LTA1 and dmLT enterotoxin-based proteins activate antigen-presenting cells independent of PKA and despite distinct cell entry mechanisms

Abstract

Enterotoxin-based proteins are powerful manipulators of mucosal immunity. The A1 domain of heat-labile enterotoxin from E. coli, or LTA1, is a newer adjuvant from this family under investigation for intranasal vaccines. Although LTA1 has been examined in mouse vaccination studies, its ability to directly stimulate immune cells compared to related adjuvant proteins has not been well explored. Here, we perform the first rigorous examination of LTA1's effect on antigen presenting cells (APC) using a human monocyte cell line THP-1. To better understand LTA1's stimulatory effects, we compared it to dmLT, or LT-R192G/L211A, a related AB 5 adjuvant in clinical trials for oral or parenteral vaccines. LTA1 and dmLT both activated APCs to upregulate MHC-II (HLA-DR), CD86, cytokine secretion (e.g., IL-1β) and inflammasome activation. The effect of LTA1 on surface marker changes (e.g., MHC-II) was highly dose-dependent whereas dmLT exhibited high MHC-II expression regardless of dose. In contrast, cytokine secretion profiles were similar and dose-dependent after both LTA1 and dmLT treatment. Cellular activation by LTA1 was independent of ganglioside binding, as pretreatment with purified GM1 blocked the effect of dmLT but not LTA1. Unexpectedly, while activation of the inflammasome and cytokine secretion by LTA1 or dmLT was blocked by the protein kinase A inhibitor H89 (similar to previous reports), these responses were not inhibited by a more specific PKA peptide inhibitor or antagonist; thus Indicating that a novel and unknown mechanism is responsible for inflammasome activation and cytokine secretion by LT proteins. Lastly, LTA1 stimulated a similar cytokine profile in primary human monocytes as it did in THP1 cells, including IL-1β, IL-6, IL-8, MIP-1α, MIP-1β, and TNFα. Thus, we report that LTA1 protein programs a dendritic cell-like phenotype in APCs similar to dmLT in a mechanism that is independent of PKA activation and GM1 binding and entry.

Mucosal vaccines — fortifying the frontiers

“…Interaction of cholera toxin with GM1 on gut dendritic cells is required for its oral adjuvanticity 146 , although the precise mechanisms of action are not fully resolved. NF-κB activation was required for the adjuvanticity of mmCT, with cyclic AMP–protein kinase A (cAMP–PKA) signalling proposed to be required for NF-κB activation in mmCT-stimulated dendritic cells in vitro 186 , although PKA may be dispensable for dendritic cell activation by dmLT 187 . Both dmLT and mmCT required cAMP–PKA-dependent inflammasome activation to promote human T H 17-type responses 188 .…”

L-DBF Elicits Cross Protection Against Different Serotypes of Shigella spp

“…Th17 responses are known to be especially important for protection against mucosal pathogens, including Shigella (13,14). Unfortunately, recent studies showed that dmLT, when delivered IN, can cause Bell's palsy (11,15,16), however, the development of Bell's palsy from dmLT is related to the ability of the B subunit to bind to the gangliosides of neuron cells. It is the LTA1 (heat-labile enterotoxin A1) portion of the A subunit that is responsible for generating the Th17 response (15,17,18).…”

“…Unfortunately, recent studies showed that dmLT, when delivered IN, can cause Bell's palsy (11,15,16), however, the development of Bell's palsy from dmLT is related to the ability of the B subunit to bind to the gangliosides of neuron cells. It is the LTA1 (heat-labile enterotoxin A1) portion of the A subunit that is responsible for generating the Th17 response (15,17,18). To simplify and lower the costs of producing a Shigella spp.…”

Fentanyl conjugate vaccine by injected or mucosal delivery with dmLT or LTA1 adjuvants implicates IgA in protection from drug challenge

“…In addition, dmLT enhanced antibody responses to conjugate antigens using a polysaccharide-protein conjugate against V. cholerae 47 . We have also developed a B-subunit free adjuvant called LTA1 based on the A1 domain 39 , 48 , 49 . LTA1 was specifically developed for safe intranasal (IN) use to overcome the risk of Bell’s palsy with AB 5 LT proteins 48 , 50 , 51 .…”

“…LTA1 was specifically developed for safe intranasal (IN) use to overcome the risk of Bell’s palsy with AB 5 LT proteins 48 , 50 , 51 . Like dmLT, LTA1 also activates antigen presenting cells 49 and stimulates immunity to parenteral injection of vaccine antigen 52 . However, the ability of dmLT, LTA1, or any bacterial-enterotoxin based adjuvant for vaccines targeting substances associated with use disorders has not been explored (though the cholera toxin binding B-subunit has been used as a carrier for cocaine vaccines 16 ).…”