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F4(+) enterotoxigenic Escherichia coli (ETEC) infections are an important cause of postweaning diarrhoea in piglets and an oral immunization of piglets with purified F4 fimbriae protects them from a subsequent F4(+) ETEC infection. However, oral immunization of suckling piglets is hampered due to the immature status of their immune system. Targeting of antigens to Fcgamma receptors (FcgammaR) on human and murine dendritic cells (DC) has been shown to enhance DC maturation and both humoral and cellular immune responses. To investigate the effect of F4 fimbriae incorporated in immune complexes (F4-IC) on porcine DC, we used porcine monocytic-derived DC (MoDC) as a model system. The results in this study demonstrate that FcgammaRI, II and III mRNA is expressed by porcine MoDC. Furthermore, we show that FcgammaRII and III are expressed on the cell surface and that F4-IC are internalized by MoDC via FcgammaR. This FcgammaR ligation induced a significantly enhanced expression of Major Histocompatibility complex (MHCII) class II and the costimulatory molecules CD80/86 and CD40 by MoDC compared with immature MoDC. Furthermore, the phagocytic capacity of F4-IC stimulated MoDC was reduced as evidenced by a reduced uptake of DQ-ovalbumin and FITC-dextran. In an allogenic and autologous mixed lymphocyte reaction, these F4-IC-activated MoDC showed an improved T cell stimulatory capacity in comparison with immature MoDC. The F4-IC induced DC maturation correlated with significant higher expression levels of several pro-inflammatory cytokines such as interleukine (IL) 1beta, IL-6 and Tumor necrosis factor alpha, the chemokine IL-8 and IL-12p40 in comparison with immature MoDC. Altogether, these results clearly demonstrate that FcgammaR engagement enhances the maturation of porcine MoDC, which may suggest that antigen targeting to FcgammaR on DC could improve vaccine design against infections.

We previously demonstrated that intradermal (ID) delivery of plasmid DNA encoding the porcine granulocyte–macrophage colony-stimulating factor (GM-CSF) 7 days before DNA vaccination enhances both cellular and humoral responses in pigs. In the present work, we studied the effect of the GM-CSF gene on antigen-presenting cells (APC) in pigs. We demonstrated that ID delivery of this gene significantly increased the number of epidermal CD1+ cells (Langerhans’ cells, skin dendritic cells) at the injection site at day 7. This was accompanied by an enhanced percentage of APC at the immune induction site following DNA vaccination, whereas a positive effect on APC maturation could not be demonstrated. Taken together, our data suggest that both DC recruitment to the immunization site and expansion of APC in the draining LN following DNA vaccination might contribute to the immune enhancing effect of plasmid encoded GM-CSF in pigs.

Very few antigens have been described that induce an intestinal immunity when given orally. Our laboratory demonstrated that oral administration of isolated F4 (K88) fimbriae of Escherichia coli to F4-receptor positive (F4R + ) pigs induces protective mucosal immunity against challenge infection. However, presence of F4-receptors (F4R) on villous enterocytes is a prerequisite for inducing the immune response, as no F4-specific antibody-secreting cells (ASC) can be induced in F4R − pigs. In this study, the in vivo binding of isolated F4 fimbriae (F4) to the gut epithelium was examined in F4R + and F4R − pigs. It was further investigated whether binding of F4 to the F4R results in endocytosis in and translocation across the gut epithelium using microscopy. F4 did not adhere to the intestinal epithelium of F4R − pigs, whereas it strongly adhered to the villous epithelium and the follicle-associated epithelium (FAE) of the jejunum and ileum of F4R + pigs. Following binding to F4R, F4 was endocytosed by villous enterocytes, follicle-associated enterocytes and M cells. Transcytosis of F4 across the epithelium resulted in the appearance of F4 in the lamina propria and dome region of the jejunal and ileal PP. This is the first study showing transcytosis of fimbriae across the gut epithelium. This receptor-dependent transcytosis can explain the success of F4 fimbriae as oral immunogen for inducing protective immunity in F4R + pigs strengthening the importance of receptor-dependent endocytosis and translocation in oral vaccine strategies. Further identification of the receptor responsible for this transport is in progress.

We previously showed that an intradermal (i.d.) FaeG DNA prime (2x)-oral F4 protein boost immunization induces a systemic response and weakly primes a mucosal IgG response in pigs, especially when plasmid vectors encoding the A and B subunit of the E. coli thermo-labile enterotoxin (LT) are added to the DNA vaccine. In the present study, we evaluated whether addition of 1alpha,25-dihydroxyvitamin D(3) (vitD(3)) to the DNA vaccine could further enhance this mucosal priming and/or modulate the antibody response towards IgA. To further clarify priming of systemic and mucosal responses by the i.d. DNA vaccination, we firstly compared the localization of the F4-specific antibody response in pigs that were orally boosted with F4 to that in pigs that received a third i.d. DNA immunization and secondly evaluated cytokine mRNA expression profiles after i.d. DNA vaccination. The i.d. DNA prime (2x)-oral F4 boost immunization as well as the 3 i.d. DNA vaccinations induced mainly a systemic response, with a higher response observed following the heterologous protocol. Co-administration of vitD(3), and especially of the LT vectors, enhanced this response. Furthermore, only the heterologous immunization resulted in a weak mucosal priming, which appeared to require the presence of the LT vectors or vitD(3) as adjuvants. In addition, the LT vectors strongly enhanced the FaeG-specific lymphocyte proliferation and this was accompanied by the absence of a clear IL-10 response. However, despite two DNA immunizations in the presence of these adjuvants and an oral F4 boost, we failed to demonstrate the secretory IgA response needed to be protective against enterotoxigenic E. coli.

Oral immunization of both humans and animals with non-replicating soluble antigens often results in the induction of oral tolerance. However, receptor-dependent uptake of orally administered soluble antigens can lead to the induction of an antigen-specific immune response. Indeed, oral immunization of pigs with recombinant FaeG (rFaeG), the adhesin of the F4(K88) fimbriae of enterotoxigenic Escherichia coli (ETEC), induces an F4-specific humoral and cellular immune response. This response is accompanied with a reduction in the excretion of F4 + E. coli following challenge. To improve the immune response against F4, rFaeG was orally co-administered with the mucosal adjuvant cholera toxin (CT). Oral immunization of pigs with rFaeG and CT significantly improved the induction of an F4-specific humoral and cellular immune response and also significantly reduced the faecal F4 + E. coli excretion following F4 + ETEC challenge as compared to rFaeG-immunized pigs. Therefore, the present study demonstrates that CT can act in pigs as a mucosal adjuvant for antigens that bind to the intestinal epithelium by a CT-receptor-independent mechanism.

The intestinal mucosal immune system can discriminate actively between harmful pathogenic agents and harmless food antigens resulting in different immune responses namely IgA production and oral tolerance, respectively. Recently, a pig model has been developed for studying intestinal mucosal immune responses in which F4 fimbrial antigens of enterotoxigenic Escherichia coli (F4 ETEC) are used as oral antigens. A unique feature of this model is that soluble F4 antigens can be administered to pigs which have a receptor for this fimbriae (F4R(+)) on their small intestinal villous enterocytes and pigs which do not have this receptor (F4R(-)). Oral administration of F4 to the F4R(+) pigs results in an intestinal mucosal immune response that completely protects the pigs against a challenge infection. In F4R(-) pigs such an intestinal mucosal immune response does not occur. However, a priming of the systemic immune system can be seen similar to the priming in pigs fed with the same dose of a food antigen, suggesting that F4 in F4R(-) pigs behaves as a food antigen. The fact that different mucosal immune responses can be induced with soluble F4, makes it an interesting model to study mucosal immune mechanisms in the pig.

In this study, the effect of two oligodeoxynucleotide (ODN) sequences 5'GCT-AGA-CGT-TAG-CGT-3' (CpG-ODN) and 5'-GCT-AGA-GCT-TAG-GCT-3' (GpC-ODN) on the antigen-specific antibody and cellular immune response after intramuscular immunizations with OVA was analyzed in pigs. Pigs immunized with OVA supplemented with these ODNs showed a significantly enhanced primary antibody response in comparison with the control group which received OVA without ODN. This enhanced primary antibody response appeared ODN-sequence-independent as similar effects were seen in both ODN-groups. The OVA-specific antibody titers obtained after a single injection of antigen combined with either of both ODNs were as high as the titers in the control group after two injections. Furthermore, the ODN-supplemented animals showed significantly higher OVA-specific IgA antibodies in their saliva and nasal secretions at some time points after the first immunization. Proliferation assays showed that CpG- as well as GpC-ODN significantly enhanced the antigen-specific as well as the mitogen-induced proliferation in different lymphoid tissues. Furthermore, 48h after the third immunization the CpG-group showed a significantly decreased IL-6 mRNA expression in cells of the local draining lymph node but no significant difference in TGF-beta (Th3-like) and IL-10 (Th2-like). The ODN injected animals showed the tendency to have higher IFN-gamma (Th1-like) mRNA-expression in comparison with the control group. To our knowledge, these are the first in vivo studies in pigs, which demonstrate the appropriateness of CpG-ODN as immunostimulating adjuvants in vaccines for farm animals.