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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.


Related in: MedlinePlus

D35 modification with dAs40 at 5′ and/or 3′ ends and their SPGylation effect on cytokine production from human PBMC. Human PBMCs were stimulated with ODNs (D35-dAs40, dAs40-D35, and D35-dAs40-D35; 1 μM each) or their SPGylated ODNs (D35-SPG, SPG-D35, and D35-SPG-D35; 1 μM each ODN amount), and then 24 hours later, IFN-α and IL-6 secretion in supernatants were determined by ELISA. Bar graph shows mean ± SEM in triplicate. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
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fig10: D35 modification with dAs40 at 5′ and/or 3′ ends and their SPGylation effect on cytokine production from human PBMC. Human PBMCs were stimulated with ODNs (D35-dAs40, dAs40-D35, and D35-dAs40-D35; 1 μM each) or their SPGylated ODNs (D35-SPG, SPG-D35, and D35-SPG-D35; 1 μM each ODN amount), and then 24 hours later, IFN-α and IL-6 secretion in supernatants were determined by ELISA. Bar graph shows mean ± SEM in triplicate. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Mentions: We then attempted to improve the immunostimulatory profile of D35-derived ODNs by complexing them with SPG (SPGylation; see Materials and Methods), performed similarly with previously reported K3-SPG [16]. D35-SPG, SPG-D35, and D35-SPG-D35 were made by complexing SPG with D35-dAs40, dAs40-D35, and dAs40-D35-dAs40, respectively. Complexation efficiency was evaluated by a MultiNA microchip electrophoresis system and the result was as follows: D35-SPG (99.4%), SPG-D35 (96.7%), and D35-SPG-D35 (49.8%). This indicated that ODNs in either D35-SPG or SPG-D35 solution were almost completely complexed, whereas only 50% of ODN was complexed in the D35-SPG-D35 solution. Human PBMCs were stimulated with the SPGylated ODNs, and IFN-α and IL-6 secretion was determined by ELISA (Figure 10). In contrast to K3-SPG [16], 5′ or 3′ SPGylation of D35 did not improve cytokine production but reduced IFN-α and IL-6 secretion compared with the non-SPGylated ODNs (Figure 10). Among them, D35-SPG had a greater immunostimulatory effect than SPG-D35 (Figure 10). Of note, the non-SPGylated ODNs such as D35-dAs40 and dAs40-D35 with a 5′ end addition of dAs40 and 3′ end addition of dAs40 showed comparable immunostimulatory activities (Figure 10). However, we observed slightly better cytokine production with D35-dAs40 compared to with dAs40-D35 in other experiments. Interestingly, dAs40-D35 also did not develop visible large aggregates formation in PBS (Figure 11). D35-SPG-D35 substantially enhanced IFN-α and IL-6 secretion from PBMCs, although the complexation efficiency was only about 50%. Although D35-SPG-D35 showed a cytokine profile improvement similar to that for K3-SPG [16], we did not pursue D35-SPG-D35 development further in this study, because of the ODN size (80 base pairs) and difficulties in achieving full complexation with SPG. Further experiments are required to understand the mechanisms and biological characteristics of these SPGylated D type CpG ODNs.


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)

D35 modification with dAs40 at 5′ and/or 3′ ends and their SPGylation effect on cytokine production from human PBMC. Human PBMCs were stimulated with ODNs (D35-dAs40, dAs40-D35, and D35-dAs40-D35; 1 μM each) or their SPGylated ODNs (D35-SPG, SPG-D35, and D35-SPG-D35; 1 μM each ODN amount), and then 24 hours later, IFN-α and IL-6 secretion in supernatants were determined by ELISA. Bar graph shows mean ± SEM in triplicate. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4562176&req=5

fig10: D35 modification with dAs40 at 5′ and/or 3′ ends and their SPGylation effect on cytokine production from human PBMC. Human PBMCs were stimulated with ODNs (D35-dAs40, dAs40-D35, and D35-dAs40-D35; 1 μM each) or their SPGylated ODNs (D35-SPG, SPG-D35, and D35-SPG-D35; 1 μM each ODN amount), and then 24 hours later, IFN-α and IL-6 secretion in supernatants were determined by ELISA. Bar graph shows mean ± SEM in triplicate. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
Mentions: We then attempted to improve the immunostimulatory profile of D35-derived ODNs by complexing them with SPG (SPGylation; see Materials and Methods), performed similarly with previously reported K3-SPG [16]. D35-SPG, SPG-D35, and D35-SPG-D35 were made by complexing SPG with D35-dAs40, dAs40-D35, and dAs40-D35-dAs40, respectively. Complexation efficiency was evaluated by a MultiNA microchip electrophoresis system and the result was as follows: D35-SPG (99.4%), SPG-D35 (96.7%), and D35-SPG-D35 (49.8%). This indicated that ODNs in either D35-SPG or SPG-D35 solution were almost completely complexed, whereas only 50% of ODN was complexed in the D35-SPG-D35 solution. Human PBMCs were stimulated with the SPGylated ODNs, and IFN-α and IL-6 secretion was determined by ELISA (Figure 10). In contrast to K3-SPG [16], 5′ or 3′ SPGylation of D35 did not improve cytokine production but reduced IFN-α and IL-6 secretion compared with the non-SPGylated ODNs (Figure 10). Among them, D35-SPG had a greater immunostimulatory effect than SPG-D35 (Figure 10). Of note, the non-SPGylated ODNs such as D35-dAs40 and dAs40-D35 with a 5′ end addition of dAs40 and 3′ end addition of dAs40 showed comparable immunostimulatory activities (Figure 10). However, we observed slightly better cytokine production with D35-dAs40 compared to with dAs40-D35 in other experiments. Interestingly, dAs40-D35 also did not develop visible large aggregates formation in PBS (Figure 11). D35-SPG-D35 substantially enhanced IFN-α and IL-6 secretion from PBMCs, although the complexation efficiency was only about 50%. Although D35-SPG-D35 showed a cytokine profile improvement similar to that for K3-SPG [16], we did not pursue D35-SPG-D35 development further in this study, because of the ODN size (80 base pairs) and difficulties in achieving full complexation with SPG. Further experiments are required to understand the mechanisms and biological characteristics of these SPGylated D type CpG ODNs.

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.


Related in: MedlinePlus