Circular Permutation of the <i>E. coli</i> Heat-Labile Enterotoxin Pentameric B Subunit for Mucosal Vaccine Adjuvant Design

The heat-labile enterotoxins (LTs) of <i>Escherichia coli</i> structurally belong to the AB₅ toxin, which consists of an A subunit and a pentameric B subunit. LTs have been extensively reported for their potent mucosal adjuvant activities. The B subunit of type II LT (LT-IIb-B₅) recognizes the GD1a ganglioside, which is associated with the internalization of the toxic A subunit and meanwhile involved in the recognition of the Toll-like receptor (TLR-2/1) and downstream NF-κB activation. In developing LT-IIb-B₅ as a safe adjuvant, we intend to prevent concerns derived from the intrinsic toxicity of LT. A strategy of circular permutation (CP) is used to design a new LT-IIb-B₅ pentamer. By replacing the backbone opening at the GD1a-binding site, the GD1a-binding site becomes deficient and GD1a-binding ability is removed, yet TLR-2/1 activation is maintained. The novel LT-IIb-B₅ pentamer has no GD1a-mediated cell toxicity but maintains sufficient immunoreactivity. We identified a potent CP candidate (CP_13-14) with a backbone opening at position 13-14, exhibiting greater protective potential against an influenza viral challenge than the native LT-IIb-B₅ in a mouse model. In the intranasal vaccination using trivalent neuraminidase proteins formulated with LT-IIb-B₅ CP_13-14, we demonstrated complete protection against heterologous H1N1 and H3N2 viral challenge infections. This study purposes for a new design for LT-IIb-B₅ protein adjuvant and addresses the urgent need for a novel, efficient, and safer mucosal adjuvant for respiratory diseases.