ABSTRACT Hepione Therapeutics, Inc. is a biotech startup with access to a novel, proprietary platform to generate antibodies and vaccines to small molecules and peptides for a wide range of research, diagnostic, and therapeutic needs. The focus of this SBIR is to use this platform to raise antibodies to fentanyl in mice, and to extend from mice to rats our animal model proof-of-concept for a fentanyl vaccine. Our long-term goal is to make an FDA-approved vaccine that conveys long-term immunity to fentanyl overdose. The mortality of fentanyl overdose is not merely restricted to the use of fentanyl, a synthetic opioid 100 times more potent than morphine, or even the use of opioids generally, but rather is a crisis that has profoundly impacted the overdose rates of almost every addictive substance used today. The National Institute on Drug Abuse (NIDA) reports that over 70,000 Americans died of drug overdoses in 2017, a two-fold increase since 2007. Of these, 67% of these deaths involved the use of fentanyl. In the cases where overdoses were attributed to other elicit substances, co-involvement of fentanyl significantly contributed to increased mortality in the last decade. Heroin mortalities saw a 622- fold increase in deaths that involved fentanyl between 2007 and 2017, compared to the 3.1-fold increase in heroin-related overdoses without fentanyl involvement. Psychostimulant overdoses involving fentanyl saw a 73-fold increase, cocaine a 33.1-fold increase, Rx opioids a 9.1-fold increase, and even benzodiazepines and antidepressants saw an 11.2-fold and 4.8-fold increase in fentanyl involved deaths in the last decade, respectively. Medical interventions to prevent fentanyl overdose are desperately needed to combat this rise in fatalities. Just 2-3 mg of fentanyl can be lethal, causing sedation, vomiting, and respiratory depression. Overdose is more common for patients who relapse early in recovery, where abstinence upregulates endogenous opioid receptors and lowers tolerance. Fentanyl overdose is treated with mixed results by opioid blockers such as naloxone (NARCAN®). Patient revival requires multiple doses, often not delivered in enough time. Fentanyl also causes rigidity in the chest wall muscles, rendering CPR chest compressions ineffectual. For these reasons, prophylactic approaches, such as vaccines or immunotherapies, would be highly desirable interventions. We have developed a viable immunotherapy platform to block fentanyl overdose. This platform can be used without adjuvants and at low antigen doses to robustly and reliably generate specific antibodies to small molecule compounds. Our preliminary data establish that this vaccine is wholly protective against fentanyl intoxication in mice. To proceed to clinical trials, we aim to show efficacy in additional animal models, specifically the rat in this application (Aim 1). We also aim to analyze the immune response elicited by the vaccine at the repertoire level, which will allow us to ca...