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Adjuvants are often essential additions to vaccines that enhance the activation of innate immune cells, leading to more potent and protective T and B cell responses. Only a few vaccine adjuvants are currently used in approved vaccine formulations in the United States. Combinations of one or more adjuvants have the potential to increase the efficacy of existing and next-generation vaccines. In this study, we investigated how the nontoxic double mutant Escherichia coli heat-labile toxin R192G/L211A (dmLT), when combined with the TLR4 agonist monophosphoryl lipid A (MPL-A), impacted innate and adaptive immune responses to vaccination in mice. We found that the combination of dmLT and MPL-A induced an expansion of Ag-specific, multifaceted Th1/2/17 CD4 T cells higher than that explained by adding responses to either adjuvant alone. Furthermore, we observed more robust activation of primary mouse bone marrow–derived dendritic cells in the combination adjuvant–treated group via engagement of the canonical NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome complex. This was marked by a multiplicative increase in the secretion of active IL-1β that was independent of classical gasdermin D–mediated pyroptosis. Moreover, the combination adjuvant increased the production of the secondary messengers cAMP and PGE2 in dendritic cells. These results demonstrate how certain adjuvant combinations could be used to potentiate better vaccine responses to combat a variety of pathogens.

Adjuvants are important mediators of immune responses and enhance the activation of innate immune cells, leading to protective T and B cell responses. Only a few vaccine adjuvants are currently approved for use in the US and, along with the discovery of novel adjuvants, combination adjuvants are likely to have a major impact on future vaccine development. Here, we investigated the ability of both the double mutant Escherichia coli heat labile toxin R192/L211A (dmLT) and the TLR4 agonist monophosphoryl lipid A (MPL-A) to drive innate and adaptative immune responses independently and in combination. We found that the combination adjuvant induces more robust activation of dendritic cells (DCs) than either adjuvant alone, leading to the formation of the NLRP3 inflammasome complex and increased secretion of IL-1β, as well as other hallmark pro-inflammatory cytokines. Furthermore, RNA sequencing and ingenuity pathway analysis support that the combination adjuvant may allow these DCs to polarize a multi-faceted Th1/2/17 CD4 T cell response. To determine how this adjuvant combination might regulate CD4 T cell responses, we intradermally immunized mice with a model T cell antigen in conjunction with dmLT or MPL-A alone or combined and, using MHC class II tetramers, found that vaccination with the combination demonstrated the greatest expansion and activation of endogenous, vaccine-specific CD4+ T cells in all lymphoid tissues assessed which included a Th1/Th17 response. These results reveal how combination adjuvants can be used to not only potentiate better vaccine responses, but to potentially bias the immune response to one that is more capable of combating infection.

Improvements are described in the method of following temperature changes, and thus alterations in vasomotor tone, in exposed mucous membranes. Invention of an applicator holder, by means of which more sure and stable apposition of the thermopile terminals to the mucous surface may be effected has been the chief advance. Minor improvements have been the use of a saliva ejector and of better calibration technique. The palatine tonsils, like the palate, pharynx, and skin, react to chilling of the body surface with reflex vasoconstriction and ischemia. On rewarming the subject the tonsils quickly more than recover their former blood supply, actually becoming hyperemic; the skin returns to about its control condition; the pharynx and palate remain somewhat ischemic. The hypothesis is advanced that one factor in the beneficial hardening effect of cold bathing and outdoor living, with its incident heightened immunity to respiratory infection, may be the training of the vasomotor system in the direction of development in the pharynx of a reaction of hyperemia following chilling, similar to that observed in the tonsils of the present subjects. With inhalation of amyl nitrite, the skin temperature has always shown a sharp transient rise. The mucous membrane, if relatively ischemic, responds by a rise corresponding to the skin flush. If already hyperemic, local vasodilation in the mucous membrane with amyl nitrite is more than counterbalanced by the lowering of the general blood pressure, and the temperature falls. The flora of the pharynx and tonsils, studied by the unsatisfactory method described, showed, in several instances, after experimentation changes apparently due to proliferation of one of the microorganisms already present. In one case Streptococcus hæmolyticus, in one Micrococcus catarrhalis, and in a third Bacillus influenzæ was the organism showing a relative increase in numbers. The first two instances were associated with sore throat, the third with slight constitutional symptoms. The chilling in the experiments in no instance produced albuminuria or glycosuria, although a more dilute urine was apparently excreted during the experiments. A fall in hydrogen ion concentration, referable to the forced respiration, was noted.

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