The aim of this project is the development of a vaccine that could mitigate the effects of an unintended exposure to fentanyl, an event that is responsible for more than 30,000 deaths per year in the United States. The project is a collaboration between two investigators: (1) Ronald G. Crystal, MD, Professor and Chair of Genetic Medicine at Weill Cornell Medicine, a pioneer in the use of disrupted adenovirus capsids as a carrier for small molecule haptens that can raise immune responses against addictive drugs; and (2) Tristan H. Lambert, PhD, Professor and Associate Chair of Chemistry and Chemical Biology at Cornell University, an expert in synthetic organic chemistry whose lab is producing novel analogs of fentanyl for use in rational vaccine design strategies. The project has two aims: (1) produce novel fentanyl analogs; and (2) test the potential of the analogs to elicit a strong humoral immune response capable of altering the phenotype of a fentanyl exposure in a mouse model.  The first aim involves two stages: (1) to optimize the linkage position of novel chemical constructs for conjugation to the disrupted adenovirus capsids; three candidate linkage positions were proposed; and (2) to modify the structure of the fentanyl analog with a goal of raising antibodies with higher affinity; four novel analogs were proposed for this stage. The second aim is the evaluation of the potency of these novel structures by assessing the capacity to evoke high titer anti-fentanyl antibodies, and to assess the ability of the resulting vaccine to block systemically administered fentanyl from gaining access to the brain leading thereby protecting against fentanyl-mediated death. To date, the project has succeeded in synthesizing three fentanyl analogs with the carboxyl conjugation group and alkyl chain extension positioned at three different points in the fentanyl molecule. The carboxy group enables conjugation to disrupted adenovirus under mild conditions and the alkyl chain provides improved flexibility for immune recognition access to the fentanyl analog after the conjugation to the adenovirus capsid proteins. Of the three conjugate variants, analogs A and B successfully demonstrated conjugation to the capsid proteins of disrupted adenovirus constituting viable vaccine candidates. These vaccine candidates were evaluated in parallel with our current candidate fentanyl vaccine that utilizes carfentanil as the covalently-ligated fentanyl analog. Analogs A and B were compared with carfentanil for immunogenicity at a dose of 4 micrograms for each of 3 administrations over 6 weeks. After 8 wk, the anti-fentanyl titers evoked by Analog B were approximately equal to the titers evoked by carfentanil while the titer from Analog A was within a factor of 10. In contrast, anti-fentanyl antibodies evoked by both analog A and analog B demonstrated a higher specificity for fentanyl than carfentanil with analog B showing the highest specificity. Due to the capacity to evoke both higher titer and affinity to fentanyl, analog B was chosen for further characterization. Vaccination with this construct significantly reduce fentanyl-stimulated open field movement as well as reducing the antinociception property of fentanyl in mice in a dose-dependent manner. Further characterization of the first set of fentanyl analogs is ongoing. In addition, the synthesis of the second class of analogs derived from analog B of is underway.