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.

No abstract

Telomeric regions of mammalian chromosomes contain suppressive TTAGGG motifs that inhibit several proinflammatory and Th1-biased immune responses. Synthetic oligodeoxynucleotides (ODN) expressing suppressive motifs can reproduce the down-regulatory activity of mammalian telomeric repeats and have proven effective in the prevention and treatment of several autoimmune and autoinflammatory diseases. Endotoxin-induced uveitis (EIU) is an established animal model of acute ocular inflammation induced by LPS administration. Augmented expression of proinflammatory cytokines/chemokines such as TNF␣, IL-6, and MCP1 and bactericidal nitric oxide production mediated by LPS contribute to the development of EIU. Suppressing these mediators using agents that are devoid of undesirable systemic side effects may help prevent the development of EIU. This study demonstrates the selective down-regulatory role of suppressive ODN after (i) local or (ii) systemic treatment in EIU-induced rabbits and mice. Our results indicate that suppressive ODN down-regulate at both the transcript and protein levels of several proinflammatory cytokines and chemokines as well as nitric oxide and co-stimulatory surface marker molecules when administrated prior to, simultaneously with, or even after LPS challenge, thereby significantly reducing ocular inflammation in both rabbit and mouse eyes. These findings strongly suggest that suppressive ODN is a potent candidate for the prevention of uveitis and could be applied as a novel DNAbased immunoregulatory agent to control other autoimmune or autoinflammatory diseases.DNA and RNA are the essential components of all living organisms. Accumulated evidence strongly suggests that these nucleic acids have multiple and complex effects on the immune system and are more than a blueprint of life (1, 2). On one hand, due to their high unmethylated CpG motif frequency, bacterial DNAs are recognized as "non-self" via TLR9 (Toll-like receptor 9) and trigger an innate immune response characterized by the proliferation and maturation of B cells, natural killer cells, and plasmacytoid dendritic cells and the secretion of T-helper 1-type cytokines, chemokines, and/or multivalent immunoglobulins (3-8). On the other hand, telomeric regions of mammalian chromosomes contain suppressive TTAGGG motifs that can inhibit several TLR-dependent and TLR-independent Th1-mediated immune responses. Of note, these motifs are underrepresented in the prokaryotic genome. Synthetic singlestranded oligodeoxynucleotides (ODN) 3 containing repetitive TTAGGG motifs mimic this effect (1, 9 -11). Previous studies revealed that deleterious inflammatory responses to a host can be down-regulated by suppressive ODN. In vitro, suppressive ODN inhibits the production of several proinflammatory cytokines and chemokines induced by bacteria (1,(12)(13)(14). Furthermore, in vivo suppressive ODN administration reduces the frequency and severity of several autoimmune and inflammatory diseases such as arthritis, systemic lupus erythematosus, pulmonary inflam...

Our study shows that a ten base pair insertion in the 5'UTR region of AMCase gene may modify gene expression and thus may affect the severity of asthma. However, its effects appear to be different in different populations.