The Macrophage-Inducible C-type Lectin receptor (Mincle) plays a critical role in innate immune recognition and pathology, and therefore represents a promising target for vaccine adjuvants. Innovative trehalose-based Mincle agonists with improved pharmacology and potency may prove useful in the development of Th17-mediated adaptive immune responses. Herein, we report on in vitro and in silico investigations of specific Mincle ligand–receptor interactions required for the effective receptor engagement and activation of Th17-polarizing cytokines. Specifically, we employed a library of trehalose benzoate scaffolds, varying the degree of aryl lipidation and regiochemistry that produce inflammatory cytokines in a Mincle-dependent fashion. In vitro interleukin-6 (IL-6) cytokine production by human peripheral blood mononuclear cells (hPBMCs) indicated that the lipid regiochemistry is key to potency and maximum cytokine output, with the tri-substituted compounds inducing higher levels of IL-6 in hPBMCs than the di-substituted derivatives. Additionally, IL-6 production trended higher after stimulation with compounds that contained lipids ranging from five to eight carbons long, compared to shorter (below five) or longer (above eight) carbon chains, across all the substitution patterns. An analysis of the additional cytokines produced by hPBMCs revealed that compound 4d, tri-substituted and five carbons long, induced significantly greater levels of interleukin-1β (IL-1β), tumor necrosis factor- α (TNF-α), interleukin-23 (IL-23), and interferon- γ (IFN-γ) than the other compounds tested in this study. An in silico assessment of 4d highlighted the capability of this analogue to bind to the human Mincle carbohydrate recognition domain (CRD) efficiently. Together, these data highlight important structure–activity findings regarding Mincle-specific cytokine induction, generating a lead adjuvant candidate for future formulations and immunological evaluations.

In the United States, the majority of drug fatal overdoses are attributed to synthetic opioids such as fentanyl and its analogs. The opioid receptor antagonist naloxone is the leading treatment for opioid overdose, but it is limited in its ability to reverse overdoses of long‐acting, highly potent opioids, and can only be administered post‐exposure. Vaccines consisting of fentanyl‐based haptens conjugated to immunogenic carrier proteins offer a novel strategy to combat fentanyl use disorder and overdose as they can be administered both prophylactically and therapeutically, and have the potential to offer long‐lasting protection against fentanyl's pharmacological effects and overdose in pre‐clinical models. Anti‐fentanyl vaccines stimulate the immune system to produce fentanyl‐specific antibodies which sequester the drug in serum and prevent fentanyl distribution to the brain. Vaccination selectively reduces fentanyl‐induced bradycardia, respiratory depression, antinociception, and fentanyl self‐administration. To improve the clinical potential of anti‐drug vaccines, adjuvants can be added to stimulate the immune system to increase the quality and quantity of the antibody response against the target drug. In this study, mice (n=6/group) and rats (n=6/group) were vaccinated with a lead anti‐fentanyl vaccine (F‐CRM) formulated in a series of Toll‐like receptor (TLR) agonists. After immunization, mice and rats were challenged with fentanyl (0.05‐0.45 mg/kg). Antinociception, bradycardia, respiratory depression, and distribution of fentanyl in the blood and brain were measured post‐challenge. Selected TLR agonists increased the vaccine's ability to induce fentanyl‐specific antibodies in both mice and rats, and increased its efficacy in reducing fentanyl‐induced effects. Co‐administration of F‐CRM formulated in selected TLR adjuvants also offered greater protection against fentanyl‐induced respiratory arrest induced by overdose. These data support further development of anti‐fentanyl vaccines formulated in novel TLR adjuvants and warrant future studies exploring their applicability to other vaccine formulations against substance use disorders.

The U.S. opioid epidemic is an extraordinary public health crisis that started in 1990 and significantly accelerated in the last decade. Since 2020, over 100,000 fatal drug overdoses have been reported annually, and 75% of those involved fentanyl and its analogs (F/FA). Accelerating the translation of innovative, effective, and safe treatments is needed to augment existing measures to counteract such a crisis. Active immunization against F/FA and other opioids represents a promising therapeutic and prophylactic strategy for opioid use disorder (OUD) and opioid-induced overdose toxicity. Previously we demonstrated that the anti-fentanyl vaccine comprising a fentanyl-based hapten (F) conjugated to the diphtheria cross-reactive material (CRM), admixed with the novel lipidated toll-like receptor 7/8 (TLR7/8) agonist INI-4001 adsorbed on Alhydrogel ^® (alum) induced high-affinity fentanyl-specific polyclonal antibodies that protected against fentanyl-induced pharmacological effects in mice, rats, and mini-pigs. Here, INI-4001 was formulated into liposomes with different surface charges, and their impact on F-CRM adsorption, INI-4001 adjuvanticity, and vaccine efficacy were explored. Additionally, as the role of innate immunity in mediating the efficacy of addiction vaccines is largely unknown, we tested these formulations on the activation of innate immunity in vitro . Cationic INI-4001 liposomes surpassed other liposomal and aluminum-based formulations of INI-4001 by enhancing the efficacy of fentanyl vaccines and protecting rats against bradycardia and respiratory depression by blocking the distribution of fentanyl to the brain. Fentanyl vaccines adjuvanted with either cationic INI-4001 liposomes or the aqueous INI-4001 adsorbed to alum induced significant surface expression of co-stimulatory molecules and maturation markers in a murine dendritic cell line (JAWS II), while the former was superior in enhancing the macrophages surface expression of CD40, CD86 and inducible nitric oxide synthase (iNOS), indicative of maturation and activation. These results warrant further investigation of liposome-based formulations of TLR7/8 agonists for improving the efficacy of vaccines targeting F/FA and other opioids of public health interest. Graphical