Vaccines may offer a new treatment strategy for opioid use disorders and opioid-related overdoses. To speed translation, this study evaluates opioid conjugate vaccines containing components suitable for pharmaceutical manufacturing and compares analytical assays for conjugate characterization. Three oxycodone-based haptens (OXY) containing either PEGylated or tetraglycine [(Gly)4] linkers were conjugated to a keyhole limpet hemocyanin (KLH) carrier protein via carbodiimide (EDAC) or maleimide chemistry. The EDAC-conjugated OXY(Gly)4-KLH was most effective in reducing oxycodone distribution to the brain in mice. Vaccine efficacy was T cell-dependent. The lead OXY hapten was conjugated to the KLH, tetanus toxoid, diphtheria cross-reactive material (CRM), as well as the E. coli-expressed CRM (EcoCRM) and nontoxic tetanus toxin heavy chain fragment C (rTTHc) carrier proteins. All vaccines induced early hapten-specific B cell expansion and showed equivalent efficacy against oxycodone in mice. However, some hapten-protein conjugates were easier to characterize for molecular weight and size. Finally, heroin vaccines formulated with either EcoCRM or KLH were equally effective in reducing heroin-induced antinociception and distribution to the brain of heroin and its metabolites in mice. This study identifies vaccine candidates and vaccine components for further development

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During αβ T cell development, T cell antigen receptor (TCR) engagement transduces biochemical signals through a protein-protein interaction (PPI) network that dictates dichotomous cell fate decisions. It remains unclear how signal specificity is communicated, instructing either positive selection to advance cell differentiation or death by negative selection. Early signal discrimination might occur by PPI signatures differing qualitatively (customized, unique PPI combinations for each signal), quantitatively (graded amounts of a single PPI series), or kinetically (speed of PPI pathway progression). Using a novel PPI network analysis, we found that early TCR-proximal signals distinguishing positive from negative selection appeared to be primarily quantitative in nature. Furthermore, the signal intensity of this PPI network was used to find an antigen dose that caused a classic negative selection ligand to induce positive selection of conventional αβ T cells, suggesting that the quantity of TCR triggering was sufficient to program selection outcome. Because previous work had suggested that positive selection might involve a qualitatively unique signal through CD3δ, we reexamined the block in positive selection observed in CD3δ0 mice. We found that CD3δ0 thymocytes were inhibited but capable of signaling positive selection, generating low numbers of MHC-dependent αβ T cells that expressed diverse TCR repertoires and participated in immune responses against infection. We conclude that the major role for CD3δ in positive selection is to quantitatively boost the signal for maximal generation of αβ T cells. Together, these data indicate that a quantitative network signaling mechanism through the early proximal TCR signalosome determines thymic selection outcome.