Study on Molecular Immunity Mechanism of Porcine Rotavirus VP6 Proteins expressed in Lactobacillus plantarum NC8

Rotaviruses are the main cause of animal and infant diarrhea and are widely distributed worldwide. In the pig industry, porcine rotavirus infection is a significant cause of mortality and morbidity; therefore, the optimization and well-organized distribution of vaccines for infection prevention is needed. The molecularimmunological mechanisms of recombinant Lactobacillus plantarum NC8-pSIP409-pgsA-VP6-Dcpep, NC8-pSIP409-pgsA-VP7-Dcpep and NC8-pSIP409-pgsA-VP6-VP7-Dcpep strains against porcine rotavirus were explored. At 12 h after the co-incubation of L. plantarum expressing rotavirus proteins with BMDCs, the effects of the strains on the differentiation of BMDCs were detected by FCM. The results showed that the recombinant strains could significantly promote the activation of BMDCs. The expression of cytokines in the above cells supernatants were detected by ELISA and the results suggested that the recombinant strains could significantly increase the production of IL-12P70 and inhibit the secretion of IL-6. CD4 + and CD8 + T cells from the spleen of non-immunized mice were sorted and cultured with the above activated BMDCs for 48h, and the expressions of IFN-γ + and perforin in the cells and the contents of IFN-γ in the supernatants were determined by FCM and ELISA, respectively. The results showed that the recombinant strains increased the expression of IFN-γ + (4.33%) ofCD4 + T cells and IFN-γ + (7.68%) and perforin (17.50%) of CD8 + T cells, as well as the secretion of cytokines IFN-γ. The expression of IFN-γ + (P<0.001) and perforin (P<0.001) from VP6/VP7-specific CD8 + T cells of spleen and MLN were detected in vivo and the recombinant groups were significantly increased. Moreover, the recombinant groups significantly promoted the proliferation of T cells in the spleen (P<0.001). Our results confirmed that recombinant recombinant L. plantarum strains can effectively induce cellular immune response. the process of cellular immunity and humoral immunity is exerted. In vitro, we used magnetic bead sorting reagent to sort CD4 + and CD8 + T cells from spleens of unimmunized BALB/c mice, and co-cultured with the above-mentioned groups of activated BMDCs for 48 hours to detect IFN-γ in CD4 + T cells. And changes in IFN-γ and perforin in CD8 + T cells, including detection of secreted cytokines. From the results, we can see that compared with the 409p' group, the expression of IFN-γ in CD4 + T cells was significantly increased were significantly improved (P < 0.05). <0.001), while in the MLN, the the NC8-pSIP409-pgsA group increased significantly (P < 0.05), and the the NC8-pSIP409-pgsA-VP6-Dcpep, NC8-pSIP409-pgsA-VP7-Dcpep, and NC8-pSIP409-pgsA-VP6-VP7-Dcpep groups increased significantly (P < 0.01). The above results indicate that both IFN-γ and perforin in CD8 + T cells have antiviral effects, and the NC8-pSIP409-pgsA-VP6-Dcpep, NC8-pSIP409-pgsA-VP7-Dcpep, and NC8-pSIP409-pgsA-VP6-VP7-Dcpep groups group is superior to the the NC8-pSIP409-pgsA, groups group mainly in IFN-γ.


Introduction
Rotaviruses are members of the family Reoviridae. Depending on their particular inner capsid protein sequences, porcine rotaviruses are categorized into groups A, B and C [1].
Rotaviruses have a nonenveloped, double-stranded RNA genome that is composed of 11 segments enclosed by a triple-layered icosahedral capsid, [2][3][4][5]. The main source of acute diarrhea in piglets is porcine rotaviruses, which can cause high rates of mortality and morbidity [6][7][8][9][10][11]. In both pre-and post-weaning pigs, rotavirus Group A is the main source of rotavirus-associated diarrhea and accounts for 54% and 45% of the diarrhea experienced in those populations of pigs, respectively [1]. Some research has reported that in commercial pig farms, 89% of all rotavirus-associated diarrhea can be attributed to group A rotavirus infections [12]. Rotavirus outbreaks are difficult to prevent because they are transmitted via the fecal oral route and can survive in the environment for a long period of time. Replication of the virion takes place in the intestinal villi in epithelial cells and destroys enterocytes mainly in the ileum and jejunum, leading to villous atrophy [13][14][15][16]. In addition, in the affected regions, nutrients cannot be absorbed or digested, causing severe malabsorption [13][14][15][16]. The control of rotavirus infections requires a solid understanding of the epidemiology of rotaviruses, which will contribute to prevention programs and improve the current vaccines. Presently, the obtainable vaccines cannot provide adequate protection. To activate immunocompetence, repeated administrations and a large vaccine dose are generally needed. This repeatedly causes unwanted clinical signs. To overcome these weaknesses and deliver antigens to the mucosal immune system, possible improvements to lactic acid bacteria (LAB) have been proposed.
Mucosal immunity plays an important role in protective immunity because rotaviruses are enteric pathogens. In the gut, innate immune responses initiate acquired immune responses and offer the first line of protection against pathogenic microorganisms. In 4 addition, the only appropriate way of eliciting gut immunity is to prompt the immune response via oral immunization because this oral route assists in gut-associated lymphoid tissue (GALT) stimulation, improving anti-viral IgA production [17,18].
Lactobacillus is a food-grade bacterium. Lactobacillus plantarum (L. plantarum) is a common lactic acid bacterium and has a wide range of applications in industrial lactic acid fermentation and health care. Expression of a foreign antigen using a lactic acid bacteria strain induces mucosal (sIgA) and systemic immune responses [19]. Lactic acid bacteria have the following advantages: (1) easy to culture, and some strains can construct a new expression vector system; (2) genetic manipulation method is simple, reproducible and efficient; (3) can guarantee genetic engineering products The relative safety of the foodgrade expression system for direct oral administration; (4) a highly regulatable promoter system capable of expressing toxin genes; (5) exogenous proteins can obtain good expression inside and outside the cell. (6) can effectively cause the body's immune response, and lasts for a long time; (7) lactic acid bacteria are safe, the expressed foreign protein can be taken directly together with the bacteria without purification. Therefore, it can be used as an ideal antigen presentation vector [20]. Live vaccines stimulate the most efficient defensive responses, unlike heat-killed or recombinant antigen formulations, because they elicit both mucosal and systemic immunity [17,18]. Repeated vaccinations and large vaccination doses are needed due to the challenges posed by oral vaccination, such as the fact that the gut environment habitually deactivates and/or denatures potential vaccinogens, causing fever and diarrhea, with the live vaccine often being shed in the feces [17,18]. With regard to the stimulation of mucosal immunity, lactic acid bacteria (LAB) can be used to overcome these challenges [21]. Furthermore, several LAB strains are capable of colonizing and surviving the intestinal tract, stimulating nonspecific immunoadjuvant consequences [21]. It is essential to boost the immunogenicity of genetically engineered vaccines by combining them with suitable adjuvants because they are poorly immunogenic and composed of a single recombinant antigen. The dendritic cells play a central role in targeting peptide by directing innateand regulating adaptive/acquired immunity. Out of the three peptide pep3 later was renamed Dcpep, for the reason that Dcpep bound to both immature and mature DCs in a saturable way, than pep12 or pep18 and had the fascinating property to bind both human Monocyte-Derived Dendritic CellsmdDCs and mouse CD11c + I-A + DCs [22] Herein, we constructed recombinant L. plantarum NC8 strains expressing porcine rotavirus VP6 to test the effectiveness of the expression of the VP6 porcine rotavirus protein by L. BMDCs activated by NC8-pSIP409-pgsA-VP6-Dcpep, NC8-pSIP409-pgsA-VP7-Dcpep and NC8-pSIP409-pgsA-VP6-VP7-Dcpep were cultured in vitro with the primary T cells sorted by magnetic beads to study the effects of in vitro antigens on DCs, T cells and intracellular cytokines, and sorting NC8-pSIP409-pgsA-VP6-Dcpep, NC8-pSIP409-pgsA-VP7-Dcpep and NC8-pSIP409-pgsA-VP6-VP7-Dcpep by magnetic bead sorting. The CD8 + T cells of the mice were used to explore the molecular immune mechanism of the mouse T cell subsets in antiviral. The BMDCs of the mice were isolated as follows bilateral femur and tibia of mice was obtained aseptically. BMDCs of mice were isolated and cultured according to Ye et al. [23].
LPS was used as a positive control, 1 μL (final concentration 1 μg/mL) was added to each well; 1000 g was centrifuged for 10 min, cultured at 37 ° C, 5% CO 2 incubator for 3 h,  Table 1 below. Each mouse was administered with 109 CFU/100 μL, and immunization was continued for 3 days at intervals of 14 days, followed by 3 consecutive days of booster immunization, and the following experiment was performed 7 days later.

Effect of in vitro activated BMDCs on T cells
In order to simulate the in vivo DCs to obtain antigen and then deliver it to T cells, the process of cellular immunity and humoral immunity is exerted. In vitro, we used magnetic bead sorting reagent to sort CD4 + and CD8 + T cells from spleens of unimmunized BALB/c mice, and co-cultured with the above-mentioned groups of activated BMDCs for 48 hours to detect IFN-γ in CD4 + T cells. And changes in IFN-γ and perforin in CD8 + T cells, including detection of secreted cytokines. From the results, we can see that compared with the 409p' group, the expression of IFN-γ in CD4 + T cells was significantly increased 13 in the HA group (P < 0.05), and the increase in the aCD11c-HA group was extremely significant (P < 0.01). As shown in Fig. 6, compared with the NC8-pSIP409-pgsA group, the NC8-pSIP409-pgsA-VP6-Dcpep, NC8-pSIP409-pgsA-VP7-Dcpep, and NC8-pSIP409-pgsA-VP6-VP7-Dcpep groups group significantly increased the expression of IFN-γ in CD8 + T cells (P < 0.001), as shown in Fig. 7; the NC8-pSIP409-pgsA group group significantly increased

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Availability of data and materials: will be provided after acceptance Competing interests: Author1 declares that she has no conflict of interest.
Author 2 declares that he has no conflict of interest.
Author 3 declares that she has no conflict of interest.
Author 4 declares that he has no conflict of interest the content of supernatant IFN-γ after CD4+ T cells were co-cultured with BMDCs Figure 10 The content of supernatant IFN-γ after CD8+ T cells were co-cultured with BMDCs 29 Figure 11 The percent of CD4+IFN-γ+ T cells in response to VP6 and VP7 in mouse spleen 30 Figure 12 the percent of CD4+IFN-γ+ T cells in response to VP6 and VP7 in mouse MLN 31 Figure 13 the percent of CD8+IFN-γ+ T cells in response to VP6 and VP7 in mouse spleen 32 Figure 14 the percent of CD8+IFN-γ+ T cells in response to VP6 and VP7 in mouse MLN 33 Figure 15 the percent of CD8+CD107a+ T cells in response to VP6 and VP7 in mouse spleen 34 Figure 16 the percent of CD8+CD107a+ T cells in response to VP6 and VP7 in mouse MLN 35 Figure 17 the result of CD4+ proliferating T cells in mouse spleen 36 Figure 18 the result of CD8+ proliferating T cells in mouse spleen