No significant pathology was identified in other organs (Suppl. 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. Introduction Vaccines are the single most effective intervention ever devised in medical practice1. Vaccines have the potential to protect against a variety of communicable and non-communicable diseases, including material use disorders associated with abuse of stimulants and opioids. Worldwide, PK 44 phosphate over 33 million people abuse drugs, whose health effects include blood-borne pathogen transmission, cancer, and death2. In the US, 2.6 million people are dependent on heroin and prescription opioids including oxycodone and hydrocodone, costing more than $95 billion in crime, lost work productivity and health care annually3. Over 50,000 opioid-related fatal overdoses occur each year, making opioid abuse the leading cause of accidental death in the US, and prompting recent increases in stringency of prescription guidelines3. These data suggest that current interventions are insufficient to curb opioid abuse, and that more therapeutic options are needed4. Vaccines and other biologics against material use disorders have shown TRIB3 pre-clinical and clinical proof of concept, but no product has reached the market5C10. A major limitation to the translation of dependency vaccines is usually that clinical efficacy is observed only in the portion of immunized subjects that display high levels of drug-specific antibodies5,11. This is a common PK 44 phosphate shortcoming of vaccines consisting of purified protein subunits, or of proteins conjugated to peptides, carbohydrates, or synthetic small molecule haptens. For instance, the increased incidence in whooping cough is partially attributable to switching from your whole-cell pertussis vaccine to formulations made up of the purified acellular pertussis subunit12. To increase efficacy, synthetic vaccines against material use disorders are mixed with adjuvants or delivered via particle platforms5. However, a limited quantity of adjuvants or delivery systems are approved for human use because of potential side effects, and available adjuvants trigger innate immunity, but do not directly stimulate adaptive immunity13,14, suggesting that more effective immunization strategies are needed. Screening of bioconjugates with varying hapten design, conjugation chemistry, carrier choice, adjuvant and immunization regimen recognized a candidate vaccine consisting of an oxycodone-based hapten conjugated to keyhole limpet hemocyanin (KLH) through a tetraglycine linker (OXY-KLH)15C19. The OXY-KLH vaccine?generates antibodies that selectively bind oxycodone and hydrocodone in serum, block their distribution to the PK 44 phosphate brain, and prevent their behavioral effects, including oxycodone intravenous self-administration17. The OXY-KLH vaccine is also effective in preventing oxycodone-induced respiratory depressive PK 44 phosphate disorder, and it does not interfere with naloxone reversal of opioid toxicity in rats20. The pre-clinical efficacy profile of OXY-KLH supports its translation, but also warrants its use as a model to test novel strategies to improve efficacy of vaccines against material use disorders. After immunization, generation of antibodies (Ab) results from T cell-dependent B cell activation and germinal center (GC) formation in secondary lymphoid organs21,22. Within the GC, specialized CD4+ T follicular helper (Tfh) cells help cognate antigen-specific B cells differentiate into long-lived switched immunoglobulin memory cells and antibody-secreting B cells21,22. We found that frequency of na?ve and early antigen-specific B or T cells correlates with individual vaccine efficacy against nicotine or opioids23C25. Greater vaccine efficacy also PK 44 phosphate correlated with increased GC activation23. These data suggest that vaccine efficacy can be enhanced by strategies that modulate activation and differentiation of vaccine-specific B and T cell populations. The canonical Th1 signature cytokines IL-2 and IL-7 inhibit GC formation by limiting Tfh differentiation26,27, whereas IL-4 drives Th2 responses and suppresses IgG subclass shift from IgG1 to IgG2a28. Immunization success or failure hinges on the fine balance between these cytokines and other co-stimulatory factors, and their downstream effects on B and T cells. This study tested interleukins as a pharmacological target to enhance vaccine efficacy against drugs of abuse and other targets. Immune checkpoint inhibitors, interleukins, and monoclonal antibodies (mAb) against interleukins or interleukin receptors have been shown to increase efficacy of CD8+ T cell-mediated immunotherapies29,30. Combining a HIV gag/pol vaccine with IL-4 receptor antagonism induced both HIV-specific CD8+ T cells, and gag-specific B cell immunity paired to enhancement of IgG2a class switching in mice31. However, the effect of immunomodulators on subunit or conjugate vaccines that rely on CD4+ T cell-dependent B cell activation to generate antibodies remains largely unexplored. Since small molecules and biologic immunomodulators of IL-4, IL-2.