Author ORCID Identifier
Semester
Spring
Date of Graduation
2026
Document Type
Dissertation
Degree Type
PhD
College
School of Pharmacy
Department
Pharmaceutical Sciences
Committee Chair
Sharan Bobbala
Committee Member
Werner J. Geldenhuys
Committee Member
Lori A. Hazlehurst
Committee Member
Ahmad Hanif
Committee Member
Michael Vincent
Abstract
Adjuvants function as agonists of pattern recognition receptors (PRRs) and enhance the immune response against pathogens by stimulating antigen-presenting cells (APCs). Among the PRRs, toll-like receptors (TLRs) are the most extensively studied adjuvant targets; however, stimulating TLRs located within various subcellular compartments of APCs remains challenging. For instance, PRRs such as TLR7, TLR9 in the endosome, and TLR4 on the cell membrane are key targets in the development of prophylactic and therapeutic vaccines. Activation of these TLR receptors generates strong cellular immune responses to fight against notorious pathogens and cancers. Adjuvants such as Cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODN), Monophosphoryl Lipid A (MPLA), and CL347 activate TLR9, TLR4, and TLR7 receptors, respectively. Specifically, CpG ODN is a hydrophilic molecule with poor in vivo stability, whereas MPLA and CL347 are hydrophobic adjuvants. Lipid-based formulations, such as liposomes and lipid nanoparticles (LNPs), have been extensively studied as vaccine delivery platforms due to their versatility in encapsulating vaccine components and inherent immunostimulatory properties. Of note, ionizable LNPs are of significant interest due to their ability to efficiently encapsulate nucleic acid vaccines and deliver them precisely to intracellular compartments. Ionizable LNPs remain neutral at physiological pH but become protonated and positively charged under low pH conditions, such as in endolysosomes, causing membrane instability and enhancing payload release from LNPs. We envisioned that ionizable LNP technology could improve the encapsulation of adjuvants, including negatively charged CpG ODN, and hydrophobic MPLA and CL347, while facilitating their precise intracellular delivery. Firstly, I developed a novel method to fabricate ionizable LNPs using the flash nanoprecipitation (FNP) technique. This is the first demonstration to economically produce LNPs at the lab scale with superior morphological properties. Secondly, for subunit vaccine applications, I formulated CpG ODN and MPLA dual-adjuvanted ionizable LNPs using FNP and showcased their ability to induce both in vitro and in vivo immune responses using a model antigen, OVA. Finally, for mRNA vaccine applications, a TLR7-adjuvanted LNPs containing OVA mRNA and CL347 adjuvant was prepared and tested both in vitro and in vivo for immune activity. Overall, this dissertation work establishes the formulation development and the use of ionizable LNPs for adjuvant delivery, which can be beneficial in augmenting immune responses induced by subunit and mRNA vaccines.
Recommended Citation
Misra, Bishal, "Adjuvanted Ionizable Lipid Nanoparticles for Vaccine Delivery" (2026). Graduate Theses, Dissertations, and Problem Reports. 13373.
https://researchrepository.wvu.edu/etd/13373