CpG ODNs Classes

CpG ODNs are synthetic oligonucleotides that contain unmethylated CpG dinucleotides in particular sequence contexts (CpG motifs). These CpG motifs are present at a 20-fold greater frequency in bacterial DNA compared to mammalian DNA [1]. CpG ODNs are recognized by Toll-like receptor 9 (TLR9) leading to strong immunostimulatory effects [2].

CpG ODNs possess a partially or fully phosphorothioate-modified (PS) backbone for nuclease resistance, as opposed to the natural phosphodiester (PO) backbone found in genomic bacterial DNA.

Classically, three major classes of stimulatory CpG ODNs, Class A (Type D), Class B (Type K) and Class C, are defined based on structural characteristics and activity on human peripheral blood mononuclear cells (PBMCs), in particular B cells and plasmacytoid dendritic cells (pDCs). A fourth, Class P, was introduced later that combines the properties of Class A and Class B CpG ODNs and induces strong type-I IFN and inflammatory responses.

Cellular activity of the three major classes of CpG ODNs.

Class A (type D) CpG ODNs (CpG-A ODNs) are characterized by a PO central CpG-containing palindromic motif and a PS-modified 3’ poly-G string. 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 [3].

CpG-A ODNs are also strong activators of NK cells through indirect cytokine signaling [4, 5]. 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.

Class B (type K) CpG ODNs (CpG-B ODNs) are 18-28 mer linear oligodeoxynucleotides. They contain a fully phosphorothioated backbone with one or more 6 mer CpG motifs. The optimal motif is GTCGTT [6] in humans and GACGTT in mice [7]. 
CpG-B ODNs strongly activate B cells and TLR9-dependent NF-κB signaling but weakly stimulate IFN-α secretion [8, 9]. They display anti-tumor activity and are potent Th1 adjuvants.

Class C CpG ODNs (CpG-C ODNs) combine features of both classes A and B [3]. 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. 
 

Chemical structure of Class A, Class B, and Class C ODNs.

Characteristics of the three major classes of CpG ODNs

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)
   

Quality check

 Table showing the various levels of quality checked of standard grade and VacciGrade™ TLR9 products. All CpG ODNs undergo rigorous quality assessment. InvivoGen’s VacciGrade™ line includes additional checks for endotoxin levels and sterility.

References

1.    Hemmi H. et al., 2000. A Toll-like receptor recognizes bacterial DNA. Nature. 408(6813):740-5.
2.    Krieg AM. et al., 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature. 374(6522):546-9.
3.    Vollmer J. et al., 2004. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. Eur J Immunol. 34(1):251-62.
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.    Verthelyi D. et al., 2001. Human peripheral blood cells differentially recognize and respond to two distinct CPG motifs. J Immunol. 166(4):2372-7.
6.    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.
7.    Krieg AM. et al., 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature. 374(6522):546-9.
8.    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.
9.    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.