In addition, to assess whether DC activation could occur earlier than 12 h, we analyzed the expression of costimulatory molecules 6 h after injection, and observed the same pattern of CD80, CD86, and CD40 expression in both DCs subsets (Figures S4ACC, respectively). costimulatory molecules and DCs maturation (27C30). Mouse standard DC subsets differentially express a Corticotropin Releasing Factor, bovine broad repertoire of TLRs that result in different activating phenotypes and adaptive immunity (31). The co-delivery Corticotropin Releasing Factor, bovine of Corticotropin Releasing Factor, bovine TLR ligands and DEC205 targeted antigens has been shown to significantly improve vaccine immunogenicity in mice and in non-human primates (16). Polyinosinic:polycytidylic acid [poly(I:C)] is usually a synthetic analog of viral double-stranded RNAs (dsRNAs) that activates TLR3 and RIG-I-like receptors (retinoic acid-inducible gene -I- like receptors, or RLRs) (32). Poly(I:C) is the most commonly administered Vegfa adjuvant in mice in the context of DC-targeted vaccines using DEC205 mAbs fused with proteins of interest (18). This strategy has already been tested with chimeric mAbs made up of proteins derived from dengue computer virus (33), (34), sp (26, 35, 36), (37), (22), (23), HIV (21, 38, 39) and also from tumors (40). The excellent results obtained with this adjuvant, justified its use in clinical trials. To improve poly I:C stability (32) in humans, a modified version (poly-ICLC) was developed and used in different trials (41, 42). Monophosphoryl lipid A (MPL), a chemically derivative of bacterial lipopolysaccharide (LPS), is usually a Corticotropin Releasing Factor, bovine TLR4 agonist that preferentially activates the TIR-domain-containing adapter-inducing interferon- (TRIF) signaling pathway to drive the production of Th1 cytokines and activate CD4+ T cells (43) (44, 45). MPL is the first and only TLR ligand licensed in a human vaccine (Melacine?, approved as a melanoma vaccine). More recently, other MPL-containing vaccines became available (Fendrix? and Cervarix?, both from GSK) (46). CpG oligodeoxynucleotides (ODN) are unmethylated CpG motifs that interact with endosomal TLR9 and lead to proinflammatory cytokine production by DCs (47). B type ODN has a protective phosphorothioate backbone that protects it from nuclease digestion and enhances its half-life (48). Several clinical trials were conducted and CpG ODN emerged as a potent adjuvant to induce high antibody titers more quickly and after fewer doses (49, 50). Moreover, CpG ODN has been used along with DEC205 mAb to target HIV and proteins (51, 52). Here, we used eight promiscuous HIV-derived CD4+ T cell epitopes (HIVBr8) fused with DEC205 to target CD11c+ CD8+ DCs in the presence of different TLR ligands. The hierarchy of adjuvant potency shows that poly(I:C) is a superior adjuvant for the multiepitope DC-targeted vaccine in magnitude, breadth, and longevity. Materials and Methods Generation of the Fusion Monoclonal Antibody (mAb) Plasmids encoding the light and heavy chain of the mouse DEC205 antibody were kindly provided by Dr. Michel C. Nussenzweig (The Rockefeller University or college, New York, USA). The plasmid encoding the heavy chain of the mouse DEC205 fused to eight HIV-1 epitopes was previously explained and contains the following epitopes: p6 (32-46), p17 (73-89), pol (785-799), gp160 (188-201), rev (11-27), vpr (65-82), vif (144-158), and nef (180-194) (39). Expression and Purification of DECHIVBr8 mAb The production of DECHIVBr8 mAb [initial clone NLDC145 (24)] was performed after transient transfection of human embryonic kidney (HEK) 293T cells (ATCC, CRL-11268) exactly as explained elsewhere (33). Briefly, 293T cells were cultured in 150 mm plates (Sarstedt) under standard conditions in Dulbecco’s altered Eagle’s medium (Invitrogen) supplemented with 1% (v/v) antibiotic-antimycotic (Invitrogen), 1% (v/v) l-glutamine (Invitrogen), and 5% (v/v) Ultra low IgG Fetal Bovine Serum (Invitrogen). When cell confluence reached 70%, 293T cells were transfected using Corticotropin Releasing Factor, bovine 10 g of the plasmids encoding the light and the heavy chains in the presence of 150.