CpG ODN Nanorings Induce IFNα from Plasmacytoid Dendritic Cells and Demonstrate Potent Vaccine Adjuvant Activity

Abstract

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.

A novel RNA‐based adjuvant combines strong immunostimulatory capacities with a favorable safety profile

“…Moreover, the CpG dependent effects come at the cost of frequent adverse effects, such as splenomegaly, and despite successes in pre-clinical models low efficacy in clinical trials have been observed, possibly reflecting different expression patterns of TLR9 in humans and mice. 13,46,47 One of the natural receptors for single-stranded RNA is TLR7 20 ; however, our data showing that RNAdjuvant V R induces type I interferon in both pDCs (expressing TLR7) and mDCs (expressing TLR8) suggest that this molecule can be recognized by both receptors. Additionally, enhanced expression of genes crucial for RIG-I-mediated activity after intradermal injection of our adjuvant suggests that also RIG-I-like receptors participate in the local sensing of RNAdjuvant V R .…”

“…8,9 However, only modest effects have been observed in clinical trials 10-12 presumably due to the restricted cellular distribution of TLR9 in humans. 13 Poly(I:C) has been shown to rapidly induce secretion of inflammatory cytokines and to elicit cytotoxic T-cell responses, resulting in strong anti-tumor effects in pre-clinical models. [14][15][16] As Poly(I:C) has limiting toxicity, several modified versions such as PolyI:PolyC12U 17 and Poly(IC:LC) 18 have been developed to reduce toxicity.…”

CpG oligodeoxynucleotide nanomedicines for the prophylaxis or treatment of cancers, infectious diseases, and allergies

“…For instance, the induction level of IFN-α in the nanoringstructured complex of CpG-B ODN with Tat-derived peptide was similar to that of free CpG-A ODN in human PBMCs. 90 Although the induction level of IFN-α in the nanoringstructured complex did not show greater potential than free CpG-A ODN, the nanoring-structured complex has an advantage. This implies that the nanoring-structured complex has a narrow size distribution, 90 whereas free CpG-A ODN forms uncontrolled higher order structures of various sizes by self-assembly, 18,19 which makes it impossible to provide free CpG-A ODN for clinical application.…”

“…There was correlation between the level of IFN-α secreted from PBMCs because of these complexes and the zeta potential of the complex; the higher the positive charge density, the higher was the IFN-α induction level. 90 The highest IFN-α inducer was a complex of CpG-B ODN and Tat-derived peptide, prepared with a molecular ratio of 1:16 and having a zeta potential of ~40 mV. This complex had a nanoring structure, with segment particles ~40 nm in diameter arranged in a doughnut shape ( Figure 2B).…”

Double-stranded phosphodiester cytosine-guanine oligodeoxynucleotide complexed with calcium phosphate as a potent vaccine adjuvant for activating cellular and Th1-type humoral immunities

“…However, these CpG ODNs form uncontrollable higher-order structures due to their palindromic and/or poly-G sequences, 21,25,26 which precludes their clinical application. 27 One way to overcome this problem is complexation of CpG-B with other materials such as cationic peptides, polycations, and polysaccharides, [28][29][30][31][32] because multimerization of conventional CpG-B converts it from an inducer of proinflammatory cytokines to one of type-I IFN. However, complexation can be costly and complicated; this limits the use of CpG-B complexes as an adjuvant for vaccines with high coverage such as influenza vaccine.…”