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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.


Length of the tail affects ODN's immunostimulatory activities. 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.
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fig5: Length of the tail affects ODN's immunostimulatory activities. 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.

Mentions: We further performed a couple of more detailed experiments to understand the requirement of the phosphorothioate polynucleotide tailing for D35 CpG ODNs, such as the length (Figure 5), the amount of phosphorothioation (Figure 6), and the nucleotides compositions (Figure 7) of the tail. First, D35core with different number of dAs was tested for the induction of IFN-α and IL-6 (Figure 5). No IFN-α induction was observed in D35core with less than dAs6; then the amount of IFN-α was increased by the dA length reaching dAs60. Interestingly, further prolongation of dAs-tail up to 100 did not improve but rather decreased the induction of IFN-α. In contrast, IL-6 production was increased and sustained up to dAs100. Second, by using D35core-dAs40, we changed the amount of phosphorothioation from 100% to 17.5% as indicated in Figure 6. Even with 50% reduction of phosphorothioation in dA40 tail abrogated the IFN-α inducing activities. IL-6 production was less sensitive but also rapidly decreased as the reduction of phosphorothioation amount. We did not observe any IL-6 production with less than 25% phosphorothioation. Third, the requirement of nucleotides compositions was examined by using D35-dNs40. Totally random dNs40 tailed ODNs did not induce substantial IFN-α production (Figure 7; sample 10). We also controlled guanosine amount in the dNs40-tail from 10% to 90%, expecting the increase of the Hoogsteen base pairing formation in the dNs40 tail. As the guanosine amount increased, the IFN-α production increased but D35-dNs40 (91% G) (Figure 7; sample 9) induced relatively small amount of IFN-α. All together these results suggested that (1) total length, (2) phosphorothioation amount, and (3) the nucleotide composition (A-polymer is better than G-rich randomer) were all important factors affecting the immunostimulating activities of phosphorothioate polynucleotide tailed ODNs. Based on these observations, we selected D35-dAs40 and D35core-dAs40 as prototypes orienting for clinical application.


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)

Length of the tail affects ODN's immunostimulatory activities. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Length of the tail affects ODN's immunostimulatory activities. 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.
Mentions: We further performed a couple of more detailed experiments to understand the requirement of the phosphorothioate polynucleotide tailing for D35 CpG ODNs, such as the length (Figure 5), the amount of phosphorothioation (Figure 6), and the nucleotides compositions (Figure 7) of the tail. First, D35core with different number of dAs was tested for the induction of IFN-α and IL-6 (Figure 5). No IFN-α induction was observed in D35core with less than dAs6; then the amount of IFN-α was increased by the dA length reaching dAs60. Interestingly, further prolongation of dAs-tail up to 100 did not improve but rather decreased the induction of IFN-α. In contrast, IL-6 production was increased and sustained up to dAs100. Second, by using D35core-dAs40, we changed the amount of phosphorothioation from 100% to 17.5% as indicated in Figure 6. Even with 50% reduction of phosphorothioation in dA40 tail abrogated the IFN-α inducing activities. IL-6 production was less sensitive but also rapidly decreased as the reduction of phosphorothioation amount. We did not observe any IL-6 production with less than 25% phosphorothioation. Third, the requirement of nucleotides compositions was examined by using D35-dNs40. Totally random dNs40 tailed ODNs did not induce substantial IFN-α production (Figure 7; sample 10). We also controlled guanosine amount in the dNs40-tail from 10% to 90%, expecting the increase of the Hoogsteen base pairing formation in the dNs40 tail. As the guanosine amount increased, the IFN-α production increased but D35-dNs40 (91% G) (Figure 7; sample 9) induced relatively small amount of IFN-α. All together these results suggested that (1) total length, (2) phosphorothioation amount, and (3) the nucleotide composition (A-polymer is better than G-rich randomer) were all important factors affecting the immunostimulating activities of phosphorothioate polynucleotide tailed ODNs. Based on these observations, we selected D35-dAs40 and D35core-dAs40 as prototypes orienting for clinical application.

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.