Limits...
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.


G-hexamer-less D35 ODNs required DOTAP for their immunostimulatory activities. Human PBMCs were stimulated with the indicated synthetic ODNs (1 μM) with or without DOTPA for 24 hours. IFN-α and IL-6 productions in the supernatants were examined by ELISA. DOTAP revived the immunostimulatory activities of D35A, D35T, and D35C; those are A/D type ODNs which do not contain G-hexamer. These ODNs did not show any immunostimulatory activities without DOTAP. The bar graphs indicate the concentrations from a single well of each stimulation. IFN-α production overed the ELISA measurement maximum (ca. 5000 pg/mL) with DOTAP in Exp5 and Exp6.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4562176&req=5

fig2: G-hexamer-less D35 ODNs required DOTAP for their immunostimulatory activities. Human PBMCs were stimulated with the indicated synthetic ODNs (1 μM) with or without DOTPA for 24 hours. IFN-α and IL-6 productions in the supernatants were examined by ELISA. DOTAP revived the immunostimulatory activities of D35A, D35T, and D35C; those are A/D type ODNs which do not contain G-hexamer. These ODNs did not show any immunostimulatory activities without DOTAP. The bar graphs indicate the concentrations from a single well of each stimulation. IFN-α production overed the ELISA measurement maximum (ca. 5000 pg/mL) with DOTAP in Exp5 and Exp6.

Mentions: We also tested the immunostimulatory activities of A-, T-, and C-hexamers containing D35 without phosphorothioate polynucleotide tails on human PBMCs (Figure 2). Consistent with previous reports [5, 22, 23], the immunostimulatory activity of original D35 was dependent on the presence of a G-hexamer (Figure 2). A-, T-, and C-hexamer-containing D35 (D35A, D35T, and D35C) had no effect on human PBMCs (Figure 2; black bar). In contrast, the same ODNs became comparatively active when they were added with DOTAP (Figure 2; white bar), a cationic lipid (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate) that has been used for targeting CpG-ODNs to certain endosome compartments where TLR9-mediated signaling starts [24, 25]. This result indicated that the biological activity of A/D type ODNs does not require a G-hexamer sequence if ODNs are targeted to cellular uptake and appropriate intracellular compartments by DOTAP.


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)

G-hexamer-less D35 ODNs required DOTAP for their immunostimulatory activities. Human PBMCs were stimulated with the indicated synthetic ODNs (1 μM) with or without DOTPA for 24 hours. IFN-α and IL-6 productions in the supernatants were examined by ELISA. DOTAP revived the immunostimulatory activities of D35A, D35T, and D35C; those are A/D type ODNs which do not contain G-hexamer. These ODNs did not show any immunostimulatory activities without DOTAP. The bar graphs indicate the concentrations from a single well of each stimulation. IFN-α production overed the ELISA measurement maximum (ca. 5000 pg/mL) with DOTAP in Exp5 and Exp6.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: G-hexamer-less D35 ODNs required DOTAP for their immunostimulatory activities. Human PBMCs were stimulated with the indicated synthetic ODNs (1 μM) with or without DOTPA for 24 hours. IFN-α and IL-6 productions in the supernatants were examined by ELISA. DOTAP revived the immunostimulatory activities of D35A, D35T, and D35C; those are A/D type ODNs which do not contain G-hexamer. These ODNs did not show any immunostimulatory activities without DOTAP. The bar graphs indicate the concentrations from a single well of each stimulation. IFN-α production overed the ELISA measurement maximum (ca. 5000 pg/mL) with DOTAP in Exp5 and Exp6.
Mentions: We also tested the immunostimulatory activities of A-, T-, and C-hexamers containing D35 without phosphorothioate polynucleotide tails on human PBMCs (Figure 2). Consistent with previous reports [5, 22, 23], the immunostimulatory activity of original D35 was dependent on the presence of a G-hexamer (Figure 2). A-, T-, and C-hexamer-containing D35 (D35A, D35T, and D35C) had no effect on human PBMCs (Figure 2; black bar). In contrast, the same ODNs became comparatively active when they were added with DOTAP (Figure 2; white bar), a cationic lipid (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate) that has been used for targeting CpG-ODNs to certain endosome compartments where TLR9-mediated signaling starts [24, 25]. This result indicated that the biological activity of A/D type ODNs does not require a G-hexamer sequence if ODNs are targeted to cellular uptake and appropriate intracellular compartments by DOTAP.

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.