Fully synthetic arecoline and arecaidine vaccine candidates with α-Galactosylceramide as built-in adjuvant.

Arecoline and arecaidine, the primary alkaloids in betel nut, are responsible for betel-quid chewing addiction. Vaccination against small molecules is a promising approach but limited by their weak immunogenicity. Invariant NKT cells (iNKT cells) serve as a key bridge between innate and adaptive immunity, providing cognate B cell help and enhancing antibody responses. Herein, we developed two fully synthetic, structurally defined arecoline (Arec) and arecaidine (Areca) vaccine candidates with NKT cell agonist α-Galactosylceramide (αGalCer) as a built-in adjuvant. Both these vaccine candidates efficiently promoted class switching from IgM to IgG, with a response dominated by the IgG1 and IgG3 subclasses. Cross-reactivity analysis found that antibodies elicited by Arec-αGalCer recognized both arecoline and guvacoline but with reduced affinity for the latter. Meanwhile, antibodies induced by Areca-αGalCer specifically recognized arecaidine, and the affinity was comparable to that of Arec-αGalCer antibodies for arecoline. In vivo, Arec-αGalCer vaccination caused a modest mitigation of arecoline-induced hypothermia. In addition, a mixed vaccine formulation elicited a broader serological response than the single vaccine.