Hamid, F. A.; Le, N.-M. N.; Song, D.; Amin, H.; Hicks, L.; Bird, S.; Siram, K.; Hoppe, B.; Demeler, B.; Evans, J. T.; Burkhart, D.; Pravetoni, M.

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 (registered trademark) (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.