Abstract
Enterotoxigenic Escherichia coli (ETEC) infections have been identified as a major cause of acute diarrhoea in children in developing countries, associated with substantial morbidity and mortality rates. Additionally, ETEC remains the most common cause of acute diarrhea of international travellers to endemic areas. The heat-labile toxin (LT) is a major virulence factor of ETEC, with a significant correlation between the presence of antibodies against LT and protection in infected patients. In the present work, we constructed a recombinant LTB unit (rLTB) and studied the capacity of this toxoid incorporated in microneedles (rLTB-MN) to induce a specific immune response in mice. MN were prepared from aqueous blends of the polymer Gantrez AN® [poly (methyl vinyl ether-co-maleic anhydride)], which is not cytotoxic and has been shown to possess immunoadjuvant properties. The mechanical and dissolution properties of rLTB-MNs were evaluated in an in vitro Parafilm M® model and in mice and pig skin ex vivo models. The needle insertion ranged between 378 µm and 504 µm in Parafilm layers, and MNs fully dissolved within 15 min of application inside porcine skin. Moreover, female and male BALB/c mice were immunized through ear skin with one single dose of 5 μg·rLTB in MNs, eliciting significant fecal anti-LT IgA antibodies, higher in female than in male mice. Moreover, we observed an enhanced production of IL-17A by spleen cells in the immunized female mice, indicating a mucosal non-inflammatory and neutralizing mediated response. Further experiments will now be required to validate the protective capacity of this new rLTB-MN formulation against this deadly non-vaccine-preventable disease.
Highlights
Once the bacteria colonizes the small intestine via adhesins, Enterotoxigenic Escherichia coli (ETEC) produces enterotoxins that lead to diarrhea
Coding LTB was cloned into a pET21 vector for expression of recombinant LTB unit (rLTB) in E. coli BL21 (DE3)
Mice of IL-4, IFN-γ, and IL-17 compared to both male mice immunized with rLTB-MN and immunized by intradermal route (Figure mice immunized by intradermal route (Figure 6)
Summary
Enterotoxigenic Escherichia coli (ETEC) strains cause severe diarrheal illness and are a leading cause of death among children under five in low incoming countries [1]. ETEC strains are the leading cause of traveller’s diarrhea [2]. There is a critical need for a vaccine against this pathotype [1]. The development of an effective vaccine against ETEC poses a great challenge, due to its pronounced genetic and antigenic variability [3]. Once the bacteria colonizes the small intestine via adhesins, ETEC produces enterotoxins that lead to diarrhea. Virulent ETEC strains mainly produce heat-labile toxin (LT), which causes the release of electrolytes and the secretion of water, resulting in the typical watery diarrhea [4,5,6].
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