GM1 Binding-Deficient Exotoxin Is a Potent Noninflammatory Broad Spectrum Intradermal Immunoadjuvant

Abstract

Intradermal (i.d.) immunization is a promising route of vaccine administration. Suitable i.d. adjuvants are important to increase vaccine efficacy in poorly responding populations such as the elderly or for dose-sparing strategies in the face of vaccine shortages. Bacterial exotoxins, such as Escherichia coli heat-labile enterotoxin (LT), exert strong immunostimulatory effects through binding to monosialoganglioside (GM1) cell surface receptors; however, injection is hampered by local inflammation. We demonstrate that the injection of LT formulations deficient in GM1 binding by mutation (LT(G33D)) or in vitro ligand coupling does not cause localized edema and inflammation in mice, yet these formulations retain potent adjuvant activity by enhancing functional Ab and cellular immune responses to coadministered Ags. Complete protection against in vivo lethal tetanus toxin challenge and the induction of Ag-specific CTL responses capable of killing target cells in vivo indicated in vivo efficacy of the induced immune responses. LT(G33D) proved superior to standard alum adjuvant regarding the magnitude and breadth of the induced immune responses. Immunizations in complex ganglioside knockout mice revealed a GM1-independent pathway of LT adjuvanticity. Immunostimulation by i.d. LT(G33D) is explained by its ability to induce migration of activated APCs to the proximal draining lymph nodes. LT(G33D) is a promising candidate adjuvant for human trials of parenteral vaccines in general and for current i.d. vaccine development in particular.

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

“…S8), effectively excluding this possibility. The EPI adjuvant properties of CT thus depend on the availability of the gangliosides capable of binding CT-B on host cells, in contrast to studies using intradermal delivery of CT and LT (30).…”

“…The A subunit displays ADP ribosylation activity, whereas the pentameric B subunit acts as a receptor for cell entry by high-affinity binding to GM1 ganglioside present at the cell surface (10-12) However, one study reported robust intradermal adjuvant activities of both CT and LT occur in the absence of GM1 binding (30), indicating alternative mechanisms through which CT may provoke the immune response. To determine whether the EPI adjuvant effects of CT require recognition of GM1, we utilized mice (GalT −/− ) deficient in the two glycosyltransferases involved in GM1 biosynthesis.…”

Distinctive Immunomodulatory and Inflammatory Properties of the Escherichia coli Type II Heat-Labile Enterotoxin LT-IIa and Its B Pentamer following Intradermal Administration

“…However, when delivered via the i.d. route, LT-I induced intense and persistent inflammatory reactions in mice (44). In contrast, no information is available for the inflammatory effects of LT-II enterotoxins, particularly LT-IIa, when they are administered by the i.d.…”

“…For evaluation of cytokine production, spleen cells were harvested from animals euthanized 2 weeks after the immunization protocol (44). Spleen cells from mice of the same immunization group were pooled (1 ϫ 10 7 viable cells/ml) and cultured in a CO 2 -containing atmosphere at 37°C in the presence or absence of OVA (5 g/ml), the OVA class )-restricted CD4 ϩ T cell epitope synthetic peptide ( 265 TEWTSSNVMEERKIKV 280 , denoted OVA 265-280 ) (1.2 g/ml).…”

“…Nonetheless, while a plethora of studies have characterized the adjuvant properties of LT-I when it is delivered via parenteral routes, particularly with the transcutaneous (3) and intradermal (i.d.) (44) routes, there are no reports describing the use of type II heat-labile toxins by these routes of administration.…”