Active immunization is an emerging potential modality to combat fatal overdose amid the opioid epidemic. In this study, we described the design, synthesis, formulation, and animal testing of an efficacious vaccine against fentanyl. The vaccine formulation is composed of a novel fentanyl hapten conjugated to tetanus toxoid (TT) and adjuvanted with liposomes containing monophosphoryl lipid A adsorbed on aluminum hydroxide. The linker and hapten N -phenyl- N -(1-(4-(3-(tritylthio)propanamido)phenethyl)piperidin-4-yl)propionamide were conjugated sequentially to TT using amine- N -hydroxysuccinimide-ester and thiol–maleimide reaction chemistries, respectively. Conjugation was facile, efficient, and reproducible with a protein recovery of >98% and a hapten density of 30–35 per carrier protein molecule. In mice, immunization induced high and robust antibody endpoint titers in the order of >10 6 against the hapten. The antisera bound fentanyl, carfentanil, cyclopropyl fentanyl, para -fluorofentanyl, and furanyl fentanyl in vitro with antibody-drug dissociation constants in the range of 0.36–4.66 nM. No cross-reactivity to naloxone, naltrexone, methadone, or buprenorphine was observed. In vivo , immunization shifted the antinociceptive dose–response curve of fentanyl to higher doses. Collectively, these preclinical results showcased the desired traits of a potential vaccine against fentanyl and demonstrated the feasibility of immunization to combat fentanyl-induced effects.
Glycosphingolipids are essential biomolecules widely distributed across biological kingdoms yet remain relatively underexplored owing to both compositional and structural complexity. While the glycan head group has been the subject of most studies, there is paucity of reports on the lipid moiety, particularly the location of unsaturation. In this paper, ozone-induced dissociation mass spectrometry (OzID-MS) implemented in a traveling wave-based quadrupole time-of-flight (Q-ToF) mass spectrometer was applied to study unsaturated glycosphingolipids using shotgun approach. Resulting high resolution mass spectra facilitated the unambiguous identification of diagnostic OzID product ions. Using [M+Na]+ adducts of authentic standards, we observed that the long chain base and fatty acyl unsaturation had distinct reactivity with ozone. The reactivity of unsaturation in the fatty acyl chain was about eight-fold higher than that in the long chain base, which enables their straightforward differentiation. Influence of the head group, fatty acyl hydroxylation and length of fatty acyl chain on the oxidative cleavage of double bonds was also observed. Application of this technique to bovine brain galactocerebrosides revealed co-isolated isobaric and regioisomeric species which otherwise would be incompletely identified using contemporary collision-induced dissociation (CID) alone. These results highlight the potential of OzID-MS in glycosphingolipids research, which not only provides complementary structural information to existing CID technique but also facilitates de novo structural determination of these complex biomolecules.
The Picornaviridae are a diverse family of positive-strand RNA viruses that includes numerous human and veterinary pathogens 1 . Among these, hepatitis A virus (HAV), a common cause of acute hepatitis in humans, is unique in that it is hepatotropic and released from hepatocytes without lysis in small vesicles resembling exosomes 2 , 3 . These quasi-enveloped virions (eHAV) are infectious and the only form of virus detected in blood during acute infection 2 . By contrast, non-enveloped, naked virions (nHAV) are shed in feces, stripped of membranes by bile salts during passage through bile ducts to the gut 4 . How these two distinct types of infectious hepatoviruses enter cells to initiate infection is enigmatic. Here we describe a genome-wide forward screen that identified glucosylceramide synthase (UGCG) and other components of the ganglioside synthetic pathway as crucial host factors required for cellular entry by hepatoviruses. We show that gangliosides, preferentially disialogangliosides, function as essential endolysosome receptors required for infection by both naked and quasi-enveloped virions. In the absence of gangliosides, both virion types are efficiently internalized through endocytosis, but capsids fail to uncoat and accumulate within LAMP1 + endolysosomes. Gangliosides relieve this block, binding the capsid at low pH and facilitating a late step in entry involving uncoating and delivery of the RNA genome to the cytoplasm. These results reveal an atypical cellular entry pathway for hepatoviruses that is unique among picornaviruses.
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