Cholera toxin activates nonconventional adjuvant pathways that induce protective CD8 T-cell responses after epicutaneous vaccination

Abstract

The ability to induce humoral and cellular immunity via antigen delivery through the unbroken skin (epicutaneous immunization, EPI) has immediate relevance for vaccine development. However, it is unclear which adjuvants induce protective memory CD8 T-cell responses by this route, and the molecular and cellular requirements for priming through intact skin are not defined. We report that cholera toxin (CT) is superior to other adjuvants in its ability to prime memory CD8 T cells that control bacterial and viral challenges. Epicutaneous immunization with CT does not require engagement of classic toll-like receptor (TLR) and inflammasome pathways and, surprisingly, is independent of skin langerin-expressing cells (including Langerhans cells). However, CT adjuvanticity required type-I IFN sensitivity, participation of a Batf3-dependent dendritic cell (DC) population and engagement of CT with suitable gangliosides. Chemoenzymatic generation of CT-antigen fusion proteins led to efficient priming of the CD8 T-cell responses, paving the way for development of this immunization strategy as a therapeutic option. M ost current vaccination methods involve intramuscular or intradermal injection of antigen with suitable adjuvants. However, this approach produces biohazardous needle waste, requires trained personnel for its implementation, and evokes needle phobia, a significant complication that reduces compliance (1). Transdermal or epicutaneous immunization (EPI) strategies aim to avoid these concerns through application of antigen and adjuvants to the unbroken skin. This approach yields both antibody and T-cell responses, including priming of CD8 T cells (2-5). However, little is known about the capacity of EPI to prime memory CD8 T cells capable of protection against pathogen challenge, nor do we understand how adjuvants operate when applied to the intact skin.Several adjuvants can induce CD8 T-cell priming through EPI. These include toll-like receptor (TLR) agonists such as imiquimod and CpG (6-9), but also the ADP ribosylating bacterial exotoxins such as cholera toxin (CT) and the closely related Escherichia coli heat-labile enterotoxin (LT) (10-12). Application of such toxins to the skin is safe and effective in priming humoral and cellular responses in both mice and humans (4, 10, 13-15). However, previous studies failed to define which adjuvants afford optimal priming of CD8 T-cell responses and durable protective memory. Furthermore, whereas TLR pathways are well characterized, the basis for adjuvanticity of bacterial exotoxins remains mysterious.Here we compared multiple adjuvants for epicutaneous priming and found that CT was superior in effective induction of CD8 T-cell responses, resulting in protective immunity against pathogen challenge. We find that CT-mediated adjuvanticity occurs in the absence of typical TLR and inflammasome signaling pathways and that langerin-expressing cells (including Langerhans cells) are dispensable for EPI using CT. The adjuvant properties of CT were, however, dependent on host sensi...

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