ODN 2007 - TLR9 ligand

Class B CpG oligonucleotide - Bovine / porcine / chicken TLR9-preferred ligand

TLR9 activation with ODN 2007

ODN 2007 is a Class B CpG oligonucleotide (ODN). 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. ODN 2007 is a ligand of choice for bovine and porcine Toll-like receptor 9 (TLR9) [1-2]. It is also extensively studied as a vaccine adjuvant against the avian virus in chicken [3].

 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 [4-6]. Unmethylated CpG dinucleotides are a hallmark of microbial (bacterial, viral, fungal, and parasite) DNA, as well as mitochondrial self-DNA [6,7]. Class B (also called Type K) CpG ODNs, such as ODN 2007, contain a full phosphorothioate backbone with one or more CpG dinucleotides. They strongly activate B cells but weakly stimulate IFN-α secretion in plasmacytoid dendritic cells [8]. 

InvivoGen's ODN 2007 is a strong TLR9 agonist verified using our HEK-Blue™ reporter cell lines expressing human or mouse TLR9 (see figure). 

Key features of ODN 2007

• Potent activator of TLR9 in cattle, pigs, and chickens.
• Synthetic ODN with unmethylated CpG motifs
• Each lot is functionally tested

Get more information about CpG ODNs Classes.

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

References

1. Vordermeier HM, et al., 2005. Synthetic peptide vaccination in cattle: induction of strong cellular immune responses against peptides derived from the Mycobacterium bovis antigen Rv3019c. Vaccine. ;23(35):4375-84.
2. Ouyang K, et al., 2016. Comparative analysis of routes of immunization of a live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in a heterologous virus challenge study. Vet Res. ;47:45.
3. Raj S, et al., 2023. Treatment with Toll-like Receptor (TLR) Ligands 3 and 21 Prevents Fecal Contact Transmission of Low Pathogenic H9N2 Avian Influenza Virus (AIV) in Chickens. Viruses. ;15(4):977.
4. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.
5. Heinz L.X. et al., 2021. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9. Nature. 581(7808):316-322.
6. Kayraklioglu N. et al., 2021. CpG oligonucleotides as vaccine adjuvants. DNA Vaccines: Methods and Protocols. Methods in Molecular Biology. Vol. 2197. p51-77.
7. Kumar V., 2021. The trinity of cGAS, TLR9, and ALRs: guardians of the cellular galaxy against host-derived self-DNA. Front. Immunol. 11:624597.
8. Krieg A.M. et al., 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature. 374(6522):546-9.

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 2007. 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).

Specifications

Specificity: Bovine / porcine TLR9 agonist

Working concentration: 5 µM (10 μg/ml)

Solubility:  5 mg/ml in water

ODN 2007 sequence
5’- tcg tcg ttg tcg ttt tgt cgt t -3’ (22 mer)
Note: Bases are phosphorothioate (nuclease resistant).

Quality control:

• TLR9 activity has been tested using cellular assays.

• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cells.

Contents

ODN 2007 is provided lyophilized and is available in two quantities.

tlrl-2007-1 (formerly tlrl-podnb-1):

• 1 mg (141.65 nmol) lyophilized ODN 2007
• 1.5 ml sterile endotoxin-free water

tlrl-2007-5 (formerly tlrl-podnb-5):

• 5 mg (708.25 nmol) lyophilized ODN 2007
• 10 ml sterile endotoxin-free water

ODN 2007 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.