[PMC free article] [PubMed] [CrossRef] [Google Scholar] 37. of doses ranging from 0-1000 ng/mL. TNF supernatant concentrations were measured 24 hours after pDC activation. In all experiments, dual delivery was performed with a single nanoparticle system. Nanoparticle mass was fixed across all PUUC doses. Statistical significance was evaluated with AHU-377 (Sacubitril calcium) one-way ANOVA followed by Tukeys test for multiple comparisons. *P0.05, ** P0.01, ***P0.001. NIHMS1645380-supplement-7.tif (5.8M) GUID:?4C272A0F-AE0A-48DD-9923-B41402ABA13F 8: SI Figure 3. Characterization of nanoparticles for different surface densities of PUUC loading. A) Nanoparticles with and without encapsulated R848 (4.5 g/mg) were loaded with PUUC adjuvant densities ranging from 0-11.6 g/mg of particle, corresponding to the same densities depicted in Figure 4B and SI Figure 2. Surface zeta potential and B) particle size (diameter peak intensity) were determined by DLS measurements at 0.2 mg NP/mL in 10 mM KCl on a Malvern Zetasizer Nano ZS. Spectroscopic analysis of supernatant RNA concentration confirmed 100% loading efficiency of PUUC at these loading densities. Mean standard deviation for surface zeta potential and diameter are shown. NIHMS1645380-supplement-8.tif (2.0M) GUID:?24D92B56-DD55-443E-9AD3-A8D41C07393A 9: SI Figure 4. Representative flow gating scheme to identify Class II tetramer positive CD3+CD8? T-cells in the splenocytes. NIHMS1645380-supplement-9.tif (4.9M) GUID:?7517A18D-EBB6-43F9-B47D-E7CDB42534B4 10: SI Figure 5. Antibody and cell-mediated memory responses to NPs with HA, R848, and PUUC. A) Percentage of popliteal lymphocytes with high expression of B220. B) IgG1 and IgG2a antibody titers were measured from serum. Populations of C) CD4+ central memory cells (CD3+ CD4+ CD44+ CD62L+ CCR7+), D) CD4+ effector memory cells (CD3+ CD4+ CD44+ CD62L? CCR7?), and E) CD8+ central memory cells (CD3+ CD8+ CD62L+ CD127+ KLRG1+) were measured in spleen. F) CD3+ CD8+ live splenocytes producing IFN-, IL-4, and TNF- and G) polyfunctional CD4+ and CD8+ T-cells producing both IFN- and TNF- after restimulation with H1N1 antigen for 6 hours. Error bars represent SD of the mean. Statistical significance was determined by one-way ANOVA followed by Tukeys test for multiple comparisons for normal datasets. *P0.05, **P 0.01, ***P0.001, ****P0.0001. NIHMS1645380-supplement-10.tif (7.3M) GUID:?26814690-5734-45F2-AB83-885D21F9373E 11: SI Figure 6. Flow plots showing CD4+ and CD8+ T-cells in pre- and post-stimulated splenocytes with H1N1 antigen for single and dual adjuvant groups. CD4+ and CD8+ T-cell population for A) all experimental groups and B) for dual-adjuvant (showing individual mice) treatment group before and after restimulation with H1N1 antigen for 6 hours. One replicate of the dual-adjuvant treatment group was omitted due to low staining. The anti-CD4 antibody was not included in the pre-stimulation flow cytometry panel, and therefore, the CD4 T-cell was presumably gated on CD3+CD8? population. NIHMS1645380-supplement-11.tif (12M) GUID:?C2CAA879-282A-410F-8CD6-56A3AE1B168D Abstract Although the existing flu vaccines elicit strong antigen-specific antibody responses, they fail to provide effective, long term protection C 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, MGC33570 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-B and AHU-377 (Sacubitril calcium) interferon signaling. Interferon responses AHU-377 (Sacubitril calcium) 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 restimulation with dual adjuvants, was observed, indicating highly antigen-reactive T cells. 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. Graphical Abstract INTRODUCTION It is estimated that every year, globally, between 294,000 and 518,000 people die of influenza and associate complications. In the previous century, three global pandemic outbreaks of influenza occurred, with the AHU-377 (Sacubitril calcium) largest killing an estimated 50-100 million.1-3 While vaccines have controlled the spread of other deadly diseases, such as smallpox, polio, and measles, vaccines against the flu, although somewhat effective in the short-term, do not offer long-term protection. In the elderly, it is estimated that flu vaccines only protect 46% AHU-377 (Sacubitril calcium) of patients against the 2009 2009 pandemic H1N1 virus.4 High affinity antibodies generated.