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-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. VHH-mediated targeting of antigens to APN to generate protective immunity at the mucosal surface remains to be further validated.
Keywords: Antibody engineering; BioXP; Gibson cloning; Intestinal infection; Library screening; Nanobodies; Nanobody characterization; Production; Protein expression; Recombinant antibody; Single-domain antibodies; VHH; VHH-Fc; Vaccine targeting.
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Conflict of interest statement
Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work and there is no conflict of interest regarding the publication of this paper.
Orally fed seeds producing designer IgAs protect weaned piglets against enterotoxigenic Escherichia coli infection.Proc Natl Acad Sci U S A. 2013 Jul 16;110(29):11809-14. doi: 10.1073/pnas.1301975110. Epub 2013 Jun 25. Proc Natl Acad Sci U S A. 2013. PMID: 23801763 Free PMC article.
Oral Delivery of Probiotics Expressing Dendritic Cell-Targeting Peptide Fused with Porcine Epidemic Diarrhea Virus COE Antigen: A Promising Vaccine Strategy against PEDV.Viruses. 2017 Oct 25;9(11):312. doi: 10.3390/v9110312. Viruses. 2017. PMID: 29068402 Free PMC article.
Neutralization of Clostridium difficile toxin B with VHH-Fc fusions targeting the delivery and CROPs domains.PLoS One. 2018 Dec 12;13(12):e0208978. doi: 10.1371/journal.pone.0208978. eCollection 2018. PLoS One. 2018. PMID: 30540857 Free PMC article.
Crossing the barrier: Targeting epithelial receptors for enhanced oral vaccine delivery.J Control Release. 2012 Jun 28;160(3):431-9. doi: 10.1016/j.jconrel.2012.02.006. Epub 2012 Feb 13. J Control Release. 2012. PMID: 22353618 Review.
Induction of mucosal immune responses and protection against enteric viruses: rotavirus infection of gnotobiotic pigs as a model.Vet Immunol Immunopathol. 2002 Sep 10;87(3-4):147-60. doi: 10.1016/s0165-2427(02)00046-6. Vet Immunol Immunopathol. 2002. PMID: 12072229 Free PMC article. Review.