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Development of Nonaggregating Poly-A Tailed Immunostimulatory A/D Type CpG Oligodeoxynucleotides Applicable for Clinical Use.

Aoshi T, Haseda Y, Kobiyama K, Narita H, Sato H, Nankai H, Mochizuki S, Sakurai K, Katakai Y, Yasutomi Y, Kuroda E, Coban C, Ishii KJ - J Immunol Res (2015)

Bottom Line: We found that two modified ODNs including D35-dAs40 and D35core-dAs40 were immunostimulatory, similar to original D35 in human PBMCs, resulting in high IFN-α secretion in a dose-dependent manner.Physical property analysis by dynamic light scattering revealed that both D35-dAs40 and D35core-dAs40 did not form aggregates in saline, which is currently impossible for the original D35.These results suggested that D35-dAs40 and D35core-dAs40 are two promising prototypes of nonaggregating A/D type ODN with advantages of ease of drug preparation for clinical applications as vaccine adjuvants or IFN-α inducing immunomodifiers.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Adjuvant Innovation, National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka 567-0085, Japan ; Laboratory of Vaccine Science, Immunology Frontier Research Center (iFReC), Osaka University, Suita, Osaka 565-0871, Japan ; Vaccine Dynamics Project, BIKEN Innovative Vaccine Research Alliance Laboratories, Research Institute for Microbial Diseases (RIMD), Osaka University, Suita, Osaka 565-0871, Japan.

ABSTRACT
Immunostimulatory CpG ODNs have been developed and utilized as TLR9-dependent innate immune activators and vaccine adjuvants. Four different types of immunostimulatory CpG ODNs (A/D, B/K, C, and P type) have been reported. A/D type ODNs are characterized by high IFN-α production but intrinsically form aggregates, hindering its good manufacturing practice grade preparation. In this study, we developed several D35-derived ODNs (a commonly used A/D type ODN), which were modified with the addition of a phosphorothioate polynucleotide tail (such as dAs40), and examined their physical properties, solubility in saline, immunostimulatory activity on human PBMCs, and vaccine adjuvant potential in monkeys. We found that two modified ODNs including D35-dAs40 and D35core-dAs40 were immunostimulatory, similar to original D35 in human PBMCs, resulting in high IFN-α secretion in a dose-dependent manner. Physical property analysis by dynamic light scattering revealed that both D35-dAs40 and D35core-dAs40 did not form aggregates in saline, which is currently impossible for the original D35. Furthermore, D35-dAs40 and D35core-dAs40 worked as better vaccine adjuvant in monkeys. These results suggested that D35-dAs40 and D35core-dAs40 are two promising prototypes of nonaggregating A/D type ODN with advantages of ease of drug preparation for clinical applications as vaccine adjuvants or IFN-α inducing immunomodifiers.

No MeSH data available.


Screening of human PBMC with several different polynucleotide tailed A/D type ODNs. Human PBMCs were stimulated with the indicated synthetic ODNs (1 μM) (Table 1) for 24 hours. IFN-α and IL-6 production in supernatants was measured by ELISA. (a) The requirement of CpG-motifs and G-hexamers for cytokine secretion from human PBMCs. Both IFN-α and IL-6 were secreted in a CpG motif dependent but G-hexamer sequence independent manner. (b) Comparison of tail backbones (phosphorothioate or phosphodiester). Cytokine secretion was dependent on phosphorothioate 40-mer tails (1, 3, and 5). ODNs with phosphodiester 40-mer tails showed almost no biological activity (2, 4, 6). The bar graphs indicate the concentrations from a single well of each stimulation. The results are representative of three different experiments consisting of two different donors (Exp-1 and Exp-2, Exp-3 and Exp-4).
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fig1: Screening of human PBMC with several different polynucleotide tailed A/D type ODNs. Human PBMCs were stimulated with the indicated synthetic ODNs (1 μM) (Table 1) for 24 hours. IFN-α and IL-6 production in supernatants was measured by ELISA. (a) The requirement of CpG-motifs and G-hexamers for cytokine secretion from human PBMCs. Both IFN-α and IL-6 were secreted in a CpG motif dependent but G-hexamer sequence independent manner. (b) Comparison of tail backbones (phosphorothioate or phosphodiester). Cytokine secretion was dependent on phosphorothioate 40-mer tails (1, 3, and 5). ODNs with phosphodiester 40-mer tails showed almost no biological activity (2, 4, 6). The bar graphs indicate the concentrations from a single well of each stimulation. The results are representative of three different experiments consisting of two different donors (Exp-1 and Exp-2, Exp-3 and Exp-4).

Mentions: We previously developed K3-SPG, a second generation B/K type CpG adjuvant, which is a particulate soluble complex of K3 CpG-ODN and schizophyllan (SPG) [16, 17]. To form complexes between K3-ODN and SPG, K3 ODNs have to be modified by adding a phosphorothioate 40-mer of deoxyadenylic acids (dAs40 tail) at the 3′ ends where SPG and dAs40 tails form triple-helix complexes [16]. In parallel with these experiments, we have also synthesized similarly tailed D35 ODNs, and tested their immunostimulatory activities (IFN-α and IL-6 induction) on human PBMCs. Cytokine ELISAs revealed that D35 (A/D type CpG-ODN) with an additional dAs40 tail at the 3′ end was similarly active as the original D35 (Figure 1(a); sample 1 versus D35). Each ODN sequence used in this study is shown in Table 1. Subsequent experiments further revealed that some D35-derived ODNs with a dAs40 tail were also biologically active, even with replacement of the 3′ G-hexamer to A-, T-, and C-hexamer (Figure 1(a); samples 5, 9, and 13). This is relatively unexpected, because the G-hexamer formed quadruplex structures via Hoogsteen base pairing, resulting in self-aggregates [10, 11], and it is believed that G-hexamer-mediated aggregation is an obligatory requirement for the biological activity of A/D type CpG ODNs [5, 12–14]. On the other hand, the immunostimulatory activities of D35-derived ODNs on human PBMCs are totally dependent on the presence of the CpG sequence. Replacement of CpG motif to GC, TG, and CT completely suppressed the ODNs' immunostimulatory activities (Figure 1(a); sample 9 versus samples 10–12), consistent with the unmethylated CpG-motif theory [18]. These results indicated that the presence of the G-hexamer sequence is not a necessary requirement for A/D type ODN immunostimulatory activities, whereas the activities of D35-derived ODNs was dominantly dependent on the presence of the CpG motif sequence.


Development of Nonaggregating Poly-A Tailed Immunostimulatory A/D Type CpG Oligodeoxynucleotides Applicable for Clinical Use.

Aoshi T, Haseda Y, Kobiyama K, Narita H, Sato H, Nankai H, Mochizuki S, Sakurai K, Katakai Y, Yasutomi Y, Kuroda E, Coban C, Ishii KJ - J Immunol Res (2015)

Screening of human PBMC with several different polynucleotide tailed A/D type ODNs. Human PBMCs were stimulated with the indicated synthetic ODNs (1 μM) (Table 1) for 24 hours. IFN-α and IL-6 production in supernatants was measured by ELISA. (a) The requirement of CpG-motifs and G-hexamers for cytokine secretion from human PBMCs. Both IFN-α and IL-6 were secreted in a CpG motif dependent but G-hexamer sequence independent manner. (b) Comparison of tail backbones (phosphorothioate or phosphodiester). Cytokine secretion was dependent on phosphorothioate 40-mer tails (1, 3, and 5). ODNs with phosphodiester 40-mer tails showed almost no biological activity (2, 4, 6). The bar graphs indicate the concentrations from a single well of each stimulation. The results are representative of three different experiments consisting of two different donors (Exp-1 and Exp-2, Exp-3 and Exp-4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: Screening of human PBMC with several different polynucleotide tailed A/D type ODNs. Human PBMCs were stimulated with the indicated synthetic ODNs (1 μM) (Table 1) for 24 hours. IFN-α and IL-6 production in supernatants was measured by ELISA. (a) The requirement of CpG-motifs and G-hexamers for cytokine secretion from human PBMCs. Both IFN-α and IL-6 were secreted in a CpG motif dependent but G-hexamer sequence independent manner. (b) Comparison of tail backbones (phosphorothioate or phosphodiester). Cytokine secretion was dependent on phosphorothioate 40-mer tails (1, 3, and 5). ODNs with phosphodiester 40-mer tails showed almost no biological activity (2, 4, 6). The bar graphs indicate the concentrations from a single well of each stimulation. The results are representative of three different experiments consisting of two different donors (Exp-1 and Exp-2, Exp-3 and Exp-4).
Mentions: We previously developed K3-SPG, a second generation B/K type CpG adjuvant, which is a particulate soluble complex of K3 CpG-ODN and schizophyllan (SPG) [16, 17]. To form complexes between K3-ODN and SPG, K3 ODNs have to be modified by adding a phosphorothioate 40-mer of deoxyadenylic acids (dAs40 tail) at the 3′ ends where SPG and dAs40 tails form triple-helix complexes [16]. In parallel with these experiments, we have also synthesized similarly tailed D35 ODNs, and tested their immunostimulatory activities (IFN-α and IL-6 induction) on human PBMCs. Cytokine ELISAs revealed that D35 (A/D type CpG-ODN) with an additional dAs40 tail at the 3′ end was similarly active as the original D35 (Figure 1(a); sample 1 versus D35). Each ODN sequence used in this study is shown in Table 1. Subsequent experiments further revealed that some D35-derived ODNs with a dAs40 tail were also biologically active, even with replacement of the 3′ G-hexamer to A-, T-, and C-hexamer (Figure 1(a); samples 5, 9, and 13). This is relatively unexpected, because the G-hexamer formed quadruplex structures via Hoogsteen base pairing, resulting in self-aggregates [10, 11], and it is believed that G-hexamer-mediated aggregation is an obligatory requirement for the biological activity of A/D type CpG ODNs [5, 12–14]. On the other hand, the immunostimulatory activities of D35-derived ODNs on human PBMCs are totally dependent on the presence of the CpG sequence. Replacement of CpG motif to GC, TG, and CT completely suppressed the ODNs' immunostimulatory activities (Figure 1(a); sample 9 versus samples 10–12), consistent with the unmethylated CpG-motif theory [18]. These results indicated that the presence of the G-hexamer sequence is not a necessary requirement for A/D type ODN immunostimulatory activities, whereas the activities of D35-derived ODNs was dominantly dependent on the presence of the CpG motif sequence.

Bottom Line: We found that two modified ODNs including D35-dAs40 and D35core-dAs40 were immunostimulatory, similar to original D35 in human PBMCs, resulting in high IFN-α secretion in a dose-dependent manner.Physical property analysis by dynamic light scattering revealed that both D35-dAs40 and D35core-dAs40 did not form aggregates in saline, which is currently impossible for the original D35.These results suggested that D35-dAs40 and D35core-dAs40 are two promising prototypes of nonaggregating A/D type ODN with advantages of ease of drug preparation for clinical applications as vaccine adjuvants or IFN-α inducing immunomodifiers.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Adjuvant Innovation, National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka 567-0085, Japan ; Laboratory of Vaccine Science, Immunology Frontier Research Center (iFReC), Osaka University, Suita, Osaka 565-0871, Japan ; Vaccine Dynamics Project, BIKEN Innovative Vaccine Research Alliance Laboratories, Research Institute for Microbial Diseases (RIMD), Osaka University, Suita, Osaka 565-0871, Japan.

ABSTRACT
Immunostimulatory CpG ODNs have been developed and utilized as TLR9-dependent innate immune activators and vaccine adjuvants. Four different types of immunostimulatory CpG ODNs (A/D, B/K, C, and P type) have been reported. A/D type ODNs are characterized by high IFN-α production but intrinsically form aggregates, hindering its good manufacturing practice grade preparation. In this study, we developed several D35-derived ODNs (a commonly used A/D type ODN), which were modified with the addition of a phosphorothioate polynucleotide tail (such as dAs40), and examined their physical properties, solubility in saline, immunostimulatory activity on human PBMCs, and vaccine adjuvant potential in monkeys. We found that two modified ODNs including D35-dAs40 and D35core-dAs40 were immunostimulatory, similar to original D35 in human PBMCs, resulting in high IFN-α secretion in a dose-dependent manner. Physical property analysis by dynamic light scattering revealed that both D35-dAs40 and D35core-dAs40 did not form aggregates in saline, which is currently impossible for the original D35. Furthermore, D35-dAs40 and D35core-dAs40 worked as better vaccine adjuvant in monkeys. These results suggested that D35-dAs40 and D35core-dAs40 are two promising prototypes of nonaggregating A/D type ODN with advantages of ease of drug preparation for clinical applications as vaccine adjuvants or IFN-α inducing immunomodifiers.

No MeSH data available.