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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

D35-dAs40, D35core-dAs40, and D35-SPG showed better adjuvanticity with influenza SV vaccine in cynomolgus monkeys. (a) Immunization and assay schedule. Six groups of monkeys (n = 2 or 3) were immunized with SV vaccine (A/New Caledonia/20/99, 5 μg/head) s.c. in a total volume of 500 μL with or without the indicated adjuvants (4.7 nmol each: K3; 30 μg, D35; 30 μg, D35-dAs40; 92 μg, D35-SPG; 92 μg as D35-dAs40 amount) twice in 2-week intervals. (b) Anti-SV total IgG in sera was determined by ELISA. X-axis indicates the serum dilutions. Each line indicates an individual monkey. (c) In a separate experiment, two groups of monkeys (n = 3) were immunized with D35 (4.7 nmol = 30 μg) or D35core-dAs40 (4.7 nmol = 80 μg) as in (a). Four weeks after the first immunization, anti-SV total IgG titers were determined by ELISA. Bar indicates the mean ± SEM.
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fig12: D35-dAs40, D35core-dAs40, and D35-SPG showed better adjuvanticity with influenza SV vaccine in cynomolgus monkeys. (a) Immunization and assay schedule. Six groups of monkeys (n = 2 or 3) were immunized with SV vaccine (A/New Caledonia/20/99, 5 μg/head) s.c. in a total volume of 500 μL with or without the indicated adjuvants (4.7 nmol each: K3; 30 μg, D35; 30 μg, D35-dAs40; 92 μg, D35-SPG; 92 μg as D35-dAs40 amount) twice in 2-week intervals. (b) Anti-SV total IgG in sera was determined by ELISA. X-axis indicates the serum dilutions. Each line indicates an individual monkey. (c) In a separate experiment, two groups of monkeys (n = 3) were immunized with D35 (4.7 nmol = 30 μg) or D35core-dAs40 (4.7 nmol = 80 μg) as in (a). Four weeks after the first immunization, anti-SV total IgG titers were determined by ELISA. Bar indicates the mean ± SEM.

Mentions: We finally tested the in vivo adjuvant potency of D35-dAs40, D35core-dAs40, and D35-SPG in a monkey vaccine model and compared them with K3 and original D35 (Figure 12). Six groups of monkeys (n = 2 or 3) were immunized subcutaneously twice (at 2-week intervals) with the indicated SV plus adjuvants, and, 8 weeks after the first immunization, SV-specific IgG responses in sera were examined by ELISA (Figure 12(a)). D35-dAs40 and D35-SPG showed better and more consistent adjuvanticity than K3 and original D35 (Figure 12(b)). We also performed another set of monkey experiments to compare original D35 and D35core-dAs40 and found that D35core-dAs40 also showed better adjuvanticity than original D35 (Figure 12(c)). These results suggested that D35-dAs40, D35core-dAs40, and D35-SPG function as comparable or better adjuvants compared with K3 and original D35 in vivo in monkeys, at least for influenza SV vaccination. D35-SPG result in monkey also suggested that IFN-α and IL-6 profiles in vitro were not always correlated with the in vivo adjuvanticity. We performed more detailed cytokine profiling with a 26-cytokine multiplex using human PBMCs (instead of monkey PBMCs, owing to the limitation of obtaining sufficient amounts of monkey PBMCs for assay) stimulated with D35, D35-dAs40, or D35-SPG (Figure 13) and did not observe apparent correlation between cytokines and in vivo adjuvanticity among the 18 detected cytokines (Figure 13).


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, D35core-dAs40, and D35-SPG showed better adjuvanticity with influenza SV vaccine in cynomolgus monkeys. (a) Immunization and assay schedule. Six groups of monkeys (n = 2 or 3) were immunized with SV vaccine (A/New Caledonia/20/99, 5 μg/head) s.c. in a total volume of 500 μL with or without the indicated adjuvants (4.7 nmol each: K3; 30 μg, D35; 30 μg, D35-dAs40; 92 μg, D35-SPG; 92 μg as D35-dAs40 amount) twice in 2-week intervals. (b) Anti-SV total IgG in sera was determined by ELISA. X-axis indicates the serum dilutions. Each line indicates an individual monkey. (c) In a separate experiment, two groups of monkeys (n = 3) were immunized with D35 (4.7 nmol = 30 μg) or D35core-dAs40 (4.7 nmol = 80 μg) as in (a). Four weeks after the first immunization, anti-SV total IgG titers were determined by ELISA. Bar indicates the mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4562176&req=5

fig12: D35-dAs40, D35core-dAs40, and D35-SPG showed better adjuvanticity with influenza SV vaccine in cynomolgus monkeys. (a) Immunization and assay schedule. Six groups of monkeys (n = 2 or 3) were immunized with SV vaccine (A/New Caledonia/20/99, 5 μg/head) s.c. in a total volume of 500 μL with or without the indicated adjuvants (4.7 nmol each: K3; 30 μg, D35; 30 μg, D35-dAs40; 92 μg, D35-SPG; 92 μg as D35-dAs40 amount) twice in 2-week intervals. (b) Anti-SV total IgG in sera was determined by ELISA. X-axis indicates the serum dilutions. Each line indicates an individual monkey. (c) In a separate experiment, two groups of monkeys (n = 3) were immunized with D35 (4.7 nmol = 30 μg) or D35core-dAs40 (4.7 nmol = 80 μg) as in (a). Four weeks after the first immunization, anti-SV total IgG titers were determined by ELISA. Bar indicates the mean ± SEM.
Mentions: We finally tested the in vivo adjuvant potency of D35-dAs40, D35core-dAs40, and D35-SPG in a monkey vaccine model and compared them with K3 and original D35 (Figure 12). Six groups of monkeys (n = 2 or 3) were immunized subcutaneously twice (at 2-week intervals) with the indicated SV plus adjuvants, and, 8 weeks after the first immunization, SV-specific IgG responses in sera were examined by ELISA (Figure 12(a)). D35-dAs40 and D35-SPG showed better and more consistent adjuvanticity than K3 and original D35 (Figure 12(b)). We also performed another set of monkey experiments to compare original D35 and D35core-dAs40 and found that D35core-dAs40 also showed better adjuvanticity than original D35 (Figure 12(c)). These results suggested that D35-dAs40, D35core-dAs40, and D35-SPG function as comparable or better adjuvants compared with K3 and original D35 in vivo in monkeys, at least for influenza SV vaccination. D35-SPG result in monkey also suggested that IFN-α and IL-6 profiles in vitro were not always correlated with the in vivo adjuvanticity. We performed more detailed cytokine profiling with a 26-cytokine multiplex using human PBMCs (instead of monkey PBMCs, owing to the limitation of obtaining sufficient amounts of monkey PBMCs for assay) stimulated with D35, D35-dAs40, or D35-SPG (Figure 13) and did not observe apparent correlation between cytokines and in vivo adjuvanticity among the 18 detected cytokines (Figure 13).

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