Document Type

Dissertation - Open Access

Award Date

2025

Degree Name

Doctor of Philosophy (PhD)

Department / School

Pharmaceutical Sciences

First Advisor

Wenfeng An

Abstract

This dissertation presents the design, formulation, and immunogenicity evaluation of a novel mRNA vaccine targeting the L1 ORF1p protein, a tumor-associated antigen. The vaccine was synthesized using in vitro transcription and encapsulated in lipid nanoparticles (LNPs), with both unmodified uridine and N1-methylpseudouridine variants tested. Physicochemical characterization confirmed optimal particle size (160–220 nm), acceptable zeta potential, and encapsulation efficiency ranging from 74% to 92%, with preserved RNA integrity post-formulation. These results validate the robustness of the formulation workflow and its suitability for antigen delivery. Immunogenicity studies in C57BL/6 mice revealed modest but promising immune responses. While early activation markers (CD69) and memory T cell populations (CD44⁺CD62L⁺) showed upward trends, they did not reach statistical significance likely due to timing of sample collection and limited cohort size. Notably, the low-dose uridine-formulated group demonstrated significant increases in IFN-γ and TNF-α production in both CD4⁺ and CD8⁺ T cells upon stimulation, indicating functional T cell activation. ELISpot assays supported these findings, though elevated background responses in controls suggest non-specific activation. Despite some inconclusive data, this study establishes a strong foundation for future research. It highlights the importance of dose optimization, timing of analysis, and assay refinement. The results support the potential of L1 ORF1p as a viable target for cancer immunotherapy and provide a reproducible framework for mRNA-LNP vaccine development.

Publisher

South Dakota State University

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Rights Statement

In Copyright