RT Journal Article SR Electronic T1 TLR7 and RIG-I dual-adjuvant loaded nanoparticles drive broadened and synergistic responses in dendritic cells in vitro and generate unique cellular immune responses in influenza vaccination JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.07.17.207423 DO 10.1101/2020.07.17.207423 A1 Randall Toy A1 M. Cole Keenum A1 Pallab Pradhan A1 Katelynn Phang A1 Patrick Chen A1 Chinwendu Chukwu A1 Anh Nguyen A1 Jiaying Liu A1 Sambhav Jain A1 Gabrielle Kozlowski A1 Justin Hosten A1 Mehul S. Suthar A1 Krishnendu Roy YR 2020 UL http://biorxiv.org/content/early/2020/09/24/2020.07.17.207423.abstract AB Although the existing flu vaccines elicit strong antigen-specific antibody responses, they fail to provide effective, long term protection – partly due to the absence of robust cellular memory immunity. We hypothesized that co-administration of combination adjuvants, mirroring the flu-virus related innate signaling pathways, could elicit strong cellular immunity. Here, we show that the small molecule adjuvant R848 and the RNA adjuvant PUUC, targeting endosomal TLR7s and cytoplasmic RLRs respectively, when delivered together in polymer nanoparticles (NP), elicits a broadened immune responses in mouse bone marrow-derived dendritic cells (mBMDCs) and a synergistic response in both mouse and human plasmacytoid dendritic cells (pDCs). In mBMDCs, NP-R848-PUUC induced both NF-κB and interferon signaling. Interferon responses to co-delivered R848 and PUUC were additive in human peripheral blood mononuclear cells (PBMCs) and synergistic in human FLT3-differentiated mBMDCs and CAL-1 pDCs. Vaccination with NPs loaded with H1N1 Flu antigen, R848, and PUUC increased percentage of CD8+ T-cells in the lungs, percentage of antigen-specific CD4+T-cells in the spleen, and enhanced overall cytokine-secreting T cell percentages upon antigen restimulation. Also in the spleen, T lymphopenia, especially after in vitro restimulation, was observed. Our results demonstrate that simultaneous engagement of TLR7 and RIG-I pathways using particulate carriers is a potential approach to improve cellular immunity in flu vaccination.Competing Interest StatementThe authors have declared no competing interest.