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Abstract
PEDV In Newborn Piglets
Jialu Wang, Lulu Huang, Chunxiao Mou, En Zhang, Yongheng Wang, Yanan Cao And Qian Yang*
Introduction
Reagents
Animals & Vaccination Programs
Immunohistochemistry
Cytokine Detection
Statistical Analyses
Results
Discussion
Competing Interests
AUTHOR CONTRIBUTIONS
FUNDING
FIGURE LEGEND
Abstract
Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. Porcine epidemic diarrhea virus (PEDV) spreads by fecal-oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carriers has been used for antigen delivery and proved well immune effect and safety. The present study evaluated the immunogenicity of the recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies to against PEDV infection, through enlarging the area of Peyer's patches (PPs) and increasing the number of ileum IgA+ secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). Besides, we observed that oral administration of B. subtilis-RC significantly increased CD3+T lymphocytes numbers and up-regulating the ratio of CD4+/CD8+T cells. Furthermore, high titer of specific serum immunoglobulin G (IgG) revealed that satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger high level of local and systemic immune responses and would be a promising candidate vaccine to against PEDV infection in piglets.
Mucosal Immune Responses Induced by Oral Administration Recombinant Bacillus subtilis expressing the COE antigen of
PEDV in newborn piglets
Jialu Wang, Lulu Huang, Chunxiao Mou, En Zhang, Yongheng Wang, Yanan Cao and Qian Yang*
MOE Joint International Research Laboratory of Animal Health and Food Safety, College of veterinary medicine, Nanjing Agricultural University. Weigang 1 Nanjing, Jiangsu, 210095, PR China. Correspondence and requests for materials should address to Q.Yang. (Email: zxbyq@njau.edu.cn) Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. Porcine epidemic diarrhea virus (PEDV) spreads by fecal-oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carriers has been used for antigen delivery and proved well immune effect and safety. The present study evaluated the immunogenicity of the recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies to against PEDV infection, through enlarging the area of Peyer's patches (PPs) and increasing the number of ileum IgA+ secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). Besides, we observed that oral administration of B. subtilis-RC significantly increased CD3+T lymphocytes numbers and up-regulating the ratio of CD4+/CD8+T cells. Furthermore, high titer of specific serum immunoglobulin G (IgG) revealed that satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger high level of local and systemic immune responses and would be a promising candidate vaccine to against PEDV infection in piglets. Keywords: Porcine epidemic diarrhea virus; recombinant Bacillus subtilis; piglets; mucosal immunity; vaccine
Introduction
Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, resulting in enormous losses in the worldwide pig industry [1]. The causative agent porcine epidemic diarrhea virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through digestive tract [2], and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3]. Thus A C C E P T E D M A N U S C R IP T 10.1042/BSR20182028 . Please cite using the DOI 10.1042/BSR20182028http://dx.doi.org/up-to-date version is available at encouraged to use the Version of Record that, when published, will replace this version. The most this is an Accepted Manuscript, not the final Version of Record. You are:Bioscience Reports ). http://www.portlandpresspublishing.com/content/open-access-policy#ArchivingPolicy of Portland Press ( which the article is published. Archiving of non-open access articles is permitted in accordance with the Archiving Use of open access articles is permitted based on the terms of the specific Creative Commons Licence under enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4]. Currently, traditional vaccines (intramuscular route or subcutaneous injection) have developed and applied widely in the market [5]. These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal to against PEDV infection [6]. Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7]. As a superior way of mucosal immunization, the oral administration can protect the gut and stimulate the common mucosal immune system [8]. Besides, oral immunization has several attractive features which include safety, straightforward inexpensive and needle-free approach [9]. Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10]. Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the gastrointestinal tract) [11]. It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12]. Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from severe environment in the gastrointestinal tract and deliver antigens to intestinal mucosa [13]. Currently, Bacillus subtilis (B. subtilis) is widely used as vaccine delivery system for its unique characteristics. As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotics and food additive in humans and animals [14]. The B. subtilis has adjuvant activity and can germinate to delivery of heterologous antigens to the gastrointestinal tract, providing the additional immunity stimulation [15]. Besides, research had shown that oral administration B. subtilis also could enhance immune regulation and gut health in pigs [16]. Moreover, oral administration B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17]. DCs are the most important professional antigen-presenting cells and can effectively regulate the antibody titer [18]. DCs naturally exist in the gutassociated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs) and scattered throughout the subepithelial lamina propria (LP)of in the small intestine and colon [19]. Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20]. Therefore, B. subtilis has widely used as the effective vaccine delivery system to induce mucosal immune responses and shows unique effect on immune system. In the present report, we explored the immune effect of a recombinant Bacillus subtilis (B. subtilis-RC) which had successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies to against PEDV infection, suggesting a potential approach for preventing PEDV infection. Materials and Methods Virus, bacterium & cell lines The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao [21]. Recombinant Bacillus subtilis strain (B. subtilis RC) previously constructed in our laboratory was able to express the gene COE (499–638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 °C for 12 h, and then washing bacterial twice with PBS, and suspending in Phosphate-buffered saline (PBS) to a final concentration of 1×1010 CFU/ml. The PEDV Zhejiang08 strain virus was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22]. The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm2 for 24 h to achieve a complete loss of infectivity [23]. The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, USA).
Reagents
FACS: 647 Mouse anti-Pig CD3ε (BB23-8E6-8C8), FITC Mouse anti-Pig CD4a (74-12-4), PE Mouse Anti-Pig CD8a (76-2-11) were purchased from BD. IHC: Rabbit anti-pig CD3 (SP7) mAbs were purchased from Abcam, Hong Kong. FITC Rabbit antipig CD4a were purchased from Santa. Mouse anti-Pig CD8 alpha Antibody (76-2-11) were purchased from Novus. ELISA: Rabbit anti-pig IgG (HRP), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABCbased system (biotinylated goat anti rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from BOSTER, Wuhan, China.
Animals & vaccination programs
Specific-pathogen-free (SPF) DLY piglets (Duroc & Landrace & Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health’s guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets each group), and housed under similar conditions in different stables in order to avoid probiotic cross contamination. The piglets orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets 5 day-old (Figure 1c), and signed as 0 day. Then booster immunizations were administered on 5 days. Specimen collection was then performed every seven days post boost immunization (Figure 1c). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at -20 °C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for one month, including nasal, oral and feces swabs for the ELISA assay. The piglets sacrificed in 33 day. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn’s liquid and 4% paraformaldehyde. Hematoxylin-eosin staining assay and intraepithelial lymphocytes (IELs) number The small intestine and ileum tissues in same location were fixed with Bouin Fixative Solution for 24 hours, embedded in paraffin and sectioned at 4 μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of IELs were counted in every one hundred epithelial cells under the same multiple light microscope from ten pictures each group [24].
Immunohistochemistry
The immunohistochemistry detection was performed with the SABC kit (BOSTER Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10mM citric acid, 2mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4°C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. Adding biotin labeled secondary antibody to the slides followed by adding horseradish peroxidase (HRP) labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25]. Isolation of intestinal lymphocytes and detect by Flow Cytometry The isolated intestines with Peyer’s patches were transferred into ice-cold PBS. Then, removing remaining fat and connective tissue and washing thoroughly by ice-cold PBS. Next, cutting the intestine longitudinally into 0.5 cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% fetal calf serum, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's Phosphate Buffered Saline (DPBS) for 20 min at 37 by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the intraepithelial lymphocytes (IELs) were separated by intensive vortex and passed through a 70μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40μm cell strainer. Supernatants was washed by precooled RPMI Medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 percoll gradient, overlaid on 5 ml of the 80% fraction in a 15 ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. Lamina propria lymphocytes (LPLs) were collected at the interphase of the percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3 -APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and wash with PBS twice, and analyzed by fluorescence-activated cell sorting (FACS) [26].
Cytokine Detection
Cytokines IL-10 and IL-1β (Abcam) were measured by ELISA according to the manufacturer’s instructions. Data acquired on an automated ELISA plate reader at 450 nm immediately. Plaque reduction neutralization test (PRNT) PEDV neutralizing antibodies were measured in intestine washing liquid by PRNT. The test was performed as previously described with minor modifications [27]. 450 μl of intestine washing liquid were two-fold serially diluted and mixed with 50 μl viral suspension containing 103 TCID50 PEDV virus for 1 h at 37 °C in 12-well flatbottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 °C and 5 % CO2. Then, the mixture was inoculated with Vero cells suspension (ca.1.0 × 106 ml-1) for another 3–4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect.
Statistical analyses
Datas were obtained as the means ± standard errors of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, USA) used to perform statistical analyses. Tukey’s multiple comparison tests and one-way analysis of variance (ANOVA) used to analyze the significance of the difference between means. P-Values less than 0.05 (P < 0.05) were considered significant and p-values less than 0.01 (P < 0.01) as highly significant.
Results
1 Oral administration of B. subtilis-RC significantly promoted intestinal development in piglets Peyer's patches (PPs) are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28]. The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P < 0.01) increased the area of PPs compared with two control groups as shown in Figure 1a. In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P < 0.01) than the other two groups (Figure 1b). These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure intestine. Intestinal intraepithelial lymphocytes (IELs) are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29]. IELs are important part of the gut mucosal immune system. No matter B. subtilis-RC or inactivated PEDV significantly (P < 0.01) increased the number of ileum IELs in per one hundred IECs (Figures 2). These results further proved the strength of immune enhancement by oral administration B. subtilisRC. 2. Oral immunization with B. subtilis-RC changed the local and systemic immune responses The level of specific anti-PEDV SIgA antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3a, b, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P < 0.05 or P < 0.01). As expected, the mouth had the highest levels of SIgA than other site. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3c. The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 days to 33 days than inactivated PEDV group (P < 0.05 or P < 0.01). 3. Oral immunization with B. subtilis-RC increased ileum IgA+ secreting (SIgA) cells and CD3+ T lymphocytes CD3+ T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3+ T lymphocytes could represent the quantity of T lymphocytes. Consequently, we made an analysis on the number of CD3+ T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P < 0.05) increase CD3+ T lymphocytes compared with PBS group (Figure 4a). These changes showed confident evidence that oral administration with B. subtilis-RC had a well influence on the intestinal mucosal immunity in piglets. SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30]. SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen than other two groups (P < 0.05) (Figure 4b). These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection. 4. Oral immunization with B. subtilis-RC stimulated differentiation of lymphocytes in the small intestinal mucosa. A great deal of immune cells are scattered in the epithelial cells. Intestinal epithelial cells indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31]. Consequently, learning about how the lymphocytes distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data’s had shown that CD3+ T lymphocytes significantly (P < 0.05) increased (Figure 4a), so we further analyzed the immunological classification of CD3+ T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4 and CD8 were analyzed by three colors flow cytometry. These results showed that CD3+CD4+ T cells have obviously (P < 0.01) increased (Figure 5b), nevertheless the CD3+CD8+T cells remarkably (P < 0.05) declined (Figure 5c). After being calculated, the ratio of CD4+ /CD8+T cells increased (Figure 5d). This ratio could also further measure the immunity levels of piglets. 5. Oral immunization with B. subtilis-RC regulated cytokine responses Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6a,b. As we can see from the diagram, significant (P < 0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than other two groups. These all revealed that B. subtilis-RC could stimulate releasing cytokines to mediate communication with and between cells of the immune system, improving the mucosal immune response to against PEDV infection. 6. Oral immunization with B. subtilis-RC increased the titer of PEDV neutralizing antibodies The PEDV neutralizing antibodies was detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P < 0.01) compared with other two groups in Figure 7. This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies to against PEDV infection.
Discussion
While the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfied. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32]. Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreaks [33]. It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34]. SIgA is the basic of mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During the PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35]. Our data showed a strong and long-lasting anti-PEDV IgG response that was detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titer declined a little, it still stayed on overhead than control groups and accorded with the antibody changeable tendency. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36]. The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37]. We further explored the species and amounts distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38]. The result showed that the number of CD3+ T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T cell maturation. According to the molecules express on the cell surface, T lymphocytes can further divide into T helper cells (CD4+ T cells) and cytotoxic T cells (CD8+ T cells) [39]. Furthermore, we observed the ratio of CD4+/CD8+ T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4+/CD8+ T cells. Small intestine morphology can directly reflect the intestinal health and play an important role in maintaining the intestine immune system [40]. In the early stage of PEDV infection, frequently accompanies that of necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41]. Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of Peyer's patches (PPs) expanding significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogenic in the intestines. Furtherly, an increase in the number of IELs also demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road to against PEDV infection. The several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and finally form the comprehensive protection. As previously mentioned, oral administration with B. subtilis-RC could stimulate T cells proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42]. As a cell-signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43]. Therefore, cytokine is a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed IL-1β and IL-10 increasing (P < 0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokine is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up regulate the local and systemic immune tissues post microbial infection [44]. In addition, Interleukin 10 (IL-10) is a potent antiinflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45]. In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL1β and IL-10. In conclusion, The present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, in the meantime, enhancing and regulating the immune function by raising the ratio of CD4+ /CD8+T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets.
Competing interests
The authors had no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance utilized in the production of this manuscript.
AUTHOR CONTRIBUTIONS
Jialu Wang performed the experiments and wrote the first draft of the manuscript. Lulu Huang and En Zhang help the animal experiment. Chunxiao Mou constructed the recombinant Bacillus subtilis. Yongheng Wang and Yanan Cao helped the immunological assays. Qian Yang designed and coordinated the study, supervised all experimental steps, and revised and finalized the manuscript.
FUNDING
This work supported by 31772777 from the National Science Grant of China and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
FIGURE LEGEND
Figure 1. Oral administration B. subtilis-RC significantly promoted piglets intestinal development and Schematic diagram of the immunization. Panel (a, b): the area of Peyer's patches (PPs) and the length of ileum villus were counted from eight discontinuous HE staining slices which were selected from each group. Panel (c): the schematic of the immunization, the red arrows indicated the time points of primary immunization and booster immunization, the blue triangle (under the line) indicated the time point of sampling three swabs (including nasal, mouth and feces swabs) and serum, the red triangle (under the line) represent sacrificing the piglets and sampling the three swabs, serum and intestinal. Data were shown as the mean ± SD. Two individual experiments were performed, four piglets were used in each group for each individual protocol and each animal was analyzed individually. P-value: * P < 0.05, ** P < 0.01. Figure 2. Oral administration B. subtilis-RC increased the number of intraepithelial lymphocytes (IELs). Each group choose eight discontinuous HE staining slice to count and average the numbers of IELs among one hundred epithelial cells in ileum and jejunum. Data were shown as the mean ± SD. P-value: * P < 0.05, ** P < 0.01. Figure 3. B. subtilis-RC changed the local and systemic immune responses after oral immunization. Panel (a, b): Determination of anti-porcine epidemic diarrhea virus (PEDV) specific mucosal SIgA antibody in mouth (a) and feces (b) by ELISA using PEDV as the coating antigen. Panel (c): Measurement of anti-PEDV IgG antibody in serum from immunized piglets by ELISA using PEDV as the coating antigen. Data were shown as the mean ± SD. P-value: # P < 0.05, ## P < 0.01 compared to inactivated PEDV; * P < 0.05, ** P < 0.01 compared to PBS. Figure 4. Oral administration B. subtilis-RC increased ileum IgA+ secreting (SIgA) cells and CD3+ T lymphocytes. Immunohistochemistry was used to detect ileum IgA+ secreting (SIgA) cells and CD3+ T lymphocytes. The integrated optic density (IOD) of eight discontinuous slices were counted each group. Data were shown as the mean ± SD. P-value: * P < 0.05, ** P < 0.01. Figure 5. B. subtilis-RC stimulated differentiation of lymphocytes in the small intestinal mucosa via oral administration. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4 and CD8 analyzed by three colors flow cytometry. Data were shown as the mean ± SD. P-value: * P < 0.05, ** P < 0.01. Figure 6. Oral administration B. subtilis-RC regulated cytokine responses. Panel (a, b): Cytokines IL-10 (a) and IL-1β (b) were measured by ELISA. Data were shown as the mean ± SD. P-value: * P < 0.05, ** P < 0.01. Figure 7. B. subtilis-RC induced the production of the PEDV neutralizing antibodies. PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). Data were shown as the mean ± SD. P-value: * P < 0.05, ** P < 0.01.
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