TLR9-/- and TLR9+/+ mice display similar immune responses to a DNA vaccine

Abstract

Plasmid DNA continues to attract interest as a potential vaccine-delivery vehicle. However, the mechanisms whereby immune responses are elicited by plasmids are not fully understood. Although there have been suggestions regarding the importance of CpG motifs in plasmid immunogenicity, the molecular mechanisms by which CpG motifs enhance immune responses to DNA vaccines are not well understood. As Toll-like receptor 9-deficient (TLR9-/-) mice fail to respond to the adjuvant effects of CpG oligonucleotides, we used these mice to determine the effect of CpG motifs in plasmids used for DNA immunization. In the study described below, we report that DNA immunization was as effective in eliciting antigen-specific antibody and at stimulating antigen-specific interferon-gamma (IFN-gamma)-secreting cells in TLR9-/- mice as in TLR9+/+ mice. This study illustrates that DNA vaccines elicit immune responses by multiple mechanisms and demonstrates that TLR9 is not essential for the induction of immune responses following DNA immunization.

Figure 1

Glycoprotein D (gD)-specific antibody isotypes in mice immunized with gD. Toll-like receptor 9 (TLR9)^–/– and TLR^+/+ mice were immunized with gD protein (1 µg) plus CpG 1826 (10 µg). Immunizations were performed on day 1 and 4 weeks later. Two weeks after the secondary immunization, mouse sera were assessed for gD-specific immunoglobulin G (IgG)1 and IgG2a by enzyme-linked immunosorbent assay (ELISA). n=5. *P < 0·05, Student's t-test.

Figure 2

Glycoprotein D (gD)-specific antibody isotypes in mice immunized with pgD (plasmid vector pCAN1 expressing the full-length glycoprotein D protein from bovine herpesvirus type 1 under the cytomegalovirus promoter). Toll-like receptor 9 (TLR9)^–/– and TLR^+/+ mice were immunized with pgD (at doses of 0·1 µg, 1 µg or 10 µg) or gD protein (1 µg) plus CpG 1826 (10 µg). Primary immunization was on day 1 and the secondary immunization was 4 weeks later. Two weeks after the secondary immunization, mouse sera were assessed for gD-specific immunoglobulin G (IgG)1 and IgG2a titres by enzyme-linked immunosorbent assay (ELISA). Data presented are values for individual mice and the bar represents the group mean (n = 5). P < 0·001: TLR9^–/– and TLR^+/+ mice versus control for both IgG1 and IgG2a by one-way analysis of variance (anova) followed by Tukey's multiple comparison test. P < 0·05: IgG2a titre. P < 0·01: IgG1/IgG2a ratio by t-test comparing gD protein with CpG oligonucleotide (ODN)-immunized TLR9^–/– mice versus gD protein with CpG ODN-immunized TLR9^+/+ mice. Unvaccinated control mice remained negative (titre < 1). There were no significant differences in antibody titres between TLR9^–/– and TLR^+/+ mice.

Figure 3

Bovine herpesvirus type 1 (BHV-1) neutralization titre of sera from Toll-like receptor 9 (TLR9)^–/– and TLR9^+/+ mice immunized with 10 µg of pgD (plasmid vector pCAN1 expressing the full-length glycoprotein D protein from BHV-1 under the cytomegalovirus promoter) on day 1 and 4 weeks later. Two weeks after the secondary immunization, mouse sera were assayed for BHV-1 neutralization. Data presented are values for individual mice and the bar represents the group mean. n = 4 for TLR9^–/– mice and n = 5 for TLR9^+/+ mice. TLR9^–/– and TLR^+/+ versus control, P < 0·05, by one-way analysis of variance (anova) followed by Tukey's multiple comparison test.

Figure 4

Glycoprotein D-specific cytokine-secreting cells. Toll-like receptor 9 (TLR9)^–/– and TLR^+/+ mice were immunized with 10 µg of pgD (plasmid vector pCAN1 expressing the full-length glycoprotein D protein from bovine herpesvirus type 1 under the cytomegalovirus promoter) on day 1 and 4 weeks later. Two weeks after the secondary immunization, mouse splenocytes were assessed for the frequency of interferon-γ (IFN-γ)- and interleukin-4 (IL-4)-secreting cells. Error bars represent the standard deviation (SD). n = 4 for TLR9^–/– mice and n = 5 for TLR^+/+ mice. IFN-γ-secreting cells of TLR9^–/– and TLR^+/+ mice versus control, P < 0·05. IL-4-secreting cells, TLR9^–/– versus control, P < 0·01, using Kruskal–Wallis one-way analysis of variance (anova) and Dunn's multiple comparison test.

Figure 5

Luciferase gene expression in skin following the administration of plasmids encoding luciferase in Toll-like receptor 9 (TLR9)^–/– and TLR^+/+ mice. Plasmids encoding luciferase and containing different numbers of CpG motifs were administered intradermally and, 2 days later, skin samples were assessed for expression of the luciferase gene. Data represent luciferase expression for injection sites of individual mice and the bar represents the median. All plasmids induced significant gene expression versus naive tissue (P < 0·05), as determined using one-way analysis of variance (anova) followed by Tukey's multiple comparison test. There were no significant differences among the different plasmids or between TLR9^–/– and TLR^+/+ mice. LUs, light units.