A novel double antibody sandwich quantitative ELISA for detecting porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea (PED), a contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), has caused significant economic losses to the global pig farming industry due to its rapid course and spread and its high mortality among piglets. In this study, we prepared rabbit polyclonal antibody and monoclonal antibody 6C12 against the PEDV nucleocapsid (N) protein using the conserved and antigenic PEDV N protein as an immunogen. A double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) was established to detect PEDV using rabbit polyclonal antibodies as capture antibodies and horseradish peroxidase (HRP)-labeled 6C12 as the detection antibody. Using DAS-qELISA, recombinant PEDV N protein, and virus titer detection limits were approximately 0.05 ng/mL and 103.02 50% tissue culture infective dose per mL (TCID50/mL), respectively. There was no cross-reactivity with porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (PoRV), porcine pseudorabies virus (PRV), porcine deltacoronavirus (PDCoV), or porcine circovirus (PCV). The reproducibility of DAS-qELISA was verified, and the coefficient of variation (CV) for intra- and inter-batch replicates was less than 10%, indicating good reproducibility. When testing anal swab samples from PEDV-infected piglets using DAS-qELISA, the coincidence rate was 92.55% with a kappa value of 0.85 when using reverse transcription-polymerase chain reaction (RT-PCR) and 94.29% with a kappa value of 0.88 when using PEDV antigen detection test strips, demonstrating the reliability of the method. These findings provide fundamental material support for both fundamental and practical studies on PEDV and offer a crucial diagnostic tool for clinical applications. KEY POINTS: • A new anti-PEDV N protein monoclonal antibody strain was prepared • Establishment of a more sensitive double antibody sandwich quantitative ELISA • DAS-qELISA was found to be useful for controlling the PEDV spread.

A Nanobody of PEDV S1 Protein: Screening and Expression in Escherichia coli.

Porcine epidemic diarrhea virus (PEDV) has caused significant economic losses to the pig farming industry in various countries for a long time. Currently, there are no highly effective preventive or control measures available. Research into the pathogenic mechanism of PEDV has shown that it primarily causes infection by binding the S protein to the CD13 (APN) receptor on the membrane of porcine intestinal epithelial cells. The S1 region contains three neutralization epitopes and multiple receptor-binding domains, which are closely related to viral antigenicity and ad-sorption invasion. Nanobodies are a type of single-domain antibody that have been discovered in recent years. They can be expressed on a large scale through prokaryotic expression systems, which makes them cost-effective, stable, and less immunogenic. This study used a phage display library of nanobodies against the PEDV S1 protein. After three rounds of selection and enrichment, the DNA sequence of the highly specific nanobody S1Nb1 was successfully obtained. To obtain soluble nanobody S1Nb1, its DNA sequence was inserted into the vector Pcold and a solubility-enhancing SUMO tag was added. The resulting recombinant vector, Pcold-SUMO-S1Nb1, was then transformed into E. coli BL21(DE3) to determine the optimal expression conditions for the nanobody. Following purification using Ni-column affinity chromatography, Western blot analysis confirmed the successful purification of S1Nb1 carrying the solubility-enhancing tag. ELISA results demonstrated a strong affinity between the S1Nb1 nanobody and PEDV S1 protein.

Intranasal delivery of a recombinant adenovirus vaccine encoding the PEDV COE elicits potent mucosal and systemic antibody responses in mice.

Porcine epidemic diarrhea virus (PEDV) is an enteropathogenic coronavirus that causes substantial economic loss to the global pig industry. The emergence of PEDV variants has increased the need for new vaccines, as commercial vaccines confer inferior protection against currently circulating strains. It is well established that the induction of mucosal immunity is crucial for PEDV vaccines to provide better protection against PEDV infection. In this study, we constructed a recombinant adenovirus expressing the core neutralization epitope (COE) of G2b PEDV based on human adenovirus serotype 5 (Ad5). We evaluated the effects of different administration routes and doses of vaccine immunogenicity in Balb/c mice. Both intramuscular (IM) and intranasal (IN) administration elicited significant humoral responses, including COE-specific IgG in serum and mucosal secretions, along with serum-neutralizing antibodies. Moreover, IN delivery was more potent than IM in stimulating IgA in serum and mucosal samples and in dampening the immune response to the Ad5 vector. The immune response was stronger after high versus low dose IM injection, whereas no significant difference was observed between high and low IN doses. In summary, our findings provide important insights for developing novel PEDV vaccines.IMPORTANCEPorcine epidemic diarrhea (PED) is a highly contagious disease that has severe economic implications for the pork industry. Developing an effective vaccine against PEDV remains a necessity. Here, we generated a recombinant adenovirus vaccine based on Ad5 to express the COE protein of PEDV (rAd5-PEDV-COE) and systematically evaluated the immunogenicity of the adenovirus-vectored vaccine using different administration routes (intramuscular and intranasal) and doses in a mouse model. Our results show that rAd5-PEDV-COE induced potent systemic humoral response regardless of the dose or immunization route. Notably, intranasal delivery was superior to induce peripheral and mucosal IgA antibodies compared with intramuscular injection. Our data provide valuable insights into designing novel PEDV vaccines.

Porcine epidemic diarrhea virus: A review of detection, inhibition of host gene expression and evasion of host innate immune.

As one of the most important swine enteropathogenic coronavirus, porcine epidemic diarrhea virus (PEDV) is the causative agent of an acute and devastating enteric disease that causes lethal watery diarrhea in suckling piglets. Recent progress in studying PEDV has revealed many intriguing findings on its prevalence and genetic evolution, rapid diagnosis, suppression of host gene expression, and suppression of the host innate immune system. Due to the continuous mutation of the PEDV genome, viral evasions from innate immune defenses and mixed infection with other coronaviruses, the spread of the virus is becoming wider and faster, making it even more necessary to prevent the infections caused by wild-type PEDV variants. It has also been reported that PEDV nsp1 is an essential virulence determinant and is critical for inhibiting host gene expression by structural and biochemical analyses. The inhibition of host protein synthesis employed by PEDV nsp1 may contribute to the regulation of host cell proliferation and immune evasion-related biological functions. In this review, we critically evaluate the recent studies on these aspects of PEDV and assess prospects in understanding the function of PEDV proteins in regulating host innate immune response and viral virulence.

Epitopes screening and vaccine molecular design of PEDV S protein based on immunoinformatics.

Porcine epidemic diarrhea virus (PEDV) is a serious disease that poses a significant threat to the pig industry. This study focused on analyzing the Spike protein of PEDV, which harbors crucial antigenic determinants, in identifying dominant epitopes. Immunoinformatics tools were used to screen for B-cell, CD4+ and CD8+ predominance epitopes. These epitopes were then connected to the N-terminal of ferritin to form a self-assembled nanoparticle vaccine. Various physical and chemical properties of the candidate vaccine were analyzed, including secondary structure prediction, tertiary structure modeling, molecular docking, immune response simulation and computer cloning. The results demonstrated that the candidate vaccine was antigenic, soluble, stable, non-allergic, and formed a stable complex with the target receptor TLR-3. Immune simulation analysis showed that the candidate vaccine effectively stimulated both cellular and humoral reactions, leading to increased related cytokines production. Furthermore, efficient and stable expression of the candidate vaccine was achieved through reverse translation in the Escherichia coli K12 expression system following codon optimization and in silico cloning. The developed nanoparticle candidate vaccine in this study holds promise as an effective PEDV vaccine candidate, offering a new approach for the research, development and improvement of vaccines targeting porcine enteric diarrhea coronavirus.

AhR ligands from LGG metabolites promote piglet intestinal ILC3 activation and IL-22 secretion to inhibit PEDV infection.

In maintaining organismal homeostasis, gut immunity plays a crucial role. The coordination between the microbiota and the immune system through bidirectional interactions regulates the impact of microorganisms on the host. Our research focused on understanding the relationships between substantial changes in jejunal intestinal flora and metabolites and intestinal immunity during porcine epidemic diarrhea virus (PEDV) infection in piglets. We discovered that Lactobacillus rhamnosus GG (LGG) could effectively prevent PEDV infection in piglets. Further investigation revealed that LGG metabolites interact with type 3 innate lymphoid cells (ILC3s) in the jejunum of piglets through the aryl hydrocarbon receptor (AhR). This interaction promotes the activation of ILC3s and the production of interleukin-22 (IL-22). Subsequently, IL-22 facilitates the proliferation of IPEC-J2 cells and activates the STAT3 signaling pathway, thereby preventing PEDV infection. Moreover, the AhR receptor influences various cell types within organoids, including intestinal stem cells (ISCs), Paneth cells, and enterocytes, to promote their growth and development, suggesting that AhR has a broad impact on intestinal health. In conclusion, our study demonstrated the ability of LGG to modulate intestinal immunity and effectively prevent PEDV infection in piglets. These findings highlight the potential application of LGG as a preventive measure against viral infections in livestock.IMPORTANCEWe observed high expression of the AhR receptor on pig and human ILC3s, although its expression was negligible in mouse ILC3s. ILC3s are closely related to the gut microbiota, particularly the secretion of IL-22 stimulated by microbial signals, which plays a crucial regulatory role in intestinal immunity. In our study, we found that metabolites produced by beneficial gut bacteria interact with ILC3s through AhR, thereby maintaining intestinal immune homeostasis in pigs. Moreover, LGG feeding can enhance the activation of ILC3s and promote IL-22 secretion in the intestines of piglets, ultimately preventing PEDV infection.

[Fusion expression and immunogenicity of receptor-binding domains of porcine deltacoronavirus and porcine epidemic diarrhea virus].

This study aims to develop an effective bivalent subunit vaccine that is promising to prevent both porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV). The receptor-binding domains (RBDs) of PDCoV and PEDV were fused and cloned into the eukaryotic expression vector pCDNA3.1(+). The fusion protein PDCoV-RBD-PEDV-RBD (pdRBD-peRBD) was expressed by the ExpiCHOTM expression system and purified. Mice were immunized with the fusion protein at three different doses (10, 20, and 30 µg). The humoral immune response and cellular immune response induced by the fusion protein were evaluated by ELISA and flow cytometry. The neutralization titers of the serum of immunized mice against PDCoV and PEDV were determined by the microneutralization test. The results showed that high levels of IgG antibodies were induced in the three different dose groups after booster immunization, and there was no significant difference in the antibody level between different dose groups, indicating that the immunization dose of 10 µg could achieve the fine immune effect. The results of flow cytometry showed that the immunization groups demonstrated increased proportion of CD3+CD4+ T cells and decreased proportion of CD3+CD8+ T cells, which was consistent with the expectation about the humoral immune response induced by the subunit vaccine. At the same time, the levels of interleukin (IL)-2, IL-4, and interferon (IFN)-γ in the serum were determined. The results showed that the fusion protein induced both humoral immune effect and cellular immune response. The results of the neutralization test showed that the antibody induced by 10 µg fusion protein neutralized both PDCoV and PEDV in vitro, with the titers of 1:179.25 and 1:141.21, respectively. The above results suggested that the pdRBD-peRBD could induce a high level of humoral immune response at a dose of 10 µg, and the induced antibody could neutralize both PDCoV and PEDV. Therefore, the fusion protein pdRBD-peRBD is expected to be an effective subunit vaccine that can simultaneously prevent PDCoV and PEDV.

Monovalent, bivalent and biparatopic nanobodies targeting S1 protein of porcine epidemic diarrhea virus efficiently neutralized the virus infectivity.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe diarrhea and death in neonatal piglets, which has brought huge economic losses to the pork industry worldwide since its first discovery in the early 1970s in Europe. Passive immunization with neutralizing antibodies against PEDV is an effective prevention measure. To date, there are no effective therapeutic drugs to treat the PEDV infection. RESULTS: We conducted a screening of specific nanobodies against the S1 protein from a phage display library obtained from immunized alpacas. Through competitive binding to antigenic epitopes, we selected instead of chose nanobodies with high affinity and constructed a multivalent tandem. These nanobodies were shown to inhibit PEDV infectivity by the neutralization assay. The antiviral capacity of nanobody was found to display a dose-dependent pattern, as demonstrated by IFA, TCID50, and qRT-PCR analyses. Notably, biparatopic nanobody SF-B exhibited superior antiviral activity. Nanobodies exhibited low cytotoxicity and high stability even under harsh temperature and pH conditions, demonstrating their potential practical applicability to animals. CONCLUSIONS: Nanobodies exhibit remarkable biological properties and antiviral effects, rendering them a promising candidate for the development of anti-PEDV drugs.

Surface display of the COE antigen of porcine epidemic diarrhoea virus on Bacillus subtilis spores.

Porcine epidemic diarrhoea virus (PEDV) infects pigs of all ages by invading small intestine, causing acute diarrhoea, vomiting, and dehydration with high morbidity and mortality among newborn piglets. However, current PEDV vaccines are not effective to protect the pigs from field epidemic strains because of poor mucosal immune response and strain variation. Therefore, it is indispensable to develop a novel oral vaccine based on epidemic strains. Bacillus subtilis spores are attractive delivery vehicles for oral vaccination on account of the safety, high stability, and low cost. In this study, a chimeric gene CotC-Linker-COE (CLE), comprising of the B. subtilis spore coat gene cotC fused to the core neutralizing epitope CO-26 K equivalent (COE) of the epidemic strain PEDV-AJ1102 spike protein gene, was constructed. Then recombinant B. subtilis displaying the CLE on the spore surface was developed by homologous recombination. Mice were immunized by oral route with B. subtilis 168-CLE, B. subtilis 168, or phosphate-buffered saline (PBS) as control. Results showed that the IgG antibodies and cytokine (IL-4, IFN-γ) levels in the B. subtilis 168-CLE group were significantly higher than the control groups. This study demonstrates that B. subtilis 168-CLE can generate specific systemic immune and mucosal immune responses and is a potential vaccine candidate against PEDV infection.

Immunoinformatics approach for designing a multiepitope subunit vaccine against porcine epidemic diarrhea virus genotype IIA spike protein.

Background: Porcine epidemic diarrhea (PED), caused by the porcine epidemic diarrhea virus (PEDV), is associated with high mortality and morbidity rates, especially in neonatal pigs. This has resulted in significant economic losses for the pig industry. PEDV genotype II-based vaccines were found to confer better immunity against both heterologous and homologous challenges; specifically, spike (S) proteins, which are known to play a significant role during infection, are ideal for vaccine development. Aim: This study aims to design a multi-epitope subunit vaccine targeting the S protein of the PEDV GIIa strain using an immunoinformatics approach. Methods: Various bioinformatics tools were used to predict HTL, CTL, and B-cell epitopes. The epitopes were connected using appropriate linkers and conjugated with the CTB adjuvant and M-ligand. The final multiepitope vaccine construct (fMEVc) was then docked to toll-like receptor 4 (TLR4). The stability of the fMEVc-TLR4 complex was then simulated using GROMACS. C-immsim was then used to predict the in vitro immune response of the fMEVc. Results: Six epitopes were predicted to induce antibody production, ten epitopes were predicted to induce CTL responses, and four epitopes were predicted to induce HTL responses. The assembled epitopes conjugated with the CTB adjuvant and M-ligand, fMEVc, is antigenic, non-allergenic, stable, and soluble. The construct showed a favorable binding affinity for TLR4, and the protein complex was shown to be stable through molecular dynamics simulations. A robust immune response was induced after immunization, as demonstrated through immune stimulation. Conclusion: In conclusion, the multi-epitope subunit vaccine construct for PEDV designed in this study exhibits promising antigenicity, stability, and immunogenicity, eliciting robust immune responses and suggesting its potential as a candidate for further vaccine development.

High-affinity monoclonal antibodies against the porcine epidemic diarrhea virus S1 protein.

The porcine epidemic diarrhea virus (PEDV) infection inflicted substantial economic losses upon the global pig-breeding industry. This pathogen can infect all pigs and poses a particularly high fatality risk for suckling piglets. The S1 subunit of spike protein is a crucial target protein for inducing the particularly neutralizing antibodies that can intercept the virus-host interaction and neutralize virus infectivity. In the present study, the HEK293F eukaryotic expression system was successfully utilized to express and produce recombinant S1 protein. Through quantitative analysis, five monoclonal antibodies (mAbs) specifically targeting the recombinant S1 protein of PEDV were developed and subsequently evaluated using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and flow cytometry assay (FCA). The results indicate that all five mAbs belong to the IgG1 isotype, and their half-maximal effective concentration (EC50) values measured at 84.77, 7.42, 0.89, 14.64, and 7.86 pM. All these five mAbs can be utilized in ELISA, FCA, and IFA for the detection of PEDV infection. MAb 5-F9 exhibits the highest sensitivity to detect as low as 0.3125 ng/mL of recombinant PEDV-S1 protein in ELISA, while only 0.096 ng/mL of mAb 5-F9 is required to detect PEDV in FCA. The results from antigen epitope analysis indicated that mAb 8-G2 is the sole antibody capable of recognizing linear epitopes. In conclusion, this study has yielded a highly immunogenic S1 protein and five high-affinity mAbs specifically targeting the S1 protein. These findings have significant implications for early detection of PEDV infection and provide a solid foundation for further investigation into studying virus-host interactions.

Calmodulin-like 5 promotes PEDV replication by regulating late-endosome synthesis and innate immune response.

The infection caused by porcine epidemic diarrhea virus (PEDV) is associated with high mortality in piglets worldwide. Host factors involved in the efficient replication of PEDV, however, remain largely unknown. Our recent proteomic study in the virus-host interaction network revealed a significant increase in the accumulation of CALML5 (EF-hand protein calmodulin-like 5) following PEDV infection. A further study unveiled a biphasic increase of CALML5 in 2 and 12 â€‹h after viral infection. Similar trends were observed in the intestines of piglets in the early and late stages of the PEDV challenge. Moreover, CALML5 depletion reduced PEDV mRNA and protein levels, leading to a one-order-of-magnitude decrease in virus titer. At the early stage of PEDV infection, CALML5 affected the endosomal trafficking pathway by regulating the expression of endosomal sorting complex related cellular proteins. CALML5 depletion also suppressed IFN-ß and IL-6 production in the PEDV-infected cells, thereby indicating its involvement in negatively regulating the innate immune response. Our study reveals the biological function of CALML5 in the virology field and offers new insights into the PEDV-host cell interaction.

Maternal immunization and vitamin A sufficiency impact sow primary adaptive immunity and passive protection to nursing piglets against porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea virus (PEDV) causes a highly contagious enteric disease with major economic losses to swine production worldwide. Due to the immaturity of the neonatal piglet immune system and given the high virulence of PEDV, improving passive lactogenic immunity is the best approach to protect suckling piglets against the lethal infection. We tested whether oral vitamin A (VA) supplementation and PEDV exposure of gestating and lactating VA-deficient (VAD) sows would enhance their primary immune responses and boost passive lactogenic protection against the PEDV challenge of their piglets. We demonstrated that PEDV inoculation of pregnant VAD sows in the third trimester provided higher levels of lactogenic protection of piglets as demonstrated by >87% survival rates of their litters compared with <10% in mock litters and that VA supplementation to VAD sows further improved the piglets' survival rates to >98%. We observed significantly elevated PEDV IgA and IgG antibody (Ab) titers and Ab-secreting cells (ASCs) in VA-sufficient (VAS)+PEDV and VAD+VA+PEDV sows, with the latter maintaining higher Ab titers in blood prior to parturition and in blood and milk throughout lactation. The litters of VAD+VA+PEDV sows also had the highest serum PEDV-neutralizing Ab titers at piglet post-challenge days (PCD) 0 and 7, coinciding with higher PEDV IgA ASCs and Ab titers in the blood and milk of their sows, suggesting an immunomodulatory role of VA in sows. Thus, sows that delivered sufficient lactogenic immunity to their piglets provided the highest passive protection against the PEDV challenge. Maternal immunization during pregnancy (± VA) and VA sufficiency enhanced the sow primary immune responses, expression of gut-mammary gland trafficking molecules, and passive protection of their offspring. Our findings are relevant to understanding the role of VA in the Ab responses to oral attenuated vaccines that are critical for successful maternal vaccination programs against enteric infections in infants and young animals.

Display of porcine epidemic diarrhea virus spike protein B-cell linear epitope on Lactobacillus mucosae G01 S-layer surface induce a robust immunogenicity in mice.

The Porcine epidemic diarrhea virus (PEDV) presents a substantial risk to the domestic pig industry, resulting in extensive and fatal viral diarrhea among piglets. Recognizing the mucosal stimulation triggered by PEDV and harnessing the regulatory impact of lactobacilli on intestinal function, we have developed a lactobacillus-based vaccine that is carefully designed to elicit a strong mucosal immune response. Through bioinformatics analysis, we examined PEDV S proteins to identify B-cell linear epitopes that meet the criteria of being non-toxic, soluble, antigenic, and capable of neutralizing the virus. In this study, a genetically modified strain of Lactobacillus mucosae G01 (L.mucosae G01) was created by utilizing the S layer protein (SLP) as a scaffold for surface presentation. Chimeric immunodominant epitopes with neutralizing activity were incorporated at various sites on SLP. The successful expression of SLP chimeric immunodominant epitope 1 on the surface of L.mucosae G01 was confirmed through indirect immunofluorescence and transmission electron microscopy, revealing the formation of a transparent membrane. The findings demonstrate that the oral administration of L.mucosae G01, which expresses the SLP chimeric immunodominant gene epitope1, induces the production of secreted IgA in the intestine and feces of mice. Additionally, there is an elevation in IgG levels in the serum. Moreover, the levels of cytokines IL-2, IL-4, IFN-γ, and IL-17 are significantly increased compared to the negative control group. These results suggest that L. mucosae G01 has the ability to deliver exogenous antigens and elicit a specific mucosal immune response against PEDV. This investigation presents new possibilities for immunoprophylaxis against PEDV-induced diarrhea.

Combination of S1-N-Terminal and S1-C-Terminal Domain Antigens Targeting Double Receptor-Binding Domains Bolsters Protective Immunity of a Nanoparticle Vaccine against Porcine Epidemic Diarrhea Virus.

Variants of coronavirus porcine epidemic diarrhea virus (PEDV) frequently emerge, causing an incomplete match between the vaccine and variant strains, which affects vaccine efficacy. Designing vaccines with rapidly replaceable antigens and high efficacy is a promising strategy for the prevention of infection with PEDV variant strains. In our study, three different types of self-assembled nanoparticles (nps) targeting receptor-binding N-terminal domain (NTD) and C-terminal domain (CTD) of S1 protein, named NTDnps, CTDnps, and NTD/CTDnps, were constructed and evaluated as vaccine candidates against PEDV. NTDnps and CTDnps vaccines mediated significantly higher neutralizing antibody (NAb) titers than NTD and CTD recombinant proteins in mice. The NTD/CTDnps in varying ratios elicited significantly higher NAb titers when compared with NTDnps and CTDnps alone. The NTD/CTDnps (3:1) elicited NAb with titers up to 92.92% of those induced by the commercial vaccine. Piglets immunized with NTD/CTDnps (3:1) achieved a passive immune protection rate of 83.33% of that induced by the commercial vaccine. NTD/CTDnps (3:1) enhanced the capacity of mononuclear macrophages and dendritic cells to take up and present antigens by activating major histocompatibility complex I and II molecules to stimulate humoral and cellular immunity. These data reveal that a combination of S1-NTD and S1-CTD antigens targeting double receptor-binding domains strengthens the protective immunity of nanoparticle vaccines against PEDV. Our findings will provide a promising vaccine candidate against PEDV.

Construction and immune effect evaluation of the S protein heptad repeat-based nanoparticle vaccine against porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV), a highly virulent enteropathogenic coronavirus, is a significant threat to the pig industry. High frequency mutations in the PEDV genome have limited the effectiveness of current vaccines in providing immune protection. Developing efficient vaccines that can quickly adapt to mutant strains is a challenging but crucial task. In this study, we chose the pivotal protein heptad repeat (HR) responsible for coronavirus entry into host cells, as the vaccine antigen. HR-Fer nanoparticles prepared using ferritin were evaluated them as PEDV vaccine candidates. Nanoparticle vaccines elicited stronger neutralizing antibody responses in mice compared to monomer vaccines. Additionally, HR protein delivered via nanoparticles increased antigen uptake by antigen-presenting cells in vitro by 2.75-fold. The collective results suggest that HR can be used as antigens for vaccines, and the HR vaccine based on ferritin nanoparticles significantly enhances immunogenicity.

Oral administration of PEDV-dissolved Alg-CS gel induces high and sustained mucosal immunity in mice.

Porcine epidemic diarrhea (PED) is a serious disease in piglets that leads to high mortality. An effective measure that provides higher IgA levels in the intestine and milk is required to decrease losses. Porcine epidemic diarrhea virus (PEDV) was dissolved in calcium alginate (Alg) and combined with chitosan (CS) via electrostatic interactions between cationic chitosan and anionic alginate to create a porous gel (Alg-CS+PEDV). The gel was used to immunize mice orally or in combination with subcutaneous injections of inactivated PEDV vaccine. At 12 and 24 days after immunization, levels of IgA and IgG in Alg-CS+PEDV were higher than with normal PEDV oral administration. At 24 days after immunization, the concentration of IFN-γ in Alg-CS+PEDV was higher than with normal PEDV oral administration. Furthermore, oral administration combining subcutaneous immunization induced higher levels of IgG and IgA than oral administration alone. Our study provides a new method for the preparation and administration of oral vaccines to achieve enhanced mucosal immunity against PEDV.

Enteric coronavirus nsp2 is a virulence determinant that recruits NBR1 for autophagic targeting of TBK1 to diminish the innate immune response.

Non-structural protein 2 (nsp2) exists in all coronaviruses (CoVs), while its primary function in viral pathogenicity, is largely unclear. One such enteric CoV, porcine epidemic diarrhea virus (PEDV), causes high mortality in neonatal piglets worldwide. To determine the biological role of nsp2, we generated a PEDV mutant containing a complete nsp2 deletion (rPEDV-Δnsp2) from a highly pathogenic strain by reverse genetics, showing that nsp2 was dispensable for PEDV infection, while its deficiency reduced viral replication in vitro. Intriguingly, rPEDV-Δnsp2 was entirely avirulent in vivo, with significantly increased productions of IFNB (interferon beta) and IFN-stimulated genes (ISGs) in various intestinal tissues of challenged newborn piglets. Notably, nsp2 targets and degrades TBK1 (TANK binding kinase 1), the critical kinase in the innate immune response. Mechanistically, nsp2 induced the macroautophagy/autophagy process and recruited a selective autophagic receptor, NBR1 (NBR1 autophagy cargo receptor). NBR1 subsequently facilitated the K48-linked ubiquitination of TBK1 and delivered it for autophagosome-mediated degradation. Accordingly, the replication of rPEDV-Δnsp2 CoV was restrained by reduced autophagy and excess productions of type I IFNs and ISGs. Our data collectively define enteric CoV nsp2 as a novel virulence determinant, propose a crucial role of nsp2 in diminishing innate antiviral immunity by targeting TBK1 for NBR1-mediated selective autophagy, and pave the way to develop a new type of nsp2-based attenuated PEDV vaccine. The study also provides new insights into the prevention and treatment of other pathogenic CoVs.Abbreviations: 3-MA: 3-methyladenine; Baf A1: bafilomycin A1; CoV: coronavirus; CQ: chloroquine; dpi: days post-inoculation; DMVs: double-membrane vesicles; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; GIGYF2: GRB10 interacting GYF protein 2; hpi: hours post-infection; IFA: immunofluorescence assay; IFIH1: interferon induced with helicase C domain 1; IFIT2: interferon induced protein with tetratricopeptide repeats 2; IFITM1: interferon induced transmembrane protein 1; IFNB: interferon beta; IRF3: interferon regulatory factor 3; ISGs: interferon-stimulated genes; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; NBR1: NBR1 autophagy cargo receptor; nsp2: non-structural protein 2; OAS1: 2'-5'-oligoadenylate synthetase 1; PEDV: porcine epidemic diarrhea virus; PRRs: pattern recognition receptors; RIGI: RNA sensor RIG-I; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TCID50: 50% tissue culture infectious doses; VSV: vesicular stomatitis virus.

Effects of porcine epidemic diarrhea virus infection on CD21+ B cells activation.

Porcine epidemic diarrhea virus (PEDV) is a devastating pathogen of acute- gastrointestinal infectious diseases, which can cause vomiting, diarrhea, dehydration and high morbidity and mortality among neonatal piglets. Humoral immunity plays a vital role in the host anti-PEDV infection process, but the mechanism of PEDV-induced B-cell immune response remains unknown. In this study, the effects of PEDV infection on CD21+ B cell activation were systematically analyzed through animal experiments. Enzyme-linked immunosorbent assays (ELISA) revealed that low levels of serum-specific IgA, IgM, or IgG were detected in piglets after PEDV infection, respectively. Serum interleukin (IL)-6 levels increased significantly at 4 d after infection, and the levels of IL-4, B-cell activating factor (BAFF), interferon (IFN)-γ, transforming growth factor (TGF)-ß and IL-10 decreased at 7 d after infection. Fluorescence-activated cell sorting (FACS) showed that expression levels of CD21, MHC Ⅱ, CD40, and CD38 on B cell surfaces were significantly higher. In contrast, the proportions of CD21+IgM+ B cells were decreased in peripheral blood mononuclear cells (PBMCs) from the infected piglets. No differences were found in the percentage of CD21+CD80+ and CD21+CD27+ B cells in PBMCs from the infected piglets. In addition, the number of CD21+B cells in PBMCs stimulated with PEDV in vitro was significantly lower. No significant change in the mRNA expression of BCR molecules was found while the expression levels of paired immunoglobulin-like receptor B (PIR-B), B cell adaptor molecule of 32 kDa (Bam32) and BAFF were decreased. In conclusion, our research demonstrates that virulent strains of PEDV profoundly impact B cell activation, leading to alterations in phenotypic expression and BCR signaling molecules. Furthermore, this dysregulation results in compromised specific antibody secretion and perturbed cytokine production, highlighting the intricate immunological dysfunctions induced by PEDV infection.

TLR9 mediates IgA production in the porcine small intestine during PEDV infection.

IgA plays a vital role in defending against the infectious pathogens. However, the specific regulatory pathways involved in IgA secretion in the context of PEDV infection have remained elusive. Therefore, in this study, we explore the molecular mechanisms underlying IgA secretion in response to infection, with a particular focus on PEDV, a devastating enteric virus affecting global swine production. Our investigation begins by examining changes in IgA concentrations in both serum and small intestinal contents following PEDV infection in 2- and 4-week-old pigs. Remarkably, a significant increase in IgA levels in these older pigs post-infection were observed. To delve deeper into the regulatory mechanisms governing IgA secretion in response to PEDV infection, isolated porcine intestinal B cells were co-cultured with monocytes derived DCs (Mo-DCs) in vitro. In the intestinal DC-B cell co-cultures, IgA secretion was found to increase significantly after PEDV infection, as well as upregulating the expression of AID, GLTα and PSTα reflecting isotype switching to IgA. In addition, the expression of TLR9 was upregulated in these cultures, as determined by RT-qPCR and western blotting. Moreover, our findings extend to in vivo observations, where we detected higher levels of TLR9 expression in the ileum of pig post PEDV infection. Collectively, our results highlight the ability of PEDV to stimulate the generation of IgA, particularly in elder pigs, and identify TLR9 as a critical mediator of IgA production within the porcine intestinal microenvironment during PEDV infection.