Type B CpG oligodeoxynucleotides induce Th1 responses to peanut antigens: modulation of sensitization and utility in a truncated immunotherapy regimen in mice

Abstract

Scope: Peanut allergy stems from a Th2-biased immune response to peanut allergens leading to IgE production and allergic reactions upon ingestion.

Methods and results: A model of peanut allergy in C3H/HeJ mice was used to assess whether type A, B, or C CpG oligodeoxynucleotide (ODN) molecules would be effective in: (i) a prophylactic approach to prevent peanut allergy when administered simultaneously with a Th2-skewing adjuvant, and (ii) a therapeutic model to allow for shortened immunotherapy. Type B ODNs were extremely effective in inhibiting anaphylaxis in the sensitization protocol as evidenced by differences in symptom scores, body temperature, and mouse mast cell protease 1 release compared to sham treatment. In the therapeutic model, co-administration of type B ODN plus peanut proteins was highly effective in reducing anaphylactic reactions in mice with established peanut allergy. The therapeutic effect was accompanied by an increase in IFN-γ and peanut-IgG2a, without a significant decrease in peanut IgE or IL-4 responses.

Conclusion: CpG ODNs, especially type B, were highly effective in inducing Th1 responses in mice undergoing induction of peanut allergy, as well as in mice undergoing therapy for established peanut allergy. Interestingly, the IgE response was not significantly altered, suggesting that IgG antibodies may be enough to prevent peanut-induced anaphylaxis.

Figure 1

Secreted Th1-type cytokines from C3H/HeJ mouse dendritic cell cultures. DCs were enriched from splenocytes and cultured for 24 h in the presence of TLR9 agonist CpG ODNs. (A) IL-12 (p40) and (B) TNF-α were measured in the culture supernatant by ELISA. Bars represent mean values with standard deviation shown. Data are from duplicates with two mice each.

Figure 2

Prevention of anaphylaxis through CpG ODN co-administration during sensitization. (A) Mice were sensitized to peanut proteins using aluminum hydroxide with or without CpG ODNs then challenged with peanut. (B) Symptom scores, (C) body temperature change, and (D) MMCP-1 were assessed for mice following peanut challenge on day 36. Closed circles represent individual mice with the median shown as a line; bars represent mean values with standard deviation shown. *, **, and *** indicate p<0.05, 0.01, and 0.001, respectively. Data is combined from two separate experiments.

Figure 3

Th1- and Th2-responses in the prophylactic model. (A) IFN-γ and (B) IL-4 were measured from splenocytes cultured with peanut proteins for 96 hours. (C) Peanut protein-specific IgG2a, (D) IgE, and (E) IgG1 were measured from serum collected following the sensitization period. Bars represent means with standard deviation shown. *, **, and *** indicate p<0.05, 0.01, and 0.001, respectively as compared to the sham and ODN 1585 groups. Other statistically significant differences are indicated by brackets. Data is combined from two separate experiments from the mice shown in Figure 2.

Figure 4

Therapeutic effects of CpG ODN-adjuvanted immunotherapy. (A) Mice were sensitized to peanut then underwent 3 weeks of immunotherapy with or without CpG ODNs and were challenged 10 days later. (B) Symptom scores, (C) body temperature change, and (D) MMCP-1 were assessed for mice following peanut challenge on day 63. Closed circles represent individual mice with the median shown as a line; bars represent mean values with standard deviation shown. *, **, and *** indicate p<0.05, 0.01, and 0.001, respectively. Data is representative of two separate experiments.

Figure 5

Th1-, Th2, and regulatory-cytokine responses in the therapeutic model. (A) IFN-γ, (B) IL-4, (C) IL-13, (D) IL-5, and (E) IL-10 were measured from splenocytes (from mice depicted in Figure 4) cultured with peanut proteins for 96 hours. Bars represent means with standard deviation shown. *, **, and *** indicate p<0.05, 0.01, and 0.001, respectively as compared to the placebo and peanut alone groups. Other statistically significant differences are indicated by brackets. Data is representative of two separate experiments.

Figure 6

Peanut protein-specific antibody responses in the therapeutic model. (A) Peanut protein- specific IgG2a, (B) IgE, and (C) IgG1 were measured from serum samples collected following the immunotherapy phase. Bars represent means with standard deviation shown. *** indicate p< 0.001 as compared to the placebo group.