QS-21 Modulates Macrophage Activation Through Synergistic Crosstalk with Pattern Recognition Receptors 2258356

Abstract

Abstract Introduction QS-21, a saponin-based adjuvant, is a key component of several licensed vaccines and is commonly co-formulated with monophosphoryl lipid A (MPLA) to enhance adaptive immune responses. Although its adjuvant efficacy is well established, the broader potential of QS-21 in combination with other pattern recognition receptor (PRR) agonists remains insufficiently characterized. In this work, we systematically investigated the immunomodulatory properties of QS-21 paired with five different PRR agonists: MPLA (TLR4 agonist), L18-MDP (NOD2 agonist), R848 (TLR7/8 agonist), CpG ODN (TLR9 agonist), and Poly I:C (TLR3 agonist). Methods Cytotoxicity assays were first conducted to define the lowest concentrations of QS-21 and each agonist, that were non-toxic yet capable of stimulating cells. Differentiated THP-1 macrophages were subsequently treated with individual agonists, QS-21 combinations, or MPLA combinations, and activation was assessed through surface activation marker expression and cytokine/chemokine secretion profiles. To further delineate the molecular mechanisms underlying these responses, we performed RNA sequencing and metabolomics profiling to identify distinct immunological signatures associated with each treatment. Results Our findings reveal that, although the PRR agonists exhibited variable activation capacities, the combinations of QS-21+MPLA with either Poly I:C or L18-MDP produced the strongest macrophage activation, surpassing the effects of single agents. Conclusion Collectively, these results expand the understanding of QS-21’s synergistic potential with diverse PRR agonists and suggest new avenues for adjuvant design. Notably, Poly I:C emerges as a particularly promising co-adjuvant with QS-21, capable of engaging complementary innate immune pathways to enhance vaccine-induced immunity. Funding Source NIH Topic Categories Vaccines and Immunotherapy (VAC)