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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

3′ addition of dAs40 sequence to A2216 and A2336 improves direct solubilization in saline. (a) Lyophilized A2216 vial (10 mg/vial) (upper left) was completely solubilized with 1 mL of saline directly at room temperature (RT) (lower left). The solution turned to a gel at 4°C (upper right) and reliquefied at 37°C (lower right). (b) Lyophilized A2336 vial (10 mg/vial) (upper) formed many visible gelatinous aggregation with 1 mL of saline at RT (lower). (c) Lyophilized A2216-dAs40 (0.5 mg/vial) and A2336-dAs40 (0.5 mg/vial) were easily and completely dissolved with 50 μL of saline at RT. (d) Human PBMCs were stimulated with the indicated synthetic ODNs (1 μM) for 24 hours. IFN-α and IL-6 production in the supernatants were examined by ELISA. Bar indicates the mean ± SEM.
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fig9: 3′ addition of dAs40 sequence to A2216 and A2336 improves direct solubilization in saline. (a) Lyophilized A2216 vial (10 mg/vial) (upper left) was completely solubilized with 1 mL of saline directly at room temperature (RT) (lower left). The solution turned to a gel at 4°C (upper right) and reliquefied at 37°C (lower right). (b) Lyophilized A2336 vial (10 mg/vial) (upper) formed many visible gelatinous aggregation with 1 mL of saline at RT (lower). (c) Lyophilized A2216-dAs40 (0.5 mg/vial) and A2336-dAs40 (0.5 mg/vial) were easily and completely dissolved with 50 μL of saline at RT. (d) Human PBMCs were stimulated with the indicated synthetic ODNs (1 μM) for 24 hours. IFN-α and IL-6 production in the supernatants were examined by ELISA. Bar indicates the mean ± SEM.

Mentions: We also examined the direct solubility of other A/D type ODNs such as A2216 and A2336 in saline, with and without the addition of dAs40 tail. Unexpectedly A2216 was gradually but completely dissolved in saline at room temperature. However, the solution turned into a gel at 4°C (Figure 9(a)). This gelation was reliquefied at 37°C incubation (Figure 9(a)). A2336 was not dissolved in saline and formed gelatinous aggregations, similar to D35 (Figure 9(b)). Addition of dAs40 tail to A2216 and A2336 greatly improved their solubility in saline. Both A2216-dAs40 and A2336-dAs40 readily dissolved in saline (Figure 9(c)) and did not show gelation at the 4°C. A2216-dAs40 and A2336-dAs40 also kept the IFN-α inducing abilities (Figure 9(d)). These results suggested that the addition of dAs40 tail is also useful modification for improving the other A/D type ODNs' manageabilities.


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)

3′ addition of dAs40 sequence to A2216 and A2336 improves direct solubilization in saline. (a) Lyophilized A2216 vial (10 mg/vial) (upper left) was completely solubilized with 1 mL of saline directly at room temperature (RT) (lower left). The solution turned to a gel at 4°C (upper right) and reliquefied at 37°C (lower right). (b) Lyophilized A2336 vial (10 mg/vial) (upper) formed many visible gelatinous aggregation with 1 mL of saline at RT (lower). (c) Lyophilized A2216-dAs40 (0.5 mg/vial) and A2336-dAs40 (0.5 mg/vial) were easily and completely dissolved with 50 μL of saline at RT. (d) Human PBMCs were stimulated with the indicated synthetic ODNs (1 μM) for 24 hours. IFN-α and IL-6 production in the supernatants were examined by ELISA. Bar indicates the mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig9: 3′ addition of dAs40 sequence to A2216 and A2336 improves direct solubilization in saline. (a) Lyophilized A2216 vial (10 mg/vial) (upper left) was completely solubilized with 1 mL of saline directly at room temperature (RT) (lower left). The solution turned to a gel at 4°C (upper right) and reliquefied at 37°C (lower right). (b) Lyophilized A2336 vial (10 mg/vial) (upper) formed many visible gelatinous aggregation with 1 mL of saline at RT (lower). (c) Lyophilized A2216-dAs40 (0.5 mg/vial) and A2336-dAs40 (0.5 mg/vial) were easily and completely dissolved with 50 μL of saline at RT. (d) Human PBMCs were stimulated with the indicated synthetic ODNs (1 μM) for 24 hours. IFN-α and IL-6 production in the supernatants were examined by ELISA. Bar indicates the mean ± SEM.
Mentions: We also examined the direct solubility of other A/D type ODNs such as A2216 and A2336 in saline, with and without the addition of dAs40 tail. Unexpectedly A2216 was gradually but completely dissolved in saline at room temperature. However, the solution turned into a gel at 4°C (Figure 9(a)). This gelation was reliquefied at 37°C incubation (Figure 9(a)). A2336 was not dissolved in saline and formed gelatinous aggregations, similar to D35 (Figure 9(b)). Addition of dAs40 tail to A2216 and A2336 greatly improved their solubility in saline. Both A2216-dAs40 and A2336-dAs40 readily dissolved in saline (Figure 9(c)) and did not show gelation at the 4°C. A2216-dAs40 and A2336-dAs40 also kept the IFN-α inducing abilities (Figure 9(d)). These results suggested that the addition of dAs40 tail is also useful modification for improving the other A/D type ODNs' manageabilities.

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