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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

Biological activities of D35-dAs40 and D35core-dAs40. (a) Dose-dependent IFN-α production from human PBMC by the indicated ODNs. (b) Effect of the adjacent sequence of cytokine inducing activity. D35core plus dAs40 is sufficient to induce IFN-α secretion from human PBMC. Bar graph indicates mean ± SEM in triplicate. (c) dAs tail length affects the biological activity of D35core-dAs type ODNs. Human PBMC stimulated with the indicated ODNs (final concentration = 1 μM), and after 24 h cytokine concentration was determined by ELISA. Bar graph indicates mean ± SEM in triplicate. (d) The tail length and ODN dose relations of the indicated ODNs. The bar graph shows dose-dependent IFN-α and IL-6 production from a single well by each stimulation. ∗P < 0.05; ∗∗P < 0.01.
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fig4: Biological activities of D35-dAs40 and D35core-dAs40. (a) Dose-dependent IFN-α production from human PBMC by the indicated ODNs. (b) Effect of the adjacent sequence of cytokine inducing activity. D35core plus dAs40 is sufficient to induce IFN-α secretion from human PBMC. Bar graph indicates mean ± SEM in triplicate. (c) dAs tail length affects the biological activity of D35core-dAs type ODNs. Human PBMC stimulated with the indicated ODNs (final concentration = 1 μM), and after 24 h cytokine concentration was determined by ELISA. Bar graph indicates mean ± SEM in triplicate. (d) The tail length and ODN dose relations of the indicated ODNs. The bar graph shows dose-dependent IFN-α and IL-6 production from a single well by each stimulation. ∗P < 0.05; ∗∗P < 0.01.

Mentions: We further evaluated the immunostimulatory activities of these ODNs by dose titration (Figure 4(a)). All ODNs including D35, D35-dAs40 (containing G-hexamer sequence), and D35core-dAs40 (without G-hexamer sequence) induced increased IFN-α and IL-6 responses from human PBMC in a dose-dependent manner (Figure 4(a)). In contrast, a recently reported P-type ODN, 21889, consisting of a phosphorothioate backbone containing two tandem palindromic sequences that promote the formation of dimeric structure or aggregates [4] showed decreased IFN-α and IL-6 responses when higher concentrations were used (Figure 4(a)).


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)

Biological activities of D35-dAs40 and D35core-dAs40. (a) Dose-dependent IFN-α production from human PBMC by the indicated ODNs. (b) Effect of the adjacent sequence of cytokine inducing activity. D35core plus dAs40 is sufficient to induce IFN-α secretion from human PBMC. Bar graph indicates mean ± SEM in triplicate. (c) dAs tail length affects the biological activity of D35core-dAs type ODNs. Human PBMC stimulated with the indicated ODNs (final concentration = 1 μM), and after 24 h cytokine concentration was determined by ELISA. Bar graph indicates mean ± SEM in triplicate. (d) The tail length and ODN dose relations of the indicated ODNs. The bar graph shows dose-dependent IFN-α and IL-6 production from a single well by each stimulation. ∗P < 0.05; ∗∗P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Biological activities of D35-dAs40 and D35core-dAs40. (a) Dose-dependent IFN-α production from human PBMC by the indicated ODNs. (b) Effect of the adjacent sequence of cytokine inducing activity. D35core plus dAs40 is sufficient to induce IFN-α secretion from human PBMC. Bar graph indicates mean ± SEM in triplicate. (c) dAs tail length affects the biological activity of D35core-dAs type ODNs. Human PBMC stimulated with the indicated ODNs (final concentration = 1 μM), and after 24 h cytokine concentration was determined by ELISA. Bar graph indicates mean ± SEM in triplicate. (d) The tail length and ODN dose relations of the indicated ODNs. The bar graph shows dose-dependent IFN-α and IL-6 production from a single well by each stimulation. ∗P < 0.05; ∗∗P < 0.01.
Mentions: We further evaluated the immunostimulatory activities of these ODNs by dose titration (Figure 4(a)). All ODNs including D35, D35-dAs40 (containing G-hexamer sequence), and D35core-dAs40 (without G-hexamer sequence) induced increased IFN-α and IL-6 responses from human PBMC in a dose-dependent manner (Figure 4(a)). In contrast, a recently reported P-type ODN, 21889, consisting of a phosphorothioate backbone containing two tandem palindromic sequences that promote the formation of dimeric structure or aggregates [4] showed decreased IFN-α and IL-6 responses when higher concentrations were used (Figure 4(a)).

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