Abstract
As emerging evidence supports the immune stimulating capability of the CpG oligodeoxynucleotides (ODN), CpG-based adjuvants have been widely used. For efficient induction of immune responses, current issues affecting the use of nucleic acid-based adjuvants, e.g. stability in physiological conditions, delivery to immune cells, and successful release within the phagolysosome, should be addressed. Here, we present CpG-based DNA microparticles (DNA-MPs) fabricated by complementary rolling circle amplification (cRCA) as adjuvants for enhancing immune response and production of selective antibody production. Using cRCA method, the sizes of CpG-based DNA-MPs were finely controlled (0.5 and 1 μm) with superior and provided mismatched single stranded form of CpG ODN region for specific cleavage site by DNase II within the phagolysosome. Fabricated CpG-based 1 μm DNA-MPs (DNA-MP-1.0) were successfully internalized into primary macrophages and macrophage cell line (RAW264.7 cells), and elicited superior cytokine production e.g. TNF-α and IL-6, compared to conventional CpG ODNs. After in vivo administration of DNA-MP-1.0 with model antigen ovalbumin (OVA), significantly elevated OVA-specific antibody production was observed. With this in mind, DNA-MP-1.0 could serve as a novel type of adjuvant for the activation of macrophages and the following production of selective antibodies without any noticeable toxicity in vitro and in vivo.
Highlights
Efficient and selective immune stimulation against diseaserelated antigens have been noticed as emerging therapeutic approaches for the treatment of cancers, allergies, and immunerelated diseases.[1,2] diverse immune-eliciting biomaterials including alum salts, monophosphoryl lipid a (MPLA), and liposomes have been investigated to provide efficient induction of immune responses with a low dose of vaccine antigens, fast immune responses, induction of a variety of antibodies, and wide range of humoral and cell-mediated immune responses.[3,4] Still, several antigens showed poor stimulation and antibody production by current adjuvant systems.[5,6] safe and selectively strong immune stimulators need to be developed
Micron sized particles have been mainly internalized into antigen presenting cells (APCs), such as macrophages and dendritic cells via phagocytosis, which could be facilitated as targeting strategies for vaccines and adjuvants to APCs.[24]
It should be noted that circle amplification (cRCA)-based formulation of DNA microparticles (DNA-MPs) resulted in greatly homogeneous microparticles with a narrow size distribution in this study
Summary
Efficient and selective immune stimulation against diseaserelated antigens have been noticed as emerging therapeutic approaches for the treatment of cancers, allergies, and immunerelated diseases.[1,2] diverse immune-eliciting biomaterials including alum salts, monophosphoryl lipid a (MPLA), and liposomes have been investigated to provide efficient induction of immune responses with a low dose of vaccine antigens, fast immune responses, induction of a variety of antibodies, and wide range of humoral and cell-mediated immune responses.[3,4] Still, several antigens showed poor stimulation and antibody production by current adjuvant systems.[5,6] safe and selectively strong immune stimulators need to be developed. As a novel type of immune stimulating adjuvant system, CpG oligodeoxynucleotide (ODN), a ligand for Toll-like receptor-9 (TLR-9), has been noted in vivo.[7,8] Unlike methylated CpG sequences in mammalian cells, unmethylated CpG sequences in bacteria could be recognized by TLR-9, which triggers strong humoral and cell-mediated immune responses via macrophages, dendritic cells, NK cells, and B lymphocytes.[8,9,10] CpG ODN with its negative charge hinders efficient internalization into cells with poor serum stability, which limits efficient induction of immune responses for antigen presenting cells (APCs).[11] To improve the stability of CpGs, chemical modi cations and double-stranded CpG structures could be considered. Particle-based delivery systems have been studied to protect CpG ODN from serum proteins and enzymes and to improve intracellular delivery without any loss of biological activity and side effects.[14]
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