Mechanisms of interleukin 4 mediated increase in efficacy of vaccines against opioid use disorders.

Opioid use disorders (OUD) affect over 27 million people worldwide. Anti-opioid vaccines offer a promising strategy to treat OUD and prevent overdose. Using immunomodulation of cytokine signaling to increase vaccine efficacy, this study found that blocking IL-4 improved the efficacy of vaccines targeting oxycodone and fentanyl in male and female mice. Genetic deletion of the IL-4 receptor, STAT6, or antibody-based depletion of IL-13, did not increase vaccine efficacy against opioids, suggesting the involvement of type I IL-4 receptors. Enhancement of vaccine efficacy with blockade of IL-4 was associated with improved germinal center formation in secondary lymphoid organs and selective transcriptome signatures in the activated CD4+ T cell population subset. These data suggest that IL-4 is both a pharmacological target and a potential biomarker of vaccine efficacy against OUD.

Polymer-mediated delivery of vaccines to treat opioid use disorders and to reduce opioid-induced toxicity.

Vaccines offer a potential strategy to treat opioid use disorders (OUD) and to reduce the incidence of opioid-related overdoses. Vaccines induce opioid-specific polyclonal antibodies that selectively and effectively bind the target opioid and prevent its distribution across the blood-brain barrier. Because antibody-mediated reduction of drug distribution to the brain reduces drug-induced behavior and toxicity, vaccine efficacy depends on the quantity and quality of the antibody response. This study tested whether polymer-mediated delivery could improve vaccine efficacy against opioids as well as eliminate the need for booster injections normally required for a successful immunization. A series of novel biodegradable biocompatible thermogelling pentablock co-polymers were used to formulate a candidate vaccine against oxycodone in mice and rats. Polymer-based delivery of the anti-oxycodone vaccine was equally or more effective than administration in aluminum adjuvant in generating oxycodone-specific antibodies and in reducing oxycodone-induced effects and oxycodone distribution to the brain in mice and rats. The composition and release kinetics of the polymer formulations determined vaccine efficacy. Specifically, a formulation consisting of three simultaneous injections of the anti-oxycodone vaccine formulated in three different polymers with slow, intermediate, and fast release kinetics was more effective than an immunization regimen consisting of three sequential injections with the vaccine adsorbed on aluminum. The novel three-phased polymer vaccine formulation was effective in blocking oxycodone-induced antinociception, respiratory depression and bradycardia in rats.

Preclinical Efficacy and Characterization of Candidate Vaccines for Treatment of Opioid Use Disorders Using Clinically Viable Carrier Proteins.

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.

Blocking interleukin-4 enhances efficacy of vaccines for treatment of opioid abuse and prevention of opioid overdose.

Vaccines offer an option to treat heroin and prescription opioid abuse and prevent fatal overdoses. Opioid vaccines elicit antibodies that block opioid distribution to the brain and reduce opioid-induced behavioral effects and toxicity. The major limitation to the translation of addiction vaccines is that efficacy is observed only in subjects achieving optimal drug-specific serum antibody levels. This study tested whether efficacy of a vaccine against oxycodone is increased by immunomodulators targeting key cytokine signaling pathways involved in B and T cell lymphocyte activation. Blockage of IL-4 signaling increased vaccine efficacy in blocking oxycodone distribution to the brain and protection against opioid-induced behavior and toxicity in mice. This strategy generalized to a peptide-protein conjugate immunogen, and a tetanus-diphtheria-pertussis vaccine. These data demonstrate that cytokine-based immunomodulators increase efficacy of vaccines against small molecules, peptides and proteins, and identify IL-4 as a pharmacological target for improving efficacy of next-generation vaccines.

Opioid Dose- and Route-Dependent Efficacy of Oxycodone and Heroin Vaccines in Rats.

Heroin and oxycodone abuse occurs over a wide range of drug doses and by various routes of administration characterized by differing rates of drug absorption. The current study addressed the efficacy of a heroin vaccine [morphine hapten conjugated to keyhole limpet hemocyanin (M-KLH)] or oxycodone vaccine [oxycodone hapten conjugated to keyhole limpet hemocyanin (OXY-KLH)] for reducing drug distribution to brain after intravenous heroin or oxycodone, or subcutaneous oxycodone. Rats immunized with M-KLH or keyhole limpet hemocyanin (KLH) control received an intravenous bolus dose of 0.26 or 2.6 mg/kg heroin. Vaccination with M-KLH increased retention of heroin and its active metabolites 6-acetylmorphine (6-AM) and morphine in plasma compared with KLH controls, and reduced total opioid (heroin + 6-AM + morphine) distribution to brain but only at the lower heroin dose. Immunization also protected against respiratory depression at the lower heroin dose. Rats immunized with OXY-KLH or KLH control received 0.22 or 2.2 mg/kg oxycodone intravenously, the molar equivalent of the heroin doses. Immunization with OXY-KLH significantly reduced oxycodone distribution to brain after either oxycodone dose, although the magnitude of effect of immunization at the higher oxycodone dose was small (12%). By contrast, vaccination with OXY-KLH was more effective when oxycodone was administered subcutaneously rather than intravenously, reducing oxycodone distribution to brain by 44% after an oxycodone dose of 2.3 mg/kg. Vaccination also reduced oxycodone-induced antinociception. These data suggest that the efficacy of OXY-KLH and M-KLH opioid vaccines is highly dependent upon opioid dose and route of administration.

Protein Expression Analysis of Melanocyte Differentiation Antigen TRP-2.

Melanocyte differentiation antigens, such as gp100, tyrosinase, and Melan-A and their corresponding antibodies HMB45, T311, and A103, are major diagnostic tools in surgical pathology. Little is known about tyrosinase-related protein 2 (TRP-2, or dopachrome tautomerase/DCT) another melanocyte differentiation antigen, which is an enzymatic component of melanogenesis. We identified a commercial reagent to TRP-2, monoclonal antibody (mAb) C-9 and undertook a comprehensive analysis to assess its specificity and usefulness for surgical pathology. Subsequently, we analyzed panels of normal tissues and tumors. We show that TRP-2 is regularly expressed in melanocytes of the normal skin. In cutaneous nevi, TRP-2 is present in junctional as well as in dermal nevocytes. In malignant tumors, C-9 reactivity is restricted to melanocytic and related lesions and present in 84% and 58% of primary and metastatic melanomas, respectively. Ten primary melanomas of the anorectal mucosa were all positive. Like the other melanocyte differentiation antigens, TRP-2 was absent in 6 desmoplastic melanomas. Also, only 2 of 9 angiomyolipomas were TRP-2 positive. We conclude that mAb C-9 is a valuable reagent for the analysis of TRP-2 expression in archival surgical pathology material. The expression pattern of TRP-2 in melanocytic and related lesions appears to parallel other melanocyte differentiation antigens, although the overall incidence is lower than other antigens, such as Melan-A or gp100.

The frequency of naive and early-activated hapten-specific B cell subsets dictates the efficacy of a therapeutic vaccine against prescription opioid abuse.

Translation of therapeutic vaccines for addiction, cancer, or other chronic noncommunicable diseases has been slow because only a small subset of immunized subjects achieved effective Ab levels. We hypothesize that individual variability in the number of naive and early-activated hapten-specific B cells determines postvaccination serum Ab levels and vaccine efficacy. Using a model vaccine against the highly abused prescription opioid oxycodone, the polyclonal B cell population specific for an oxycodone-based hapten (6OXY) was analyzed by flow cytometry paired with Ag-based magnetic enrichment. A higher frequency of 6OXY-specific B cells in either spleen biopsies or blood, before and after immunization, correlated to subsequent greater oxycodone-specific serum Ab titers and their efficacy in blocking oxycodone distribution to the brain and oxycodone-induced behavior in mice. The magnitude of 6OXY-specific B cell activation and vaccine efficacy was tightly correlated to the size of the CD4(+) T cell population. The frequency of enriched 6OXY-specific B cells was consistent across various mouse tissues. These data provide novel evidence that variations in the frequency of naive or early-activated vaccine-specific B and T cells can account for individual responses to vaccines and may predict the clinical efficacy of a therapeutic vaccine.

Effect of currently approved carriers and adjuvants on the pre-clinical efficacy of a conjugate vaccine against oxycodone in mice and rats.

Vaccination against the highly abused prescription opioid oxycodone has shown pre-clinical efficacy for blocking oxycodone effects. The current study further evaluated a candidate vaccine composed of oxycodone derivatized at the C6 position (6OXY) conjugated to the native keyhole limpet hemocyanin (nKLH) carrier protein. To provide an oxycodone vaccine formulation suitable for human studies, we studied the effect of alternative carriers and adjuvants on the generation of oxycodone-specific serum antibody and B cell responses, and the effect of immunization on oxycodone distribution and oxycodone-induced antinociception in mice and rats. 6OXY conjugated to tetanus toxoid (TT) or a GMP grade KLH dimer (dKLH) was as effective as 6OXY conjugated to the nKLH decamer in mice and rats, while the 6OXY hapten conjugated to a TT-derived peptide was not effective in preventing oxycodone-induced antinociception in mice. Immunization with 6OXY-TT s.c. absorbed on alum adjuvant provided similar protection to 6OXY-TT administered i.p. with Freund's adjuvant in rats. The toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) adjuvant, alone or in combination with alum, offered no advantage over alum alone for generating oxycodone-specific serum antibodies or 6OXY-specific antibody secreting B cells in mice vaccinated with 6OXY-nKLH or 6OXY-TT. The immunogenicity of oxycodone vaccines may be modulated by TLR4 signaling since responses to 6OXY-nKLH in alum were decreased in TLR4-deficient mice. These data suggest that TT, nKLH and dKLH carriers provide consistent 6OXY conjugate vaccine immunogenicity across species, strains and via different routes of administration, while adjuvant formulations may need to be tailored to individual immunogens or patient populations.