In vivo safety characterization of injectable amidated TEMPO-Oxidized cellulose nanofiber hydrogel vaccine formulations in farmed Atlantic salmon (Salmo salar L.)

Authors: TURNER, SARAH - University Of Maine; MASON, MICHAEL - University Of Maine; HOLBROOK, JACOB - University Of Maine; HONG, JEONGWHUI - University Of Maine; SIDOR, INGA - University Of Maine; BOUCHARD, DEBORAH - University Of Maine

Submitted to: Vaccines
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/30/2026
Publication Date: 3/31/2026
Citation: Turner, S.M., Mason, M., Holbrook, J.A., Hong, J., Sidor, I., Bouchard, D. 2026. In vivo safety characterization of injectable amidated TEMPO-Oxidized cellulose nanofiber hydrogel vaccine formulations in farmed Atlantic salmon (Salmo salar L.). Vaccines. 14(4):313. https://doi.org/10.3390/vaccines14040313.
DOI: https://doi.org/10.3390/vaccines14040313

Interpretive Summary: This study looked at whether a new type of gel-like material made from modified plant fibers could be used safely in fish vaccines. Fish farmers rely on vaccines to prevent disease, but the oil-based vaccines commonly used today can be expensive and sometimes cause unwanted side effects. Because of this, researchers are exploring new materials that might work as safer or cheaper alternatives. In this experiment, Atlantic salmon were injected with vaccines containing the new plant-fiber gel, and their health was compared with fish given a standard oil-based vaccine or simple control injections. The fish were monitored for several months. The results showed that the new gel formulations caused more deaths and more health problems than either the standard vaccine or the control treatments. Fish that received the gel sometimes developed internal swelling, tissue damage, bleeding, and infections. In contrast, the control groups remained healthy. Growth and immune measurements did not show clear or consistent differences across groups. Overall, the study found that the tested gel materials were not safe enough to be used as vaccine ingredients in salmon. The findings suggest that the material needs significant improvement before it could be considered for future vaccine development and highlight the importance of testing safety thoroughly before focusing on vaccine effectiveness.

Technical Abstract: Background: Disease outbreaks remain a major constraint on aquaculture production, making vaccination essential for disease management in farmed fish. However, injectable oil-adjuvanted vaccines can be costly and may induce adverse inflammatory reactions and welfare concerns, motivating investigations into alternative injectable adjuvant materials. Methods: We conducted an in vivo safety evaluation of shear-thinning, amidated TEMPO-oxidized cellulose nanofiber (TO-CNF) hydrogels formulated with an inactivated Vibrio anguillarum bacterin. Formulations were administered intraperitoneally to Atlantic salmon (Salmo salar L.) using a common garden design with cohabitated treatment groups across triplicate tanks. Fish were monitored and sampled at pre-injection baseline and at 300-, and 600-degree days post-injection. Safety endpoints included mortality, macroscopic and histopathological outcomes, and growth evaluated relative to sham controls, unmodified TO-CNF, and a commercial oil-adjuvanted vaccine. Results: Amidated TO-CNF formulations were associated with increased mortality (up to 16–18% in higher reagent-loading groups) compared to commercial oil-adjuvanted vaccine, material, and sham controls. Affected fish exhibited adverse outcomes, including adhesions, proliferative lesions, ascites, edema, hemorrhage, and secondary opportunistic infections. In contrast, controls showed minimal mortality and pathology. Growth and immune response endpoints were variable and did not demonstrate consistent treatment-associated effects. Physicochemical analyses indicated differences in formulation stability and qualitative compositional differences across modification levels, but these were not quantified nor linked to specific causal mechanisms in this study. Conclusions: The amidated TO-CNF formulations tested here were associated with formulation-dependent safety risks under the conditions evaluated and are not yet suitable as injectable vaccine adjuvants in Atlantic salmon. These findings define important safety constraints for this material class and highlight the need for improved modification and purification strategies. More broadly, this work underscores the importance of establishing in vivo safety boundaries prior to efficacy optimization for emerging biomaterial-based vaccine adjuvants.