CpG Oligodeoxynucleotide
Encyclopedia
CpG oligodeoxynucleotides (or CpG ODN) are short single-stranded synthetic DNA
molecules that contain a cytosine
"C" followed by a guanine
"G". The "p" refers to the phosphodiester backbone of DNA, however some ODN have a modified phosphorothioate
(PS) backbone. When these CpG motifs are unmethlyated, they act as immunostimulants. CpG motifs are considered pathogen-associated molecular patterns (PAMPs) due to their abundance in microbial genomes but their rarity in vertebrate genomes. The CpG PAMP is recognized by the pattern recognition receptor (PRR
) Toll-Like Receptor 9 (TLR9
), which is constitutively expressed only in B cells and plasmacytoid dendritic cells (pDCs) in humans and other higher primates.
s, but it was not until 1983 that Tokunaga et al. specifically identified bacterial DNA as the underlying component of the lysate that elicited the response. Then, in 1995 Krieg et al. demonstrated that the CpG motif within bacterial DNA was responsible for the immunostimulatory effects and developed synthetic CpG ODN. Since then, synthetic CpG ODN have been the focus of intense research due to the Type I pro-inflammatory
response they elicit and their successful use as vaccine adjuvants
.
s). The five classes are Class A (Type D), Class B (Type K), Class C, Class P, and Class S. It is important to note that during the discovery process, the "Classes" were not defined until much later when it became evident that ODN with certain characteristics elicited specific responses. Because of this, most ODN referred to in the literature use numbers (i.e., ODN 2006, ODN 2007, ODN 2216, ODN D35, ODN K3, etc.). The numbers are arbitrary and come from testing large numbers of ODN with slight variations in attempts to find the optimal sequence. In addition, some papers will give different names to previously described ODN, complicating the naming convention even more.
, the most important one being IFNα, and induced the maturation of pDCs. Class A ODN are also strong activators of NK cells
through indirect cytokine
signaling.
Structural features defining Class A ODN:
Class A ODN typically contain 7 to 10 PS-modified bases at one or both ends that resist degradation by nucleases and increase the longevity of the ODN. The above rules strictly define the class, but variability of the sequence within these "rules" is possible. It should also be noted that changes to the sequence will affect the magnitude of the response. For example, the internal palindrome sequence can be 4 to 8 base pairs in length and vary in the order of bases, however the pattern, 5'-Pu
Pu CG Pu Py
CG Py Py-3', was found to be the most active when compared to several other sequences. The poly G tail found at either end of the DNA strand can vary in length and even number (Type D only have a poly G sequence on the 3'end), but its presence is critical to the activity of the molecule.
Structural features defining Class B ODN:
The strongest ODN in this class have three 6mer sequences. B ODN have been studied extensively as therapeutic agents because of their ability to induce a strong humoral immune response, making them ideal as a vaccine
adjuvant
.
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
molecules that contain a cytosine
Cytosine
Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine . It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached . The nucleoside of cytosine is cytidine...
"C" followed by a guanine
Guanine
Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine . In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with...
"G". The "p" refers to the phosphodiester backbone of DNA, however some ODN have a modified phosphorothioate
Thiophosphate
A thiophosphate is a family of compounds and anions with the general chemical formula PS4-xOx3- . The state of protonation is usually not specified. They could be bound to as many as three protons for the neutral H3PS4-xOx species. Two protons correspond to the related monoanions, and one...
(PS) backbone. When these CpG motifs are unmethlyated, they act as immunostimulants. CpG motifs are considered pathogen-associated molecular patterns (PAMPs) due to their abundance in microbial genomes but their rarity in vertebrate genomes. The CpG PAMP is recognized by the pattern recognition receptor (PRR
Pattern recognition receptor
Pattern recognition receptors are a primitive part of the immune system. They are proteins expressed by cells of the innate immune system to identify pathogen-associated molecular patterns , which are associated with microbial pathogens or cellular stress, as well as damage-associated molecular...
) Toll-Like Receptor 9 (TLR9
TLR9
Toll-like receptor 9 is a protein that in humans is encoded by the TLR9 gene. TLR9 has also been designated as CD289 .- Function :...
), which is constitutively expressed only in B cells and plasmacytoid dendritic cells (pDCs) in humans and other higher primates.
History
Since 1893, it has been recognized that Coley's toxin, a mixture of bacterial cell lysate, has immunostimulatory properties that could reduce the progression of some carcinomaCarcinoma
Carcinoma is the medical term for the most common type of cancer occurring in humans. Put simply, a carcinoma is a cancer that begins in a tissue that lines the inner or outer surfaces of the body, and that generally arises from cells originating in the endodermal or ectodermal germ layer during...
s, but it was not until 1983 that Tokunaga et al. specifically identified bacterial DNA as the underlying component of the lysate that elicited the response. Then, in 1995 Krieg et al. demonstrated that the CpG motif within bacterial DNA was responsible for the immunostimulatory effects and developed synthetic CpG ODN. Since then, synthetic CpG ODN have been the focus of intense research due to the Type I pro-inflammatory
Inflammation
Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process...
response they elicit and their successful use as vaccine adjuvants
Immunologic adjuvant
In immunology, an adjuvant is an agent that may stimulate the immune system and increase the response to a vaccine, without having any specific antigenic effect in itself. The word “adjuvant” comes from the Latin word adiuvare, meaning to help or aid...
.
Structural Features
Synthetic CpG ODN differ from microbial DNA in that they have a partially or completely phosphorothioated (PS) backbone instead of the typical phosphodiester backbone and a poly G tail at the 3' end, 5' end, or both. PS modification protects the ODN from being degraded by nucleases such as DNase in the body and poly G tail enhances cellular uptake. The poly G tails form intermolecular tetrads that result in high molecular weight aggregates. These aggregates are responsible for the increased activity the poly G sequence impart; not the sequence itself. Numerous sequences have been shown to stimulate TLR9 with variations in the number and location of CpG dimers, as well as the precise base sequences flanking the CpG dimers. This led to the creation of five unofficial classes or categories of CpG ODN based on their sequence, secondary structures, and effect on human peripheral blood mononuclear cells (PBMCPBMC
A peripheral blood mononuclear cell is any blood cell having a round nucleus. For example: a lymphocyte, a monocyte or a macrophage. These blood cells are a critical component in the immune system to fight infection and adapt to intruders. The lymphocyte population consists of T cells , B cells...
s). The five classes are Class A (Type D), Class B (Type K), Class C, Class P, and Class S. It is important to note that during the discovery process, the "Classes" were not defined until much later when it became evident that ODN with certain characteristics elicited specific responses. Because of this, most ODN referred to in the literature use numbers (i.e., ODN 2006, ODN 2007, ODN 2216, ODN D35, ODN K3, etc.). The numbers are arbitrary and come from testing large numbers of ODN with slight variations in attempts to find the optimal sequence. In addition, some papers will give different names to previously described ODN, complicating the naming convention even more.
Class A
One of the first Class A ODN ,ODN 2216, was described in 2001 by Krug et al. This class of ODN was distinctly different from the previously described Class B ODN (i.e., ODN 2006) in that it stimulated the production of large amounts of Type I interferonsInterferon type I
Human type I interferons comprise a vast and growing group of IFN proteins.All type I IFNs bind to a specific cell surface receptor complex known as the IFN-α receptor that consists of IFNAR1 and IFNAR2 chains....
, the most important one being IFNα, and induced the maturation of pDCs. Class A ODN are also strong activators of NK cells
Natural killer cell
Natural killer cells are a type of cytotoxic lymphocyte that constitute a major component of the innate immune system. NK cells play a major role in the rejection of tumors and cells infected by viruses...
through indirect cytokine
Cytokine
Cytokines are small cell-signaling protein molecules that are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication...
signaling.
Structural features defining Class A ODN:
- The presences of a poly G sequence at the 5' end, the 3' end, or both
- An internal palindromePalindromeA palindrome is a word, phrase, number, or other sequence of units that can be read the same way in either direction, with general allowances for adjustments to punctuation and word dividers....
sequence - GC dinucleotides contained within the internal palindrome
- A partially PS-modified backbone
Class A ODN typically contain 7 to 10 PS-modified bases at one or both ends that resist degradation by nucleases and increase the longevity of the ODN. The above rules strictly define the class, but variability of the sequence within these "rules" is possible. It should also be noted that changes to the sequence will affect the magnitude of the response. For example, the internal palindrome sequence can be 4 to 8 base pairs in length and vary in the order of bases, however the pattern, 5'-Pu
Purine
A purine is a heterocyclic aromatic organic compound, consisting of a pyrimidine ring fused to an imidazole ring. Purines, including substituted purines and their tautomers, are the most widely distributed kind of nitrogen-containing heterocycle in nature....
Pu CG Pu Py
Pyrimidine
Pyrimidine is a heterocyclic aromatic organic compound similar to benzene and pyridine, containing two nitrogen atoms at positions 1 and 3 of the six-member ring...
CG Py Py-3', was found to be the most active when compared to several other sequences. The poly G tail found at either end of the DNA strand can vary in length and even number (Type D only have a poly G sequence on the 3'end), but its presence is critical to the activity of the molecule.
Class B
Krieg et al. was the first to describe Class B ODN in 1995. Class B ODN (i.e. ODN 2007) are strong stimulators of human B cell and monocyte maturation. They also stimulate the maturation of pDC but to a lesser extent than Class A ODN and very small amounts of IFNα.Structural features defining Class B ODN:
- One or more 6mer CpG motif 5'-Pu Py C G Py Pu-3'
- A fully phosphorothioated (PS-modified) backbone
- Generally 18 to 28 nucleotides in length
The strongest ODN in this class have three 6mer sequences. B ODN have been studied extensively as therapeutic agents because of their ability to induce a strong humoral immune response, making them ideal as a vaccine
Vaccine
A vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe or its toxins...
adjuvant
Adjuvant
An adjuvant is a pharmacological or immunological agent that modifies the effect of other agents, such as a drug or vaccine, while having few if any direct effects when given by itself...
.