<p>Opioid use disorder (OUD) remains a global public health concern, exerting a significant economic burden on societies worldwide. To combat the rise of substance use disorder, a complementary vaccine-based intervention was developed to support and strengthen the existing treatment modalities. Hapten-protein conjugate vaccines are employed as a promising novel strategy for neutralizing nonimmunogenic small-molecule drugs such as opioids by eliciting drug-specific antibodies that sequester opioids in circulation before reaching the brain. In this study, multiple parameters to optimize the efficacy of the TT-6-AmHap heroin conjugate vaccine were investigated in mice and rats, focusing on dose optimization, hapten density, booster timing, and cross-reactivity with other prescription opioids. Binding antibody responses were quantified, and vaccine efficacy was assessed using nociceptive assays following a drug challenge. Immunization with TT-6-AmHap vaccine in combination with adjuvants ALF43 and Alhydrogel^® (ALFA), demonstrated a robust in-vivo efficacy across multiple opioid challenges including heroin, hydrocodone, and hydromorphone. Both antibody titers to 6-AmHap and protective efficacy were maintained following repeated heroin challenges up to 54 weeks. Notably, these repeat heroin challenges did not induce any measurable decline in antibody titers. Further characterization of the vaccine responses indicated a strong correlation between hapten density on the carrier protein and both binding antibody titers and overall vaccine efficacy. These findings support the continued development of hapten-based conjugate vaccines for OUD and demonstrate that an optimized vaccine can provide long-lasting protection against the antinociceptive effects of opioids in animals, laying the groundwork for future translational studies.</p>