Deciphering immunological mechanisms underlying the efficacy of nanoparticle-based vaccines and novel adjuvants against Opioid Use Disorder (OUD)

Abstract

<b>Abstract ID 28305</b> <b>Poster Board 255</b> The highly complex OUD and overdose epidemic poses a public health and economic burden. In the United States, the Opioid crisis was further accelerated by COVID-19 pandemic, with more than 72,000 deaths attributed to opioid overdose in 2020, the highest number of overdose deaths ever recorded in a year. Moreover 2,7 million people were diagnosed with OUD which led to a substantial impact on the U.S economy, with the total cost of the opioid epidemic in 2020 estimated at $1,5 trillion. There is an urgent need for safe and more effective therapeutic options to augment existing measures. Vaccines offer a highly promising strategy for OUD treatment and prevention of overdose. Vaccinating with drug-based hapten-carrier protein conjugates formulated with adjuvants, generate drug-specific polyclonal antibodies (Ab) that selectively bind targeted opioids, blocking its distribution to the brain and thus protects against opioid induced rewards, pharmacological and side effects. Preclinical testing demonstrated anti-opioid vaccines as a highly selective long-lasting treatment and prophylactic strategy for OUD and overdose in animal models. Previous clinical trials of addiction vaccines showed proof of efficacy in those subjects who achieved the highest Ab titers, highlighting the need to design more effective vaccines. Therefore, we focus on developing an improved carrier system and investigating novel adjuvants and understanding the immunological mechanisms underlying their efficacy. The generation of effective anti-drug polyclonal antibodies relies on CD4+ T cell-dependent B cell activation, highlighting the importance of dendritic cells (DC) in initiating CD4+ T cells priming and adaptive immune responses. We previously developed a lipid-poly(lactic-co-glycolic acid) (PLGA) hybrid nanoparticle (LPNP) platform that improved the efficacy of conjugate vaccines against nicotine and oxycodone. Additionally, we investigated Toll-like receptor (TLR) 7/8 agonists such as the clinically approved resiquimod (R848) and other TLR7/8 agonists, that significantly improved anti-opioid conjugate vaccine efficacy when combined with alum. Nanocarriers and TLR agonists can be exploited to potentiate antigen recognition by antigen presenting cells (APCs) and subsequently vaccination outcome. Here using an <i>in&nbsp;vitro</i>-flow cytometry-based activation assay, we investigated the efficacy of vaccine formulations in inducing macrophage and DCs activation, maturation and phenotypic differentiation. Using RAW264.7 and JAWS II cell line, we demonstrated that LPNP- based oxycodone vaccine is more effective in inducing macrophages activation marker (iNOS) and DCs co-stimulatory molecules and maturation markers (CD86, CD40, and MHC II) compared to conventional vaccines. Furthermore, adding TLR7/8 agonists to conjugate vaccines outperformed the alum adjuvant in inducing maturation and activation of APCs. These findings were further confirmed with RT-qPCR assays quantifying the mRNA expression of above-mentioned markers. <i>In vivo</i> flow cytometry studies are ongoing to dissect the APCs dynamics upon vaccination to identify key APCs subsets contributing to efficacy and thus a significant vaccine target.