Editing DNA vaccine (section)

=== Immunostimulatory CpG motifs ===

Plasmid DNA itself appears to have an adjuvant effect on the immune system.<ref name=Alarcon1999 /><ref name=Robinson2000 /> Bacterially derived DNA can trigger innate immune defence mechanisms, the activation of dendritic cells and the production of TH1 cytokines.<ref name=Jakob1998 /><ref name=Krieg1995>{{cite journal | vauthors = Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, Koretzky GA, Klinman DM | display-authors = 6 | title = CpG motifs in bacterial DNA trigger direct B-cell activation | journal = Nature | volume = 374 | issue = 6522 | pages = 546–549 | date = April 1995 | pmid = 7700380 | doi = 10.1038/374546a0 | s2cid = 4261304 | bibcode = 1995Natur.374..546K }}</ref> This is due to recognition of certain CpG dinucleotide sequences that are immunostimulatory.<ref name=Sato1996 /><ref name=Klinman1997>{{cite journal | vauthors = Klinman DM, Yamshchikov G, Ishigatsubo Y | title = Contribution of CpG motifs to the immunogenicity of DNA vaccines | journal = Journal of Immunology | volume = 158 | issue = 8 | pages = 3635–3639 | date = April 1997 | doi = 10.4049/jimmunol.158.8.3635 | pmid = 9103425 | s2cid = 41861994 | url = http://www.jimmunol.org/cgi/content/abstract/158/8/3635 | doi-access = free }}</ref> CpG stimulatory (CpG-S) sequences occur twenty times more frequently in bacterially-derived DNA than in eukaryotes. This is because eukaryotes exhibit "CpG suppression" – i.e. CpG dinucleotide pairs occur much less frequently than expected. Additionally, CpG-S sequences are hypomethylated. This occurs frequently in bacterial DNA, while CpG motifs occurring in eukaryotes are methylated at the cytosine nucleotide. In contrast, nucleotide sequences that inhibit the activation of an immune response (termed CpG neutralising, or CpG-N) are over represented in eukaryotic genomes.<ref name=Krieg1998>{{cite journal | vauthors = Krieg AM, Wu T, Weeratna R, Efler SM, Love-Homan L, Yang L, Yi AK, Short D, Davis HL | display-authors = 6 | title = Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 95 | issue = 21 | pages = 12631–12636 | date = October 1998 | pmid = 9770537 | pmc = 22882 | doi = 10.1073/pnas.95.21.12631 | doi-access = free | bibcode = 1998PNAS...9512631K }}</ref> The optimal immunostimulatory sequence is an unmethylated CpG dinucleotide flanked by two 5’ [[purine]]s and two 3’ [[pyrimidine]]s.<ref name=Sato1996 /><ref name=Krieg1995 /> Additionally, flanking regions outside this immunostimulatory [[Oligomer|hexamer]] must be [[guanine]]-rich to ensure binding and uptake into target cells.

The innate system works with the adaptive immune system to mount a response against the DNA encoded protein. CpG-S sequences induce polyclonal B-cell activation and the upregulation of cytokine expression and secretion.<ref name=Klinsman1996>{{cite journal | vauthors = Klinman DM, Yi AK, Beaucage SL, Conover J, Krieg AM | title = CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 93 | issue = 7 | pages = 2879–2883 | date = April 1996 | pmid = 8610135 | pmc = 39727 | doi = 10.1073/pnas.93.7.2879 | doi-access = free | bibcode = 1996PNAS...93.2879K }}</ref> Stimulated macrophages secrete IL-12, [[IL18BP|IL-18]], TNF-α, IFN-α, IFN-β and IFN-γ, while stimulated B-cells secrete IL-6 and some IL-12.<ref name=Lewis1999 /><ref name=Klinsman1996 /><ref name=Yi1996>{{cite journal | vauthors = Yi AK, Chace JH, Cowdery JS, Krieg AM | title = IFN-gamma promotes IL-6 and IgM secretion in response to CpG motifs in bacterial DNA and oligodeoxynucleotides | journal = Journal of Immunology | volume = 156 | issue = 2 | pages = 558–564 | date = January 1996 | doi = 10.4049/jimmunol.156.2.558 | pmid = 8543806 | s2cid = 42145608 | url = http://www.jimmunol.org/cgi/content/abstract/156/2/558 | doi-access = free }}</ref>

Manipulation of CpG-S and CpG-N sequences in the plasmid backbone of DNA vaccines can ensure the success of the immune response to the encoded antigen and drive the immune response toward a TH1 phenotype. This is useful if a pathogen requires a TH response for protection. CpG-S sequences have also been used as external adjuvants for both DNA and recombinant protein vaccination with variable success rates. Other organisms with hypomethylated CpG motifs have demonstrated the stimulation of polyclonal B-cell expansion.<ref>{{cite journal | vauthors = Barwick BG, Scharer CD, Martinez RJ, Price MJ, Wein AN, Haines RR, Bally AP, Kohlmeier JE, Boss JM | display-authors = 6 | title = B cell activation and plasma cell differentiation are inhibited by de novo DNA methylation | journal = Nature Communications | volume = 9 | issue = 1 | pages = 1900 | date = May 2018 | pmid = 29765016 | pmc = 5953949 | doi = 10.1038/s41467-018-04234-4 | bibcode = 2018NatCo...9.1900B }}</ref> The mechanism behind this may be more complicated than simple methylation – hypomethylated murine DNA has not been found to mount an immune response.

Most of the evidence for immunostimulatory CpG sequences comes from murine studies. Extrapolation of this data to other species requires caution – individual species may require different flanking sequences, as binding specificities of scavenger receptors vary across species. Additionally, species such as ruminants may be insensitive to immunostimulatory sequences due to their large gastrointestinal load.