Date of Award

Spring 5-3-2024

Level of Access Assigned by Author

Open-Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Interdisciplinary Program

Advisor

Deborah A. Bouchard

Second Committee Member

Michael Mason

Third Committee Member

James Beaupre

Additional Committee Members

Matthew Hawkyard

Sarah Barker

Abstract

This applied interdisciplinary project capitalized upon the University of Maine’s research expertise, facilities, and industry partnerships in aquaculture, fish health and cellulose nanomaterial (CNM) science and engineering towards the development of a safe and efficacious new generation of CNM adjuvanted vaccines for commercial aquaculture. Disease outbreaks in aquaculture cause significant production losses, necessitating vaccines for disease management. However, vaccines can be expensive, vary in effectiveness, and produce adjuvant-induced adverse effects, causing fish welfare issues and negative economic impacts. The hypothesis driving this research was that CNM, a renewable wood fiber, could be tuned to act as depots/adjuvants in vaccine formulations to achieve biocompatible, environmentally friendly, and cost-effective disease protection in the extensively farmed species, Atlantic salmon (Salmo salar L.). First, in vivo safety of various unmodified CNM formulations demonstrated the biopolymer was a low risk of harm evidenced by minimal gross reactions in Atlantic salmon post-injection. However, Atlantic salmon vaccinated with unmodified CNM formulations demonstrated indeterminate serum IgM antibody response to inactivated strains of Aeromonas salmonicida using an indirect enzyme-linked immunosorbent assay (ELISA). This suggested a need for immobilization of antigens to CNM for efficacious immunogenicity. In collaboration with the Mason Laboratory in the University of Maine’s Biomedical Engineering Department, the surface carboxyl group on TOCNF was leveraged to investigate methods of physically crosslinking TOCN fibers into a matrix as one way to achieve high antigen loading for controlled delivery and immunomodulation. Citric acid cross-linked TOCNF hydrogels were produced with a Vibrio anguillarum bacterin acting as the antigen and examined after 600- degree days post-implantation with a modified passive integrated transponder tagging device. Scored gross and microscopic pathologies demonstrated a strong innate immune reaction known as the Foreign Body Response (FBR) in the presence of the biopolymer hydrogel vaccine compared to negative sham controls. These results highlighted the need to reduce wound trauma during delivery and/or tune hydrogel surface properties towards more anti-adhesive surfaces to achieve biocompatibility. Next, injectable shear- thinning amidated TOCNF formulations were produced for in vivo examination. Acute toxicity after intraperitoneal injection was evident by high mortalities and severe adverse reactions in Atlantic salmon vaccinated with amidated TOCNF formulations compared to oil-based adjuvanted controls. However, increased IgM antibody response as determined by RT-qPCR and ELISA analysis suggested improved immunogenicity. In vitro characterization was performed using rheology (mechanical), scanning electron microscopy (structural), and Fourier-Transform infrared spectroscopy (chemical) analysis. In vitro analysis demonstrated sonication produced a more homogenous shear-thinning formulation, however trace unconjugated octadecylamine (ODA) likely remained within the amidated TOCNF matrix causing toxicity. From this work, “green” chemistry was used to produce an injectable salt cross-linked shear-thinning TOCNF hydrogel that is safe in vivo. This could be auspicious with a challenge model needed to confirm vaccine efficacy. Overall, this extensive project informs both aquaculture and biopolymer science communities on the potential for CNM as an adjuvant for a novel aquaculture vaccine technology.

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