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Innovative approach to characterize complex colloidal TB vaccine H4-IC31 without desorption.PALS differentiates H4-IC31 vaccine vs IC31® adjuvant.Increase in β-sheet secondary structure content detected in H4 protein adsorbed to IC31® adjuvant.Tuberculosis (TB) is one of the leading causes of death worldwide, making the development of effective TB vaccines a global priority. A TB vaccine consisting of a recombinant fusion protein, H4, combined with a novel synthetic cationic adjuvant, IC31®, is currently being developed. The H4 fusion protein consists of two immunogenic mycobacterial antigens, Ag85B and TB10.4, and the IC31® adjuvant is a mixture of KLK, a leucine-rich peptide (KLKL5KLK), and the oligodeoxynucleotide ODN1a, a TLR9 ligand. However, efficient and robust methods for assessing these formulated components are lacking. Here, we developed and optimized phase analysis light scattering (PALS), electrical sensing zone (ESZ), and Raman, FTIR, and CD spectroscopy methods to characterize the H4-IC31 vaccine formulation. PALS-measured conductivity and zeta potential values could differentiate between the similarly sized particles of IC31® adjuvant and the H4-IC31 vaccine candidate and could thereby serve as a control during vaccine formulation. In addition, zeta potential is indicative of the adjuvant to antigen ratio which is the key in the immunomodulatory response of the vaccine. ESZ was used as an orthogonal method to measure IC31® and H4-IC31 particle sizes. Raman, FTIR, and CD spectroscopy revealed structural changes in H4 protein and IC31® adjuvant, inducing an increase in both the β-sheet and random coil content as a result of adsorption. Furthermore, nanoDSF showed changes in the tertiary structure of H4 protein as a result of adjuvantation to IC31®. Our findings demonstrate the applicability of biophysical methods to characterize vaccine components in the final H4-IC31 drug product without the requirement for desorption.