CpG ODNs Classes

TLR9 Agonists

CpG oligodeoxynucleotides (CpG ODN) or bacterial DNA
Potent stimulators of immune cell maturation
Purity and activity thoroughly validated
Provided with endotoxin-free water

CpG ODNs are short synthetic single-stranded DNA molecules containing unmethylated CpG dinucleotides in particular sequence contexts (CpG motifs).

CpG ODNs possess a partially or completely phosphorothioated (PS) backbone, as opposed to the natural phosphodiester (PO) backbone found in genomic bacterial DNA.

Three major classes of stimulatory CpG ODNs have been identified based on structural characteristics and activity on human peripheral blood mononuclear cells (PBMCs), in particular B cells and plasmacytoid dendritic cells (pDCs). These three classes are Class A (Type D), Class B (Type K) and Class C.

Classes of CpG ODNs
Class A ODNs
Class B ODNs
Class C ODNs
Choose the right class
Quality check

Classes of CpG ODNs

CpG-A ODNs are characterized by a PO central CpG-containing palindromic motif and a PS-modified 3’ poly-G string. They induce high IFN-α production from pDCs but are weak stimulators of TLR9-dependent NF-κB signaling and pro-inflammatory cytokine (e.g. IL-6) production. CpG-B ODNs contain a full PS backbone with one or more CpG dinucleotides. They strongly activate B cells and TLR9-dependent NF-κB signaling but weakly stimulate IFN-α secretion. CpG-C ODNs combine features of both classes A and B. They contain a complete PS backbone and a CpG-containing palindromic motif. C-Class CpG ODNs induce strong IFN-α production from pDC as well as B cell stimulation.
Effect of ODNs on the activation of B cells
The effect of ODNs on the activation of B cells derived from human PBMCs Human PBMCs were incubated with CpG ODNs or their controls at a final concentration of 1.6µg/ml for 24 hours then labeled with CD19, CD80, and CD86 mAbs, for analysis of B cell activation by flow cytometry.
The effect of ODNs on the activation of B cells derived from murine splenocytes Murine splenocytes were incubated with CpG ODNs or their controls at a final concentration of 3.3µg/ml for 48 hours and stained for CD19, CD80, and CD86, for analysis of B cell activation by flow cytometry.

Characteristics of the three major classes of CpG ODNs

Class	Backbone	B cells	pDCs	IFN-α	Il-6
A	PS-PO	+	++++	++++	+
B	PS	++++	++++	+	+++
C	PS	+++	+++	+++	++

Class A ODNs

Class A (type D) CpG ODNs (CpG-A ODNs) contain a central palindromic phosphodiester (PO) CpG sequence and a PS-modified 3’ poly-G tail.

The poly G tails form intermolecular tetrads that result in high molecular weight ordered structures. These structures confer enhanced stability and increased endosomal uptake contributing to the production of large amounts of IFN-α and the maturation of pDCs.
CpG-A ODNs are also strong activators of NK cells through indirect cytokine signaling.

Ballas ZK. et al., 1996. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J Immunol. 157(5):1840-5.
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.
Verthelyi D. et al., 2001. Human peripheral blood cells differentially recognize and respond to two distinct CPG motifs. J Immunol. 166(4):2372-7.

	Response of HEK-Blue™ hTLR9 cells to Class A CpG ODNs. HEK-Blue™ hTLR9 cells, which express human TLR9 and an NF-κB-inducible SEAP reporter gene, were incubated in HEK-Blue Detection medium and stimulated with increasing concentrations of A-class CpG ODNs, ODN 1585, ODN 2216 or ODN 2236, and their corresponding GC control ODNs. After 24h incubation, the levels of NF-ΚB-induced SEAP were determined by reading the OD at 655 nm.
	Response of HEK-Blue™ mTLR9 cells to Class A CpG ODNs. HEK-Blue™ mTLR9 cells, which express mouse TLR9 and an NF-κB-inducible SEAP reporter gene, were incubated in HEK-Blue Detection medium and stimulated with increasing concentrations of A-class CpG ODNs, ODN 1585, ODN 2216 or ODN 2236, and their corresponding GC control ODNs. After 24h incubation, the levels of NF-kB-induced SEAP were determined by reading the OD at 655 nm.
	The effect of a class A ODN on the activation of B cells derived from human PBMCs Human PBMCs were incubated with ODN 2216 or its control at a final concentration of 1.6µg/ml for 24 hours then labeled with CD19, CD80, and CD86 mAbs, for analysis of B cell activation by flow cytometry.
	The effect of a class A ODN on the activation of B cells derived from murine splenocytes Murine splenocytes were incubated with ODN 1585 or its control at a final concentration of 3.3µg/ml for 48 hours and stained for CD19, CD80, and CD86, for analysis of B cell activation by flow cytometry.
	Secretion of IL-6 and IFN-α after stimulation of human PBMCs with a class A ODN Isolated PBMCs were stimulated with ODN 2216 or its control at a final concentration of 1.6µg/ml for 24 hours. The secretion of IL-6 and IFN-α was measured by ELISA.
	Secretion of IL-6 and IFN-alpha after stimulation of murine splenocytes with a class A ODN Murine splenocytes were stimulated with ODN 1585 or its control at a final concentration of 3.3µg/ml for 24 hours. The secretion of IL-6 and IFN-α was measured by ELISA.

Class B ODNs

Class B (type K) CpG ODNs (CpG-B ODNs) are 18-28mer linear oligodeoxynucelotides. They contain a fully phosphorothioated backbone with one or more 6mer CpG motifs. The optimal motif is GTCGTT [1] in human and GACGTT in mouse [2].
CpG-B ODNs stimulate strong B cell and NK cell activation but are weak activators of IFN-α secretion [3, 4]. They display anti-tumor activity and are potent Th1 adjuvants.

1- Hartmann G & Krieg AM., 2000. Mechanism and function of a newly identified CpG DNA motif in human primary B cells. J Immunol. 164(2):944-53.
2- Krieg AM. et al., 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature. 374(6522):546-9.
3- Hartmann G. et al., 2000. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J Immunol. 164(3):1617-24.
4- Gürsel M. et al., 2002. Differential and competitive activation of human immune cells by distinct classes of CpG oligodeoxynucleotide. J Leukoc Biol. 71(5):813-20.

	Response of HEK-Blue™ hTLR9 cells to Class B CpG ODNs. HEK-Blue hTLR9™ cells, which express human TLR9 and an NF-κB-inducible SEAP reporter gene, were incubated in HEK-Blue™ Detection medium and stimulated with increasing concentrations of B-class CpG ODNs, ODN 1668, ODN 1826, ODN 2006 or ODN 2007, and their corresponding GC control ODNs. After 24h incubation, the levels of NF-κB-induced SEAP were determined by reading the OD at 655 nm.
	Response of HEK-Blue mTLR9 cells to Class B CpG ODNs. HEK-Blue mTLR9 cells, which express mouse TLR9 and an NF-kB-inducible SEAP reporter gene, were incubated in HEK-Blue Detection medium and stimulated with increasing concentrations of B-class CpG ODNs, ODN 1668, ODN 1826, ODN 2006 or ODN 2007, and their corresponding GC control ODNs. After 24h incubation, the levels of NF-kB-induced SEAP were determined by reading the OD at 655 nm.
	The effect of a class B ODN on the activation of B cells derived from human PBMCs Human PBMCs were incubated with ODN 2006 or its control at a final concentration of 1.6µg/ml for 24 hours then labeled with CD19, CD80, and CD86 mAbs, for analysis of B cell activation by flow cytometry.
	The effect of a class B ODN on the activation of B cells derived from murine splenocytes Murine splenocytes were incubated with ODN 1826 or its control at a final concentration of 3.3µg/ml for 48 hours and stained for CD19, CD80, and CD86, for analysis of B cell activation by flow cytometry.
	Secretion of IL-6 and IFN-α after stimulation of human PBMCs with a class B ODN Isolated PBMCs were stimulated with ODN 2006 or its control at a final concentration of 1.6µg/ml for 24 hours. The secretion of IL-6 and IFN-α was measured by ELISA.
	Secretion of IL-6 and IFN-alpha after stimulation of murine splenocytes with a class B ODN Murine splenocytes were stimulated with ODN 1826 or its control at a final concentration of 3.3µg/ml for 24 hours. The secretion of IL-6 and IFN-α was measured by ELISA.

Class C ODNs

Class C CpG ODNs (CpG-C ODNs) combine features of both classes A and B. They contain a complete PS backbone and a CpG-containing palindromic motif.
C-Class CpG ODNs very strongly stimulate B cells as well as type I IFN secretion. CpG-C ODNs are strong Th1-inducing adjuvants.

Vollmer J. et al., 2004. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. Eur J Immunol. 34(1):251-62.

	Response of HEK-Blue hTLR9 cells to Class C CpG ODNs. HEK-Blue hTLR9 cells, which express human TLR9 and an NF-κB-inducible SEAP reporter gene, were incubated in HEK-Blue Detection medium and stimulated with increasing concentrations of C-class CpG ODNs, ODN 2395 or ODN M362, and their corresponding GC control ODNs. After 24h incubation, the levels of NF-kB-induced SEAP were determined by reading the OD at 655 nm.
	Response of HEK-Blue mTLR9 cells to Class C CpG ODNs. HEK-Blue mTLR9 cells, which express mouse TLR9 and an NF-kB-inducible SEAP reporter gene, were incubated in HEK-Blue Detection medium and stimulated with increasing concentrations of C-class CpG ODNs, ODN 2395 or ODN M362, and their corresponding GC control ODNs. After 24h incubation, the levels of NF-kB-induced SEAP were determined by reading the OD at 655 nm.
	The effect of a class C ODN on the activation of B cells derived from human PBMCs Human PBMCs were incubated with ODN 2395 or its control at a final concentration of 1.6µg/ml for 24 hours then labeled with CD19, CD80, and CD86 mAbs, for analysis of B cell activation by flow cytometry.
	The effect of a class C ODN on the activation of B cells derived from murine splenocytes Murine splenocytes were incubated with ODN 2395 or its control at a final concentration of 3.3µg/ml for 48 hours and stained for CD19, CD80, and CD86, for analysis of B cell activation by flow cytometry.
	Secretion of IL-6 and IFN-α after stimulation of human PBMCs with a class C ODN Isolated PBMCs were stimulated with ODN 2395 or its control at a final concentration of 1.6µg/ml for 24 hours. The secretion of IL-6 and IFN-α was measured by ELISA.
	Secretion of IL-6 and IFN-alpha after stimulation of murine splenocytes with a class C ODN Murine splenocytes were stimulated with ODN 2395 or its control at a final concentration of 3.3µg/ml for 24 hours. The secretion of IL-6 and IFN-α was measured by ELISA.

Choose the right class

Class A CpG ODNs

Applications:
- stimulation of pDCs
- induction of IFN-α production
Prototypes:
- ODN 2216 (human)
- ODN 1585 (mouse)

Class B CpG ODNs

Applications:
- stimulation and proliferation of B cells
- NF-κB activation in TLR9-expressing recombinant cells
- vaccine adjuvant
Prototypes:
- ODN 2006 (human)
- ODN 1826 (mouse)

Class C CpG ODNs

Applications:
- combined applications of A-class and B-class CpG ODNs
Prototype:
- ODN 2395 (human/mouse)

	The effect of ODNs on the activation of B cells derived from human PBMCs Human PBMCs were incubated with CpG ODNs or their controls at a final concentration of 1.6µg/ml for 24 hours then labeled with CD19, CD80, and CD86 mAbs, for analysis of B cell activation by flow cytometry.
	Secretion of IL-6 and IFN-α after stimulation of human PBMCs with ODNs Isolated PBMCs were stimulated with CpG ODNs or their controls at a final concentration of 1.6µg/ml for 24 hours. The secretion of IL-6 and IFN-α was measured by ELISA.
	The effect of ODNs on the activation of B cells derived from murine splenocytes Murine splenocytes were incubated with CpG ODNs or their controls at a final concentration of 3.3µg/ml for 48 hours and stained for CD19, CD80, and CD86, for analysis of B cell activation by flow cytometry.
	Secretion of IL-6 and IFN-α after stimulation of murine splenocytes with ODNs Murine splenocytes were stimulated with CpG ODNs or their controls at a final concentration of 3.3µg/ml for 24 hours. The secretion of IL-6 and IFN-α was measured by ELISA.

Quality check

Table showing the various levels of quality checked of standard grade and VacciGrade TLR9 products

Quality check	Method	InvivoGen Standard Grade	InvivoGen VacciGrade™
Appearance	Visual inspection	✓	✓
Mass	Mass spectrometry analysis	✓	✓
Purity	AX-HPLC analysis	✓	✓
Solubility	Resuspension in endotoxin-free water	✓	✓
TLR9 activity	Cellular assay	✓	✓
TLR2 contaminating activity	Cellular assay	✓	✓
TLR4 contaminating activity	Cellular assay	✓	✓
Endotoxin level	Chromogenic LAL assay or in-house proprietary monocyte activation assay	Not checked	✓
Sterility	Direct inoculation or membrane filtration according to USP	Not checked	✓