are the inventors of a patent application based on the findings of this study. dmLT elicited the highest levels of anti-fentanyl antibodies, whereas alum achieved highest levels against the carrier protein. Vaccination with sublingual dmLT or intranasal LTA1 provided the most robust blockade of fentanyl-induced analgesia and CNS penetration correlating strongly to anti-FEN IgA. In conclusion, this study demonstrates dmLT or LTA1 adjuvant as well as mucosal delivery may be attractive strategies for improving the efficacy of vaccines against SUD. Subject terms: Adjuvants, Conjugate vaccines Introduction The United States is in the midst of a nationwide opioid crisis, with an estimated 2.1 million individuals suffering from an opioid use disorder (OUD)1. Particularly alarming are the increasing fatal overdoses resulting from illicit fentanyl (FEN). Higher incidence of drug overdose deaths was Rabbit polyclonal to GHSR proportionally associated with the number of FEN seizures in a given community2. Pyrithioxin FEN is believed to specifically account for nearly 30,000 of the 50,000 deaths involving opioids annually, due to its high potency, ease of manufacturing, and/or addition to other illicit drugs unknowing to the user3C5. Since the 2020 COVID-19 pandemic, there is new evidence that FEN use and opioid overdoses may also be increasing6. FEN and FEN-analogs rapidly penetrate the central nervous system and are 50C100 times more potent than heroin and morphine respectively. Because of this potency as well as its half-life, higher or repeated doses of the mu opioid receptor antagonist naloxone (Narcan?) are needed to be administered quickly to reverse lethal overdoses7. One option to help combat this opioid epidemic is vaccination. A vaccine specifically targeting illicit opioid(s) could attenuate the reinforcing effects of FEN and prevent overdose deaths by preventing the drug from penetrating the CNS, particularly in individuals recovering from OUD during critical periods when relapse is especially common. Such a vaccination strategy would induce serum antibodies that diminish drug activity by assembling an antibody-drug complex that is too large to cross the bloodCbrain barrier, but not prevent other pain medications (e.g., tramadol, morphine) from working. Vaccines for substance use disorders (SUDs) have been under development for decades, including for cocaine, nicotine, heroin, amphetamines, and other synthetic opioids but without commercial Pyrithioxin success and FDA approval8C13. As observed with cocaine and nicotine vaccines in Phase III clinical trials, a lack of sustainable antibody levels and consistent levels of antibodies in vaccinated subjects has been a persistent problem14C18. Yet subjects who attained high levels of antibodies against Pyrithioxin nicotine or cocaine following vaccination showed significantly reduced drug use compared to those that only achieved low levels of antibodies and the placebo group14,19. FEN is not naturally immunogenic, so the drug must be conjugated to a protein in order to create an immunogenic vaccine antigen. Recent animal studies with various opioid conjugate immunogens have demonstrated development of antibodies to FEN or heroin resulting in decreasing potency of heroin and FEN distribution to the brain after experimental exposure, reducing the drug-induced antinociception (or blocking of pain)9C13,20C24. However, a frequent problem of these conjugate vaccines is still short duration of immunity and/or insufficient antibody magnitudes even with an added adjuvant. As recently reviewed25, no single adjuvant has emerged as ideal for vaccines against SUDs, though alum, TLR-based agonist, or combinations thereof have been tested. Alum, or aluminum salts, are the most commonly used adjuvants in licensed vaccines; evaluations of alum in drug abuse or addiction vaccines thus far has had limited success in inducing an enduring antibody response13,25C27. Few studies have shown efficacy for routes other than injected delivery (e.g., by intramuscular (IM), subcutaneous, and intraperitoneal routes), with a few exceptions in vaccines for cocaine28. Mucosal vaccination has an advantage of needle-free delivery, lack of injection-related infections, possibility of non-medical personnel administration or self-administration, and induction of strong systemic and mucosal immunity29C31. In addition, mucosal vaccination can alter the quality of antibody isotypes, particularly IgA, and may better protect against mucosal drug exposures, (e.g., snorting or smoking), than parenteral vaccination. A newer class Pyrithioxin of adjuvants becoming developed are derived from heat-labile enterotoxins from (LT) or ideals between 0.52 and 0.76). However, limit of detection, background (estimated from na?ve.