1. Abstract: Opioid addiction is a national medical crisis that is expected to worsen due to the socioeconomic fallout caused by the SARS-CoV-2 pandemic. Opioid vaccines are a promising treatment strategy for preventing opioid overdose and could enhance drug treatment when combined with existing modalities. The aim of vaccination is to generate opioid-specific antibodies (-opioid)s that bind to the drugs and sequester them in the serum, stopping opioids from entering the brain and other organs, and thus preventing overdose and the sensation sought by the user. While monomeric opioid hapten conjugate vaccines composed of opioid monomers conjugated to carrier proteins admixed with experimental adjuvants are efficacious in rodents; the design of current monomeric opioid vaccines is based on monomeric vaccines for cocaine and nicotine that generated relatively weak, short-lived antibody responses in humans. The goal of this proposal is to develop a polymeric opioid conjugate vaccine that induces robust, long-lasting -opioid responses in the absence of toxicity. Durable antibody responses are maintained by long-lived antibody-secreting cells (LLASCs), which are generated upon B cell activation in the presence of B cell receptor (BCR) crosslinking, recognition of pathogen derived immunostimulatory signals, and ligation of co-stimulatory signals from helper T cells. To elicit the signals necessary for opioid-specific B cell activation, our polymeric opioid vaccine against fentanyl—the cause of most opioid overdose deaths—is composed of a water-soluble copolymer that targets and activates fentanyl-specific B cells, termed p(Fent-co-TLR7), conjugated to the immunogenic carrier protein tetanus toxin (TT) via a self-immolative linkage that, when cleaved, releases unmodified TT. We will synthesize p(Fent-co-TLR7) as a random copolymer from a monomer decorated with fentanyl and a second monomer that activates B cells via toll-like receptor 7 (TLR7). Thus, multivalent TT-p(Fent-co-TLR7) conjugates are designed to target and crosslink opioid-specific B cell receptors (BCRs), causing B cell activation and BCR-mediated internalization. Once internalized, TT-p(Fent-co-TLR7) will activate endosomal TLR7, amplifying B cell activation, and release TT. Once released from p(Fent-co-TLR7) polymers, TT can be efficiently processed and its peptides presented on the surface of B cells to TT-specific T cells, resulting in optimal T cell activation and the production of signals that drive B cells to differentiate into LLASCs. In this proposal we will (1) optimize the formulation and demonstrate the mechanisms responsible for the efficacy of TT-p(Fent-co-TLR7), (2) compare the immunogenicity and durability of the immune response generated by TT-p(Fent-co-TLR7) to that of monomeric fentanyl-TT conjugate vaccines, and (3) demonstrate the ability of TT-p(Fent-co-TLR7) to inhibit the behavior effects and lethality of fentanyl. Completion of this proposal will validate the precl...