Mucosal and systemic antibody responses after peroral or intranasal immunization: Effects of conjugation to enterotoxin B subunits and/or of co‐administration with free toxin as adjuvant

Abstract

The mucosa-binding molecules cholera toxin (CT) from Vibrio cholerae and heat-labile enterotoxin (LT) from Escherichia coli have previously been used as mucosal adjuvants and carriers for many types of antigen. However, since these molecules are toxic and cannot be used in human vaccines, it is important to study whether their non-toxic mucosa-binding B subunits, CTB and LTB, can be used as alternative safe mucosal adjuvants and/or carrier molecules. We have as a model protein antigen used human gammaglobulin (HGG) for admixture with or chemical conjugation to recombinantly produced CTB and LTB, respectively, and measured antigen-specific local secretory IgA antibodies in saponin extracts from intestine and lung tissue by ELISA following intra-nasal (i.n.) or per-oral (p.o.) immunization. The results show that local antibody formation against HGG was increased after immunization with conjugated as compared to free HGG. However, while the conjugates alone gave rise to significant immune responses in the lung and also, to a lesser degree, in the intestine after i.n. immunization, co-administration of a small amount of free CT/LT as adjuvant was needed to induce a significant immune response in the intestine after p.o. immunization. We also found that following i.n. immunization, the addition of CTB to HGG, without coupling, increased the mucosal immune response to some extent, indicating that CTB by itself can work as an adjuvant by the i.n. route of immunization. A striking finding was that, as a carrier, CTB was superior to LTB when the conjugates were used by the oral but not by the i.n. route of immunization. In conclusion, conjugation of an antigen to mucosa-binding molecules such as CTB and/or LTB can dramatically increase their mucosal immunogenicity. This approach may thus be useful in the preparation of mucosal vaccines.

Nanogram Doses of Alum-Adjuvanted HBs Antigen Induce Humoral Immune Response in Mice When Orally Administered

“…No humoral response was observed when mice were fed on seven consecutive days with plant-based HBsAg in a total dose of 1 lg (data not shown), which also suggests that multiple and high-dose antigen oral administration is conducive to tolerance rather than immunity. The importance of low dosage and extended timing for oral immunization could also be confirmed for CT-B or LT-B which, despite being soluble proteins and untypical mucosal antigens, are absorbed by M cells thanks to their ganglioside-binding properties and then elicit an immune response in the intestinal mucosa (Holmgren et al 1994;Rask et al 2000). Some boost effect was observed when CT-B was delivered as late as 40 days post priming (Arakawa et al 1998).…”

Chimeras of Labile Toxin One and Cholera Toxin Retain Mucosal Adjuvanticity and Direct Th Cell Subsets Via Their B Subunit

“…This possibility is currently under investigation. The CT-B conjugated to an Ag was recently shown to more effectively enhance Ag-specific immunity than did LT-B-Ag conjugates after oral administration (23). The mechanism underlying this difference in adjuvant activity is still unknown.…”

“…In addition, studies have now shown that neither CT-B nor LT-B enhances immune responses to mucosally coadministered protein Ags (2,14,21,22). However, some reports have suggested that CT-B and LT-B display mucosal adjuvant activity when large doses are given with proteins by the nasal route (2,13,23,24) or when enterotoxin B subunits are directly conjugated to the Ag itself (2,23). Previous studies have shown that nCT as adjuvant elicits potent mucosal and systemic CD4…”

Vibrio cholerae and Cholera biotypes

“…This additive boosts short-term protection against V. cholerae infection (Clemens et al, 1989), acting as a potent mucosal immunogen and as an immunoadjuvant (Rask et al, 2000). The two major types of oral vaccines against cholera are the killed whole cell-based and the genetically attenuated live vaccines (Shin et al, 2011).…”