Year of Award

2024

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Pharmaceutical Sciences and Drug Design

Department or School/College

Department of Biomedical and Pharmaceutical Sciences

Committee Chair

David Burkhart

Commitee Members

Erica Woodahl, Travis Hughes, Hélène Bazin-lee, Victor DeFilippis

Keywords

conjugation, self-adjuvanting, Toll-like receptor, vaccine

Publisher

University of Montana

Abstract

Toll-like receptor 7/8 (TLR7/8) agonists represent a promising vaccine adjuvant target as they directly activate innate Th1-type proinflammatory responses inducing both humoral and cell mediated immunity. However, co-delivery of the small molecule agonists with subunit antigens to the target dendritic cells (DCs) is difficult due to their highly divergent physical and chemical properties resulting in rapid dissociation of the two components. Conjugation of adjuvant to antigen can co-deliver to DCs, mimicking the natural infection these cells have evolved to detect though successful formulation can be difficult due to poor aqueously soluble adjuvants and antigen instability. We have previously shown and continue to demonstrate here that our polyethylene glycol linker technology enhances conjugate responses even at low doses of TLR7/8 agonist with a number of different antigens. The objective of this work was to advance this technology into unstudied indications and to incorporate more advanced materials such as cleavable linkers and dendrimers with the potential to further enhance responses and adjuvant delivery. First, our established technology was applied to a hapten based vaccine for fentanyl use disorders. The conjugates demonstrated a robust increase in anti-fentanyl responses, protecting mice in a challenge study with or without the use of alum adjuvant. The conjugated TLR7/8 adjuvant, while relatively large compared to the co-conjugated fentanyl hapten and might have masked the fentanyl epitope, still increased desirable antibody titers and opens the door to larger, multivalent constructs. Second, conjugates with or without Cathepsin B cleavable linkers were advanced into a porcine model of reactogenicity. Both types of conjugates reduced injection site reactions, an important milestone for addressing vaccine hesitancy. Additionally, the cleavable moiety boosted immune responses over non-cleavable counterparts to further enhance the therapeutic index of TLR7/8 adjuvants. Third, adjuvants were conjugated to a model antigen using novel dendrimer-based linkers, an alternate conjugation strategy suitable for future mechanistic studies of immune responses to conjugates. While in vitro evaluation of these conjugates was inconclusive, the successful conjugation of multivalent, poorly water-soluble adjuvants represents a great advancement of this technology which is useful for multiple applications. In particular, conjugation of adjuvants to antigens with low numbers of available lysine residues to increase copy number, conjugation of multiple haptens without overmodifying the antigen carrier, or mechanistic studies of B cell maturation phenomena such as B cell receptor clustering. Further, ongoing in vivo studies may yet reveal desirable vaccine responses such as increased antibody titers or enhanced Th1 biased T cell responses. Collectively, this work advances TLR7/8 self-adjuvanting vaccine antigens by demonstrating reduced vaccine side effects in a human-like porcine immune system as well as opening further avenues for research of large, complex conjugates for multiple indications.

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