ODN 2216 VacciGrade™

CpG ODN, type A (human) - TLR9-based vaccine adjuvant

TLR9 activation with ODN 2216

ODN 2216 VacciGrade™ is a pre-clinical Class A CpG oligonucleotide (ODN) with a preference for the human Toll-like receptor 9 (TLR9). CpG ODNs are short synthetic single-stranded DNA molecules containing unmethylated CpG dinucleotides (CpG motifs). These unmethylated CpG motifs mimic microbial DNA and act as immunostimulants via TLR9. 

 More details

Class A CpG ODNs, such as ODN 2216, are characterized by a phosphodiester central CpG-containing palindromic motif and a phosphorothioate 3’ poly-G string. ODN 2216 induces robust production of type I interferon by plasmacytoid dendritic cells (pDCs) and highly stimulates natural killer (NK) cells. However, it has little stimulatory effect on B cells [1].

In HEK-Blue™ hTLR9 reporter cells, ODN 2216 efficiently activates hTLR9 compared to the control ODN 2216 (ODN 2243). Moreover, ODN 2216 activates the hTLR9-dependent NF-κB and IRF pathways, as assessed using THP1-Dual™ hTLR9 cells expressing both NF-κB-inducible SEAP and IRF-inducible Lucia luciferase reporters (see figures). 

VacciGrade™ is a high-quality pre-clinical grade, suitable for in vivo use.
A standard grade ODN 2216 for in vitro assays is also available.

ODN 2216 VacciGrade™​ is for research use only, and not for human or veterinary use. It is not a pharmaceutical preparation fit for vaccine manufacturing.

Get more information about CpG ODNs Classes.

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

Reference

1. Krug A., et al., 2001. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur J Immunol. 31:2154-63.

Figures

Dose-dependent NF-κB response in HEK-Blue™ hTLR9 cells. HEK-Dual™ hTLR9 cells were incubated with increasing concentrations of ODN 2216 or the control ODN 2216 (ODN 2243). After 24h, the hTLR9-induced NF‑κB response was 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 2216. 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

ODN 2216 sequence:5’-ggGGGACGA:TCGTCgggggg-3’ (20 mer)
Note: Bases in capital letters are phosphodiester, and those in lowercase are phosphorothioate (nuclease resistant). Palindrome is underlined.

Description: TLR9 agonist VacciGrade™

CAS number: 332437-00-0

Polarization of adaptive immune response: Th1 response

Solubility: 2 mg/ml in physiological water

Working concentration: 50 µg/mouse; 30 µg - 200 µg/neonatal cotton rat

Quality control:

• Sterility guaranteed
• The absence of bacterial contamination (lipoproteins & endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cells
• Endotoxin level < 1 EU/mg (measurement by kinetic chromogenic LAL assay)

Contents

• 1 mg of sterile lyophilized ODN 2216 VacciGrade™
• 10 ml sterile endotoxin-free physiological water (NaCl 0.9%)

ODN 2216 VacciGrade™ is shipped at room temperature.

ODN 2216 VacciGrade™ should be stored at -20°C.

The product, once resuspended, is stable for 6 months at -20°C when properly stored.

Avoid repeated freeze-thaw cycles.

VacciGrade™

VacciGrade™ is a high-quality pre-clinical grade. VacciGrade™ products are filter-sterilized (0.2 µm) and filled under strict aseptic conditions in a clean room*. The absence of bacterial contamination is assessed by a sterility test using a pharmacopeia-derived assay. The level of bacterial contaminants (endotoxins and lipoproteins) in each lot is verified using a LAL assay and/or a TLR2 and TLR4 reporter assay.
*Except for LPS VacciGrade™, which is prepared in a laminar flow hood dedicated to LPS.

Details

CpG ODNs

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODNs), such as ODN 2216, 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.