Project Summary. In the United States, there are over 2.5 million people diagnosed with opioid use disorder (OUD) and over 47,000 opioid-related fatal overdoses occur annually. Current FDA-approved medications against OUD and overdose consist of opioid receptor agonists and antagonists, which overall show limited efficacy, adverse effects, suboptimal patient access and compliance, abuse liability, and illicit diversion. As an alternative or complimentary strategy to small molecule-based pharmacotherapies, vaccines offer a safe and long-lasting therapeutic or prophylactic strategy to counteract OUD and fatal overdoses. Anti-opioid vaccines have shown proof of efficacy and selectivity in reducing opioid-induced antinociception, respiratory depression, bradycardia, opioid intravenous self-administration, and lethality in animal models. Despite promising pre-clinical data, vaccine clinical efficacy may still be limited to a subset of immunized individuals that achieve optimal antibody responses against the target opioid. Hence, the focus of this proposal is to accelerate translation of anti-opioid vaccines by identifying mechanisms and biomarkers correlated with optimal vaccine-induced protection against opioids. Our group has previously found that blockade of the cytokine interleukin-4 (IL-4) increased vaccine efficacy against oxycodone in mice, supporting the hypothesis that changes in IL-4 signaling modulate or predict vaccine efficacy against opioids. AIM1 tests whether IL-4 modulation underlies cellular and molecular processes involved in activation of B cell lymphocytes and generation of optimal antibody responses against opioids. AIM1 will test affinity maturation, isotype switching, antibody secretion kinetics, and transcriptomic changes in opioid-specific B cells. AIM2 tests whether IL-4 or its downstream signaling components (e.g., IL-4 receptor, STAT6) are predictive biomarkers of vaccine efficacy in both mice and human patients immunized with an oxycodone conjugate vaccine currently in Phase I clinical trials (NCT04458545). Results from these studies will provide a blueprint to develop more effective next-generation vaccine formulations and to identify putative biomarkers predictive of vaccine efficacy against opioids. Such information will accelerate pre-clinical vaccine development and support patient stratification in clinical trials and clinical implementation. Training. Completing the experiments outlined in this proposal will support my training and career goals to become an independent scientist. This hypothesis-driven project proposes multidisciplinary experiments allowing me to develop a broad range of technical skills to study a clinically relevant problem in-depth. I will also be mentored by leaders and experts in the fields of vaccinology, opioid use disorders, and immunology. Additionally, I will have the unique opportunity to gain expertise in translational science and human immunology through an ongoing clinical trial of a ...