ODN 2395 - TLR9 ligand

Class C CpG oligonucleotide - Human/Mouse TLR9-preferred ligand

TLR9 activation with ODN 2395

ODN 2395 is a Class C CpG oligonucleotide (ODN), specific for the human and murine Toll-like receptor 9 (TLR9) [1]. It is a short synthetic single-stranded DNA molecule containing unmethylated CpG dinucleotides (CpG motifs). These unmethylated CpG motifs mimic microbial DNA and act as immunostimulants via TLR9. 

 More details

Mode of action

Stimulatory CpG ODNs are internalized and activate the endosomal receptor TLR9. Activation of TLR9 triggers NF-κB- and interferon regulatory factor (IRF)-mediated pro-inflammatory responses upon the recognition of unmethylated cytosine-phosphorothioate-guanosine (CpG) forms of DNA [2-4]. Unmethylated CpG dinucleotides are a hallmark of microbial (bacterial, viral, fungal, and parasite) DNA and mitochondrial self-DNA [4, 5]. Class C CpG ODNs, such as ODN 2395, combine classes A and B features. They contain a complete phosphorothioate (PS) backbone and a CpG-containing palindromic motif. They strongly induce B cell stimulation as well as IFN-α production in plasmacytoid dendritic cells [6]. 

InvivoGen's ODN 2395 is a strong TLR9 agonist as verified using our HEK-Blue™ reporter cell lines expressing human or mouse TLR9 (see figure). Moreover, ODN 2395 activates the hTLR9-dependent NF-κB and IRF pathways, as assessed using our THP1-Dual™ hTLR9 reporter cell line expressing two reporter genes, for the NF-κB-inducible SEAP and IRF-inducible Lucia luciferase, as well as hTLR9 (see figure). 

Key features of ODN 2395

• Potent activator of human and mouse TLR9
• Synthetic ODN with unmethylated CpG motifs
• Each lot is functionally tested
• High-quality, pre-clinical ODN 2395 VacciGrade™ is also available for in vivo studies

Get more information about CpG ODNs Classes.

Read our review on TLR9 agonists: double-edged sword for immune therapies.

References

1. Roda JM. et al., 2005. CpG-containing oligodeoxynucleotides act through TLR9 to enhance the NK cell cytokine response to antibodycoated tumor cells. J Immunol. 175(3):1619-27.
2. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.
3. Heinz L.X. et al., 2021. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9. Nature. 581(7808):316-322.
4. Kayraklioglu N. et al., 2021. CpG oligonucleotides as vaccine adjuvants. DNA Vaccines: Methods and Protocols. Methods in Molecular Biology. Vol. 2197. p51-77.
5. Kumar V., 2021. The trinity of cGAS, TLR9, and ALRs: guardians of the cellular galaxy against host-derived self-DNA. Front. Immunol. 11:624597.
6. Jurk M. et al., 2004. C-Class CpG ODN: sequence requirements and characterization of immunostimulatory activities on mRNA level. Immunobiology. 209(1-2):141-54.

Figures

Dose-dependent NF-κB response in HEK-Blue™-derived cells. HEK-Blue™ hTLR9 and HEK-Blue™ mTLR9 cells were incubated with increasing concentrations of ODN 2395. After 24h, the TLR9-induced NF‑κB responses were assessed by measuring the SEAP activity using QUANTI-Blue™. Data are shown as optical density (OD) at 650 nm (mean + SEM).

Dose-dependent NF-κB and IRF responses in THP1-Dual™ hTLR9 cells. Cells were incubated with increasing concentrations of ODN 2395. After 24h, the hTLR9-induced (A) NF‑κB and (B) IRF responses were assessed by measuring SEAP and Lucia activity using QUANTI-Blue™ and QUANTI-Luc™, respectively. Data are shown as optical density (OD) at 650 nm or in fold increase over non-induced cells (mean + SEM).

Specifications

Specificity: human/murine TLR9 agonist

Working concentration: 1-5 µM

Solubility:  5 mg/ml in water

ODN 2395 sequence
5’-tcgtcgttttcggcgc:gcgccg-3’ (22 mer)
Note: Bases are phosphorothioate (nuclease resistant), palindrome is underlined.

Quality control:

• Human TLR9 (hTLR9) and murine TLR9 (mTLR9) activity has been tested using HEK-Blue™ hTLR9 and HEK-Blue™ mTLR9 cells.
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cells.

Contents

ODN 2395 is provided lyophilized and is available in three quantities:

tlrl-2395 (formerly tlrl-odnc):

• 200 μg (28.37 nmol) lyophilized ODN 2395
• 1.5 ml sterile endotoxin-free water

tlrl-2395-1 (formerly tlrl-odnc-1):

• 1 mg (141.85 nmol):  tlrl-2395-1 (formerly tlrl-odnc-1)
• 1.5 ml sterile endotoxin-free water

tlrl-2395-5 (formerly tlrl-odnc-5):

• 5 mg (709.25 nmol):  tlrl-2395-5 (formerly tlrl-odnc-5)
• 10 ml sterile endotoxin-free water

ODN 2395 is shipped at room temperature.

Upon receipt, store at -20°C.

Details

CpG ODNs

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODNs), such as ODN 1018, have been extensively studied as adjuvants [1]. These CpG motifs are present at a 20-fold greater frequency in bacterial DNA compared to mammalian DNA [2]. CpG ODNs are recognized by the Toll-like receptor 9 (TLR9), which is expressed on human B cells and plasmacytoid dendritic cells (pDCs), thereby inducing Th1-dominated immune responses [3]. Pre-clinical studies, conducted in rodents and non-human primates, as well as human clinical trials, have demonstrated that CpG ODNs can significantly improve vaccine-specific antibody responses [1]. Three types of stimulatory CpG ODNs have been identified, types A, B, and C, which differ in their immune-stimulatory activities [4-5]. 

Toll-like receptor 9

The Toll-like Receptor 9 (TLR9) is an endosomal receptor that triggers NF-κB- and interferon regulatory factor (IRF)-mediated pro-inflammatory responses upon the recognition of unmethylated cytosine-phosphorothioate-guanosine (CpG) forms of DNA [6-8]. Unmethylated CpG dinucleotides are a hallmark of microbial (bacterial, viral, fungal, and parasite) DNA, as well as mitochondrial self-DNA [8,9]. These TLR9 agonists can be mimicked by synthetic oligonucleotides containing CpG motifs (CpG ODNs), which have been extensively studied to improve adaptive immune responses in the context of vaccination [6,8].

TLR9 is mainly expressed in subsets of Dendritic Cells and B cells of all mammals. In rodents, but not in humans, TLR9 is also expressed in monocytes and macrophages [8]. The structure of the receptor varies by 24% between human TLR9 (hTLR9) and mouse TLR9 (mTLR9) [8]. They recognize different CpG motifs, the optimal sequences being GTCGTT and GACGTT for hTLR9 and mTLR9, respectively [10].
 

References:

1. Steinhagen F. et al., 2011. TLR-based immune adjuvants. Vaccine 29(17):3341-55.
2. Hemmi H. et al., 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740-5.
3. Coffman RL. et al., 2010. Vaccine adjuvants: Putting innate immunity to work. Immunity 33(4):492-503.
4. Krug A. et al., 2001. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur J Immunol, 31(7): 2154-63.
5. Marshall JD. et al., 2005. Superior activity of the type C class of ISS in vitro and in vivo across multiple species. DNA Cell Biol. 24(2):63-72.
6. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.
7. Heinz L.X. et al., 2021. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9. Nature. 581(7808):316-322.
8. Kayraklioglu N. et al., 2021. CpG oligonucleotides as vaccine adjuvants. DNA Vaccines: Methods and Protocols. Methods in Molecular Biology. Vol. 2197. p51-77.
9. Kumar V., 2021. The trinity of cGAS, TLR9, and ALRs: guardians of the cellular galaxy against host-derived self-DNA. Front. Immunol. 11:624597.
10. Bauer S. et al., 2001. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA, 98(16):9237-42.