CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Th1) immunity

Abstract

Synthetic oligodeoxynucleotides (ODN) that contain unmethylated CpG motifs (CpG ODN) induce macrophages to secrete IL-12, which induces interferon (IFN)-gamma secretion by natural killer (NK) cells. Since these cytokines can induce T helper 1 (Th1) differentiation, we examined the effects of coadministered CpG ODN on the differentiation of Th responses to hen egg lysozyme (HEL). In both BALB/c (Th2-biased) and B10.D2 (Th1-biased) mice, immunization with HEL in incomplete Freund's adjuvant (IFA) resulted in Th2-dominated immune responses characterized by HEL-specific secretion of IL-5 but not IFN-gamma. In contrast, immunization with IFA-HEL plus CpG ODN switched the immune response to a Th1-dominated cytokine pattern, with high levels of HEL-specific IFN-gamma secretion and decreased HEL-specific IL-5 production. IFA-HEL plus CpG ODN also induced anti-HEL IgG2a (a Th1-associated isotype), which was not induced by IFA-HEL alone. Control non-CpG ODN did not induce IFN-gamma or IgG2a, excepting lesser increases in B10.D2 (Th1-biased) mice. Thus, CpG ODN provide a signal to switch on Th1-dominated responses to coadministered antigen and are potential adjuvants for human vaccines to elicit protective Th1 immunity.

Figures

Figure 1

Th1-associated antigen-specific IgG2a responses are induced by immunization of BALB/c mice with IFA-HEL-CpG ODN but not IFA-HEL-non–CpG ODN. (A–C). Mice were injected i.p. with CFA-HEL (a control for a Th1-dominated response), IFA-HEL (a control for a Th2-dominated response), or IFA-HEL with 100 μg of CpG ODN 1826 or non–CpG ODN 1745. Sera were collected from mice 15–18 d after injection and assayed by ELISA for: (A) anti-HEL IgG2a, an isotype associated with Th1-dominated responses; (B) anti-HEL IgG1; and (C) anti-HEL total Ig response. A–C represent data from a single experiment representative of three similar experiments. (D) BALB/c mice were immunized as above, except that 30 μg of CpG ODN 1585, non–CpG ODN 1972, CpG ODN 1760, or non–CpG ODN 1908 was used for each mouse. Anti-HEL IgG2a antibodies were detected by serum ELISA. Data shown in D are representative of three similar experiments.

Figure 2

CpG ODN enhance HEL-specific IFN-γ production by BALB/c splenocytes. Mice were immunized as in Fig. 1 with 100 μg ODN/mouse in A and 30 μg ODN/mouse in panel B. After 3 wk, splenocytes were isolated and incubated with HEL (closed circles) or medium alone (open circles). ELISA spot assay was performed and spots were quantitated by a computerized image analysis program. Each point represents the mean number of spots per well for one mouse (assayed in duplicate); horizontal bars indicate the mean of points for each group of mice. Similar results were observed in five independent experiments with CpG- and non–CpG ODN in BALB/c mice.

Figure 3

ELISA spot assessment of IFN-γ production by splenocytes from immunized BALB/c mice. Pictures show representative images of ELISA spot wells from the experiment shown in Fig. 2A. The number of spots, as quantitated by an image analysis program, is indicated next to each well. Each well contained HEL (100 μg/ml) and 106 splenocytes isolated from mice immunized with IFA-HEL (A), IFA-HEL-non–CpG ODN 1745 (B) or IFA-HEL-CpG ODN 1826 (C).

Figure 4

CpG ODN decrease HEL-specific IL-5 production by BALB/c splenocytes. Mice were immunized as in Fig. 2 (30 μg ODN/mouse), and splenocytes were harvested for in vitro restimulation with or without HEL. ELISA spot analysis was performed for IL-5. The data are representative of five similar experiments with CpG- and non–CpG ODN in BALB/c mice.

Figure 5

Induction of HEL-specific IFN-γ responses by CpG ODN in B10.D2 mice. B10.D2 mice were immunized as in Fig. 1, except that ODN were used at 30 μg per mouse. Three weeks after immunization, HEL-specific production of IFN-γ by splenocytes was measured by ELISA spot assay as in Fig. 2. The data shown are representative of three similar experiments.