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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.


D35-dAs40 and D35core-dAs40 but not original D35 are directly solubilized in saline. (a) GMP grade lyophilized ODN vials (10 mg/vial) of K3, D35, D35-dAs40, and D35core-dAs40. White material is the lyophilized synthetic ODNs. (b) Saline (1 mL) was directly added to each vial. K3, D35-dAs40, and D35core-dAs40 were completely solubilized in saline at 5 min. D35 was not completely dissolved in saline with many visible gelatinous aggregations, and this insolubilized status was unchanged for at least 1 month.
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fig8: D35-dAs40 and D35core-dAs40 but not original D35 are directly solubilized in saline. (a) GMP grade lyophilized ODN vials (10 mg/vial) of K3, D35, D35-dAs40, and D35core-dAs40. White material is the lyophilized synthetic ODNs. (b) Saline (1 mL) was directly added to each vial. K3, D35-dAs40, and D35core-dAs40 were completely solubilized in saline at 5 min. D35 was not completely dissolved in saline with many visible gelatinous aggregations, and this insolubilized status was unchanged for at least 1 month.

Mentions: We also examined several dAs40-tailed D35-related ODNs for their physical properties with DLS (Figure 3). Good manufacturing practice (GMP) grade synthesis of clinically applicable A/D type ODNs has been hampered by G-tail dependent multimerization that results in uncontrollable polymorphisms, aggregation, and precipitation of ODN products [14] (Figures 3 and 8). Original D35 (1 mg/mL) showed variable and heterogenous aggregate formations in PBS resulting in visible turbidity within 24 hours (Figure 3(c)). Of note, this turbidity was not observed in D35 in water. DLS analysis revealed that this turbidity consisted of broadly distributed aggregates (size range from around 50 nm to more than 1 μm in mean diameter; Figures 3(a) and 3(b)). In contrast, the same concentration of D35-dAs40 and D35core-dAs40 in PBS did not form visible aggregations (Figure 3(c)), and the size of the ODNs was less than 20 nm with a sharp peak in DLS (Figures 3(a) and 3(b) and Table 2). TEM analysis confirmed the DLS results and indicated that many globular particle sizes around 50–200 nm were distributed separately or formed stringed clusters of several particles (Figure 3(d)), consistent with a previous report [10, 11]. These pieces of data indicated that the addition of dAs40 tail greatly improved the physical uniformity of A/D type ODNs in PBS, even those containing a G-hexamer sequence such as D35-dAs40 showed virtually no aggregation with the dAs40 tail. We did not observe any meaningful structures in K3, D35-dAs40, D35T-dAs40, D35core-dAs40, and D35coreT-dAs40 by TEM.


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-dAs40 and D35core-dAs40 but not original D35 are directly solubilized in saline. (a) GMP grade lyophilized ODN vials (10 mg/vial) of K3, D35, D35-dAs40, and D35core-dAs40. White material is the lyophilized synthetic ODNs. (b) Saline (1 mL) was directly added to each vial. K3, D35-dAs40, and D35core-dAs40 were completely solubilized in saline at 5 min. D35 was not completely dissolved in saline with many visible gelatinous aggregations, and this insolubilized status was unchanged for at least 1 month.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig8: D35-dAs40 and D35core-dAs40 but not original D35 are directly solubilized in saline. (a) GMP grade lyophilized ODN vials (10 mg/vial) of K3, D35, D35-dAs40, and D35core-dAs40. White material is the lyophilized synthetic ODNs. (b) Saline (1 mL) was directly added to each vial. K3, D35-dAs40, and D35core-dAs40 were completely solubilized in saline at 5 min. D35 was not completely dissolved in saline with many visible gelatinous aggregations, and this insolubilized status was unchanged for at least 1 month.
Mentions: We also examined several dAs40-tailed D35-related ODNs for their physical properties with DLS (Figure 3). Good manufacturing practice (GMP) grade synthesis of clinically applicable A/D type ODNs has been hampered by G-tail dependent multimerization that results in uncontrollable polymorphisms, aggregation, and precipitation of ODN products [14] (Figures 3 and 8). Original D35 (1 mg/mL) showed variable and heterogenous aggregate formations in PBS resulting in visible turbidity within 24 hours (Figure 3(c)). Of note, this turbidity was not observed in D35 in water. DLS analysis revealed that this turbidity consisted of broadly distributed aggregates (size range from around 50 nm to more than 1 μm in mean diameter; Figures 3(a) and 3(b)). In contrast, the same concentration of D35-dAs40 and D35core-dAs40 in PBS did not form visible aggregations (Figure 3(c)), and the size of the ODNs was less than 20 nm with a sharp peak in DLS (Figures 3(a) and 3(b) and Table 2). TEM analysis confirmed the DLS results and indicated that many globular particle sizes around 50–200 nm were distributed separately or formed stringed clusters of several particles (Figure 3(d)), consistent with a previous report [10, 11]. These pieces of data indicated that the addition of dAs40 tail greatly improved the physical uniformity of A/D type ODNs in PBS, even those containing a G-hexamer sequence such as D35-dAs40 showed virtually no aggregation with the dAs40 tail. We did not observe any meaningful structures in K3, D35-dAs40, D35T-dAs40, D35core-dAs40, and D35coreT-dAs40 by TEM.

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.