Immunostimulatory CpG oligonucleotides: Effect on gene expression and utility as vaccine adjuvants

Abstract

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs mimic the immunostimulatory activity of bacterial DNA. CpG ODN directly stimulate B cells and plasmacytoid dendritic cells (pDC), promote the production of Th1 and pro-inflammatory cytokines, and trigger the maturation/activation of professional antigen presenting cells. CpG ODN are finding use as vaccine adjuvants, where they increase the speed, magnitude and duration of vaccine-specific immune responses. For example, CpG ODN significantly prolong the protection induced by AVA (Anthrax Vaccine Adsorbed). Unexpectedly, a majority of animals immunized with CpG-adjuvanted AVA maintain resistance to anthrax infection even after their Ab titers decline to sub-protective levels. This survival is mediated by the de novo production of protective Abs by high affinity long-lived memory B cells. The immunostimulatory activity of CpG ODN was probed at the molecular level by microarray. Results show that a small group of 'inducers' rapidly up-regulated a large network genes following CpG treatment of mice. This stimulatory activity is quenched by 'suppressors' that down-regulate the expression of targeted genes, including most of the 'inducers'. These findings shed light on the mechanism underlying CpG-mediated immune activation and therapeutic activity.

Figure 1. Persistence of protective Ab titers in mice immunized with AVA + CpG ODN

A/J mice were immunized i.p. with 10 ul of AVA alone (○) or adjuvanted with 20 ug of CpG ODN (●). Serum IgG anti-PA titers were monitored individually for each of >30 mice/group over time. Data reflect the percent of animals in each group with anti-PA titers in the “protective range” (> 1:16,000) and represent the combined results of two similar but independent experiments. *; p. <.05 vs AVA alone.

Figure 2. CpG ODN induce reproducible patterns of gene expression in vivo

BALB/c mice were injected i.p. with 400 µg of CpG ODN. Gene expression in the spleen was monitored over time by microarray. Four biological replicates were independently analyzed at each time point. Up-regulated genes were identified by comparison to untreated mice using a stringency cut-off of p < 0.00001 and are displayed as % of maximum gene expression.

Figure 3. Key networks contributing to the regulation of CpG-mediated gene expression

All genes activated at 3 h (p <.00001) whose regulatory interactions could be mapped by IPA are shown. A) Network analysis identified two ‘inducers’ that each up-regulated >50% of all genes: IFNg (red network) and TNF (orange network). B) Genes that remain active through 24 h are highlighted in pink (p. <.00001) while those whose expression fell towards background are highlighted in green. This down-regulation is mediated by ‘supressors’, highlighted in blue. Green arrows identify genes down-regulated by ‘suppressors’. Note that 95% of the genes targeted by ‘suppressors’ are down-regulated (green circles) and that many of these ‘suppressors’ target the ‘inducer’ TNF.