L. plantarum surface-displayed VP6 (PoRV) protein can prevent PoRV infection in piglets

doi:10.21203/rs.3.rs-3717982/v1

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L. plantarum surface-displayed VP6 (PoRV) protein can prevent PoRV infection in piglets

https://doi.org/10.21203/rs.3.rs-3717982/v1

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Porcine rotavirus (PoRV) poses a threat to the development of animal husbandry and human health, leading to substantial economic losses. Among its components, VP6 protein constitutes a major portion of the viral particles and serves as the core structural protein of the virus. In this study, we first developed an antibiotics free and environmentally friendly vector called asd-araC-PBAD-alr (AAPA). Subsequently, we constructed recombinant Lactobacillus plantarum (L. plantarum) strains induced by arabinose to express VP6 and VP6-pFc fusion proteins. Our findings revealed that NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S strains were capable of enhancing body immunity and protecting suckling mice and piglets from rotavirus infection. The novel recombinant L. plantarum strains developed in this study hold promising potential as oral vaccines to enhance body immunity and present a new strategy for preventing PoRV infection.

porcine rotavirus

VP6 protein

recombinant

Lactobacillus plantarum

immunization

(Rotavirus) RV is one of the enteric pathogens that cause diarrhea in neonatal or young animals, whereas adult animals tend to be cryptically infected(Desselberger, 2014). Porcine Rotavirus (PoRV) is highly contagious and causes anorexia, diarrhea, and vomiting in piglets(Campanha et al., 2020). PoRV is generated through a three-layer protein capsid, with the middle layer of the capsid consisting mainly of the VP6 protein(Suzuki et al., 2014).The VP6 protein is the core structural protein of the viral particles, and is therefore often used as a target protein for RV detection(Shoja et al., 2022). Previous findings have shown that the VP6 protein is the basis of PoRV transcriptase and replicase, and is able to initiate the transcriptionally active protein trimerization structure, which is important for viral replication and protein translation(Afchangi et al., 2022). In addition, VP6 is a potent antigen that stimulates the body to produce specific immune responses..

Lactobacillus (LAB) is a probiotic that exerts great advantages in the prevention of diarrhea and immune enhancement(Reuben et al., 2019, De Filippis et al., 2020). Lactobacillus plantarum (L. plantarum) adheres to intestinal epithelial cells and colonizes the intestines(Kato et al., 1999), among which L. plantarum NC8 has a high exogenous protein expression capacity, which has a prospect for use as an oral vaccine vector(Yang et al., 2017a),(Yang et al., 2017b). It has been reported that recombinant L. plantarum-expressed rotavirus VP8 protein(Shonyela et al., 2021), DC targeting peptide protein (DCpep) (Wang et al., 2020), and porcine epidemic diarrhea virus S protein can effectively enhance the body's immunity(Huang et al., 2018). S layer protein in pCXa-S vector enables stable secretory expression of target proteins and is often applied to high expression vector (Klotz and Barrangou, 2018). Therefore, the use of NC8-pCXa-S recombinantly expressing VP6 protein to develop a new oral probiotic mucosal vaccine targeting PoRV may be a new idea for preventing PoRV infection.

Fragment crytallizable (Fc) refers to a region of the structure of immunoglobulin(lg) that lies at the upper end of the antibody molecule and is also known as the "normal region" of the antibody. Fc is mainly composed of heavy chains, which contains some of the sequences that determine the differences between the antibody classes and subclasses of lg, and It also has activities related to the immune-mediated cell biological effects. For the immune system, Fc fragments play a crucial role(Nimmerjahn and Ravetch, 2007). When an antibody binds to an antigen, the Fc fragment is recognized and bound by its own immune cell receptor (Fc receptor), which triggers a complex series of biological responses(Paudyal et al., 2022). We used the FC fragment gene (pFc) of porcine-derived lgG as an adjuvant for fusion expression with the VP6 gene to enhance immunoprotection of the organism.

The activation of B cells and T cells plays a very important role in the enhancement of body immunity(Luo and Yin, 2021). MHCII can activate B cells to recognize exogenous antigens and express a variety of cytokine receptors(Huszthy et al., 2019). After activation, CD4-T cells can differentiate into Th1, Th2, Th17, follicular helper T cells (Tfh), or regulatory T cells (Treg) to coordinate various adaptive immune responses(Butcher and Zhu, 2021). Among them, IL-2 and IFN-γ secreted by Th1 can promote the proliferation and differentiation of B cells into cells with the function of synthesizing and secreting antibodies(Romagnani, 2000), and cytokines such as IL4, IL-5, IL-6, and IL10 produced by Th2 cells play a role. In our results, mice fed recombinant Lactobacillus plantarum showed a significant increase in the expression of some of the cytokines secreted by Th1 and Th2, which are essential for improving the body's immunity.

In our study, firstly, we developed recombinant L. plantarum vaccine expressing VP6 and VP6-pFc proteins. Then we demonstrated the enhancement of organismal immunity by recombinant L. plantarum vaccine on mice. Finally, we conducted virus infection experiments on mice and pigs and evaluated the protective effect of recombinant L. plantarum vaccine's protective effect on the organism.

Animals and Ethics Statement

Mice used in this experiment were purchased from HFK Bioscience (Beijing, China) and housed in SPF-grade rat houses for the experiment, including pregnant females and 4-week-old pathogen-free female C57BL/6 mice. The SPF piglets were obtained from the Harbin Veterinary Research Institute and transported in enclosed carts designed for SPF conditions. For the duration of the experiments, all animals were accommodated in SPF-grade animal houses within a specific pathogen-free facility. The housing conditions included a 12-hour light/dark cycle and maintained appropriate ambient temperature and humidity levels. The entire animal experiments adhered to the regulations set by the Animal Management and Ethics Committee of Jilin Agricultural University. The experiments were conducted in strict accordance with the National Guidelines for Laboratory Animal Welfare.

Construction of expression vectors and recombinant L. plantarum

Reference was made to the gene sequence of asd-P23-alr on plQa-EGFP-S vector (Jilin Agricultural University) and the gene sequence of araC-PBAD of pBAD-6xHis-GST-TEV in the relevant literature(Schleif, 2010). To replace the P23 promoter in asd-P23-alr with araC-PBAD, we initially designed the gene sequence of asd-araC-PBAD-alr (AAPA). This involved adding a 100 bp homology arm at both ends of the gene sequence. Subsequently, we successfully carried out the replacement of the AAPA gene sequence, resulting in the successful construction of the Δalr-pCXa-S plasmid(GENEWIZ company assistance, Suzhou, China).

Rotavirus VP6 gene (NCBI: JN977137.1) and pFc gene (NCBI: M81771.1) were synthesized in the company with the addition of cleavage site and HA tag sequence. The target fragments of Δalr-pCXa-S plasmid were partially replaced with VP6 and VP6-pFc, and finally Δalr-pCXa-vp6-S and Δalr-pCXa-VP6-pFc-S were obtained (with the help of GenScript Biotech Corp, Nanjing, China). The plasmids obtained were correctly verified by enzyme digestion and sequencing (with the help of Sangon Biotech, Shanghai, China). Afterwards, the constructed plasmids were electroporated into NC8 cells. As a result, we obtained two final strains: NC8/Δalr-pCXa-vp6-S and NC8/Δalr-pCXa-VP6-pFc-S.

Preparation of polyclonal antibodies to VP6

The gene sequence of VP6 was ligated into the pET28a expression vector and transformed into BL21 cells, resulting in the generation of BL21-pET28a-VP6. The expression of the VP6 protein was induced by using TPTG (100 mM), followed by purification and recovery of the VP6 protein. The 6-week-old white rabbits were immunized multiple times. Following the immunization, cardiac blood was collected and serum was obtained to obtain rabbit-derived polyclonal antibodies specific to VP6.

Western Blotting

NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S were first cultured and incubated anaerobically with 50 ng / ml of the inducible peptide Sakacin P (SppIP) for 6 h. Bacterial proteins were freeze-thawed three times and then fragmented by sonication. Bacterial proteins were separated by SDS-PAGE, transferred to PVDF membranes and incubated with polyclonal rabbit anti-VP6 antibody using horseradish peroxidase-coupled goat anti-rabbit as secondary antibody (Solarbio). After washing, protein blots were displayed by enhanced chemiluminescence (ECL, Thermo Scientific) on an Amersham Imager (General Electric Company).

Immunization procedures in suckling mice

NC8/Δalr-pCXa-S, NC8/Δalr-pCXa-VP6-S, and NC8/Δalr-pCXa-VP6-pFc-S were first grown overnight (SppIP). Then thirty-two 4-week-old C57BL / 6 mice were divided into four groups: PBS, NC8/Δalr-pCXa-S, NC8/Δalr-pCXa-VP6-S, and NC8/Δalr-pCXa-VP6-pFc-S. Immunization dose was 0.5×10⁹ CFU/each. Mice were immunized by oral gavage on days 1–3, boosted on days 14–16, and again on days 28, 29, and 30.

Flow cytometry and reagents

After euthanizing the animals, single cell suspensions were prepared from the desired immune organs or tissues, and antibodies were added to tubes containing 1 × 10⁶ cells, mixed thoroughly and stained for 30 min at 4°C under dark conditions. Add 1 mL of PBS, centrifuge at 2000 r pm and 4°C for 5 min and discard the supernatant. The cells can then optionally be fixed and permeabilized, and after permeabilization the antibody can be used to continue the staining. The staining is completed and detected using a flow cytometric analyzer (BD).

BD Pharmingen: Fixable Viability Stain 780 (565388), purified rat anti-mouse CD16/CD32 (Mouse BD Fc Block) (553142), Alexa Fluor®700 Anti-mouse CD3 ( 557984), PerCP-Cy™5.5 Rat Anti-Mouse CD4 (550954), FITC anti-mouse CD8 (553030), PE Anti-Mouse IFN-γ (554412), APC anti-mouse IL-4 (554436), PE Anti- Mouse CD25 (553866), Alexa Fluor® 647 Rat Anti-Mouse Foxp3 (553486), FITC Anti-Mouse IgA (559354), PE Anti-Mouse B220 (553089), PerCP-Cy™5.5 Rat Anti- Mouse CD80(560526), PE Anti-Mouse CD86(553692), APC anti-mouse CD11c(550261). PE-cy7 mouse anti-pig CD3ε (561485), PE mouse anti-pig CD4a (559586), Alexa Fluor 647 mouse anti-pig CD8a (561475), Percp-cy5.5 Anti pig IFN-γ (561481) ,

Invitrogen: Biotin CD3 monoclonal antibody (MA5-28771), CD21 monoclonal antibody (A1-19243), FITC Anti pig CD45 (MA5-28383), PE CD163 monoclonal antibody (MA5-16476), CD172a monoclonal antibody (MA5-28299). antibody (MA5-16476), CD172a monoclonal antibody (MA5-28299).

Solarbio: CFSE (S1076), PMA (P6741), D-PBS (D1040), PBS (P1010)

GE Healthcare: Percoll (17089101).

R&D: APC Anti pig IL-4 (1644057).

Sigma: Penicillin And Streptomycin (V900929), Collagenase IV (V900893-1G), Ionomycin (56092-81-0), DNase I (10104159001), DTT (3483-12-3), HEPES ( H3375), EDTA (E8008), FBS (F8318).

PE Goat anti Mouse(SPP101), FITC Rabbit anti Goat (SPP101-100), PE anti Mouse (SHP501) purchased from 4A Biotech.,Ltd.

T-cell proliferation assay

The cell density was adjusted to 1–5×10⁶ cells/mL using complete cell culture medium. Then, 1 mL of the cell suspension was transferred to a 15 mL centrifuge tube. Next, a pre-warmed 2× CFSE working solution was added and the mixture was incubated at 37 ℃ for 10 minutes. The reaction was then blocked by immediately adding pre-warmed complete culture medium at 37 ℃. The cells were transferred to 24-well plates and incubated with VP6 antigen-purified protein at a concentration of 20 µg/mL. After incubation, the cells were retrieved into flow tubes and washed twice with PBS. Then, Alexa Fluor® 700 Anti-mouse CD3 and PerCP-Cy™ 5.5 Rat Anti-Mouse CD4 antibodies were added in a premixed staining solution and incubated at 4 ℃ for 30 minutes. Following the incubation, the cells were washed with PBS, leaving 200 µL of cell suspension, and then subjected to flow cytometry detection.

Enzyme-linked immunosorbent assay

96-well plates were coated with purified VP6 antigen at a concentration of 3 µg/µL and incubated overnight at 4°C. The plates were then washed three times with PBST (PBS with 0.1% Tween-20) and blocked with 150 µL of blocking solution for 10 hours. Serum samples were diluted 64-fold and fecal serum was diluted 32-fold. These diluted samples were added to the wells and incubated at 37°C for 2 hours. After incubation, the wells were washed three times with PBST. Next, a horseradish peroxidase (HRP)-labeled goat anti-mouse SIgA was added at a dilution of 1:4,000 and incubated at 37°C for 1 hour. The reaction was performed in a thermostat using PBST. After the incubation, the wells were washed three times with PBST. Then, the color development was initiated by adding TMB color development solution, and the reaction was carried out at 37°C for 15–30 minutes with light protection. When the blue color appeared in the well plate, 2 M concentrated sulfuric acid was added to stop the reaction, resulting in a color change to yellow. The OD450 was measured using an enzyme marker.

Immunization procedures in piglets

A total of 20 neonatal piglets, aged 3 days, were equally divided into 4 groups based on their body weight: PBS, NC8/Δalr-pCXa-S, NC8/Δalr-pCXa-VP6-S, and NC8/Δalr-pCXa-VP6-pfc-S. From day 3 to day 13, the piglets were subjected to alternate-day gavage(each feeding of L. plantarum contained a quantity of 1×10¹⁰ CFU). On day 14, the piglets were challenged with a dose of 5mL/each (10⁴ TCID₅₀). On day 21, the piglets were euthanized, and samples were collected from the same section of the small intestine for pathological examination. Serum and fecal supernatants were collected from the piglets for ELISA experiments. Additionally, lymphocyte samples will be collected from the intestinal lamina propria, peripheral blood, spleen, and mesenteric lymph nodes to perform flow cytometry analysis.

Extraction of lymphocytes from the lamina propria of piglets

First, the piglets were euthanized, and their intestines were dissected longitudinally. The dissected intestines were then rinsed with PBS and placed in 15 mL centrifuge tubes. The tubes were kept on ice and pre-warmed isolation solution(10 ml of RPMI 1640, 1% penicillin and streptomycin, 1% HEPES, 5 mM EDTA, 2 mM DTT, and 2% heat-inactivated FBS) was added. The tubes were then centrifuged at 37°C and 180 rpm for 20 minutes. Afterward, the samples were vigorously shaken for 30 to 60 seconds and filtered through a sieve. This step was repeated once before transferring the samples into EP tubes containing 5 mL of digestive solution(8 ml of RPMI 1640 medium, 1% penicillin and streptomycin, 1% HEPES, 50 mg of collagenase IV, 1 mg of DNase I, and 2% FBS). The cells were washed with serum-free medium and co-incubated with EP tubes containing 5 mL of digestive solution at 37°C and 250 rpm for 15 minutes. This step was repeated once. After passing through a cell screen, the cells were washed with 1640 medium containing 3% serum and centrifuged at 4°C and 460 g for 10 minutes. Percoll was used for cell isolation by adding 4 mL of 80% percoll to a 15 mL centrifuge tube and resuspending it with 8 mL of 40% percoll suspension. The mixture was centrifuged at 20°C and 500 g for 20 minutes, and the intermediate layer was collected. The collected cells were washed by centrifugation at 4°C and 400 g for 8 minutes in 1640 medium containing 3% serum. After cell counting, the density of intestinal lamina propria cells was adjusted to 1–5×10₆ cells/mL using complete cell culture medium.

Subsequently, the cells were stained with flow cytometry antibodies. Surface antibodies CD3, CD4, CD8, CD21, and CD45 could be premixed, including single-labeled positive flow cytometry tubes and blank flow cytometry tubes. If intracellular staining is required, BD's Fixed Broken Nuclei Reagent can be used to stain intracellular factors IL-4 and IFN-γ.

Statistical analysis of data

Flow cytometry results were obtained using FlowJo 10.8.1, statistical plots were drawn using Graph Prism 8.0.2 software for data entry, and the results were analyzed using conventional one-way ANOVA to analyze whether the control and experimental groups differed from each other. (P < 0.05 marked *; P < 0.01 marked **; P < 0.001 marked ***; P < 0.0001 marked ****).

Construction of Plasmids and In Vitro Expression of Target Genes

Firstly, we successfully replaced the araC-P_BAD promoter with P₂₃ on the original vector plasmid by seamless cloning technology, and constructed the arabinose-regulated antibiotics free vector Δalr-pCXa-S (Fig. 1A), which is unable to survive in natural conditions and has no pollution to the environment by NC8/Δalr-pCXa-S. This expression system has been granted a national invention patent in China (Patent No. ZL202211148027.6). Next we obtained the VP6 gene (1230 bp), VP6-pFc gene (1950 bp) and EGFP gene (717 bp) (Fig. s1A), and the three genes were ligated to the Δalr-pCXa-S expression plasmid (Fig. 1B) to obtain the plasmids Δalr-pCXa-EGFP-S, Δalr-pCXa-VP6-S and Δalr-pCXa-VP6-pFc-S (Fig. 1C), it was verified by enzyme digestion and sequencing (Fig. s1B). Then we transferred the plasmids into NC8 via electroconversion. Finally obtained NC8/Δalr-pCXa-EGFP-S, NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S.

To verify the surface expression of the target protein, we detected NC8/Δalr-pCXa-EGFP-S by immunofluorescence assay and found a clear expression of green fluorescence (Fig. S1C), demonstrating that the recombinant bacterium could efficiently express the target fragment. Next we constructed BL21/pET-28a-VP6 recombinant bacteria (Fig. s1D), successfully purified the expressed VP6 protein (Fig. s1E), and a rabbit-derived polyclonal antibody against VP6 protein was obtained. The results of Western blot experiments showed that the recombinant bacteria successfully expressed EGFP,VP6 and VP6-pFc proteins (Fig. 1D). Immunofluorescence results also further demonstrated the expression of VP6 and VP6-pFc proteins (Fig. 1E).

We successfully constructed novel recombinant L. plantarum NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S and demonstrated efficient expression of the target proteins.

Recombinant L. plantarum induces activation of DC cells and B cells in Pyle's Pooled Lymph Nodes (PPs)

To verify the enhancement of organismal immunity by recombinant L. plantarum, we immunized mice (Fig. s2A). Next, we examined the activation of immune cells mainly by flow cytometry, and the gating of flow cytometry in Fig. s2B.

DC cells are able to play the role of antigen presentation with the specificity of CD11c, the main cellular marker molecule on their surface, and CD86 is able to stimulate T-lymphocytes to regulate the body's immune response in synergy with CD11c. We detected significant activation of DC cells in PP junctions, and DC cells fed NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S expressed increased CD11c and CD86 signals compared with those fed NC8 and NC8/Δalr-pCXa-S groups (Fig. 2A).

The signature cytokine detected on the surface of B cells is B220, which is converted to plasma cells upon antigenic stimulation, and the secreted immunoglobulin IgM is converted to IgA capable of resisting mucosal infections and regulating the balance of intestinal flora. Recombinant L. plantarum anchored to express porcine rotavirus VP6 was found to induce B cell activation in mice. The number of B220⁺ IgA⁺ B cells was significantly higher in PPs of the recombinant L. plantarum-fed group compared with the control group, in which the expression level of B220⁺ IgA⁺ B cells was higher in the NC8/Δalr-pCXa-VP6-pFc-S group compared with that in the NC8/Δalr-pCXa-VP6-S group (Fig. 2B).

The above results indicate that feeding NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S had a significant activating effect on both DC cells and B cells in mouse PP junctions.

Recombinant L. plantarum induces activation of mouse CD4-T cells

CD4-T cells can differentiate into helper T cells (Th), Th1 type cells can secrete IFN-γ mainly involved in cellular immunity, and Th2 type cells can secrete IL-4 mainly involved in humoral immunity . Our experimental results revealed that recombinant L. plantarum could induce the activation of T cells in mesenteric lymph nodes (MLN) and spleen (SP). The increase in the number of CD4-T cells was more significant compared to CD8-T cells (Fig. 3A). Further analysis revealed that the expression level of CD4-T-expressed IFN-γ was higher in the MLN and SP of mice fed NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S compared to mice fed NC8 and NC8/Δalr-pCXa-S (Fig. 3B), while the expression of CD4-T expressed higher levels of IL-4 (Fig. 3C), which also indicated Th1 and Th2 activation.

When the body is exposed to antigenic stimuli, regulatory T cells can regulate the body's immune system by releasing a variety of cytokines to maintain the balance of Th1/Th2. In this study, we analyzed the CD25 and Foxp3 double-positive cells among the CD4-T cells, which can reflect the immune homeostasis level of the body. It was found that the number of CD4-T cells expressing CD25 and Foxp3 was significantly increased in the MLN of mice fed NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S compared with mice fed NC8 and NC8/Δalr-pCXa-S (Fig. 3D).

These results indicate that NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S significantly induced mixed Th1/Th2 immune responses in both MLN and SP.

CD4-T cell proliferation induced by recombinant L. plantarum

Proliferation of CD4-T cells can assist B cell activation to enhance the immune effect of the body. Flow cytometry examination of CD4-T cell proliferation in SP and MLN revealed that significant CD4-T proliferation occurred in MLN and SP after specific antigenic stimulation. CD4-T cells were more pronounced in mice fed NC8/Δalr-pCXa-S and NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S compared with those fed NC8 and NC8/Δalr-pCXa-S (Fig. 4A). The results of the cell proliferation assay showed that mice in the experimental group had a certain number of memory lymphocytes, which proliferated and produced specific antibodies upon antigen stimulation. The results showed that the role of memory lymphocytes in MLN was more significant.

Recombinant L. plantarum enhances immunoglobulin secretion

Secretory IgA (SIgA) is abundantly distributed among the mucosal surfaces of the digestive tract, and is the main secretory antibody in mucosal immunity. IgG is an important component of total immunoglobulin in the organism, secreted by plasma cells, with a high content and long-lasting effect. We found that the content of SIgA was similar in all groups of mice before immunization, and the content of SIgA in the intestines of mice in the experimental group increased significantly after three immunizations, while the content of SIgA in the intestines of mice in the control group mice did not increase significantly (Fig. 4B). The IgG content in serum was similar in all groups before immunization, and the IgG content in serum of experimental group mice increased significantly after three immunizations (Fig. 4C).

We found that the increase in fecal SIgA secretion was more pronounced compared to the increase in serum IgG content, suggesting that recombinant L. plantarum may be more effective in inducing intestinal mucosal immunity.

Recombinant L. plantarum for the prevention of rotavirus infection in suckling mice

We used MA104 cells for the amplification of PoRV (NCBI: DN30209), and the MA104 cells were closely arranged in a shuttle shape in the normal state, while the diseased MA104 cells were irregularly arranged, with the occurrence of pulling net, vacuoles, and a small amount of cell death (Fig. S5A).The Mock control group was non-fluorescent, and the DN30209 test group 10^-1 showed a large amount of fluorescence, and 10^-5 was non-fluorescent, and after the calculations, the viral titer was approximately 10⁴ TCID₅₀/mL (Fig. S5B).

We infected mice with PoRV (Fig. 5A) and found that oral immunization with recombinant L. plantarum had a significant protective effect on mice. Pathologic sections showed that the epithelial cells of the small intestinal villi of control mice were disorganized (black arrows), and part of the lamina propria structure disappeared (yellow arrows). The vascularization of the lamina propria and submucosa was dilated and congested (red arrows), accompanied by inflammatory cell infiltration (green arrows), with the most obvious lesions in the PBS group, which showed thinning of the intestinal wall and a decrease in the number of cup cells. The NC8/Δalr-pCXa-VP6-S group showed significantly reduced damage compared with the above two groups, and did not have significant damage to the lamina propria and inflammatory cell infiltration, A mild decrease in the number of cup-shaped cells was seen. Intestinsl tissue structure tended to be normal in the NC8/Δalr-pCXa-VP6-pFc group. The intestinal villi were neatly arranged, the epithelial cells were neatly and closely arranged, and the number of cup-shaped cells was more abundant (Fig. 5B).

ELISA results revealed that the antiviral and anti-inflammatory abilities of mice were enhanced after oral administration of VP6 recombinant L. plantarum. Compared with the control group, the experimental group showed a significant increase in the level content of IL-1β; a significant increase in the level content of IL-4; and a significant decrease in the level content of TNF-α. It indicates that oral NC8/Δalr-pCXa-VP6-pFc-S can reduce the inflammatory response during viral invasion while enhancing the immune response and clearing the virus from the body (Fig. 5C).

Prevention of rotavirus infection in piglets by recombinant L. plantarum

To further validate the protective effect of recombinant L. plantarum on piglets, we performed an attack and protection experiment on piglets (Fig. 6A). The results revealed that feeding NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S significantly alleviated diarrhea in piglets (Fig. 6B). It also significantly reduced the intestinal pathological changes caused by PoRV infection, which were slightly more severe in the PBS group, with disruption of the glandular structure, vacuolization and degeneration of the epithelial cells (blue arrows), wider spacing, dilated and congested vasculature in the lamina propria (red arrows), and scattered small amounts of inflammatory cell infiltration (green arrows). The intestinal mucosal epithelial cells in the NC8/Δalr-pCXa-S group were disorganized with degeneration and visible The epithelial cells in the NC8/Δalr-pCXa-VP6-S group were slightly disorganized (black arrowheads), and no other obvious pathologic changes were seen. No obvious pathological changes were seen in the NC8/Δalr-pCXa-VP6-pFc-S group (Fig. 6C).

Recombinant L. plantarum promotes activation of B cells and myeloid cells in piglets

We examined the activation of immune cells mainly by flow cytometry, and the gating of flow cytometry in Fig. s7A. The Flow cytometry results revealed that feeding NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S promoted significant activation of B cells in piglets LPL, MLN, and PBL compared to the control group, and the percentage of B cells expressing CD45 and CD21 were significantly increased in the percentage of B cells (Fig. 7A). Feeding NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S promoted a significant increase in the number of activated myeloid cells in piglets LPL and MLN (Fig. 7B).

Recombinant L. plantarum promotes CD4-T cell activation and expression of specific immunoglobulin in piglets

It was found that feeding recombinant L. plantarum could promote the activation of CD4-T cells in piglets' MLN and SP, especially in MLN, which could more significantly promote the secretion of IFN-γ and IL-4 by CD4-T cells (Fig. 8A), the gating of flow cytometry in Fig. s8A. After immunization of piglets against takedown, feeding NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S induced higher levels of SIgA secretion in the intestine (Fig. 8B), as well as increased the expression of specific IgG antibodies in the serum compared with the control group (Fig. 8C).

Our study demonstrated that the oral administration of recombinant L. plantarum NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S elicited robust mucosal and cellular immune responses in mice. These responses were characterized by the activation of Th1/2 cells and an increase in the secretion of cytokines such as IgA and IgG. The recombinant L. plantarum showed potential as an oral vaccine for prophylaxis, as it significantly reduced pathological changes in suckling mice and piglets while also activating the immune system of piglets. These findings provide evidence for the development of a novel oral recombinant L. plantarum vaccine.

PoRV is a zoonotic disease that can be mixed with other bacteria, limiting the development of the domestic animal husbandry industry, causing high economic losses, and greatly affecting human health development. VP6 is an important epitope marker of porcine rotavirus capable of inducing protective immunity. The VP6 protein of porcine rotavirus is highly conserved among serotypes, abundant in the structure of porcine rotavirus, and immunogenic(Jalilvand et al., 2015).

Lactobacillus modulate the body's immune system, and when stimulating the body's intestinal mucosa, the body is able to generate a host defense response that activates macrophages, natural killer (NK) cells, and neutrophils, and releases inhibitory inflammatory cytokines and IgA(Ashraf and Shah, 2014). L. plantarum NC8 is not pathogenic in intestinal symbiosis and can be used as an expression vector for a variety of antigenic genes, which are widely used in mucosal delivery systems, and oral administration of recombinant L. plantarum promotes the immune function of the organism(Bejar et al., 2013). Mucosal immunity can induce immune defense in mucosal and systemic tissues, in preventing pathogen invasion on mucosal surfaces(Li et al., 2020). Recombinant L. plantarum vaccine can effectively activate mucosal immune response and promote mucosal IgA or SIgA production after vaccination via mucosal route(Boyaka, 2017, Jin et al., 2019). Some studies have reported that mucosal anti-influenza vaccines induced higher localized humoral and cellular immune responses(Calzas and Chevalier, 2019). Mucosal vaccines constructed using HIV envelope proteins also induced HIV-specific immunity in blood and local mucosa(Chege et al., 2017). In this study, we successfully constructed novel recombinant L. plantarum strains: NC8/Δalr-pCXa-S, NC8/Δalr-pCXa-EGFP-S, NC8/Δalr-pCXa-VP6-S, and NC8/Δalr-pCXa-VP6-pFc-S. We verified VP6 and VP6-pFc protein expression using immunofluorescence and Western blot experiments, and determined the optimal feeding conditions.

The successful development of mucosal vaccines against influenza virus and rotavirus infections in the last decade has increased interest in this field and raised high hopes for new mucosal vaccines(Lycke, 2012). Immune cells play an important role in the enhancement of body immunity by recombinant L. plantarum.

CD11c can represent the maturity level of DC cells and is often used as a surface marker to detect the activation level of DC cells(Wang et al., 2020). In this experiment, the average fluorescence intensity of CD80 and CD86 in CD11c was detected. The experimental results showed that the novel recombinant L. plantarum could enhance the activation level of DC cells. As a result, the function of antigen presentation was subsequently strengthened, aiding in the clearance of pathogens. This is beneficial for the organism's ability to restore health quickly in case of viral infections.

A large number of B cells located in the germinal center are aggregated in PPs. The recombinant L. plantarum constructed in this study increased the content of B220⁺IgA⁺ cells in PPs. CD4-T cells in both MLN and SP were significantly proliferated and activated. Activated CD4-T cells mainly included Treg and Th1/2 subsets. The main marker for Treg was CD25⁺Foxp3⁺. In this study, the content of CD25⁺Foxp3⁺ cells in CD4-T cells in MLN was analyzed. The results indicated that the constructed recombinant L. plantarum increased the content of CD25⁺Foxp3⁺ cells in CD4-T cells in MLN. Further analysis revealed that the secretion of Th1 cytokine IFN-γ was significantly increased in the MLN of the experimental group compared with the control group, while the secretion of Th2 cytokine IL-4 was also significantly increased. In the SP of the experimental group of mice, we found that the secretion of cytokines by TH1/2 cells was also significantly increased. Cell proliferation experiments revealed that both MLN and SP could be promoted to produce memory lymphocytes after specific antigenic stimulation and were activated upon encountering antigenic stimulation.

IgG is the most important antibody in the body as it has antiviral, virus neutralizing, antimicrobial and immunomodulatory functions(Barabas et al., 2018). In our study, prepared VP6 antigens were used for stimulation, and a significant increase in blood-specific IgG was found after each immunization, especially on days 20 and 30, when mice fed NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S reached higher levels.

After the onset of infection, a broad and complex array of cytokines appear in the body, which interact to form a cytokine network. Among them, IL-4 is a cytokine secreted by Th2 cells, which stimulates the proliferation of activated B and T cells and the differentiation of CD4-T cells into Th2 cells. It also plays a key role in the regulation of humoral and adaptive immunity. IL-1β is an important member of the IL-1 family and is also known as a classic multifunctional cytokine. IL-1β is an important component of the host's defense against infections, activating neutrophils and macrophages to consume invading pathogens(Lopez-Castejon and Brough, 2011). TNF-α is a major inflammatory cytokine that plays a central role in the inflammatory response, and high TNF-α levels indicate that the organism is in an inflammatory state(Cruceriu et al., 2020). We found that feeding recombinant L. plantarum increased immunity and significantly alleviated PoRV infection in suckling rats, while increasing the significant secretion of IL-4 and IL-1β and decreasing the secretion of TNF-α in serum. Experimental results on piglets revealed that feeding NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S significantly increased immunity, including activation of piglet B cells, macrophage and CD4-T cells. The cytokine could promote the expression of specific IgG and SIgA. Ultimately, the pathologic changes in piglets were significantly reduced.

When PoRV stimulates the organism, the Fc receptor mainly exerts mucosal immunity and can effectively induce immune responses. In this study, a novel recombinant L. plantarum was constructed by combining both VP6 and pFc with the fusion expression of an anchored highly expressed S-layer protein. The novel oral vaccines, VP6, NC8/Δalr-pCXa-vp6-S, and NC8/Δalr-pCXa-VP6-pFc-S, have been shown to effectively enhance the organism's resistance to PoRV infection when administered to suckling mice and piglets. In conclusion, the recombinant L. plantarum targeting porcine rotavirus prepared by us has some research value and can be used as an oral vaccine to enhance the immune effect and antiviral ability of suckling mice and piglets. Our results are important for the development of a novel oral immunization vaccine to mitigate PoRV infection.

Ethics approval and consent to participate

The animal management procedures and all laboratory procedures abided by the regulations of the Animal Care and Ethics Committees of Jilin Agriculture University, China.

Consent for publication

All authors have approved the content of this manuscript and provided consent for publication.

Availability of data and materials

The data and materials in the article are guaranteed to be true. The raw data for this article were deposited in Jilin Agriculture University.

Competing interests

The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This work was supported by the National Natural Science Foundation of China (32273043, 32202890, U21A20261), the Science and Technology Development Program of Changchun City (21ZY42), the Science and Technology Development Program of Jilin Province (20200402041NC), and China Agriculture Research System of MOF and MARA (CARS-35).

Authors' contributions

Cell isolation, J.H.W., and X.X.L.; data analysis, D.L.Z., J.X.Z., and F.Y.L.; manuscript preparation and writing, M.G., M.Y.C., and Y.Y.L.; supervision and project administration, Y.Z., C.F.W., and X.C. Preparation of experimental reagent materials, H.X.W., and H.Y.B. All authors contributed to the article and approved the submitted version.

Acknowledgements

We thank Dr. Yan Zeng, Dr. Chunfeng Wang and Dr. Xin Cao provide help and guidance.

This paper does not report the original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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No competing interests reported.

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L. plantarum surface-displayed VP6 (PoRV) protein can prevent PoRV infection in piglets

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Abstract

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Introduction

Materials and methods

Animals and Ethics Statement

Preparation of polyclonal antibodies to VP6

Western Blotting

Immunization procedures in suckling mice

Flow cytometry and reagents

T-cell proliferation assay

Enzyme-linked immunosorbent assay

Immunization procedures in piglets

Extraction of lymphocytes from the lamina propria of piglets

Statistical analysis of data

Results

Conclusion

Disscussion

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References

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Supplementary Files

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