The Influence of DNA Sequence on the Immunostimulatory Properties of Plasmid DNA Vectors

Abstract

To determine the influence of DNA sequence on immunostimulatory properties of vaccine vectors, we tested the induction of in vitro and in vivo immune responses by plasmids modified to contain extended runs of dG sequences. Studies with oligonucleotides indicate that dG sequences can directly stimulate B cells as well as enhance the activity of immunostimulatory CpG motifs because of interaction with the macrophage scavenger receptor (MSR); this receptor can bind a variety of polyanions including dG sequences. To modify vectors, we introduced stretches of 20–60 dG residues into the pCMV-β and pSG5rab.gp vectors and measured the ability of these plasmids to induce IL-12 and IFN-γ production by murine splenocytes. The induction of in vivo antibody responses to rabies glycoprotein was also assessed with the pSG5rab.gp vectors. In in vitro cultures, cytokine production induced by plasmids with and without dG sequences was similar. Furthermore, the addition of dG sequences to pSG5rab.gp vectors failed to enhance the anti-rabies glycoprotein response to immunization. To assess further mechanisms by which plasmids stimulate macrophages, we measured the effects of MSR ligands on in vitro cytokine induction. In in vitro cultures, poly(G), dG₃₀, and fucoidan inhibited IL-12 induction by plasmids. IL-12 induction was also inhibited by mammalian DNA but was unaffected by polyanions that are not MSR ligands. Together, these results suggest that the addition of 20 to 60-base dG sequences to plasmids does not significantly affect their properties as immunostimulators or vaccines. Furthermore, these results suggest that MSR ligands can block cytokine induction by plasmid DNA whether or not the plasmid contains extended runs of dG.

Overview summary

The goal of these studies is to determine the influence of dG sequences on the immunostimulatory properties of plasmid vectors. dG sequences are B cell mitogens and, in addition, can promote cytokine induction by immunostimulatory oligonucleotides by binding to the macrophage scavenger receptor (MSR). As shown using two different plasmids, runs of dG did not cause major effects on the induction of IL-12 and IFN-γ in vitro. Furthermore, antibody responses induced in mice by immunization with plasmids containing dG runs did not differ from those induced by unmodified plasmids. To investigate further the interaction of plasmids with cells, the effect of MSR ligands on in vitro responses was measured. These experiments indicated that MSR ligands can block the activity of plasmids whether or not they contain dG runs. These findings thus clarify the role of DNA sequence in immunostimulation and are relevant to the host response to bacterial DNA as well as strategies for enhancing the potency of DNA vaccines.