Date of Award
2024
Degree Type
Dissertation
Degree Name
Doctor of Philosophy in Pharmaceutical Sciences
Department
Biomedical and Pharmaceutical Sciences
First Advisor
Xinyuan Chen
Abstract
An adjuvant is a pharmacological substance added to a vaccine to enhance the intensity, breadth and endurance of the immune responses. Traditional discovery and development of vaccine adjuvants mainly based on empirical experiences have been slow, but the situation has improved in the past two decades as more molecular mechanisms of vaccine adjuvants were revealed. Now it has been widely recognized that most adjuvants activate innate immune system by targeting pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) to enhance adaptive immune responses. PRRs are proteins expressed mainly on innate immune cells, such as dendritic cells (DCs) and macrophages, which can be mainly recognized by pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), nucleic acid and flagellin, or damage-associated molecular patterns (DAMPs), such as uric acid, ATP and heat shock proteins (HSP). Here, we studied the application and mechanisms of several different adjuvants including physical radiofrequency (RFA), and chemical CpG 1018 and 2’3’-cGAMP. RFA is a novel physical adjuvant developed by our group, which induces HSP70, belonging to DAMPs. CpG 1018 and 2’3’-cGAMP are a TLR9 agonist and a STING agonist respectively, which belong to PAMPs.
Our group previously found RFA can increase protein antigen-induced humoral and cellular immune responses and boost pdm09 influenza vaccinations. In MANUSCRIPT I, we further studied the use of radiofrequency on highly conserved internal antigens influenza Matrix 1 protein (M1) and Nucleoprotein (NP). We found recombinant NP/M1 immunization in the presence of RFA could elicit potent anti-NP but not anti-M1 cytotoxic T lymphocyte responses (CTLs) and confer significant protection against homologous viral challenges. Interestingly, we also found that RFA failed to induce anti-NP or anti-M1 antibody responses, which proved that RFA induced Th-1-biased immune responses for M1/NP vaccinations.
Next, we studied the molecular mechanisms of RFA using comparative tissue proteomics (MANUSCRIPT II). We found that 14 unique proteins were only upregulated by RFA, after comparing the tissue proteome changes induced by five different adjuvants. Heat shock protein (HSP) 70 was selected from the 14 proteins for further analysis and our study showed that RFA showed a significantly weakened ability to boost ovalbumin (OVA) and pandemic influenza vaccination in HSP70 knockout than wild-type mice, indicating crucial roles of HSP70 in RFA effects. We also published our detailed protocol (MANUSCRIPT III) for preparing murine tissue for comparative proteomics study of vaccine adjuvants, offering an efficient tool for vaccine adjuvant molecular mechanism study for the scientific community.
In the following three manuscripts, my research focused on the study of another category of vaccine adjuvants - pathogen-associated molecular patterns (PAMPs). CpG ODNs belong to a type of PAMP due to their resemblance to microbiome genomes, which can be recognized by TLR9. Among all CpG ODNs, CpG 1018 adjuvant has been approved for use in the hepatitis B vaccine, but few studies reported the adjuvant effects of CpG 1018 for internal antigen influenza Nucleoprotein (NP) vaccination. In MANUSCRIPT IV, we found that CpG 1018 was highly effective in enhancing NP-induced humoral and cellular immune responses and potentiated Th1-biased antibody responses. Furthermore, influenza NP immunization in the presence of CpG 1018 induced significant protection against lethal viral challenges.
To further enhance the vaccine-induced immune responses, we explored the combination of two different types of PAMPs (TLR9 agonist CpG 1018 and STING agonist 2’3’-cGAMPs) for influenza vaccination (MANUSCRIPT V). we demonstrated that CpG 1018 and 2’3’-cGAMP combination could synergistically induce robust antibody responses and production of TNF-α secreting CD4+ and CD8+ T cells, as well as provide better protection against homologous influenza virus challenge. We also found that CpG 1018 and 2’3’-cGAMP combination induced more significant dendritic cell maturation and subsequent TNF-α secreting T cell activation and could also upregulate Nos2, a protein co-regulated by CD40 and TNF-α, demonstrating TNF-α may play an important role in the adjuvant effects.
Besides influenza vaccines, we also studied the adjuvant effects of CpG 1018 for mRNA vaccination against melanoma (MANUSCRIPT VI). we demonstrated soluble CpG 1018 could enhance the production of CD8+ cytotoxic T cells after OVA mRNA vaccinations and could also significantly delay tumor growth in both immunoprophylactic and immunotherapeutic B16F10-OVA melanoma models. Moreover, we found the soluble CpG 1018/2’3’-cGAMP combination could further enhance the anti-tumor efficacy compared with CpG 1018 alone for OVA mRNA vaccinations. Finally, we also found that CpG 1018/OVA mRNA didn’t induce higher body weight loss post immunizations compared with CpG 1018/OVA protein group and the CpG 1018/2’3’-cGAMP combination showed a similar trend as CpG 1018 for OVA mRNA vaccinations.
Recommended Citation
Li, Yibo, "DEVELOPING EFFECTIVE PHYSICAL AND CHEMICAL ADJUVANTS FOR INFLUENZA AND TUMOR VACCINES" (2024). Open Access Dissertations. Paper 1646.
https://digitalcommons.uri.edu/oa_diss/1646