Integration of TLR7/8 agonists into lipid nanoparticles enhances antigen-specific immune responses to N1-methyl-Ψ-modified mRNA-LNP vaccines

Abstract

N1-methylpseudouridine (N1-methyl-'P)-modified mRNA offers a safer alternative to unmodified mRNA-based cancer immunotherapies but induces weaker innate immune responses. This study aimed to enhance the expression of N1-methyl-'P-modified mRNA and improve innate and adaptive immune responses by incorporating a toll-like receptor (TLR) 7/8 agonist (AD7/8) into a lipid nanoparticle (LNP). AD7/8 was incorporated into LNPs by partially replacing cholesterol, and the mRNA expression efficiency of various formulations was evaluated, leading to the selection of the LNP formulation containing 0.5% AD7/8 (AD03-LNP). AD03-LNP was evaluated using mRNAs encoding human papillomavirus (HPV)16 E7 and HPV18 E6 antigens, the SARS-CoV-2 Omicron spike protein (S-Omicron), and influenza hemagglutinin (HA), and it consistently enhanced antigen-specific immune responses compared with conventional LNP. In the HPV mRNA model, antigen-specific CD8+T cell and cytokine responses were significantly increased by 1.5-2.1-fold. In the S-Omicron mRNA model, IgG2a levels, indicative of a Th1-skewed response, were markedly elevated by 8-fold as measured by endpoint titers. Importantly, in the HA mRNA model, which evaluated both cellular and humoral immunity, AD03-LNP induced significantly higher CD8+ T cell responses by 2.3-2.6-fold, together with increased antibody production, with total IgG elevated by 3.6-fold as measured by endpoint titers. These findings demonstrate that AD03-LNP enhances both cellular and humoral immune responses across diverse antigens. These results provide insights into how TLR7/8 agonist-loaded LNPs influence mRNA expression and immune responses, supporting an effective formulation approach to boost the immunogenicity of mRNA-LNP vaccines. This approach may help advance the design of mRNA-based cancer immunotherapies and prophylactic vaccines that depend on strong T cell responses.