Evaluating single-domain antibodies as carriers for targeted vaccine delivery to the small intestinal epithelium.

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.

Simplified monomeric VHH-Fc antibodies provide new opportunities for passive immunization.

Simplified monomeric monoclonal antibodies consisting of a single-domain VHH, derived from camelid heavy-chain only antibodies, fused with the Fc domain of either IgG (VHH-IgG) or IgA (VHH-IgA) antibodies, are promising therapeutic proteins. These simplified single-gene encoded antibodies are much easier to manufacture and can be produced in plants and in yeast for bulk applications. These merits enable novel passive immunization applications, such as in-feed oral delivery of VHH-IgAs, which have successfully provided protection against a gastrointestinal infection in the piglet model.

A two-amino acid mutation in murine IgA enables downstream processing and purification on staphylococcal superantigen-like protein 7.

With few exceptions, all currently marketed antibody therapeutics are IgG molecules. One of the reasons that other antibody isotypes are less developed are the difficulties associated with their purification. While commercial chromatography affinity resins, like staphylococcal superantigen-like 7 (SSL7) protein-containing resin, allow purification of IgAs from many animal species, these are not useful for murine IgAs. Because the mouse model is predominantly used for preclinical evaluation of IgA-based therapeutics, there is a need to develop an effective purification method for mouse IgA. Here, we adapted the sequence of a mouse IgA by mutating two amino acid residues in the fragment crystallizable (Fc) sequence to facilitate its purification on SSL7 resin. The mutated IgA Fc (hereafter referred to as IgA*) was then genetically fused to the variable domain of a llama heavy chain-only antibody (VHH) directed against the fusion protein of human respiratory syncytial virus (HRSV), resulting in VHH-IgA*, and transiently produced in infiltrated Nicotiana benthamiana leaves. These plant-produced mouse VHH-IgA* fusions were enriched by SSL7 affinity chromatography and were found to be functional in ELISA and could neutralize RSV in vitro, suggesting no detrimental effect of the mutation on their antigen-binding properties. This approach for the purification of murine IgA will facilitate downstream processing steps when designing innovative murine IgA-based fusions.