Enterotoxigenic Escherichia coli (ETEC) are an important pathogen for humans and farm animals, like pigs. Porcine ETEC strains induce diarrhea through the production of heat-labile enterotoxin (LT) and/or heat-stable enterotoxins (pSTa/STb). Although LT secretion levels differ between porcine ETEC strains and this has been linked to virulence, it is unclear whether ST secretion levels also differ between porcine ETEC strains. In addition, the molecular mechanism underlying different LT secretion levels has not been elucidated. In this work, multiple porcine ETEC strains were assessed for their capacity to produce and secrete the enterotoxins LT, pSTa and STb. The strains differed greatly in their capacity to secrete LT, pSTa and STb. Remarkably, in some strains periplasmic production did not correlate with their ability to secrete LT, resulting in high periplasmic production and low LT secretion levels. Furthermore, the results indicated that the type two secretion system protein yghG plays a regulatory role in controlling LT secretion levels. These findings highlight yghG as an important mediator for the secretion of heat-labile enterotoxin LT by porcine ETEC strains and provide better insights in ETEC enterotoxin secretion. Importance Enterotoxigenic E. coli are a major health concern. Enterotoxins secreted by enterotoxigenic E. coli are crucial for diarrhea induction. Enterotoxin secretion levels differ between strains; however, it is currently unclear what drives these differences. The discrepancy in production and secretion capacity of enterotoxins in ETEC is important to clarify their function involved in diarrhea induction. Our results further deepen our understanding of how type II secretion system (T2SS) components of ETEC control enterotoxin secretion levels and may lay the foundation for a better understanding of ETEC molecular pathogenesis.

Targeting a vaccine to the mucosal surface has recently been recognized as a promising approach to efficiently induce mucosal immune responses against enteric pathogens. However, poor uptake and inefficient transport of orally delivered subunit vaccines across the intestinal epithelium combined with weak immune responses still present important bottlenecks for mucosal vaccination. A possible strategy suggested to surmount these hurdles is to target the selected antigen to transcytotic receptors, such as aminopeptidase N (APN) present on enterocytes and antigen-presenting cells (APCs). Therefore, we aimed to identify potent and selective VHHs against porcine aminopeptidase N (pAPN), that were fused to the fragment crystallizable (Fc) domain of the murine IgG2a, resulting in dimeric VHH-MG fusions. Out of a library of 30 VHH-2 MG fusion candidates, two fusions displaying the best binding on pAPN-expressing cells were selected and showed in vivo internalization across the porcine gut epithelium. One of these fusions triggered systemic and intestinal IgA responses upon oral administration. Our results demonstrate the potential of bivalent VHH-MG fusions as delivery vehicles for vaccine antigens. VHHmediated and APN-targeted antigens to generate protective immunity at the mucosal surface remains to be further validated.

Many pathogens enter the host via the gut, causing disease in animals and humans. A robust intestinal immune response is necessary to protect the host from these gut pathogens. Despite being best suited for eliciting intestinal immunity, oral vaccination remains a challenge due to the gastrointestinal environment, a poor uptake of vaccine antigens by the intestinal epithelium and the tolerogenic environment pervading the gut. To improve uptake, efforts have focused on targeting antigens towards the gut mucosa. An interesting target is aminopeptidase N (APN), a conserved membrane protein present on small intestinal epithelial cells shown to mediate epithelial transcytosis. Here, we aimed to further optimize this oral vaccination strategy in a large animal model. Porcine APN-specific monoclonal antibodies were generated and the most promising candidate in terms of epithelial transcytosis was selected to generate antibody fusion constructs, comprising a murine IgG1 or porcine IgA backbone and a low immunogenic antigen: the F18-fimbriated E. coli tip adhesin FedF. Upon oral delivery of these recombinant antibodies in piglets, both mucosal and systemic immune responses were elicited. The presence of the FedF antigen however appeared to reduce these immune responses. Further analysis showed that F18 fimbriae were able to disrupt the antigen presenting capacity of intestinal antigen presenting cells, implying potential tolerogenic effects of FedF. Altogether, these findings show that targeted delivery of molecules to epithelial aminopeptidase N results in their transcytosis and delivery to the gut immune systems. The results provide a solid foundation for the development of oral subunit vaccines to protect against gut pathogens.