Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Xinyuan Chen

Abstract

Vaccination is one of the most outstanding inventions in medical history and has made significant contributions to prevent various types of infectious diseases. Once highly fatal disease, smallpox, was eradicated in 1980 after decades of vaccination effort. Mounting evidence also supports vaccination as the most effective way to control the recent outbreak of COVID-19.

Different types of vaccines have been developed from the early inactivated and live-attenuated vaccines to recent subunit vaccines, and to recent emerging nucleic acid-based vaccines that vary in vaccine immunogenicity and safety. Subunit vaccines are attractive vaccine types due to its good safety profiles, flexible design, and diverse manufacturing platforms. Yet, subunit vaccines have relatively low immunogenicity and often requires the addition of vaccine adjuvants or takes advantages of vaccine delivery platforms to be effective.

Flagellin is the structural protein of bacterial flagellar and can serve as both vaccine adjuvants and delivery platforms. It activates Toll-like receptor (TLR) 5 and triggers downstream signaling pathway Myeloid differentiation factor 88 (MyD88) to enhance vaccine-induced immune responses. Foreign antigens can also be inserted into its N- or C-terminus or highly flexible D2/D3 region to develop recombinant vaccines. Despite its high immunogenicity, flagellin-based vaccines were reported to induce systemic adverse reactions that hampered its use in clinics.

In this dissertation, we developed a novel flagellin-based vaccine delivery platform by high-density display of Flagellin on Hepatitis B core virus-like particle (FH VLP) surface. FH VLPs show improved immunogenicity and safety as compared to soluble flagellin due to the conversion of soluble flagellin to particulate one and the significantly reduced innate immune system activation, while sufficient to enhance adaptive immunity.

FH VLPs also provide a versatile vaccine delivery platform considering the highly flexible D2/D3 domains can be replaced with a variety of vaccine antigens with little restriction on size and 3-dimensional structures. Vaccine antigens can also be chemically conjugated to surface of FH VLPs to develop conjugated vaccines. In this dissertation, we explored the use of FH VLPs as a highly immunogenic, safe, and versatile vaccine delivery platform for i) nicotine vaccine, ii) preventive cancer vaccine and iii) universal influenza vaccine development.

By conjugating nicotine hepten to FH VLPs, we constructed FH VLP-based nicotine vaccine (FH-Nic VLPs). FH-Nic VLP immunization showed significantly stronger ability to prevent nicotine entry into the brain as compared to nicotine conjugate vaccine based on flagellin (FljB-Nic). Furthermore, FH-Nic VLP-based nicotine vaccine efficacy can be enhanced by incorporation of Alum or CpG adjuvant. FH-Nic VLPs also induced minimal local or systemic adverse reactions, indicative of a good local and systemic safety.

By replacing the D3 domain of flagellin with cytotoxic T lymphocyte (CTL) epitope of OVA, which is a model protein antigen widely used in preclinical studies, we constructed FH-OVA VLPs as a tumor vaccine. FH-OVA VLPs in the presence of CpG induced similar OVA-specific CTLs and similarly suppressed OVA-expressing B16F10 melanoma growth to FljB-OVA in the presence of CpG. Yet, FH-OVA VLPs in the presence of CpG showed better safety than FljB-OVA in the presence of CpG as evidenced by lower systemic cytokine levels after vaccination.

By replacing the D3 domain of flagellin with four tandem copies of the highly conservative ectodomain of M2 (M2e) protein of influenza virus, we constructed FH-M2e VLPs as a universal influenza vaccine. FH-M2e VLP induced significantly higher anti-M2e antibody titer and better protection against lethal viral challenges as compared to M2e-fused flagellin (FljB-M2e) and incorporation of CpG further enhanced anti-M2e antibody titer and protection. FH-M2e VLPs also showed better safety than FljB-M2e with regard to the ability to induce systemic cytokines and increase rectal temperature.

Our data support FH VLPs to be a highly immunogenic, safe, and versatile vaccine delivery platforms to support conjugate or recombinant vaccine development.

Available for download on Sunday, August 13, 2023

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