Adjuvants are compounds supplemented into vaccinations to enhance their immunogenicity (i.e., ability to stimulate the immune response).1 Unfortunately, they often also increase their reactogenicity (i.e., production of adverse events post-vaccination) with pyrexia remaining one of the most reported adverse events of adjuvanted influenza vaccines.2 Poly(N-isopropylacrylamide) (PNIPAM) is a thermo-responsive polymer that undergoes a phase transition around a lower critical solution temperature (LCST) which can be engineered to occur within physiological range (35-39 °C).3 Its incorporation into a vaccine adjuvant may permit thermal-based attenuation of the immunological response upon onset of pyrexia, improving the safety profiles of the adjuvant and associated vaccinations. Trehalose-6,6’-dibehenate (TDB) is a MINCLE receptor agonist that can be synthetically conjugated to PNIPAM.4 Incubation of the TDB-PNIPAM polymer adjuvants with HEK-mMINCLE cells provides a method for assessing the thermal attenuation activity of the adjuvants in response to pyrexial temperatures.

LCST Determination: The cloud point (Cp) of the polymers (1 mg/mL) was determined by monitoring the change in % transmittance at 600 nm with increasing temperature. The LCST of the polymer corresponds to the temperature at which the transmittance reached 50% of its original value.  

Quanti-Blue Assay: Hek-mMINCLE cells were seeded in two 96-well plates and incubated with the polymer samples (0.01 g/mL adjuvant) for 24 hours at 35 °C and 37 °C. Stimulation of the MINCLE receptor activates the NF-κβ signaling pathway, leading to the secretion of alkaline phosphatase (SEAP). Addition of Quanti-Blue reagent permits the quantification of SEAP levels by monitoring absorbance at 620 nm, allowing for the determination of MINCLE receptor stimulation by the adjuvant samples. The procedure was repeated at 37 °C and 39 °C. 

Further studies will examine:

• Immunological activity upon completion of the phase transition (i.e. temperatures above 39 °C).

• Reversibility of the inactivation of the adjuvant upon restoration of normal physiological temperatures.

1. C. Hervé, B. Laupèze, G. Del Giudice, A.M. Didierlaurent, F. Tavares Da Silva, “The how’s and what’s of vaccine reactogenicity,” Nature Partner Journals Vaccines 4, no. 39 (2019): 1-11, https://doi.org/10.1038/s41541-019-0132-6. 

2. VAERS– Data, https://vaers.hhs.gov/data.html.

3. A.T. Hendricksen, S. Ezzatpour, A. J. Pulukuri, et al., Advanced Healthcare Materials 12, no. 19 (2023): 2202918, https://doi.org/10.1002/adhm.202202918.

4. Zhao, Y. Cai, Y.  Jiang, X. He, Y. Wei, Y. Yu, X. Tian, “Vaccine adjuvants: mechanisms and platforms,” Signal Transduction and Targeted Therapy 8, no. 283 (2023): 1-24, https://doi.org/10.1038/s41392-023-01557-7.