Novel ionic liquid adjuvant promotes a robust and antigen-specific Th1 response with biocompatible and GRAS materials

Introduction::
Adjuvants are important components in vaccines for enhancing the magnitude and durability of the immune response. However, despite their ability to promote and create a robust and tailored immune response, adjuvants are largely underdeveloped in the field. There are only six FDA approved adjuvants, with alum (aluminum salts) being the only licensed adjuvant for 70 years before the 1990s, and the most prominent adjuvant in vaccines today (Lodaya, 2021). Within the currently used adjuvants, there is a lack of those that induce a strong T-cell (Th1) response necessary for complete protection against broad infectious diseases. Alum specifically does not have the ability to promote antigen-specific T-cell responses and is instead utilized for its antibody dependent protection (Oleszycka, 2018). The lack of approved and effective adjuvants presents a gap in vaccine development. We propose that ionic liquids may address this unmet need. Ionic liquids (IL) are bulky organic salts that remain liquid below 100°C and present a high degree of tunability and manufacturability. The safety profile of ILs can be enhanced by synthesizing them from “generally regarded as safe” (GRAS) materials, which is important as many pre-clinical adjuvants have not yet been used in humans due to safety or tolerability concerns. In this study we present a novel ionic liquid adjuvant that creates a robust antigen-specific T-cell and antibody response and dissected the mechanism of action, thus providing the potential to expand the repertoire of currently available adjuvants.




Materials and Methods::
Ionic liquids were synthesized by combining choline bicarbonate (80% in water) with an acid of interest at a 1:1 molar ratio while stirring at 40°C. This mixture stirs under heat over night before undergoing rotary evaporation at 10mbar and 60°C for 3h. The formulation is dried at 60°C for 72h. The synthesized ionic liquids were then screened for adjuvant success through protein stability, activation of JAWSII and J774A cells lines, and in vivo adaptive immune response when vaccinated intramuscularly (IM) with the model antigen ovalbumin in 7-week-old female C57BL/6 Mice. The formulation displaying antigen specific immune responses, choline and sorbic acid in a 1:1 molar ratio (ChoSorb), was investigated further. For in vitro studies of ChoSorb mechanism, we utilized C2C12 skeletal muscle cells that were differentiated into myotubes and dendritic cells that were differentiated from C57BL/6 female mice bone marrow. Both cell types were evaluated for cell death mechanisms, C2C12s were also evaluated for cytokine release, and dendritic cells were evaluated for antigen uptake. For in vivo studies of ChoSorb, 7-week-old female C57BL/6 Mice were used. Mice were vaccinated IM and boosted with varying concentrations of ChoSorb in combination with ovalbumin and at separate times than ovalbumin for a dose and payload dependent evaluation of the adaptive immune response. The innate immune response was evaluated using flow cytometric analysis of the injection site, draining lymph node, and serum, 24h and 48h after IM adjuvant injection. H&E studies of the injection site 24h after ChoSorb injection was conducted to evaluate toxicity.




Results, Conclusions, and Discussions::
We synthesized seven unique ionic liquids utilizing the cation, choline, and varying anions by hydrophobicity. By screening these ionic liquids in vitro for immune cell activation and in vivo for adaptive immune response, we determined choline and sorbic acid in a 1:1 molar ratio (ChoSorb) elicits an antigen-specific CD8+ T-cells and antibody response. First, we determined the effect of dose and antigen delivery on adjuvanticity by injecting ChoSorb IM at varying concentrations in combination with ovalbumin (OVA) and before OVA injection. After booster, we found the vaccinations delivered in separate formulations displayed impressive T-cell responses, outperforming the combination vaccinations in percentage of antigen-specific CD8+ T-cells at each dose. There were similar responses across separate doses, with 2.5% ChoSorb having the highest average OVA+ percentage of CD8+ T-cell population (8.02%). However, the OVA+ CD8+ T-cell population displayed a dose dependent response in the combination formulations. We believe this indicates an impact of payload on the adjuvanticity of ChoSorb—with ratio of ChoSorb to OVA having a direct result on T-cell responses. There was no significant difference in antibody responses across treatment groups with all significantly greater than OVA alone. We next sought to analyze the mechanism of this response using two doses (2.5% and 5%). We analyzed innate immune responses to ChoSorb through immune cell profiling of the muscle and draining lymph node (dLN) 24h and 48h after IM injection. There was a significant increase in CD45+ cells in the muscle for all treatment groups, with a significant increase in CD4+ T-cells at 24h for both doses, and inflammatory monocytes increasing significantly at 48h. In the dLN there was a significant increase in inflammatory monocytes and mature resident cDC1 dendritic cells after both doses at both timepoints. In vitro, we determined ChoSorb induces ferroptotic cell death of myotubes, which could be important in recruiting immune cells to the site of injection and prompting an immune cascade.









In conclusion, we synthesized and investigated the mechanism of a novel ionic liquid formulation that displays antigen-specific CD8+ T-cell and antibody responses. This introduces a safe, scalable, and effective material to garner adjuvanticity in vaccination.




Acknowledgements (Optional): :

References (Optional): : 1. Lodaya RN, Gregory S, Amiji MM, O’Hagan DT. 2 - Overview of vaccine adjuvants. In: Kolhe P, Ohtake SBT-PA of VD, eds. Academic Press; 2022:9-25. doi:https://doi.org/10.1016/B978-0-12-814357-5.00003-9

2. Oleszycka, E., McCluskey, S., Sharp, F.A., Muñoz-Wolf, N., Hams, E., Gorman, A.L., Fallon, P.G. and Lavelle, E.C. (2018), The vaccine adjuvant alum promotes IL-10 production that suppresses Th1 responses. Eur. J. Immunol., 48: 705-715. https://doi.org/10.1002/eji.201747150