Evaluation of the immune response in piglets fed seed-based oral vaccine against verocytotoxic Escherichia Coli

The gut mucosal surface is considered the largest immunologic organ bearing specialized components of the immune system that protect the host against pathogens such as verocytotoxic Escherichia coli (VTEC), control responses to food components, and maintain tolerance to harmless external antigens. Oral vaccines can selectively elicit mucosal secretory immunoglobulins A, that play a pivotal role in the mucosal immunity, and a variety of cell-mediated immune responses, including the expression of antigen presenting molecules (major histocompatibility complex, MHC), Toll-like receptors (TLRs) and soluble factors (cytokines and chemokines). Transgenic plants are a valuable platform to produce genetically modified oral vaccines for a large number of human and animal diseases. This study focuses on the immune response elicited in piglets after administration of tobacco seeds-based oral vaccine expressing F18 adhesive fimbriae and the B subunit of verocytotoxin genes from VTEC (Rossi et al. 2014a,b). 36 weaned piglets were divided randomly into 4 experimental groups (CC, challenged control; CT, challenged treated; UC, unchallenged control; UT, unchallenged treated). Treated piglets were fed five times, on day 0, 1, 2, 7 and 14, with 20 g of engineered milled tobacco seeds (expressing 6.6-7.4 \u3bcg of F18 plus 34-37 \u3bcg of VT2eB) mixed with 20 g of milk powder. Controls received 20 g of wild type of tobacco seeds. The animals were challenged at day 20 with 10E10 CFU of O138 VTEC E. coli. Blood samples and small intestinal scrapes were collected from all sacrificed piglets and processed for ELISA determination of serum IgA and intestinal mucosa IgA, TNF-\u3b1, IL-8 and CXCL9 (MIG). Samples of jejunum and mesenteric lymph nodes were collected and processed for gene expression study. RNA was extracted, reverse transcribed and assayed with specific primer pairs by Real-Time PCR to quantify the gene expression of the innate immunity receptors TLRs 2 and 4 and the proinflammatory cytokines IFN-\u3b3 and IL-1\u3b2 in jejunum, and the expression of the antigen presenting molecules MHC type I and II in mesenteric lymph nodes. The challenge significantly increased the expression of MHC-I, which is the molecule responsible for antigen presentation to CD8+ T lymphocytes (CC had the highest expression, followed by CT). UC group showed the lowest expression of MHC-I, demonstrating that the upregulation of this gene expression occurs only in the presence of antigen, either vaccine or pathogen. No statistically significant differences were found for MHC-II and IFN-\u3b3. TLR2, TLR4, IL-1\u3b2, TNF-\u3b1 and CXCL9 were maximally expressed in UT group and minimally expressed in UC group. The proinflammatory cytokine IL-1\u3b2 resulted highly expressed in CT group in jejunum by PCR. Overall the immune parameters measured showed the highest values in the vaccinated animals versus controls; these results indicate that vaccination with both antigens stimulates the IgA and cytokine production more than the infectious agents