N-regulated, when compared with alumalumD150IFN700 600 ULOQ 400 300IP-10 ****IL-125 100IL-******** ****3 2 1 LLOQ4h**** **pg/mL100 75 50 LLOQh e 4h h Pr 48* **50 25****100e h h 4h Pr 48** *e Pr h 24 48 he4hPrhEnvEnv + alumEnv + alum / TLR4 Env + ANE / TLREnv + alum / TLR7 Env + pIC:LChEnv + MF59 Env + ISCOMEnv + ANE / TLRFigure 2. Adjuvant-specific innate gene signatures. RNA transcripts have been measured from peripheral blood sampling prevaccination and 4 hours, 24 hours, 72 hours, and 14 days immediately after the initial immunization. (A) Heatmap showing upregulation and downregulation of innate gene modules by adjuvant at 24 hours. (B) Representative plots displaying the expression degree of the IFN (variety 1) module (major panel) and Ag-presentation module (bottom panel). (C) Association networks depicting the amount of modules regulated in prevalent between every adjuvant. Similarities in upregulation (red borders) and downregulation (blue borders) are shown; width of connecting lines indicates the amount of modules in prevalent, also shown numerically. (D) Quantification of serum cytokines prevaccination, and four hours, 24 hours, or 48 hours following the first vaccination. All data points represent vaccine group medians. *P , .05; **P , .01; ****P , .1451091-01-2 web 0001 compared using the prevaccination time point by ANOVA.7-Methoxyisoquinolin-1-ol structure LLOQ, reduce limit of quantitation; ULOQ, upper limit of quantitation.PMID:23291014 with alum alone (supplemental Figure 3C). PolyIC:LC and the emulsion-based formulations elicited statistically greater mucosal IgG titers compared with alum alone, using the ANE/TLR4 formulation inducing the highest titer (supplemental Figure 3D). Mucosal IgA responses have been largely undetectable.Effect of adjuvants on Env T-cell responsesWe next assessed T helper cell 1 (TH1) and TH2 T-cell responses by enumerating IFN-g and IL-4 ELISpots, the canonical cytokines for such responses, respectively (supplemental Figure 4A). Animals28 NOVEMBER 2017 x VOLUME 1, NUMBERimmunized with Env alone or Env 1 alum had low to undetectable IFN-g or IL-4 responses. In contrast, alum/TLR7 or pIC:LC had a higher frequency of TH1/IFN-g responses ranging from ;100 to ;1000 ELISpots but low to undetectable TH2/IL-4 responses. Immunization with MF59, ANE formulations with TLR4 or 7, and ISCOMs resulted in mixed TH1/TH2 responses with comparable frequencies of IFN-gand IL-4 roducing cells. Analysis of responses by flow cytometry showed that Env-specific CD41 T cells generating IFN-g was highest in animals immunized with pIC:LC (;0.four ) (supplemental Figure 4B). NHPs immunized with alum/TLR7, pIC:LC and ISCOMs had the highestEFFECT OF CLINICAL ADJUVANTS ON HIV Env IMMUNITYACell cycle Coagulation Monocyte enr. (surface) Monocyte enr. Neutrophil enr. Immune act. (generic) Inflammation (M3.two) Inflammation (M4.13) Inflammation (M4.two) Monocytes (surface) Muscle (MF59) Muscle (Alum,MF59) Muscle (MF59,CpG+MF59) Platelets Transcription Ag. presentation DC Monocyte enr. (act.) ECM Cell death Apoptosis Signaling DCs (resting) Transmembrane DC enr. (act.) DCs (act.) Antiviral Interferon (M3.4) Interferon (M5.12) IFN (Type I) Viral sensing Interferon (M1.two) IFN (antiviral) Muscle (Alum,MF59,CpG,CpG+MF59) Muscle (PBS,Alum,MF59,CpG,CpG+MF59)BMMPCEffect of TLR 4 or 7 on alum-regulated modules alum / TLR7 fold transform (log2)three two 1 -0.5 0.5 1.r2= 0.705 p= 0.Fold transform (log2)three two 1 0 -1 –1 -1.two.EIF2AKalum / TLR4 fold change (log2)Impact of TLR 4 or 7 on alum-regulated genes alum / TLR7 fold modify (log2)6 four two -1 1 two 3Fold transform (log2)three two 1 0 -.