CpG oligodeoxynucleotides (ODN) are short single-stranded synthetic DNA molecules that activate the immune system and have been found to be effective for preventing and treating infectious diseases, allergies, and cancers. Structurally distinct classes of synthetic ODN expressing CpG motifs differentially activate human immune cells. K-type ODN (K-ODN), which have progressed into human clinical trials as vaccine adjuvants and immunotherapeutic agents, are strong activators of B cells and trigger plasmacytoid dendritic cells (pDCs) to differentiate and produce tumor necrosis factor-α (TNFα). In contrast, D-type ODN (D-ODN) stimulate large amounts of interferon-α (IFNα) secretion from pDCs. This activity depends on the ability of D-ODN to adopt nanometer-sized G quadruplex-based structures, complicating their manufacturing and hampering their progress into the clinic. In search of a D-ODN substitute, we attempted to multimerize K-ODN into stable nanostructures using cationic peptides. We show that short ODN with a rigid secondary structure form nuclease-resistant nanorings after condensation with the HIV-derived peptide Tat(47-57). The nanorings enhanced cellular internalization, targeted the ODN to early endosomes, and induced a robust IFNα response from human pDCs. Compared to the conventional K-ODN, nanorings boosted T helper 1-mediated immune responses in mice immunized with the inactivated foot and mouth disease virus vaccine and generated superior antitumor immunity when used as a therapeutic tumor vaccine adjuvant in C57BL/6 mice bearing ovalbumin-expressing EG.7 thymoma tumors. These results suggest that the nanorings can act as D-ODN surrogates and may find a niche for further clinical applications.

Nucleic acid-based Toll-like receptor (TLR) ligands are promising adjuvants and immunotherapeutic agents. Combination of TLR ligands potentiates immune response by providing synergistic immune activity via triggering different signaling pathways and may impact antigen dependent T-cell immune memory. However, their short circulation time due to nuclease attack hampers their clinical performance. Liposomes offer inclusion of protein and nucleic acid-based drugs with high encapsulation efficiency and drug loading. Furthermore, they protect cargo from enzymatic cleavage while providing stability, and enhancing biological activity. Herein, we aimed to develop a liposomal carrier system co-encapsulating TLR3 (polyinosinic-polycytidylic acid; poly(I:C)) and TLR9 (oligodeoxynucleotides (ODN) expressing unmethylated CpG motifs; CpG ODN) ligands as immunoadjuvants together with protein antigen. To demonstrate that this depot system not only induce synergistic innate immune activation but also boost antigen-dependent immune response, we analyzed the potency of dual ligand encapsulated liposomes in long-term cancer protection assay. Data revealed that CpG ODN and poly(I:C) co-encapsulation significantly enhanced cytokine production from spleen cells. Activation and maturation of dendritic cells as well as bactericidal potency of macrophages along with internalization capacity of ligands were elevated upon incubation with liposomes co-encapsulating CpG ODN and poly(I:C). Immunization with co-encapsulated liposomes induced OVA-specific Th1-biased immunity which persisted for eight months post-booster injection. Subsequent challenge with OVA-expressing tumor cell line, E.G7, demonstrated that mice immunized with liposomes co-encapsulating dual ligands had significantly slower tumor progression. Tumor clearance was dependent on OVA-specific cytotoxic memory T-cells. These results suggest that liposomes co-encapsulating TLR3 and TLR9 ligands and a specific cancer antigen could be developed as a preventive cancer vaccine.

Recognition of pathogen-derived nucleic acids by immune cells is critical for the activation of protective innate immune responses. Bacterial cyclic dinucleotides (CDNs) are small nucleic acids that are directly recognized by the cytosolic DNA sensor STING (stimulator of IFN genes), initiating a response characterized by proinflammatory cytokine and type I IFN production. Strategies to improve the immune stimulatory activities of CDNs can further their potential for clinical development. Here, we demonstrate that a simple complex of cylic-di-GMP with a cell-penetrating peptide enhances both cellular delivery and biological activity of the cyclic-di-GMP in murine splenocytes. Furthermore, our findings establish that activation of the TLR-dependent and TLR-independent DNA recognition pathways through combined use of CpG oligonucleotide (ODN) and CDN results in synergistic activity, augmenting cytokine production (IFN-α/β, IL-6, TNF-α, IP-10), costimulatory molecule upregulation (MHC class II, CD86), and antigen-specific humoral and cellular immunity. Results presented herein indicate that 3 3 -cGAMP, a recently identified bacterial CDN, is a superior stimulator of IFN genes ligand than cyclic-di-GMP in human PBMCs. Collectively, these findings suggest that the immune-stimulatory properties of CDNs can be augmented through peptide complexation or synergistic use with CpG oligonucleotide and may be of interest for the development of CDN-based immunotherapeutic agents. Keywords:Arginine peptide (nona-arginine) r cGAMP r CpG ODN r Cyclic-di-GMP r Immunostimulation See accompanying article by Temizoz et al.Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionDetection of pathogen-derived nucleic acids by innate immune cells is critical for the initiation of protective responses against bacterial, viral, and fungal pathogens. Pathogen-derived nucleic acids are sensed based on their sequence, structure, nucleotide Correspondence: Dr. Mayda Gursel e-mail: mgursel@metu.edu.tr modifications, and their intracellular localization. So far, several nucleic acid sensors have been identified, including the endosomal TLRs TLR3, TLR7/TLR8, and TLR9 that recognize dsRNA, ssRNA, and CpG-containing DNA; cytosolic dsRNA sensors RIG-I, MDA5, LGP2; and a plethora of cytosolic receptors dedicated for dsDNA recognition (DAI, AIM2, RNA polymerase III, IFI16, DEAD-box helicase DDX41) [1]. Signaling in response to cytosolic DNA depends on the expression of the adaptor protein STING (stimulator of IFN genes, also known as TMEM173, MPYS, MITA and ERIS) and proceeds through the TBK1-IRF3 axis, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2015. 45: 1170-1179 Innate immunity 1171 culminating in type I IFN production [2, 3]. STING itself is not a DNA-recognition molecule. However, it can directly recognize cyclic dinucleotides (CDNs) such as the bacteria-derived molecule cyclic-di-GMP (c-di-GMP) [4]. Although CDNs were thought to functio...

Type I IFN-mediated neutrophil activation and NET formation may contribute to inflammatory manifestations observed in patients with AT, Artemis deficiency, and SAVI. Thus, neutrophils represent a promising target to manage inflammatory syndromes in diseases with active type I IFN signature.

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