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

Eosinophils have broad and extensive immunomodulatory capacity; recent studies have focused on the roles of distinct eosinophil subsets in specific tissue microenvironments. Ly6G is a GPI‐linked leukocyte surface Ag understood primarily as a marker of mouse neutrophils, although its full function is not known. Here, we show that Ly6G/Gr1, detected by mAbs 1A8 (anti‐Ly6G) and RB6‐8C5 (anti‐Gr1), is detected prominently on a significant fraction of eosinophils from mouse bone marrow and bone marrow‐derived culture, with fractions expressing this Ag increasing in IL‐5‐enriched microenvironments. Among our findings, we identified SiglecF+Gr1+ eosinophils in bone marrow from naïve, allergen‐challenged and IL‐5 transgenic mice; SiglecF+Gr1+ eosinophils were also prominent ex vivo in bone marrow‐derived eosinophils (bmEos) in IL‐5‐enriched culture. Reducing the IL‐5 concentration 20‐fold had no impact on the rate of generation of SiglecF+ bmEos but did result in a marked increase in the Gr1− fraction (from 17.4 ± 2% to 30 ± 2.3%, ***P < 0.005). Reducing the IL‐5 concentration also enhanced chemotaxis; SiglecF+Gr1− bmEos were considerably more responsive to eotaxin‐1 than were their SiglecF+Gr1+ counterparts. These results suggest that (i) IL‐5 regulates the expression of Ly6G/Gr1, either directly or indirectly, in cells of the eosinophil lineage, (ii) eosinophils generated in response to high concentrations of IL‐5 can be distinguished from those generated under homeostatic conditions by expression of the Ly6G/Gr1 cell surface Ag, and (iii) expression of Ly6G/Gr1 may have an impact on function, directly or indirectly, including the potential to undergo chemotaxis in response to eotaxin‐1.

Respiratory virus infections can have long-term effects on lung function that persist even after the acute responses have resolved. Numerous studies have linked severe early childhood infection with respiratory syncytial virus (RSV) to the development of wheezing and asthma, although the underlying mechanisms connecting these observations remain unclear. Here, we examine airway hyperresponsiveness (AHR) that develops in wild-type mice after recovery from symptomatic but sublethal infection with the natural rodent pathogen, pneumonia virus of mice (PVM). We found that BALB/c mice respond to a limited inoculum of PVM with significant but reversible weight loss accompanied by virus replication, acute inflammation, and neutrophil recruitment to the airways. At day 21 post-inoculation, virus was no longer detected in the airways and the acute inflammatory response had largely resolved. However, and in contrast to most earlier studies using the PVM infection model, all mice survived the initial infection and all went on to develop serum anti-PVM IgG antibodies. Furthermore, using both invasive plethysmography and precision-cut lung slices, we found that these mice exhibited significant airway hyperresponsiveness at day 21 post-inoculation that persisted through day 45. Taken together, our findings extend an important and versatile respiratory virus infection model that can now be used to explore the role of virions and virion clearance as well as virus-induced inflammatory mediators and their signaling pathways in the development and persistence of post-viral AHR and lung dysfunction.