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

The mucosa-binding molecules cholera toxin (CT) from Vibrio cholerae and he at-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 impo rtant to study whether their non-toxic mucosa-binding B subunits, CTB and L TB, can be used as alternative safe mucosal adjuvants and/or carrier molecu les. We have as a model protein antigen used human gammaglobulin (HGG) for admixture with or chemical conjugation to recombinantly produced CTB and LT B, respectively, and measured antigen-specific local secretory IgA antibodi es in saponin extracts from intestine and lung tissue by ELISA following in tra-nasal(i.n.) or per-oral (p.o.) immunization. The results show that loca l antibody formation against HGG was increased after immunization with conj ugated as compared to free HGG. However, while the conjugates alone gave ri se 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 am ount of free CT/LT as adjuvant was needed to induce a significant immune re sponse in the intestine after p.o. immunization. We also found that followi ng i.n. immunization, the addition of CTB to HGG, without coupling, increas ed the mucosal immune response to some extent, indicating that CTB by itsel f call work as an adjuvant by the i.n. route of immunization. A striking fi nding was that, as a carrier, CTB was superior to LTB when the conjugates w ere used by the oral but not by the i.n. route of immunization. In conclusi on, conjugation of an antigen to mucosa-binding molecules such as CTB and/o r LTB can dramatically increase their mucosal immunogenicity. This approach may thus be useful in the preparation of mucosal vaccines.