Comparative characterization of novel lipid nanoparticle adjuvants reveals distinct immunogenicity and reactogenicity profiles

Abstract Rational design of adjuvants that simultaneously increase immunogenicity and reduce reactogenicity remains a challenge for protein vaccine development. Conventional adjuvants, like aluminum salts, and oil-in-water emulsions (like MF59, AS03, and AddaVax), work well to increase antibody titers but have variable abilities to modulate innate immune pathways, sustain long-term humoral immunity, or effectively redirect the Th2-skewed cellular responses they typically induce. Recent advances in the use of pattern-recognition receptors (PRRs), including Toll-like receptors (TLR) such as TLR4 and TLR9 agonists as adjuvants, formulated within nanoemulsions, have highlighted the potential of combination adjuvants for manipulating such responses. IVAX-1, a combination adjuvant consisting of MPLA (TLR4 agonist), CpG ODN 1018 (TLR9 agonist), and AddaVax nanoemulsion, exemplifies this tactic. In animal models, strong Th1-biased responses are elicited, and protection against infection is enhanced compared to individual adjuvant components. However, transient weight loss and enhanced systemic inflammation are observed with IVAX-1. To test the hypothesis that adjuvant delivery platform and TLR agonists influence the balance between immunogenicity and reactogenicity, we developed and characterized novel adjuvant formulations in which the model H5N1 hemagglutinin protein antigen was formulated with AddaVax or lipid nanoparticles, with or without incorporation of MPLA and or CpG. Our objective was to identify an alternative to IVAX-1 capable of co-delivering antigen and TLR agonists to reduce systemic reactogenicity without compromising immunogenicity or efficacy against H5N1 challenge. Our findings suggest that TLR agonist, adjuvant delivery platform and lipid composition play a central role in innate activation, adaptive immune polarization, and overall vaccine performance. Together, these results provide insight into the rational design of next-generation combination adjuvants for protein-based vaccines.