Many pathogens invade the host at the intestinal surface. To protect against these enteropathogens, the induction of intestinal secretory IgA (SIgA) responses is paramount. While systemic vaccination provides strong systemic immune responses, oral vaccination is the most efficient way to trigger protective SIgA responses. However, the development of oral vaccines, especially oral subunit vaccines, is challenging due to mechanisms inherent to the gut. Oral vaccines need to survive the harsh environment in the gastrointestinal tract, characterized by low pH and intestinal proteases and need to reach the gut-associated lymphoid tissues, which are protected by chemical and physical barriers that prevent efficient uptake. Furthermore, they need to surmount default tolerogenic responses present in the gut, resulting in suppression of immunity or tolerance. Several strategies have been developed to tackle these hurdles, such as delivery systems that protect vaccine antigens from degradation, strong mucosal adjuvants that induce robust immune responses and targeting approaches that aim to selectively deliver vaccine antigens towards specific immune cell populations. In this review, we discuss recent advances in oral vaccine design to enable the induction of robust gut immunity and highlight that the development of next generation oral subunit vaccines will require approaches that combines these solutions.
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.
To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.