Optimizing T Cell Priming Using TLR4/9 Combination Adjuvant For Robust Cell-Mediated Immunity Against Influenza 2307646

Abstract

Abstract Introduction Subunit vaccines are safe but rarely induce robust, long-lasting immunity, especially cell-mediated immunity (CMI). Here, we investigated the potential of IVAX-1, a combination of TLR4 (MPLA) and TLR9 (CpG 1018) agonists formulated in a squalene oil-in-water emulsion (AddaVax), to mediate protection and generate CMI. Methods We leveraged a murine H5N1 influenza model with fate-map reporters, conditional knock-out mice, multiphoton imaging, and flow cytometry to elucidate early spatiotemporal features of IVAX-1-driven immunity. Antigen-specific CMI was evaluated via ELISpot and recall assays. Results Immune priming with IVAX-1 enhanced dendritic cell (DC) maturation and recruitment of myeloid cells to the injection site, resulting in higher numbers of neutrophils, monocytes, and antigen-presenting DCs in the draining lymph node (DLN), independent of antigen clearance kinetics. This induced islands of IFN-γ+ cells in the DLN, suggesting clonal expansion. In addition, IVAX-1 generated a robust antigen-specific functional (IFN-γ+) T cell response. Deletion of Type 1 conventional DCs (XCR1+) cells abrogated IVAX-1-mediated T cell response. Furthermore, immunization with IVAX-1 generated significantly higher numbers of flu-specific memory T cells in the lungs and spleen. IVAX-1 conferred durable protection and superior viral clearance from the lungs up to 60 days after immunization. Conclusion By leveraging synergistic innate immune activation through multiple TLR pathways, the combination optimizes T cell priming without relying on antigen depot effects. Our findings identify key early innate immune events and cellular mediators that drive robust CMI and demonstrate the potential of TLR agonist combinations to elicit durable T-cell immunity. Funding Source NIH/NIAID grants U01AI160397 & R01AI168063; Adjuvant Comparison and Characterization contract BAA-DAIT-75N93020R00022; and grant from the Defense Threat Reduction Agency #HDTRA-18-1-0036; UC Irvine UROP Research Experience Fellowship; NIH-MARC U-STAR trai Topic Categories Vaccines and Immunotherapy (VAC)