Skip to main content
PMC home page
Springer Nature - PMC COVID-19 Collection logoLink to Springer Nature - PMC COVID-19 Collection
. 2007 Oct;28(10):1637–1644. doi: 10.1111/j.1745-7254.2007.00628.x

Structure-efficacy relationships of immunostimulatory activity of CpG-containing oligodeoxynucleotides on mouse spleen cells

Hai-yan Du 2, Shao-you Xia 3, Hai-feng Song 1,✉, Na Li 1, Ming-mei Shang 1, Jia Zou 1, Qing-qing Wang 1, Lun Ou 1, Xiao Sun 1, Ai-guo Ji 2, Zhong-ming Tang 1
PMCID: PMC7091584  PMID: 17883951

Abstract

Aim:

To study the relationship between primary structures of oligodeoxynucleo-tides (ODN) containing unmethylated deoxycytidyl-deoxyguanosine (CpG) dinucleotide motifs and their immunostimulatory activities in mouse spleen cells.

Methods:

A series of CpG ODN with different primary structures were synthesized. Their capabilities to stimulate mouse spleen cell proliferation were determined by [3H]thymidine incorporation assay. Cytokine (interleukin [IL]-6, IL-12, and IFN-α) secretion spectra induced by CpG ODN were assessed by ELISA. The ability of CpG ODN to activate natural killer cells was evaluated by standard 4 h 51Cr-release assay. Flow cytometry was utilized to examine the expressions of various lymphocyte surface molecules on diverse immunocytes. An effective CpG ODN for murine, ODN1826, was set as the template of modification and the positive control.

Results:

The immunostimulatory activities of CpG ODN with different sequences and compositions varied markedly, both in character and in extent. It was useless for improving the immunostimulatory activity of ODN1826 by simply increasing the functional hexameric CpG motif number, modifying the site of CpG motifs, or changing the distance between multi-CpG motifs. However, an addition of a self-complementary palindrome structure at the 3′-end, but not the 5′-end of CpG ODN, aroused marked improvement in its activity. Several designed ODN had superior comprehensive immunostimulatory properties compared to ODN1826.

Conclusion:

The immunostimulatory activity of a CpG ODN was relevant to its primary structure. It was useless for promoting immunostimulatory activity to simply change CpG motif number, space, or distance. The 3′-end palindrome structure of CpG ODN is associated with enhanced immunostimulatory activity.

Keywords: CpG, oligodeoxynucleotides, immuno-stimulation, structure-activity relationship

Footnotes

Project supported by grants from the National Natural Science Foundation of China (No 30500625) and the National High Biotechnology Development Program of China (No 2003AA2Z347B).

References

  • 1.Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature. 1995;374:546–9. doi: 10.1038/374546a0. [DOI] [PubMed] [Google Scholar]
  • 2.Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol. 2002;20:709–60. doi: 10.1146/annurev.immunol.20.100301.064842. [DOI] [PubMed] [Google Scholar]
  • 3.Hartmann G, Weeratna RD, Ballas ZK, Payette P, Blackwell S, Suparto I. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J Immunol. 2000;164:1617–24. doi: 10.4049/jimmunol.164.3.1617. [DOI] [PubMed] [Google Scholar]
  • 4.Krieg AM. An innate immune defense mechanism based on the recognition of CpG motifs in microbial DNA. J Lab Clin Med. 1996;128:128–33. doi: 10.1016/S0022-2143(96)90004-9. [DOI] [PubMed] [Google Scholar]
  • 5.Walker PS, Scharton-Kersten T, Krieg AM, Love-Homan L, Rowton ED, Udey MC. Immunostimulatory oligodeoxy-nucleotides promote protective immunity and provide systemic therapy for leishmaniasis via IL-12- and IFN-gamma-dependent mechanisms. Proc Natl Acad Sci USA. 1999;96:6970–5. doi: 10.1073/pnas.96.12.6970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Krieg AM, Yi AK, Hartmann G. Mechanisms and therapeutic applications in DNA vaccines. Trends Microbiol. 1999;6:23–7. doi: 10.1016/S0966-842X(97)01145-1. [DOI] [Google Scholar]
  • 7.Ballas ZK, Rasmussen WL, Krieg AM. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J Immunol. 1996;157:1840–5. [PubMed] [Google Scholar]
  • 8.Macfarlane DE, Manzel L. Antagonism of immunostimulatory CpG-oligodeoxynucleotides by quinacrine, chloroquine, and structurally related compounds. J Immunol. 1998;160:1122–31. [PubMed] [Google Scholar]
  • 9.Zhao Q, Matson S, Herrera CJ, Fisher E, Yu H, Kreig AM. Comparison of cellular binding and uptake of antisense phosphodiester, phosphorothioate, and mixed phosphorothioate and methylphos-phonate oligonucleotides. Antisense Res Dev. 1993;3:53–66. doi: 10.1089/ard.1993.3.53. [DOI] [PubMed] [Google Scholar]
  • 10.Dalpke AH, Zimmermann S, Albrecht I, Heeg K. Phospho-diester CpG oligonucleotides as adjuvants: polyguanosine runs enhance cellular uptake and improve immunostimulative activity of phosphodiester CpG oligonucleotides in vitro and in vivo. Immunology. 2002;106:102–12. doi: 10.1046/j.1365-2567.2002.01410.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Sester DP, Naik S, Beasley SJ, Hume DA, Stacey KJ. Phosphorothioate backbone modification modulates macrophage activation by CpG DNA. J Immunol. 2000;165:4165–73. doi: 10.4049/jimmunol.165.8.4165. [DOI] [PubMed] [Google Scholar]
  • 12.Lee SW, Song MK, Baek KH, Park Y, Kim JK, Lee CH. Effects of a hexameric deoxyriboguanosine run conjugation into CpG oligodeoxynucleotides on their immunostimulatory potentials. J Immunol. 2000;165:3631–9. doi: 10.4049/jimmunol.165.7.3631. [DOI] [PubMed] [Google Scholar]
  • 13.Verthelyi D, Ishii KJ, Gursel M, Takeshita F, Klinman DM. Human peripheral blood cells differentially recognize and respond to two distinct CpG motifs. J Immunol. 2001;166:2372–7. doi: 10.4049/jimmunol.166.4.2372. [DOI] [PubMed] [Google Scholar]
  • 14.Kadowaki N, Antonenko S, Liu YJ. Distinct CpG DNA and polyi-nosinic-polycytidylic acid double-stranded RNA, respectively, stimulate CD11c type II dendritic precursor and CD11c+ dendritic cells to produce type I IFN. J Immunol. 2001;166:2291. doi: 10.4049/jimmunol.166.4.2291. [DOI] [PubMed] [Google Scholar]
  • 15.Vollmer J, Weeratna R, Payette P, Jurk M, Schetter C, Laucht M. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. Eur J Immunol. 2004;34:251–62. doi: 10.1002/eji.200324032. [DOI] [PubMed] [Google Scholar]
  • 16.Bao MS, Zhang Y, Wan M, Dai L, Hu XP, Wu XL. Anti-SARS-CoV immunity induced by a novel CpG oligodeoxy-nucleotide. Clin Immunol. 2006;118:180–7. doi: 10.1016/j.clim.2005.09.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Hartmann G, Krieg AM. Mechanism and function of a newly identified CpG DNA motif in human primary B cells. J Immunol. 2000;164:944–53. doi: 10.4049/jimmunol.164.2.944. [DOI] [PubMed] [Google Scholar]
  • 18.Rankin R, Pontarollo R, Ioannou X, Krieg AM, Hecker R, Babiuk LA. CpG motif identification for veterinary and laboratory species demonstrates that sequence recognition is highly conserved. Antisense Nucleic Acid Drug Dev. 2001;11:333–40. doi: 10.1089/108729001753231713. [DOI] [PubMed] [Google Scholar]
  • 19.Ashman RF. CpG oligodeoxyribonucleotides rescue mature spleen B cells from spontaneous apoptosis and promote cell cycle entry. J Immunol. 1998;160:5898–906. [PubMed] [Google Scholar]
  • 20.Krug A, Rothenfusser S, Hornung V, Jahrsdorfer B, Blackwell S, Ballas ZK. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur J Immunol. 2001;31:2154–63. doi: 10.1002/1521-4141(200107)31:7<2154::AID-IMMU2154>3.0.CO;2-U. [DOI] [PubMed] [Google Scholar]
  • 21.Vollmer J, Weeratna R, Jurk M, Samulowitz U, McCluskie MJ, Payette P. Oligodeoxynucleotides lacking CpG dinucleotides mediate Toll-like receptor 9 dependent T helper type 2 biased immune stimulation. Immunology. 2004;113:212–23. doi: 10.1111/j.1365-2567.2004.01962.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Yu D, Zhu FG, Bhagat L, Wang H, Kandimalla ER, Zhang R. Potent CpG oligodeoxynucleotides containing phosphodiester linkages: in vitro and in vivo immunostimulatory properties. Biochem Biophys Res Commun. 2002;297:83–90. doi: 10.1016/S0006-291X(02)02127-7. [DOI] [PubMed] [Google Scholar]
  • 23.Zhao Q, Temsamani J, Iadarola PL, Jiang Z, Agrawal S. Effect of different chemically modified oligodeoxynucleotides on immune stimulation. Biochem Pharmacol. 1996;51:173–82. doi: 10.1016/0006-2952(95)02177-9. [DOI] [PubMed] [Google Scholar]
  • 24.Yu D, Zhao Q, Kandimalla ER, Agrawal S. Accessible 5′-end of CpG-containing phosphorothioate oligodeoxynucleotides is essential for immunostimulatory activity. Bioorg Med Chem Lett. 2000;10:2585–8. doi: 10.1016/S0960-894X(00)00537-0. [DOI] [PubMed] [Google Scholar]
  • 25.Kandimalla ER, Bhagat L, Yu D, Cong Y, Tang J, Agrawal S. Conjugation of ligands at the 5′-end of CpG DNA affects immuno-stimulatory activity. Bioconjug Chem. 2002;13:966–74. doi: 10.1021/bc0200374. [DOI] [PubMed] [Google Scholar]
  • 26.Yu D, Kandimalla ER, Bhagat L, Tang JY, Cong Y, Tang J. ′Immunomer′-novel 3′-3′-linked CpG oligodeoxynucleotides as potent immunomodulatory agents. Nucleic Acids Res. 2002;30:4460–9. doi: 10.1093/nar/gkf582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Bhagat L, Zhu FG, Yu D, Tang J, Wang H, Kandimalla ER. CpG penta- and hexadeoxyribonucleotides as potent immuno-modulatory agents. Biochem Biophys Res Commun. 2003;300:853–61. doi: 10.1016/S0006-291X(02)02943-1. [DOI] [PubMed] [Google Scholar]
  • 28.Ballas ZK, Kreig AM, Warren T, Rasmussen W, Davis HL, Waldschmidt M. Divergent therapeutic and immunologic effects of oligodeoxynucleotides with distinct CpG motifs. J Immunol. 2001;167:4878–86. doi: 10.4049/jimmunol.167.9.4878. [DOI] [PubMed] [Google Scholar]
  • 29.Vollmer J, Weeratna R, Payette P, Jurk M, Schetter C, Laucht M. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. Eur J Immunol. 2004;34:251–62. doi: 10.1002/eji.200324032. [DOI] [PubMed] [Google Scholar]
  • 30.Hemmi H, Kaisho T, Takeda K, Akira S. The roles of Toll-like receptor 9, MyD88, and DNA-dependent protein kinase catalytic subunit in the effects of two distinct CpG DNAs on dendritic cell subsets. J Immunol. 2003;170:3059–64. doi: 10.4049/jimmunol.170.6.3059. [DOI] [PubMed] [Google Scholar]
  • 31.Honda K, Ohba Y, Yanai H, Negishi H, Mizutani T, Takaoka A. Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature. 2005;434:1035–40. doi: 10.1038/nature03547. [DOI] [PubMed] [Google Scholar]

Articles from Acta Pharmacologica Sinica are provided here courtesy of Nature Publishing Group

RESOURCES