ETEC colonisation factors disrupt the antigen presenting capacity of porcine intestinal dendritic cells

Enterotoxigenic E. coli (ETEC) are not only a major cause of diarrhoea in travellers to and children in developing countries, but also cause neonatal and postweaning diarrhoea in piglets, leading to a reduced feed conversion and a higher mortality rate. As a consequence ETEC infections result in severe economic losses in the swine production industry. This intestinal pathogen displays colonisation factors or fimbriae on its surface enabling the microorganism to adhere to the intestinal epithelium (Fig. 1). In pig, F4 and F18 fimbriae are the most frequently associated with ETEC-induced diarrhoea1. As opposed to F4 fimbriae, oral immunisation with F18 fimbriae doesn’t protect piglets from a subsequent challenge infection2. F18 fimbriae bind glycosphingolipids in the apical membrane of enterocytes, but no transcytosis occurs, resulting in lower sunepithelial antigen concentrations as compared to F4 fimbriae, which bind the transcytotic receptor aminopeptidase N3,4. However, M-cell mediated transport of F18 fimbriae should still occur. Hence, besides a lower antigen concentration, these fimbriae could affect the function of intestinal antigen presenting cells. Here, we investigated the influence of purified F18 fimbriae on the antigen presentation capacity of small intestinal lamina propria dendritic cells (LPDCs)

APN-targeted β-glucan microparticles for oral immunisation

Enterotoxigenic Escherichia coli (ETEC) infections are a major cause of morbidity and mortality among both humans and pigs. Human ETEC strains affect mainly children and are also a source of traveler’s diarrhoea in regions where ETEC is endemic. Porcine ETEC cause diarrhoea in neonatal and newly weaned piglets.1-3 Pathogenicity of these bacteria is associated with fimbrial adhesins that mediate their colonisation to the microvilli of the intestinal epithelial cells in the small intestine. Porcine ETEC strains express five different fimbriae, namely the F4, F5, F6, F18 and F41 fimbriae, of which F18 and F4 fimbriae are most frequently associated with post-weaning disease in pigs.4 Once colonised, ETEC strains will secrete heat-labile enterotoxins (LT) or heat-stable enterotoxins (STa or STb). These enterotoxins will disrupt the water and electrolyte balance in the intestine which will cause severe watery diarrhoea.5 In neonatal and recently weaned piglets, ETEC infections results in severe economic losses due to growth retardation, increased drug use and elevated mortality.6 Most neonatal infections can be prevented by a passive lactogenic immunity obtained by vaccination of the sow. These maternal vaccines are mainly applied parenterally in the pregnant sow and contain inactivated ETEC bacteria with fimbriae or purified fimbriae with or without LT.7 However, this passive protection disappears at the moment of weaning.8 For the control of post-weaning diarrhoea (PWD), antibiotics are commonly used.9, 10 Besides their prophylactic usage, antibiotics were also applied for improving growth and production. The long term and extensive use of antibiotics has resulted in the development of antibiotic resistance. Therefore, since 1 January 2006 all commonly-used antimicrobial growth promoters have been banned in the EU member states. However, this caused a reduced performance and increased morbidity in post-weaning pigs and consequently, the development of alternative strategies is required.11 A wide variety of immunomodulating substances used as in-feed additives have been proposed to help post-weaning piglets to cope with feed transition and stress during this period.12, 13 The aim of these dietary substances is to help piglets develop an ‘appropriate’ innate and acquired immunity at the intestinal mucosal surface to support a microenvironment for protection against enteric infections, including ETEC.11 Among these substances, a variety of non-digestible carbohydrates are extensively studied, such as β-glucans.14 The beneficial effects of dietary β-glucans have already been demonstrated. Indeed, these polysaccharides display immunomodulatory effects upon oral administration and are also known to improve growth and general performance of the individual.15-20 Although numerous articles have tried to unravel these immunostimulatory effects, there is no consensus about their mechanism of action. Understanding β-glucan-mediated effects by elucidating the main β-glucan receptor and its signalling pathway in immune cells is important to use these powerful modulating properties in the protection of newly-weaned piglets against enteropathogens. Another strategy to prevent ETEC infections in post-weaning piglets consist of inducing an active mucosal immune response by oral vaccination of piglets. Thereby, it would be interesting to use the immunostimulatory potential of β-glucans. Interestingly, β-glucan microparticles (GPs) were recently described as promising antigen vehicle systems with inherent adjuvant capacity owing to their β-glucans. Moreover, these particles are known for their high antigen encapsulation, efficiency and safety.21-32 Unfortunately, developing oral subunit vaccines has been challenging due to numerous potential obstacles, such as the hostile environment of the gastro-intestinal tract, oral tolerance and the epithelial barrier.33-37 Many approaches have been described to overcome these limitations, including enteric coating, encapsulation in immune-stimulating antigen delivery systems and targeting to endocytotic receptors, located at the apical surface of intestinal epithelial cells.38 Chapter one will provide background information about β-glucans, their receptor usage and signalling, while the second chapter is focused on oral vaccination strategies and the potential role of β-glucans as both mucosal adjuvants and antigen vehicle system. Different methods for targeting particles to endocytotic receptors are discussed in the second chapter as well

Duality of β-glucan microparticles: antigen carrier and immunostimulants

Designing efficient recombinant mucosal vaccines against enteric diseases is still a major challenge. Mucosal delivery of recombinant vaccines requires encapsulation in potent immunostimulatory particles to induce an efficient immune response. This paper evaluates the capacity of beta-glucan microparticles (GPs) as antigen vehicles and characterizes their immune-stimulatory effects. The relevant infectious antigen FedF was chosen to be loaded inside the microparticles. The incorporation of FedF inside the particles was highly efficient (roughly 85%) and occurred without antigen degradation. In addition, these GPs have immunostimulatory effects as well, demonstrated by the strong reactive oxygen species (ROS) production by porcine neutrophils upon their recognition. Although antigen-loaded GPs still induce ROS production, antigen loading decreases this production by neutrophils for reasons yet unknown. However, these antigen-loaded GPs are still able to bind their specific beta-glucan receptor, demonstrated by blocking complement receptor 3, which is the major beta-glucan receptor on porcine neutrophils. The dual character of these particles is confirmed by a T-cell proliferation assay. FedF-loaded particles induce a significantly higher FedF-specific T-cell proliferation than soluble FedF. Taken together, these results show that GPs are efficient antigen carriers with immune-stimulatory properties

Single step spray-dried polyelectrolyte microparticles enhance the antigen cross-presentation capacity of porcine dendritic cells

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