. Global Dynamics of Porcine Enteric Coronavirus PEDV Epidemiology, Evolution, and Transmission. Mol Biol Evol. 2023 Mar 4;40(3):msad052. doi: 10.1093/molbev/msad052. Lin CM, Saif LJ, Marthaler D, Wang Q. Evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains. Virus Res. 2016 Dec 2;226:20-39. doi: 10.1016/j.virusres.2016.05.023. Pasick J, Berhane Y, Ojkic D, Maxie G, Embury-Hyatt C, Swekla K, Handel K, Fairles J, Alexandersen S. Investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in Canada. Transbound Emerg Dis. 2014 Oct;61(5):397-410. doi: 10.1111/tbed.12269. Li K, Wang H, Chen T, Wang X, Wang X, Zhong M, Gao X, Hao Z. Dehydroevodiamine inhibits PEDV through regulateing ERK1/2 MAPK pathway in Vero cells. Virology. 2024 Oct;598:110166. doi: 10.1016/j.virol.2024.110166. Ma X, Zheng H, Chen H, Ma S, Wei Z. Porcine epidemic diarrhea virus: A review of detection, inhibition of host gene expression and evasion of host innate immune. Microb Pathog. 2024 Oct;195:106873. doi: 10.1016/j.micpath.2024.106873. Shen Z, Ye G, Deng F, Wang G, Cui M, Fang L, Xiao S, Fu ZF, Peng G. Structural Basis for the Inhibition of Host Gene Expression by Porcine Epidemic Diarrhea Virus nsp1. J Virol. 2018 Feb 12;92(5):e01896-17. doi: 10.1128/JVI.01896-17. Hou Y, Meulia T, Gao X, Saif LJ, Wang Q. Deletion of both the Tyrosine-Based Endocytosis Signal and the Endoplasmic Reticulum Retrieval Signal in the Cytoplasmic Tail of Spike Protein Attenuates Porcine Epidemic Diarrhea Virus in Pigs. J Virol. 2019 Jan 4;93(2):e01758-18. doi: 10.1128/JVI.01758-18. Suzuki T, Terada Y, Enjuanes L, Ohashi S, Kamitani W. S1 Subunit of Spike Protein from a Current Highly Virulent Porcine Epidemic Diarrhea Virus Is an Important Determinant of Virulence in Piglets. Viruses. 2018 Aug 30;10(9):467. doi: 10.3390/v10090467. Gong L, Lin Y, Qin J, Li Q, Xue C, Cao Y. Neutralizing antibodies against porcine epidemic diarrhea virus block virus attachment and internalization. Virol J. 2018 Aug 30;15(1):133. doi: 10.1186/s12985-018-1042-3. Li C, Li W, Lucio de Esesarte E, Guo H, van den Elzen P, Aarts E, van den Born E, Rottier PJM, Bosch BJ. Cell Attachment Domains of the Porcine Epidemic Diarrhea Virus Spike Protein Are Key Targets of Neutralizing Antibodies. J Virol. 2017 May 26;91(12):e00273-17. doi: 10.1128/JVI.00273-17. PMID: 28381581; PMCID: PMC5446644. Zhang D, Xie Y, Liao Q, Jiao Z, Liang R, Zhang J, Zhang Y, Tan Y, Wang H, Zhang W, Xiao S, Peng G, Shi Y. Development of a safe and broad-spectrum attenuated PEDV vaccine candidate by S2 subunit replacement. J Virol. 2024 Nov 19;98(11):e0042924. doi: 10.1128/jvi.00429-24. Wood EN. An apparently new syndrome of porcine epidemic diarrhoea. Vet Rec. 1977 Mar 19;100(12):243-4. doi: 10.1136/vr.100.12.243. Jung K, Saif LJ. Porcine epidemic diarrhea virus infection: Etiology, epidemiology, pathogenesis and immunoprophylaxis. Vet J. 2015 May;204(2):134-43. doi: 10.1016/j.tvjl.2015.02.017. Wang D, Fang L, Xiao S. Porcine epidemic diarrhea in China. Virus Res. 2016 Dec 2;226:7-13. doi: 10.1016/j.virusres.2016.05.026. Pasick J, Berhane Y, Ojkic D, Maxie G, Embury-Hyatt C, Swekla K, Handel K, Fairles J, Alexandersen S. Investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in Canada. Transbound Emerg Dis. 2014 Oct;61(5):397-410. doi: 10.1111/tbed.12269. Li Y, Wu Q, Huang L, Yuan C, Wang J, Yang Q. An alternative pathway of enteric PEDV dissemination from nasal cavity to intestinal mucosa in swine. Nat Commun. 2018 Sep 19;9(1):3811. doi: 10.1038/s41467-018-06056-w. Jung K, Annamalai T, Lu Z, Saif LJ. Comparative pathogenesis of US porcine epidemic diarrhea virus (PEDV) strain PC21A in conventional 9-day-old nursing piglets vs. 26-day-old weaned pigs. Vet Microbiol. 2015 Jul 9;178(1-2):31-40. doi: 10.1016/j.vetmic.2015.04.022. Sun M, Ma J, Wang Y, Wang M, Song W, Zhang W, Lu C, Yao H. Genomic and epidemiological characteristics provide new insights into the phylogeographical and spatiotemporal spread of porcine epidemic diarrhea virus in Asia. J Clin Microbiol. 2015 May;53(5):1484-92. doi: 10.1128/JCM.02898-14. Epub 2015 Feb 18. PMID: 25694517; PMCID: PMC4400760. Gao B, He L, Bao Y, Chen Y, Lu G, Zhang Y, Xu Y, Su B, Xu J, Wang Y, Yeap LS. Repeated vaccination of inactivated SARS-CoV-2 vaccine dampens neutralizing antibodies against Omicron variants in breakthrough infection. Cell Res. 2023 Mar;33(3):258-261. doi: 10.1038/s41422-023-00781-8. Gao B, He L, Bao Y, Chen Y, Lu G, Zhang Y, Xu Y, Su B, Xu J, Wang Y, Yeap LS. Repeated vaccination of inactivated SARS-CoV-2 vaccine dampens neutralizing antibodies against Omicron variants in breakthrough infection. Cell Res. 2023 Mar;33(3):258-261. doi: 10.1038/s41422-023-00781-8. Linden RM, Berns KI. Molecular biology of adeno-associated viruses. Contrib Microbiol. 2000;4:68-84. doi: 10.1159/000060327. Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019 May;18(5):358-378. doi: 10.1038/s41573-019-0012-9. Zaiss AK, Liu Q, Bowen GP, Wong NC, Bartlett JS, Muruve DA. Differential activation of innate immune responses by adenovirus and adeno-associated virus vectors. J Virol. 2002 May;76(9):4580-90. doi: 10.1128/jvi.76.9.4580-4590.2002. Somanathan S, Breous E, Bell P, Wilson JM. AAV vectors avoid inflammatory signals necessary to render transduced hepatocyte targets for destructive T cells. Mol Ther. 2010 May;18(5):977-82. doi: 10.1038/mt.2010.40. Gaudet D, Méthot J, Déry S, Brisson D, Essiembre C, Tremblay G, Tremblay K, de Wal J, Twisk J, van den Bulk N, Sier-Ferreira V, van Deventer S. Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an open-label trial. Gene Ther. 2013 Apr;20(4):361-9. doi: 10.1038/gt.2012.43. Bommakanti N, Young BK, Sisk RA, Berrocal AM, Duncan JL, Bakall B, Mathias MT, Ahmed I, Chorfi S, Comander J, Nagiel A, Besirli CG. Classification and Growth Rate of Chorioretinal Atrophy after Voretigene Neparvovec-Rzyl for RPE65-Mediated Retinal Degeneration. Ophthalmol Retina. 2024 Jan;8(1):42-48. doi: 10.1016/j.oret.2023.08.017. Tang X, Lu H, Tarwater PM, Silverberg DL, Schorl C, Ramratnam B. Adeno-Associated Virus (AAV)-Delivered Exosomal TAT and BiTE Molecule CD4-αCD3 Facilitate the Elimination of CD4 T Cells Harboring Latent HIV-1. Microorganisms. 2024 Aug 18;12(8):1707. doi: 10.3390/microorganisms12081707. Rashid MHO, Kayesh MEH, Hashem MA, Hifumi T, Ogawa S, Miyoshi N, Tanaka Y, Kohara M, Tsukiyama-Kohara K. Adeno-associated virus 2 CRISPR/Cas9-mediated targeting of hepatitis B virus in tree shrews. Virus Res. 2025 Feb 21;354:199550. doi: 10.1016/j.virusres.2025.199550. Epub ahead of print. PMID: 39988206. Colon-Moran W, Baer A, Lamture G, Stapleton JT, Fischer JW, Bhattarai N. A short hepatitis C virus NS5A peptide expression by AAV vector modulates human T cell activation and reduces vector immunogenicity. Gene Ther. 2022 Nov;29(10-11):616-623. doi: 10.1038/s41434-021-00302-5. Yamazaki T, Chiba J, Akashi-Takamura S. Neutralizing Anti-Hemagglutinin Monoclonal Antibodies Induced by Gene-Based Transfer Have Prophylactic and Therapeutic Effects on Influenza Virus Infection. Vaccines (Basel). 2018 Jun 26;6(3):35. doi: 10.3390/vaccines6030035. Ried MU, Girod A, Leike K, Büning H, Hallek M. Adeno-associated virus capsids displaying immunoglobulin-binding domains permit antibody-mediated vector retargeting to specific cell surface receptors. J Virol. 2002 May;76(9):4559-66. doi: 10.1128/jvi.76.9.4559-4566.2002. PMID: 11932421; PMCID: PMC155067. Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd edn. New York: Cold Spring Harbor Laboratory Press; 2001. Biernacki MA, Brault M, Bleakley M. T-Cell Receptor-Based Immunotherapy for Hematologic Malignancies. Cancer J. 2019 May/Jun;25(3):179-190. doi: 10.1097/PPO.0000000000000378. Hu Z, Li Y, Zhang B, Zhao Y, Guan R, Zhou Y, Du J, Zhang Z, Li X. Serum IgA antibody level against porcine epidemic diarrhea virus is a potential pre-evaluation indicator of immunization effects in sows during parturition under field conditions. Porcine Health Manag. 2024 Sep 3;10(1):32. doi: 10.1186/s40813-024-00382-w. Liu H, Yin X, Tian H, Qiu Y, Wang Z, Chen J, Ma D, Zhao B, Du Q, Tong D, Huang Y. The S protein of a novel recombinant PEDV strain promotes the infectivity and pathogenicity of PEDV in mid-west China. Transbound Emerg Dis. 2022 Nov;69(6):3704-3723. doi: 10.1111/tbed.14740. Zhu QX, Zhang YN, Zhang HQ, Leng C, Deng CL, Wang X, Li JJ, Ye XL, Zhang B, Li XD. A single dose recombinant AAV based CHIKV vaccine elicits robust and durable protective antibody responses in mice. PLoS Negl Trop Dis. 2024 Nov 4;18(11):e0012604. doi: 10.1371/journal.pntd.0012604. Additional Declarations No competing interests reported. Supplementary Files SumplmentFiles.docx Cite Share Download PDF Status: Published Journal Publication published 06 Nov, 2025 Read the published version in npj Vaccines → Version 1 posted Editorial decision: Revision requested 08 Jul, 2025 Reviews received at journal 01 Jul, 2025 Reviewers agreed at journal 11 Jun, 2025 Reviews received at journal 28 May, 2025 Reviewers agreed at journal 08 May, 2025 Reviewers invited by journal 28 Apr, 2025 Editor assigned by journal 26 Mar, 2025 Submission checks completed at journal 19 Mar, 2025 First submitted to journal 14 Mar, 2025 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6224876","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":449567638,"identity":"d1451a8d-2b3a-4143-b68d-7229fe463115","order_by":0,"name":"Maonan Pang","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Maonan","middleName":"","lastName":"Pang","suffix":""},{"id":449567639,"identity":"5520944a-13e1-48a4-ba76-581aa5dfe936","order_by":1,"name":"Meishen Ren","email":"","orcid":"","institution":"Hong Kong Baptist University","correspondingAuthor":false,"prefix":"","firstName":"Meishen","middleName":"","lastName":"Ren","suffix":""},{"id":449567640,"identity":"edda131f-7613-4e7f-b65c-88751b5c7393","order_by":2,"name":"Dike Jiang","email":"","orcid":"","institution":"Chengdu Agricultural College","correspondingAuthor":false,"prefix":"","firstName":"Dike","middleName":"","lastName":"Jiang","suffix":""},{"id":449567641,"identity":"0e91f4fb-5308-4449-875d-4418402a6338","order_by":3,"name":"Teng Tu","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Teng","middleName":"","lastName":"Tu","suffix":""},{"id":449567642,"identity":"9beb8f2f-16b0-4219-8805-068f0bce4e19","order_by":4,"name":"You Zhou","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"You","middleName":"","lastName":"Zhou","suffix":""},{"id":449567643,"identity":"3857b24f-7109-42af-b0c7-90dcfd5e4ff9","order_by":5,"name":"Yin Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYBACNmb2ww8kKhh4oFwitPCx96QZWJwhRYsczwEDico2uKXEOEwiIcHg5rw6Gf72MwYMH8oOM/DPbiCkJfHAw5nbDvNInMkxYJxx7jCDxJ0DhG0xltx2gMeAIceAmbftMIOBRAJBLQbSf+fU8RjwvzFg/kuUFpD3JRuYeQwkgLYwEqUFFMgSx4B+ufGs4GDPuXQgg4AW+WZQVNbU2fP3J2988KPMWo5/BgEtKOAAEPOQoH4UjIJRMApGAS4AAFXzPI+y54PrAAAAAElFTkSuQmCC","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Yin","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-03-14 09:23:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6224876/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6224876/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41541-025-01248-0","type":"published","date":"2025-11-06T15:58:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82145053,"identity":"a90ff649-4b6c-43ca-8727-9e738358ee22","added_by":"auto","created_at":"2025-05-07 06:50:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":922525,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign, construction, and characterization of recombinant AAV vectors (rAAV-CMV-PEDV S1 and rAAV-CMV-EGFP). (a) \u003c/strong\u003eSchematic representation of the viral genome architecture, featuring 5’ and 3’ inverted terminal repeats (ITRs) flanking a CMV promoter-driven expression cassette containing either the PEDV S1 gene or EGFP gene, followed by a poly(A) tail. \u003cstrong\u003e(b)\u003c/strong\u003e Recombinant AAV production was achieved through triple plasmid co-transfection (pAAV-CMV-PEDV S1/pAAV-CMV-EGFP, pAAV-Helper, and pAAV-RC) in HEK293T cells, with successful EGFP expression confirming transfection efficiency. \u003cstrong\u003e(c)\u003c/strong\u003e IFA demonstrated robust S1 protein expression (red fluorescence) in rAAV-CMV-PEDV S1-infected HEK293T cells (\u003cem\u003eMOI=5\u003c/em\u003e), while intrinsic EGFP fluorescence (green) validated rAAV-CMV-EGFP functionality.\u003cstrong\u003e (d)\u003c/strong\u003e Western blot analysis further confirmed S1 protein expression (~150 kDa) in rAAV-CMV-PEDV S1-infected cells, with no cross-reactivity observed in EGFP controls. \u003cstrong\u003e(e) \u003c/strong\u003eTransmission electron microscopy (TEM) revealed both constructs formed non-enveloped, icosahedral viral particles (~22 nm diameter) with comparable morphology.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6224876/v1/f6e0fee7a1a4df781030294a.png"},{"id":82143670,"identity":"796d0c96-6d05-41eb-9059-7d3b1d329dfa","added_by":"auto","created_at":"2025-05-07 06:42:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":799811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDual immune activation profile of rAAV-CMV-PEDV S1 vaccine in murine models.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Experimental design: Six-week-old female BALB/c mice (\u003cem\u003en \u003c/em\u003e= 120) were allocated into three groups: (1) rAAV-CMV-PEDV S1 (\u003cem\u003en\u003c/em\u003e = 40, 100 μL of 1.0×10⁷ vg/dose), (2) inactivated vaccine (\u003cem\u003en\u003c/em\u003e = 40, 100 μL of 1.0×10⁷ vg equivalent), and (3) DMEM control (\u003cem\u003en\u003c/em\u003e = 40, 100 μL), all receiving intramuscular injections. Longitudinal serum sampling (0–20 weeks post-immunization) was performed to quantify anti-PEDV S1 IgG levels via ELISA. At 4 weeks post-immunization, splenocytes were harvested and subjected to flow cytometric analysis of T-cell subsets. \u003cstrong\u003e(b)\u003c/strong\u003e Serum IgG kinetics demonstrated sustained humoral responses in vaccinated groups compared to controls. \u003cstrong\u003e(c–f) \u003c/strong\u003eSpleens were collected at 4 weeks post-immunization (wpi). Single-cell suspensions were prepared and stained with antibodies against T-cell markers (CD3⁺, CD4⁺, and CD8⁺) for flow cytometry. (d, f) Representative flow cytometric plots of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells. (c, e) Statistical analysis of activated T-cell percentages. Data are presented as means ± SEMs. Significant differences between groups are indicated as follows: *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; \u003cem\u003ens\u003c/em\u003e, not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6224876/v1/b3b96bd8e65e6633c3fd3ee3.png"},{"id":82141334,"identity":"d5dc377a-696d-434f-b898-fb12cdec4ebf","added_by":"auto","created_at":"2025-05-07 06:34:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":362118,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSafety evaluation of rAAV-CMV-PEDV S1 vaccine in pregnant sows and neonatal piglets.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eExperimental design: Pregnant sows (\u003cem\u003en \u003c/em\u003e= 20, first pregnancy) were divided into four groups (\u003cem\u003en\u003c/em\u003e=5/group). Groups 1–3 received 1 mL rAAV-CMV-PEDV S1 (1.00×10⁹ vg/mL), 1.78 mL attenuated vaccine (1.00×10⁹ vg/mL), or 2.11 mL inactivated vaccine (1.00×10⁹ vg/mL), respectively; the control group received 1 mL DMEM. The inactivated vaccine group was boosted at 14 days post-immunization (d.p.i.). Blood samples from sows were collected at 0, 27, and 60 d.p.i.; Colostrum was collected within the first 12 hours after parturition and analyzed for IgA and neutralizing antibody titers. Neonatal piglets were challenged with PEDV SCJY-1 at 5 days old, and blood was collected pre-challenge. \u003cstrong\u003e(b)\u003c/strong\u003e Body temperature changes in sows monitored for 14 days post-immunization. \u003cstrong\u003e(c) \u003c/strong\u003eComprehensive health scores (feeding, drinking, mental status, fecal morphology; criteria in \u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e) recorded for 14 days post-immunization. \u003cstrong\u003e(d)\u003c/strong\u003e Average body weight of neonatal piglets post-delivery. \u003cstrong\u003e(e) \u003c/strong\u003eProportion of healthy neonatal piglets post-delivery. Data are presented as means ± SEMs. Significant differences between groups are indicated as follows: *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; \u003cem\u003ens\u003c/em\u003e, not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6224876/v1/ab89db0965efc3389b4a4fe4.png"},{"id":82141330,"identity":"b9149b28-1a6f-40e7-8472-c949a9ab5986","added_by":"auto","created_at":"2025-05-07 06:34:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":286061,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003erAAV-CMV-PEDV S1 vaccine enhances humoral immunity in sows and passive immunity in piglets. (a-c)\u003c/strong\u003e Serum anti-PEDV S IgG (a), Colostrum anti-PEDV S IgG (b) and IgA (c) levels (S/P ratio) in sows measured by ELISA at 0, 27, and 60 days post-immunization (d.p.i.). \u003cstrong\u003e(d)\u003c/strong\u003e Neutralizing antibody titers (Log\u003csub\u003e2\u003c/sub\u003e) in colostrum assessed by virus neutralization assay (\u003cem\u003en \u003c/em\u003e= 5/group). \u003cstrong\u003e(e) \u003c/strong\u003eSerum anti-PEDV S IgG levels (S/P ratio) measured by ELISA in piglets (\u003cem\u003en \u003c/em\u003e= 15/group) at 5 days after birth. Data are presented as means ± SEMs. Significant differences between groups are indicated as follows: *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; \u003cem\u003ens\u003c/em\u003e, not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6224876/v1/a6869a296c7944e11efadccf.png"},{"id":82141340,"identity":"f789e354-2531-49a2-b627-6a5434284146","added_by":"auto","created_at":"2025-05-07 06:34:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":200945,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum cytokine profiles in pregnant sows immunized with rAAV-CMV-PEDV S1 vaccine.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a-d)\u003c/strong\u003eConcentrations of Th1-associated cytokines (IL-2, IFN-γ, TNF-β) and Th2-associated cytokine (IL-4) in serum collected at 27 d.p.i. from sows (\u003cem\u003en \u003c/em\u003e= 5/group). Cytokine levels were quantified using commercial ELISA kits according to the manufacturer’s protocols. Data are presented as means ± SEMs. Significant differences between groups are indicated as follows: *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; \u003cem\u003ens\u003c/em\u003e, not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6224876/v1/fb855b8e76fbfe202154c32a.png"},{"id":82141331,"identity":"0f92e778-c960-489e-9c2d-4cd2cad32a24","added_by":"auto","created_at":"2025-05-07 06:34:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":274160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePassive immune protection conferred by rAAV-CMV-PEDV S1 vaccine in neonatal piglets post-PEDV challenge. (a-b)\u003c/strong\u003e Feed intake average scores (a) and diarrhea symptom average scores (b) in suckling piglets (\u003cem\u003en \u003c/em\u003e= 5/group) were scored every 12 hours post-challenge using standardized criteria (\u003cstrong\u003eSupplementary Table 2，3\u003c/strong\u003e). Higher scores indicate stronger feeding ability and milder diarrhea. \u003cstrong\u003e(c) \u003c/strong\u003eViral shedding in anal swabs (\u003cem\u003en \u003c/em\u003e= 5/group) was quantified by RT-qPCR at 12-hour intervals post-challenge. \u003cstrong\u003e(d)\u003c/strong\u003eSurvival rates of piglets (\u003cem\u003en \u003c/em\u003e= 5/group) were recorded daily.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6224876/v1/378fed247e4657e375bc7e71.png"},{"id":82141350,"identity":"59432219-d0ce-470a-b7f1-176cb99c259d","added_by":"auto","created_at":"2025-05-07 06:34:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2974239,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe anatomical and microscopic pathological changes of suckling piglets after challenge.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6224876/v1/d1d53bb86594d0d179e62ec6.png"},{"id":95564119,"identity":"0d50aade-15aa-42ed-a61d-c78c19aaa0af","added_by":"auto","created_at":"2025-11-10 16:08:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7242548,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6224876/v1/13f6ca85-6977-48b3-a603-15672c3d81ef.pdf"},{"id":82141336,"identity":"5f859bf9-9ccb-4a5b-b811-643091f9e878","added_by":"auto","created_at":"2025-05-07 06:34:03","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":201334,"visible":true,"origin":"","legend":"","description":"","filename":"SumplmentFiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-6224876/v1/b2a4f1791d794d9fb6b749bc.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of a Recombinant Adeno-Associated Virus Vaccine Expressing PEDV S1 Protein: Enhanced Humoral and Cellular Immunity with Superior Neonatal Protection","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePorcine epidemic diarrhea (PED), a highly detrimental intestinal infectious disease, has inflicted heavy losses on the global swine industry. The spread of its causative agent, the porcine epidemic diarrhea virus (PEDV), poses a severe threat to the health of pig populations and the sustainable development of the swine- farming sector [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePEDV belongs to the genus Alphacoronavirus within the Coronaviridae family. It is an enveloped, single -stranded positive-sense RNA virus with a diameter of approximately 95\u0026ndash;190 nm [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Its genome, around 28 kb in length (excluding the poly A tail), encodes multiple proteins, including four structural proteins (S, M, E, and N), sixteen non-structural proteins (nsp1 - nsp16), and an accessory protein ORF3 [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Among these, the S glycoprotein is a crucial structural protein on the viral surface. It can be further divided into two subunits, S1 and S2[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The S1 subunit plays a crucial role in binding to the surface receptors of host cells, thereby initiating the viral infection process [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, it encompasses multiple neutralizing epitopes. Notably, through cryo-electron microscopy (cryo-EM) structural analysis, it has been found that the S1 trimer contains 4 conserved neutralizing epitopes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This characteristic makes it an ideal antigenic target for the development of vaccines. The S2 subunit plays a vital role in the fusion of the virus with cells and cell-to-cell membranes, facilitating the virus's entry into host cells and subsequent replication [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePED was first identified in the United Kingdom in 1977. Subsequently, it spread rapidly across Europe, causing substantial damage to the local swine industry [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. With the increasing frequency of global swine trade, PED gradually disseminated to numerous Asian countries, such as Japan, South Korea, Pakistan, Malaysia, and China [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Since 2011, PED has been prevalent on a large scale in China for an extended period, bringing about a huge economic impact on the domestic swine-farming industry [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In 2013, PED erupted in the United States, resulting in a large number of piglet deaths, a sharp decline in pig inventories, and a severe blow to the entire swine-farming industry chain, and caused an estimated \u003cspan\u003e$\u003c/span\u003e1.6\u0026nbsp;billion loss in the US swine industry during 2013\u0026ndash;2014[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. PEDV has a broad host range and can infect pigs of all ages through direct or indirect fecal-oral routes[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. It is particularly devastating to suckling piglets within one week of age, with a mortality rate that can reach as high as 80\u0026ndash;90% [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This not only leads to a significant reduction in the number of piglets and an increase in breeding costs but also severely affects the production efficiency and economic benefits of pig farms [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, PEDV vaccines available on the market, such as traditional inactivated and attenuated vaccines, have several limitations in practical applications. Inactivated vaccines, although having a relatively short production cycle and high safety, are weak in stimulating the body to produce cellular and mucosal immunity, making it difficult to comprehensively and effectively resist virus invasion[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Attenuated vaccines can trigger a relatively strong immune response, but due to the instability of the PEDV genome, gene mutations and recombinations are likely to occur during the passage process. This may lead to the reversion of the virulence of attenuated strains, potentially causing harm to the pig herd. Additionally, the adaptability issues of PEDV variant strains in cell culture limit the large-scale production of traditional inactivated and attenuated vaccines.\u003c/p\u003e \u003cp\u003eAdeno-associated virus (AAV), a single-stranded DNA virus lacking self-replication ability, possesses unique advantages that render it a mature and widely used gene delivery vector [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. AAV can infect various cell types and stably express foreign proteins over an extended period. It also has low immunogenicity and causes no obvious side effects on the body [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In recent years, AAV has made remarkable progress in the field of gene therapy. In 2012, the European Medicines Agency (EMA) approved the AAV-based gene therapy drug \"Glybera\" for the treatment of inherited lipoprotein lipase deficiency (LPLD) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In 2017, the US Food and Drug Administration (FDA) approved Spark Therapeutics' LUXTURNA, a recombinant adeno- associated virus serotype 2 (rAAV2-hRPE65) expressing human retinal pigment epithelium 65 kDa protein, for the treatment of patients with confirmed biallelic RPE65 mutation-associated retinal dystrophy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. AAV vectors are also extensively applied in the development of vaccines against various viral infectious diseases, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and influenza A virus subtype (H1N1) [\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In addition, studies have shown that AAV2 demonstrated superior intestinal tropism (3.8-fold higher than AAV8 in porcine enteroids, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), which makes it more suitable for the prevention and treatment of diseases caused by intestinal-related viruses[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the current severe threat of PEDV to the swine industry and the inadequacies of existing vaccines, the development of novel, efficient, and safe PEDV vaccines is of utmost urgency. In this study, we constructed a recombinant adeno-associated virus serotype 2 vector vaccine (rAAV-CMV-PEDV S1) expressing the PEDV S1 protein by cytomegalovirus (CMV) promoter. Our objectives were to comprehensively analyze its biological characteristics, safety, and immunogenicity, evaluate the immune responses it induced in mouse and sow models, and assess its protective efficacy in neonatal piglets. This research aims to provide a theoretical basis for the development of PEDV vaccines and hopes to open up new avenues for the prevention and control of PED and the healthy development of the swine industry. If successful, this AAV-based platform could be rapidly adapted to combat emerging coronaviruses in livestock, leveraging its modular design for antigen swapping.\u003c/p\u003e"},{"header":"RESULT","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstruction, Characterization, and Genome-Based Quantification of rAAV-CMV-PEDV S1 Virions\u003c/h2\u003e \u003cp\u003eWe obtained the two genome of the recombinant adeno-associated virus (rAAV-CMV-PEDV S1/rAAV-CMV-EGFP) by individually cloning the optimized porcine epidemic diarrhea virus (PEDV) S1 gene and enhanced green fluorescent protein (EGFP) gene into the AAV backbone plasmid and incorporating the CMV promoter \u003cb\u003e(Figure.1a)\u003c/b\u003e. After separately transfecting the two backbone plasmids into HEK293T cells, successful expression of EGFP in HEK293T cells was observed in the pAAV-CMV-EGFP group, indicating the successful construction of the backbone plasmids \u003cb\u003e(Figure.1b)\u003c/b\u003e. We obtained the fully packaged recombinant adeno-associated viruses (rAAV-CMV-PEDV S1 virus and rAAV-CMV-EGFP virus), after co-transfecting the two backbone plasmids with the helper plasmid respectively into HEK293T cells and culturing the cells for another 6 days. Subsequently, we verified through indirect immunofluorescence assay (IFA) and Western blot (WB) tests that both the rAAV-CMV-PEDV S1 virus and the rAAV-CMV-EGFP virus can infect cells and express either EGFP or S1 protein \u003cb\u003e(Figure.1c,d)\u003c/b\u003e. Transmission electron microscopy (TEM) analysis revealed that both rAAV-CMV-PEDV S1 and rAAV-CMV-EGFP virions exhibited characteristic spherical morphology, with a uniform diameter of approximately 22 nm, consistent with the expected size of adeno-associated virus particles \u003cb\u003e(Figure.1e)\u003c/b\u003e. Since rAAV-CMV-PEDV S1 infection does not induce cytopathic effects (CPE), rendering the TCID\u003csub\u003e50\u003c/sub\u003e (50% tissue culture infectious dose) assay inapplicable for viral titer determination, we developed an absolute quantitative qPCR standard curve targeting the viral genome. The equation Y= -3.221log\u003csub\u003e10\u003c/sub\u003eN\u0026thinsp;+\u0026thinsp;36.441 (E\u0026thinsp;=\u0026thinsp;104.4%, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.999) was established to quantify genome copies in viral preparations, providing a robust and reproducible metric for viral stock characterization. Following purification and removal of residual free DNA, the titer of rAAV-CMV-PEDV S1 viral particles was quantified as 1.86\u0026times;10\u003csup\u003e10\u003c/sup\u003e vg/mL using an absolute qPCR standard curve, ensuring accurate measurement of encapsidated genomes. In summary, The recombinant adeno-associated virus expressing the PEDV S1 protein (rAAV-CMV-PEDV S1) was successfully constructed. To quantify antigen content in inactivated and live-attenuated PEDV vaccines and assess viral shedding loads during challenge protection experiments, we developed an absolute quantitative qPCR standard curve targeting the PEDV M gene. The regression equation Y= -3.337log\u003csub\u003e10\u003c/sub\u003eN\u0026thinsp;+\u0026thinsp;38.601 demonstrated high amplification efficiency (E\u0026thinsp;=\u0026thinsp;99.4%) and robust linearity (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.999), establishing a validated method for precise PEDV genome quantification across vaccine formulations and in vivo challenge models. The standard curves figures for absolute quantification of rAAV-CMV-PEDV S1 and PEDV genomes, including their respective regression equations (Y= -3.221log\u003csub\u003e10\u003c/sub\u003eN\u0026thinsp;+\u0026thinsp;36.441 and Y= -3.337log\u003csub\u003e10\u003c/sub\u003eN\u0026thinsp;+\u0026thinsp;38.601), are provided in \u003cb\u003eSupplementary Fig.\u0026nbsp;1, 2\u003c/b\u003e, demonstrating their high linearity (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.999 for both) and amplification efficiencies (E\u0026thinsp;=\u0026thinsp;104.4% and 99.4%, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003erAAV-CMV-PEDV S1 vaccine elicits robust and sustained humoral-cellular immunity with superior CD8\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eT-cell activation over inactivated vaccines in a murine model\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate the immunogenicity of the rAAV-CMV-PEDV S1 vaccine, we compared its efficacy in inducing serum anti-PEDV S1 IgG and activating CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e\u0026thinsp;and CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T-cells with that of a conventional inactivated PEDV vaccine in a murine model \u003cb\u003e(Figure.2a)\u003c/b\u003e. The rAAV-CMV-PEDV S1 vaccine induced significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and more durable serum anti-PEDV S1 IgG titers than the inactivated vaccine, with elevated antibody levels persisting for at least 20 weeks \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Furthermore, mice immunized with rAAV-CMV-PEDV S1 exhibited a marked increase in CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T-cells frequencies compared to both the inactivated vaccine and control groups at 4 weeks p.i. (1.39-fold increase vs. inactivated vaccine, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) \u003cb\u003e(\u003c/b\u003eFigures.\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee,f\u003cb\u003e)\u003c/b\u003e. In contrast, no significant difference in CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T-cells proportions was observed between the rAAV and inactivated vaccine groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFigures.\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec,d\u003cb\u003e)\u003c/b\u003e. These findings demonstrate that the rAAV-CMV-PEDV S1 vaccine elicits potent and sustained humoral immunity (high IgG titers) and enhances cellular immunity (CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T-cell activation), outperforming traditional inactivated vaccines in inducing a balanced adaptive immune response.\u003c/p\u003e\n\u003ch3\u003eSafety and Tolerability of rAAV-CMV-PEDV S1 Vaccine in Pregnant Sows\u003c/h3\u003e\n\u003cp\u003eTo assess the safety profile of the rAAV-CMV-PEDV S1 vaccine, we conducted a comparative study in pregnant sows, evaluating its immunogenicity alongside inactivated and attenuated PEDV vaccines \u003cb\u003e(Figure. 3a)\u003c/b\u003e. Safety metrics included rectal temperature fluctuations and clinical symptom scores in sows monitored daily for 14 days post-immunization, as well as neonatal piglet viability (health proportion) and average birth weight \u003cb\u003e(Figure.3d,e)\u003c/b\u003e. Notably, sows immunized with rAAV-CMV-PEDV S1 exhibited no significant differences in body temperature trends (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) or comprehensive score (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) compared to the inactivated vaccine, attenuated vaccine, and DMEM control groups \u003cb\u003e(\u003c/b\u003eFigures.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb,c\u003cb\u003e)\u003c/b\u003e. Similarly, neonatal piglets from the rAAV-CMV-PEDV S1 group showed comparable health proportions and average birth weights to those from other groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), with no adverse effects attributable to the vaccine \u003cb\u003e(\u003c/b\u003eFigures.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed,e\u003cb\u003e)\u003c/b\u003e. These data demonstrate that the rAAV-CMV-PEDV S1 vaccine is well-tolerated in pregnant sows and poses no detectable risks to neonatal piglets, supporting its potential for safe use in maternal immunization strategies against PEDV.\u003c/p\u003e \u003cp\u003e \u003cb\u003erAAV-CMV-PEDV S1 vaccine enhances humoral immunity in sows and confers natural passive immunity to neonatal piglets\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo comprehensively explore the impact of the rAAV-CMV-PEDV S1 vaccine impact on humoral immunity, a series of experiments were conducted. Specifically, the serum anti-PEDV S IgG titers, colostrum anti-PEDV S IgG and IgA titers, as well as the colostric neutralizing antibody titers were detected through ELISA (Enzyme-Linked Immunosorbent Assay) and the virus neutralization test. The arrangement for collecting serum and colostrum is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. The results revealed that the rAAV-CMV-PEDV S1 vaccine could prompt the body to generate higher and more stable titers of serum anti-PEDV S IgG compared to both the attenuated vaccine and the inactivated vaccine at 27 days post-immunization (d.p.i) and 60 d.p.i, (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 stability) \u003cb\u003e(Figure.4a)\u003c/b\u003e. Moreover, when contrasted with the attenuated vaccine and the inactivated vaccine, this particular vaccine was able to increase the titer of colostric anti-PEDV S IgG (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 \u003cem\u003evs.\u003c/em\u003e attenuated vaccine; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 \u003cem\u003evs.\u003c/em\u003e inactivated vaccine) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) and also induce the body to produce similar levels of colostral anti-PEDV S IgA ( \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 \u003cem\u003evs.\u003c/em\u003e attenuated vaccine and inactivated vaccine) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Through the virus neutralization test, it was further demonstrated that the rAAV-CMV-PEDV S1 vaccine could enhance the neutralizing valence in colostrum ( log\u003csub\u003e2\u003c/sub\u003e 7.08 vs. 6.06 and 5.48 in commercial vaccines \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). To evaluate the natural passive immunity, the serum anti-PEDV S IgG titer of neonatal piglets was detected by ELISA at 5 days after birth. Intriguingly, compared with the other three groups, the rAAV-CMV-PEDV S1 vaccine could significantly increase the serum anti-PEDV S IgG titer of neonatal piglets, thereby enhancing the natural passive immunity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 \u003cem\u003evs.\u003c/em\u003e attenuated vaccine; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 \u003cem\u003evs.\u003c/em\u003e inactivated vaccine) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. In conclusion, based on all these findings, it can be firmly stated that the rAAV-CMV-PEDV S1 vaccine indeed plays a crucial role in strengthening the humoral immunity of sows and enhancing maternal passive immunity of piglets.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003erAAV-CMV-PEDV S1 vaccine could improve the cellular immunity and humoral immunity of sows in a certain sense.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo assess the effects of the rAAV-CMV-PEDV S1 vaccine on cellular and humoral immunity in sows, serum concentrations of Th1-associated cytokines (IL-2, IFN-γ, TNF-β) and Th2-associated cytokine (IL-4) were analyzed across immunization groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d\u003cb\u003e)\u003c/b\u003e. Compared to the attenuated vaccine group, the rAAV-CMV-PEDV S1 vaccine induced significantly higher levels of IL-2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while IL-4, IFN-γ, and TNF-β concentrations showed non-significant increases (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d\u003cb\u003e)\u003c/b\u003e. In contrast, when compared to the inactivated vaccine group, the rAAV vaccine elicited markedly elevated concentrations of all cytokines, including IL-4 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), IL-2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), IFN-γ (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and TNF-β (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (\u003cb\u003eFigure.5a-d\u003c/b\u003e). These results, to some extent, indicate that the rAAV-CMV-PEDV S1 vaccine robustly enhances both Th1-mediated cellular immunity (via IL-2, IFN-γ, and TNF-β) and Th2-driven humoral immunity (via IL-4), demonstrating its dual immunomodulatory efficacy in sows.\u003c/p\u003e \u003cp\u003e \u003cb\u003erAAV-CMV-PEDV S1 vaccine confers superior protection in suckling piglets via passive immunity, enhancing survival and preserving intestinal integrity against PEDV challenge\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the protective efficacy of various vaccines against PEDV infection, we conducted a comprehensive assessment in suckling piglets subjected to oral PEDV challenge, measuring four critical parameters: feed intake scores, diarrhea severity, viral shedding titers, and survival rates. Comparative analysis revealed that the rAAV-CMV-PEDV S1 vaccine demonstrated superior protective efficacy over conventional attenuated and inactivated vaccines, as evidenced by significantly improved feed intake scores, diarrhea symptom score, and markedly reduced viral shedding titers in challenged suckling piglets \u003cb\u003e(Figure. 6a, b,c)\u003c/b\u003e. Post-challenge survival analysis demonstrated that piglets immunized with the rAAV-CMV-PEDV S1 vaccine achieved an 80% survival rate, significantly surpassing conventional attenuated (60%) and inactivated (40%) vaccines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eHistopathological evaluation demonstrated marked preservation of intestinal integrity in the piglets of rAAV-CMV-PEDV S1 group, with neither gross pathological alterations (e.g., distension or hemorrhage) nor microscopically detectable mucosal damage \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. In stark contrast, The piglets in the other three group exhibited progressive intestinal pathology characterized by (1) macroscopic manifestations including severe intestinal distension, hemorrhagic foci, and pronounced wall thinning; (2) microscopic evidence of mucosal necrosis, epithelial desquamation, laminar propria vascular congestion, and muscular layer disintegration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The above results confirmed that the rAAV-CMV-PEDV S1 candidate vaccine can provide highly effective protection against the infection of wild-type PEDV strains for suckling piglets through a passive immune mechanism. Moreover, its comprehensive protective efficacy is significantly superior to that of traditional attenuated vaccines and inactivated vaccines.\u003c/p\u003e "},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003ePlasmids, viruses, cells and antibodies\u003c/h2\u003e \u003cp\u003eThe plasmids T-vector containing the EGFP gene, pAAV-CMV, pAAV-Helper, and pAAV-RC were preserved at Sichuan Agricultural University. HEK293T cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Australia) at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e. The PEDV SNJ-P (GenBank No. GQ918139.1) and PEDV AJ1102-F12 (GenBank No. MK584552.1) was also preserved at Sichuan Agricultural University. Commercial reagents included Lipo293\u0026trade; Transfection Reagent, RIPA Lysis Buffer, 4% PFA Fix Solution, Triton X-100, Trypsin solution, and Cy3-labeled Goat Anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (Beyotime Biotech, China); DNAzyme I (Thermo Fisher Scientific, USA); PMSF (Solarbio, China); IL-2, IL-4, IFN-γ, and TNF-β cytokine detection kits (Zhuocai Biotechnology, China); PEDV IgA/IgG antibody test kits (IDEXX Laboratories, China; Kangbaote Biotechnology, China); APC Hamster Anti-Mouse CD3ε, FITC anti-mouse CD4, and PE-Cy\u0026trade; anti-mouse CD8α antibodies (Biolegend, China); and PEDV inactivated/attenuated vaccines (Tianjin Ruipu Biotechnology; Zhongmu Industry, China). Mouse anti-PEDV S1 polyclonal antibody was prepared in-house.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eSix-week-old female RIC mice (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;120) were obtained from Chengdu Dashuo Experimental Animal Co., Ltd. Twenty-five primiparous sows and and their neonatal piglets were purchased from Chengdu Wangjiang Agricultural and Animal Husbandry Technology Co., Ltd. (Chengdu, China). Prior to immunization, rectal swabs from the experimental sows tested negative for PEDV RNA by reverse transcription quantitative polymerase chain reaction (RT-qPCR).\u003c/p\u003e \u003cp\u003eMice and piglets were housed in the specific pathogen-free (SPF) laboratory animal facility at Sichuan Agricultural University (Chengdu, China), which underwent rigorous disinfection and sterilization prior to the trial. Sows were raised in a PEDV-negative swine farm. All animals were provided with ad libitum access to water and feed. Sows were fed commercial swine diets, while piglets received maternal milk within the first 5 days of life and were manually fed animal milk replacer during the challenge protection trial. Mice were maintained on SPF-certified feed. Throughout the experiment, animals remained conscious, and no non-experimental procedures were conducted. Piglets and mice were humanely euthanized by CO₂ (\u0026ge;\u0026thinsp;70% (v/v), 5 minutes for mice; 10 minutes for piglets.) inhalation. This study was approved by the Sichuan Provincial Experimental Animal Management Committee (License No. SYXK (Chuan) 2019\u0026thinsp;\u0026minus;\u0026thinsp;187) and conducted in compliance with the Regulations on the Administration of Laboratory Animals (Ministry of Science and Technology, Beijing, China) and the Animal Research: Reporting of In Vivo Experiments (ARRIVE guidelines).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDesign and construction of rAAV-CMV-PEDV S1 virus and rAAV-CMV-EGFP virus\u003c/h3\u003e\n\u003cp\u003eThe codon-optimized PEDV S1 gene (SNJ-P strain) and EGFP gene were cloned into the pAAV-CMV plasmid via AsiSI and MluI sites, generating pAAV-CMV-PEDV S1 and pAAV-CMV-EGFP backbone plasmid. HEK293T cells were co-transfected with plasmids pAAV-CMV-PEDV S1 (or pAAV-CMV-EGFP), pAAV-Helper, and pAAV-RC using Lipo293\u0026trade; Transfection Reagent (Beyotime Biotech, China) in Opti-MEM\u0026reg; Medium (Gibco, USA). The plasmid structures of pAAV-Helper and pAAV-RC are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. Cells were monitored for EGFP expression by fluorescence microscopy at 24 h post-transfection. Recombinant viruses were harvested at 6 days, purified via cesium chloride ultracentrifugation, and treated with DNAzyme I to remove external DNA. Viral morphology and size were confirmed by transmission electron microscopy (TEM). Note: EGFP was inserted into the backbone plasmid as a control since PEDV plasmids could not accommodate it.\u003c/p\u003e\n\u003ch3\u003eIndirect immunofluorescence (IFA) and Western blot\u003c/h3\u003e\n\u003cp\u003eThe HEK293T cells in a 6-well plate were infected with rAAV-CMV-PEDV S1 virus (100 \u0026micro;L) and rAAV-CMV-EGFP virus (100 \u0026micro;L) respectively, and cells inoculated with DMEM were used as mock. As for IFA, the cells were fixed with 4% Paraformaldehyde for 15 min after being cultured for 72 h at 37\u0026deg;C, and then stained with primary antibody of mouse anti-PEDV S1 polyclonal antibody and second antibody of Cy3-labeled Goat Anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (Beyotime, China). The fluorescent signals were observed under an inverted fluorescence microscope (Olympus, IX71, Japan). As for western blot, the HEK293T cells infected with rAAV-CMV-PEDV S1 virus, rAAV-CMV-EGFP virus or mock respectively were lysed by 200 \u0026micro;L RIPA (Solarbio, China) with 0.5 mM PMSF(Solarbio, China). The lysed cells were centrifuged in 8000 g, 10 min, and supernatants were separated by a 12% SDS-PAGE and then transferred onto PVDF membrane (Millipore, USA). That was incubated with primary antibody of mouse anti-PEDV S1 polyclonal antibody and mouse anti-GAPDH monoclonal antibody(Abclonal, China), and secondary antibody of Goat Anti-Mouse IgG (HRP) (Abcam, UK), Ultimately, which visualized by the enhanced chemiluminescent (ECL) HRP substrate (Beyotime, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEstablishment of the qPCR standard curve and determination of the rAAV-CMV-PEDV S1 Titer.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe plasmid copy numbers for the rAAV-CMV-PEDV S1 backbone plasmid and PEDV M gene T-vector were calculated using the formula: Plasmid copy number (copies/\u0026micro;L)= (\u003cem\u003eCp\u003c/em\u003e\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e\u0026times;\u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e)/(\u003cem\u003eNp\u003c/em\u003e\u0026times;648) (\u003cem\u003eCp\u003c/em\u003e: Plasmid concentration (ng/\u0026micro;L); \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e: Avogadro's constant (6.022\u0026times;10\u003csup\u003e23\u003c/sup\u003e molecules/mol); \u003cem\u003eNp\u003c/em\u003e: plasmid size (bp); 648: Average molecular weight of a dsDNA base pair (g/mol))[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Initial concentrations were determined as 1.84\u0026times;10\u003csup\u003e11\u003c/sup\u003e copies/\u0026micro;L (rAAV-CMV-PEDV S1 plasmid) and 2.21\u0026times;10\u003csup\u003e10\u003c/sup\u003e copies/\u0026micro;L (PEDV M gene plasmid). Both plasmids were serially diluted to 1.0\u0026times;10\u003csup\u003e10\u003c/sup\u003e copies/\u0026micro;L using nuclease-free dH\u003csub\u003e2\u003c/sub\u003eO (Thermo Fisher Scientific, USA), followed by eight 10-fold serial dilutions to generate standards ranging from 1.0\u0026times;10\u003csup\u003e9\u003c/sup\u003e to1.0\u0026times;10\u003csup\u003e2\u003c/sup\u003e copies/\u0026micro;L. Quantitative PCR (qPCR) was performed using optimized cycling conditions (denaturation: 95\u0026deg;C for 30 s; 40 cycles of 95\u0026deg;C for 5 s, 60\u0026deg;C for 30 s). The threshold cycle (Cq) values were plotted against log\u003csub\u003e10\u003c/sub\u003e-transformed plasmid copy numbers (x-axis) to generate standard curves. A curve was deemed valid if it met the following criteria: amplification efficiency (E)\u0026thinsp;=\u0026thinsp;95\u0026ndash;105%, coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e)\u0026thinsp;\u0026ge;\u0026thinsp;0.998, and slope between \u0026minus;\u0026thinsp;3.58 and \u0026minus;\u0026thinsp;3.10. The viral nucleic acid was extracted using a DNA extraction kit(Tiangen China). qPCR amplification was performed according to the optimized amplification conditions described above. The Cq value obtained by qPCR was brought into the standard equation, which obtain the copy number of the virus genome per milliliter of virus liquid (vg/mL).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMouse Immunization Test\u003c/h2\u003e \u003cp\u003eSix-week-old female mice were divided into 3 groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;40/group). rAAV-CMV-PEDV S1 group and inactivated vaccine group were respectively injected 100 \u0026micro;L (1.00\u0026times;10\u003csup\u003e7\u003c/sup\u003e vg/100 \u0026micro;L ) rAAV-CMV-PEDV S1 virus and 100 \u0026micro;L (1.00\u0026times;10\u003csup\u003e7\u003c/sup\u003e vg/100 \u0026micro;L) inactivated vaccine, the negative control group was injected 100 \u0026micro;L DMEM. All the mice received one inoculations and no adverse events after vaccination. The blood samples of mine were collected every two weeks from orbit at 0 to 20 weeks.p.i.\u003cb\u003e(Figure. 2a)\u003c/b\u003e, and the serum was harvested after centrifugation at 8000 g 5min. Serum anti-PEDV S1 IgG OD\u003csub\u003e450\u003c/sub\u003e value were determined with enzyme-linked immunosorbent assays (ELISA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSplenocytes Isolation and Flow Cytometry\u003c/h2\u003e \u003cp\u003eThe spleens were dissected from the immunized mice and homogenized through a 70 \u0026micro;m strainer at 4 weeks.p.i. For analysis of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells, splenocytes were stained at 4 ℃ for 30 min with APC Hamster Anti-Mouse CD3ε, FITC anti-mouse CD4, and PE-Cy\u0026trade; anti-mouse CD8α antibodies. The flow cytometry procedure was performed with a Beckman cytoflex, and the data were analyzed using CytExpert software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunity studies in sows\u003c/h2\u003e \u003cp\u003eTwenty-five primiparous sows were divided into 4 groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5/group). rAAV-CMV-PEDV S1 virus group, attenuated vaccine group and inactivated vaccine group were respectively inoculated 1 mL (1.00\u0026times;10\u003csup\u003e9\u003c/sup\u003e vg/mL) rAAV-CMV-PEDV S1 virus, 1.78 mL (1.00\u0026times;10\u003csup\u003e9\u003c/sup\u003e vg ) attenuated vaccine and 2.11 mL (1.00\u0026times;10\u003csup\u003e9\u003c/sup\u003e vg ) inactivated vaccine, the negative control group was injected 1ml DMEM in 34 days before delivery. Inactivated vaccine group was boosted once in 20 days before delivery. The immune procedure is shown in \u003cb\u003eFigure.3a\u003c/b\u003e. From 0 to 14 days after immunization the body temperature and the comprehensive score ( the feeding, drinking water, mental condition and fecal morphology ) were monitored and observed, the scoring criteria are shown in \u003cb\u003eSumplmentary Table\u0026nbsp;1\u003c/b\u003e. The health proportion and the body weight of neonatal piglets were counted after delivery. The blood of sows was collected in 0,27,60 days.p.i, and harvested after centrifugation at 8000\u0026times; g 5min. Serum anti-PEDV S IgG S/P value was detected according to the ELISA kit instructions. The colostrum was collected after delivery, and harvested after centrifugation at 8000\u0026times; g 5min. Anti-PEDV S IgG and IgA S/P value in milk serum was detected according to the ELISA kit instructions. Three 7-day-old neonatal piglets were randomly selected from each sow, and the serum anti-PEDV S IgG S/P value in piglets were detected according to the above method. The IL-2, IL-4, IFN-γ and TNF-β concentrations in serum of sows were measured at 27 days.p.i according to the instructions of cytokine ELISA detection kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePEDV neutralization assay\u003c/h2\u003e \u003cp\u003eThe colostrum was collected after delivery to determine PEDV virus neutralizing antibody (VNA) levels using end-point neutralization test. Colostrous supernatant is separated from colostrum and were inactivated at 56\u0026deg;C for 30 min. Colostrous supernatant were separated and serially diluted two-fold with DMEM from 2\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 2\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e and incubated with 100 TCID\u003csub\u003e50\u003c/sub\u003e of the PEDV AJ1102-F12 strain for 1 h at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. When the vero cells in the 96-well plate were 90% confluent, the above mixture of serum and virus or DMEM as negative control was added, and the cells were cultured for 2 h at 37\u0026deg;C. Next, DMEM containing trypsin (5 mg/mL), was added to each well, and the plate was incubated for 3 to 5 days at 37\u0026deg;C. Neutralizing antibody titers were expressed as the log\u003csub\u003e2\u003c/sub\u003e transformation of the reciprocal highest colostrous supernatant dilution that completely inhibited cytopathic effects (CPEs). Each sample was run in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eChallenge protection in piglets\u003c/h2\u003e \u003cp\u003eTwenty 5-day-old neonatal piglets from each group (n\u0026thinsp;=\u0026thinsp;5/group), and was orally infected with 1 mL(10\u003csup\u003e5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mL) PEDV AJ1102-F12. The feed intake and diarrhea symptoms in infected neonatal piglets were observed and scored every 12 hours after challenge, the scoring criteria are shown in the \u003cb\u003eSupplementary Table\u0026nbsp;2, 3\u003c/b\u003e. The higher the score, the stronger the feeding ability and the lighter the diarrhea symptoms of suckling piglets. After PEDV challenge, anal swabs of infected neonatal piglets were collected every 12 hours, the amount of virus shedding was monitored by RT-qPCR, and the survival rate of infected neonatal piglets was monitored and counted every day. Five days post-challenge, surviving piglets were euthanized for necropsy to examine intestinal lesions. Jejunal segments were collected to prepare histopathological sections for observing microscopic pathological changes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistics and reproducibility\u003c/h2\u003e \u003cp\u003eGraphPad Prism 8 statistical software (GraphPad Software, San Diego, CA, USA) was used to analyze the data and draw graphs The data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical differences among the groups were identified through one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test for multiple comparisons. A threshold of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant, with the following notation: * \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; *** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; **** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; \u003cem\u003ens\u003c/em\u003e, not significant, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study successfully constructed a recombinant adeno-associated virus (rAAV-CMV-PEDV S1) expressing the S1 subunit of PEDV and systematically evaluated its immunogenicity and protective efficacy. The results demonstrate that the rAAV-based vaccine outperforms traditional inactivated and attenuated vaccines by inducing robust, durable humoral and cellular immune responses in mice, enhancing maternal antibody transfer in sows, and conferring significant protection to piglets against lethal PEDV challenge. These findings highlight the potential of AAV vectors as a novel platform for developing vaccines against enteric coronaviruses in livestock.\u003c/p\u003e \u003cp\u003eThe rAAV-CMV-PEDV S1 vaccine induced significantly higher and longer-lasting serum IgG titers in mice compared to inactivated vaccines (\u003cb\u003eFigure.2b\u003c/b\u003e), likely due to sustained antigen expression mediated by the AAV vector. Unlike inactivated vaccines, which primarily rely on exogenous antigen presentation via MHC-II pathways, AAV-delivered antigens are synthesized intracellularly, enabling MHC-I presentation and subsequent activation of CD8\u003csup\u003e+\u003c/sup\u003e T cells. This mechanism aligns with the observed increase in CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T-cell frequencies (Figures.\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-f), suggesting enhanced cytotoxic T lymphocyte (CTL) activity critical for eliminating virus-infected cells[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Such dual humoral-cellular immunity is a distinct advantage over traditional vaccines, which often fail to elicit robust CTL responses.\u003c/p\u003e \u003cp\u003eIn pregnant sows, the rAAV vaccine generated elevated serum and colostral IgG and neutralizing antibody titers (Figures.\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-d), translating to 80% survival rates in challenged piglets (\u003cb\u003eFigure.6d\u003c/b\u003e). The high IgA levels in colostrum further suggest mucosal immune priming, which is vital for blocking PEDV entry at intestinal epithelial surfaces[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. By contrast, commercial attenuated vaccines carry risks of virulence reversion due to PEDV\u0026rsquo;s genetic instability, while inactivated vaccines lack the durability and mucosal immune induction observed here[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The modular design of AAV vectors also allows rapid antigen updates to match emerging variants, a feature critical for addressing PEDV\u0026rsquo;s high mutation rate.\u003c/p\u003e \u003cp\u003eThe rAAV-CMV-PEDV S1 vaccine exhibited an excellent safety profile in sows, with no significant differences in body temperature, clinical scores, or neonatal piglet viability compared to controls (\u003cb\u003eFigure.3b-e\u003c/b\u003e). This aligns with the inherent safety of AAV vectors, which lack pathogenicity and exhibit low immunogenicity[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, histopathological analysis revealed preserved intestinal integrity in vaccinated piglets (\u003cb\u003eFigure.7\u003c/b\u003e), underscoring the vaccine\u0026rsquo;s ability to mitigate PEDV-induced enteric damage through passive immunity. These results position the rAAV platform as a safer alternative to live-attenuated vaccines, particularly in sensitive populations such as pregnant sows.\u003c/p\u003e \u003cp\u003eWhile promising, this study has limitations. First, the experiments were conducted under controlled laboratory conditions; field trials are necessary to evaluate efficacy in diverse farming environments. Second, long-term safety assessments are needed to rule out potential risks of vector genome integration or immune tolerance after prolonged antigen exposure[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Third, the vaccine\u0026rsquo;s cross-protective efficacy against emerging PEDV variants (e.g., GIIb strains) remains untested. Future studies should explore multivalent AAV designs incorporating conserved epitopes from divergent strains to broaden protection. Additionally, optimizing production scalability and cost-effectiveness will be crucial for real-world application.\u003c/p\u003e \u003cp\u003eThe rAAV-CMV-PEDV S1 vaccine represents a significant advancement in PEDV prophylaxis, combining the safety of non-replicating vectors with the immunogenicity of live vaccines. Its ability to induce durable humoral immunity, activate CTLs, and confer passive protection to piglets addresses critical gaps in current PEDV vaccine strategies. As a modular platform, this approach could be rapidly adapted for other coronaviruses, offering a versatile tool to combat emerging zoonotic threats. Further translational research, including large-scale efficacy trials and cost-benefit analyses, will accelerate its transition from bench to farm.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author/s.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMethodology, M.P.; software,M.R.; validation,\u0026nbsp;D.J.; data curation, M.P. and\u0026nbsp;Y.Z; writing-original draft preparation,\u0026nbsp;M.P.; writing-review and editing,\u0026nbsp;T.T and Y.W; project administration, M.P. and\u0026nbsp;M.R. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author/s.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported by the Sichuan Province Science and Technology Planning Program (2021ZDZX0010), the Natural Science Foundation of Sichuan Province (2024NSFSC0374), and the Sichuan Innovation Team Project of National Modern Agricultural Industry Technology System (SCCXTD-2024-26)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Wang for his insightful comments on the design of the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary material\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Supplementary Material for this article can be found online at: https:\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang H, Zou C, Peng O, Ashraf U, Xu Q, Gong L, Fan B, Zhang Y, Xu Z, Xue C, Wei X, Zhou Q, Tian X, Shen H, Li B, Zhang X, Cao Y. Global Dynamics of Porcine Enteric Coronavirus PEDV Epidemiology, Evolution, and Transmission. Mol Biol Evol. 2023 Mar 4;40(3):msad052. doi: 10.1093/molbev/msad052. \u003c/li\u003e\n\u003cli\u003eLin CM, Saif LJ, Marthaler D, Wang Q. Evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains. Virus Res. 2016 Dec 2;226:20-39. doi: 10.1016/j.virusres.2016.05.023. \u003c/li\u003e\n\u003cli\u003ePasick J, Berhane Y, Ojkic D, Maxie G, Embury-Hyatt C, Swekla K, Handel K, Fairles J, Alexandersen S. Investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in Canada. Transbound Emerg Dis. 2014 Oct;61(5):397-410. doi: 10.1111/tbed.12269. \u003c/li\u003e\n\u003cli\u003eLi K, Wang H, Chen T, Wang X, Wang X, Zhong M, Gao X, Hao Z. Dehydroevodiamine inhibits PEDV through regulateing ERK1/2 MAPK pathway in Vero cells. Virology. 2024 Oct;598:110166. doi: 10.1016/j.virol.2024.110166. \u003c/li\u003e\n\u003cli\u003eMa X, Zheng H, Chen H, Ma S, Wei Z. Porcine epidemic diarrhea virus: A review of detection, inhibition of host gene expression and evasion of host innate immune. Microb Pathog. 2024 Oct;195:106873. doi: 10.1016/j.micpath.2024.106873. \u003c/li\u003e\n\u003cli\u003eShen Z, Ye G, Deng F, Wang G, Cui M, Fang L, Xiao S, Fu ZF, Peng G. Structural Basis for the Inhibition of Host Gene Expression by Porcine Epidemic Diarrhea Virus nsp1. J Virol. 2018 Feb 12;92(5):e01896-17. doi: 10.1128/JVI.01896-17. \u003c/li\u003e\n\u003cli\u003eHou Y, Meulia T, Gao X, Saif LJ, Wang Q. Deletion of both the Tyrosine-Based Endocytosis Signal and the Endoplasmic Reticulum Retrieval Signal in the Cytoplasmic Tail of Spike Protein Attenuates Porcine Epidemic Diarrhea Virus in Pigs. J Virol. 2019 Jan 4;93(2):e01758-18. doi: 10.1128/JVI.01758-18. \u003c/li\u003e\n\u003cli\u003eSuzuki T, Terada Y, Enjuanes L, Ohashi S, Kamitani W. S1 Subunit of Spike Protein from a Current Highly Virulent Porcine Epidemic Diarrhea Virus Is an Important Determinant of Virulence in Piglets. Viruses. 2018 Aug 30;10(9):467. doi: 10.3390/v10090467. \u003c/li\u003e\n\u003cli\u003eGong L, Lin Y, Qin J, Li Q, Xue C, Cao Y. Neutralizing antibodies against porcine epidemic diarrhea virus block virus attachment and internalization. Virol J. 2018 Aug 30;15(1):133. doi: 10.1186/s12985-018-1042-3. \u003c/li\u003e\n\u003cli\u003eLi C, Li W, Lucio de Esesarte E, Guo H, van den Elzen P, Aarts E, van den Born E, Rottier PJM, Bosch BJ. Cell Attachment Domains of the Porcine Epidemic Diarrhea Virus Spike Protein Are Key Targets of Neutralizing Antibodies. J Virol. 2017 May 26;91(12):e00273-17. doi: 10.1128/JVI.00273-17. PMID: 28381581; PMCID: PMC5446644.\u003c/li\u003e\n\u003cli\u003eZhang D, Xie Y, Liao Q, Jiao Z, Liang R, Zhang J, Zhang Y, Tan Y, Wang H, Zhang W, Xiao S, Peng G, Shi Y. Development of a safe and broad-spectrum attenuated PEDV vaccine candidate by S2 subunit replacement. J Virol. 2024 Nov 19;98(11):e0042924. doi: 10.1128/jvi.00429-24. \u003c/li\u003e\n\u003cli\u003eWood EN. An apparently new syndrome of porcine epidemic diarrhoea. Vet Rec. 1977 Mar 19;100(12):243-4. doi: 10.1136/vr.100.12.243. \u003c/li\u003e\n\u003cli\u003eJung K, Saif LJ. Porcine epidemic diarrhea virus infection: Etiology, epidemiology, pathogenesis and immunoprophylaxis. Vet J. 2015 May;204(2):134-43. doi: 10.1016/j.tvjl.2015.02.017. \u003c/li\u003e\n\u003cli\u003eWang D, Fang L, Xiao S. Porcine epidemic diarrhea in China. Virus Res. 2016 Dec 2;226:7-13. doi: 10.1016/j.virusres.2016.05.026. \u003c/li\u003e\n\u003cli\u003ePasick J, Berhane Y, Ojkic D, Maxie G, Embury-Hyatt C, Swekla K, Handel K, Fairles J, Alexandersen S. Investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in Canada. Transbound Emerg Dis. 2014 Oct;61(5):397-410. doi: 10.1111/tbed.12269. \u003c/li\u003e\n\u003cli\u003eLi Y, Wu Q, Huang L, Yuan C, Wang J, Yang Q. An alternative pathway of enteric PEDV dissemination from nasal cavity to intestinal mucosa in swine. Nat Commun. 2018 Sep 19;9(1):3811. doi: 10.1038/s41467-018-06056-w. \u003c/li\u003e\n\u003cli\u003eJung K, Annamalai T, Lu Z, Saif LJ. Comparative pathogenesis of US porcine epidemic diarrhea virus (PEDV) strain PC21A in conventional 9-day-old nursing piglets vs. 26-day-old weaned pigs. Vet Microbiol. 2015 Jul 9;178(1-2):31-40. doi: 10.1016/j.vetmic.2015.04.022. \u003c/li\u003e\n\u003cli\u003eSun M, Ma J, Wang Y, Wang M, Song W, Zhang W, Lu C, Yao H. Genomic and epidemiological characteristics provide new insights into the phylogeographical and spatiotemporal spread of porcine epidemic diarrhea virus in Asia. J Clin Microbiol. 2015 May;53(5):1484-92. doi: 10.1128/JCM.02898-14. Epub 2015 Feb 18. PMID: 25694517; PMCID: PMC4400760.\u003c/li\u003e\n\u003cli\u003eGao B, He L, Bao Y, Chen Y, Lu G, Zhang Y, Xu Y, Su B, Xu J, Wang Y, Yeap LS. Repeated vaccination of inactivated SARS-CoV-2 vaccine dampens neutralizing antibodies against Omicron variants in breakthrough infection. Cell Res. 2023 Mar;33(3):258-261. doi: 10.1038/s41422-023-00781-8. \u003c/li\u003e\n\u003cli\u003eGao B, He L, Bao Y, Chen Y, Lu G, Zhang Y, Xu Y, Su B, Xu J, Wang Y, Yeap LS. Repeated vaccination of inactivated SARS-CoV-2 vaccine dampens neutralizing antibodies against Omicron variants in breakthrough infection. Cell Res. 2023 Mar;33(3):258-261. doi: 10.1038/s41422-023-00781-8. \u003c/li\u003e\n\u003cli\u003eLinden RM, Berns KI. Molecular biology of adeno-associated viruses. Contrib Microbiol. 2000;4:68-84. doi: 10.1159/000060327. \u003c/li\u003e\n\u003cli\u003eWang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019 May;18(5):358-378. doi: 10.1038/s41573-019-0012-9. \u003c/li\u003e\n\u003cli\u003eZaiss AK, Liu Q, Bowen GP, Wong NC, Bartlett JS, Muruve DA. Differential activation of innate immune responses by adenovirus and adeno-associated virus vectors. J Virol. 2002 May;76(9):4580-90. doi: 10.1128/jvi.76.9.4580-4590.2002. \u003c/li\u003e\n\u003cli\u003eSomanathan S, Breous E, Bell P, Wilson JM. AAV vectors avoid inflammatory signals necessary to render transduced hepatocyte targets for destructive T cells. Mol Ther. 2010 May;18(5):977-82. doi: 10.1038/mt.2010.40. \u003c/li\u003e\n\u003cli\u003eGaudet D, M\u0026eacute;thot J, D\u0026eacute;ry S, Brisson D, Essiembre C, Tremblay G, Tremblay K, de Wal J, Twisk J, van den Bulk N, Sier-Ferreira V, van Deventer S. Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an open-label trial. Gene Ther. 2013 Apr;20(4):361-9. doi: 10.1038/gt.2012.43. \u003c/li\u003e\n\u003cli\u003eBommakanti N, Young BK, Sisk RA, Berrocal AM, Duncan JL, Bakall B, Mathias MT, Ahmed I, Chorfi S, Comander J, Nagiel A, Besirli CG. Classification and Growth Rate of Chorioretinal Atrophy after Voretigene Neparvovec-Rzyl for RPE65-Mediated Retinal Degeneration. Ophthalmol Retina. 2024 Jan;8(1):42-48. doi: 10.1016/j.oret.2023.08.017. \u003c/li\u003e\n\u003cli\u003eTang X, Lu H, Tarwater PM, Silverberg DL, Schorl C, Ramratnam B. Adeno-Associated Virus (AAV)-Delivered Exosomal TAT and BiTE Molecule CD4-\u0026alpha;CD3 Facilitate the Elimination of CD4 T Cells Harboring Latent HIV-1. Microorganisms. 2024 Aug 18;12(8):1707. doi: 10.3390/microorganisms12081707.\u003c/li\u003e\n\u003cli\u003eRashid MHO, Kayesh MEH, Hashem MA, Hifumi T, Ogawa S, Miyoshi N, Tanaka Y, Kohara M, Tsukiyama-Kohara K. Adeno-associated virus 2 CRISPR/Cas9-mediated targeting of hepatitis B virus in tree shrews. Virus Res. 2025 Feb 21;354:199550. doi: 10.1016/j.virusres.2025.199550. Epub ahead of print. PMID: 39988206.\u003c/li\u003e\n\u003cli\u003eColon-Moran W, Baer A, Lamture G, Stapleton JT, Fischer JW, Bhattarai N. A short hepatitis C virus NS5A peptide expression by AAV vector modulates human T cell activation and reduces vector immunogenicity. Gene Ther. 2022 Nov;29(10-11):616-623. doi: 10.1038/s41434-021-00302-5. \u003c/li\u003e\n\u003cli\u003eYamazaki T, Chiba J, Akashi-Takamura S. Neutralizing Anti-Hemagglutinin Monoclonal Antibodies Induced by Gene-Based Transfer Have Prophylactic and Therapeutic Effects on Influenza Virus Infection. Vaccines (Basel). 2018 Jun 26;6(3):35. doi: 10.3390/vaccines6030035. \u003c/li\u003e\n\u003cli\u003eRied MU, Girod A, Leike K, B\u0026uuml;ning H, Hallek M. Adeno-associated virus capsids displaying immunoglobulin-binding domains permit antibody-mediated vector retargeting to specific cell surface receptors. J Virol. 2002 May;76(9):4559-66. doi: 10.1128/jvi.76.9.4559-4566.2002. PMID: 11932421; PMCID: PMC155067.\u003c/li\u003e\n\u003cli\u003eSambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd edn. New York: Cold Spring Harbor Laboratory Press; 2001.\u003c/li\u003e\n\u003cli\u003eBiernacki MA, Brault M, Bleakley M. T-Cell Receptor-Based Immunotherapy for Hematologic Malignancies. Cancer J. 2019 May/Jun;25(3):179-190. doi: 10.1097/PPO.0000000000000378. \u003c/li\u003e\n\u003cli\u003eHu Z, Li Y, Zhang B, Zhao Y, Guan R, Zhou Y, Du J, Zhang Z, Li X. Serum IgA antibody level against porcine epidemic diarrhea virus is a potential pre-evaluation indicator of immunization effects in sows during parturition under field conditions. Porcine Health Manag. 2024 Sep 3;10(1):32. doi: 10.1186/s40813-024-00382-w. \u003c/li\u003e\n\u003cli\u003eLiu H, Yin X, Tian H, Qiu Y, Wang Z, Chen J, Ma D, Zhao B, Du Q, Tong D, Huang Y. The S protein of a novel recombinant PEDV strain promotes the infectivity and pathogenicity of PEDV in mid-west China. Transbound Emerg Dis. 2022 Nov;69(6):3704-3723. doi: 10.1111/tbed.14740. \u003c/li\u003e\n\u003cli\u003eZhu QX, Zhang YN, Zhang HQ, Leng C, Deng CL, Wang X, Li JJ, Ye XL, Zhang B, Li XD. A single dose recombinant AAV based CHIKV vaccine elicits robust and durable protective antibody responses in mice. PLoS Negl Trop Dis. 2024 Nov 4;18(11):e0012604. doi: 10.1371/journal.pntd.0012604. \u003c/li\u003e\n\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":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6224876/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6224876/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe continuous spread of porcine epidemic diarrhea virus (PEDV) has highlighted the importance and necessity of developing accessible, safe, effective, and versatile vaccine platforms. While approved inactivated and attenuated vaccines have been instrumental in reducing the burdens of prevention and control and economic impacts, the induction of antibody level remains an unmet need. Here, we constructed a recombinant adeno-associated virus (rAAV-CMV-PEDV S1) expressing regions of the PEDV S1 subunit based on Adeno-associated virus serotype 2 (AAV-2) vector). We assessed its immunogenicity, durability, and protective efficacy. In mice, rAAV-CMV-PEDV S1 induced significantly more remarkable and persistent serum-specific IgG compared with the inactivated vaccine and accompanied by robust CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells activation within 4 weeks post-immunization. In pregnant sows, compared with commercially available inactivated and attenuated vaccines, the rAAV-CMV-PEDV S1 induced significantly higher serum and colostral IgG as well as neutralizing antibody titers, which provided piglets with abundant maternal antibodies. Crucially, challenge experiments demonstrated that rAAV-CMV-PEDV S1 conferred 80% clinical protective efficacy for piglets, accompanied by significantly reduced viral shedding loads, surpassing other vaccine groups These findings suggest that rAAV-CMV-PEDV S1 candidate could be a promising PEDV vaccine for enhancing antibody levels and could strengthen the protection of piglets against PEDV infection.\u003c/p\u003e","manuscriptTitle":"Development of a Recombinant Adeno-Associated Virus Vaccine Expressing PEDV S1 Protein: Enhanced Humoral and Cellular Immunity with Superior Neonatal Protection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 06:33:58","doi":"10.21203/rs.3.rs-6224876/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-08T19:12:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-01T19:57:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98309668287895091717620144542863121477","date":"2025-06-11T19:36:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-28T12:39:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331849207563316147328792161380956615899","date":"2025-05-08T12:22:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-28T14:05:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-26T15:26:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-19T08:20:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Vaccines","date":"2025-03-14T09:21:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3263575b-85b3-43e8-9382-b42e9649783a","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47838182,"name":"Biological sciences/Immunology"},{"id":47838183,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-11-10T16:03:24+00:00","versionOfRecord":{"articleIdentity":"rs-6224876","link":"https://doi.org/10.1038/s41541-025-01248-0","journal":{"identity":"npj-vaccines","isVorOnly":false,"title":"npj Vaccines"},"publishedOn":"2025-11-06 15:58:06","publishedOnDateReadable":"November 6th, 2025"},"versionCreatedAt":"2025-05-07 06:33:58","video":"","vorDoi":"10.1038/s41541-025-01248-0","vorDoiUrl":"https://doi.org/10.1038/s41541-025-01248-0","workflowStages":[]},"version":"v1","identity":"rs-6224876","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6224876","identity":"rs-6224876","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}