CpG DNA induces protective antiviral immune responses in Atlantic salmon (Salmo salar L.)

Abstract

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN show promise as vaccine adjuvants and immunoprotective agents in animal models. Here we report that pretreatment with CpG ODN in animals induces nonspecific protection against viral infection. A panel of different synthetic CpG ODN was tested for the in vitro effects in Atlantic salmon (Salmo salar L.) leukocytes. The ODN were tested for their capacity to stimulate proliferation of peripheral blood leukocytes and to induce production of interferon-like factors in head kidney leukocytes. These studies revealed that the sequence and number of the CpG motifs as well as the lengths of the ODN contribute to their stimulatory activity. ODN with the 6-mer CpG motif (5'-GTCGTT-3') showed the highest stimulatory activity and were shown to induce protection against infectious pancreatic necrosis virus when injected in Atlantic salmon. Expression of the Mx transcript, as an indicator of alpha/beta interferon induction, was induced in the CpG-injected fish. These results suggest that CpG DNA in fish induces early, nonspecific antiviral protection.

FIG. 1.

Proliferation of Atlantic salmon PBL stimulated with CpG ODN, non-CpG ODN, calf thymus DNA, and bacterial LPS. Atlantic salmon PBL (8 × 10⁵ cells/well) were used for cell proliferation studies. The cells were cultured with various concentrations of the stimulants for 6 days, and proliferation was assessed by measuring [³H]thymidine incorporation. The data are reported as the mean counts per minute ± standard deviation (SD) of the mean for six fish or as the stimulation index ± SD and are representative of at least two independent experiments. (a) The results are presented in a semilogarithmic scale; the closed symbols represent cultures stimulated with CpG ODN 1681, and the open symbols represent cultures stimulated with control GpC ODN. (b) The stimulation with ODN 1681 and 1668 (at 2 and 8 μM [i.e., 12 and 48 μg ml⁻¹, respectively]) is compared with those with non-CpG, bacterial and calf thymus DNA (at 12.5 and 50 μg ml⁻¹), and LPS (at 10 and 50 μg ml⁻¹). **, statistical significance (P < 0.01) in comparison to the control.

FIG. 1.

Proliferation of Atlantic salmon PBL stimulated with CpG ODN, non-CpG ODN, calf thymus DNA, and bacterial LPS. Atlantic salmon PBL (8 × 10⁵ cells/well) were used for cell proliferation studies. The cells were cultured with various concentrations of the stimulants for 6 days, and proliferation was assessed by measuring [³H]thymidine incorporation. The data are reported as the mean counts per minute ± standard deviation (SD) of the mean for six fish or as the stimulation index ± SD and are representative of at least two independent experiments. (a) The results are presented in a semilogarithmic scale; the closed symbols represent cultures stimulated with CpG ODN 1681, and the open symbols represent cultures stimulated with control GpC ODN. (b) The stimulation with ODN 1681 and 1668 (at 2 and 8 μM [i.e., 12 and 48 μg ml⁻¹, respectively]) is compared with those with non-CpG, bacterial and calf thymus DNA (at 12.5 and 50 μg ml⁻¹), and LPS (at 10 and 50 μg ml⁻¹). **, statistical significance (P < 0.01) in comparison to the control.

FIG. 2.

Screening for the optimal sequence of phosphothiorate ODN to activate Atlantic salmon leukocytes. Leukocytes were incubated with different ODN (2 μM) with the sequences indicated (CG or GC dinucleotides are underlined). (A) Supernatants were harvested from HK leukocytes 48 h poststimulation, and antiviral activity was detected. The results are given as the mean IFN-like activity for pooled supernatants from six different fish and are representative of at least four different experiments. (B) Cell proliferation of Atlantic salmon PBL assessed by thymidine incorporation. The data are reported as the mean counts per minute ± standard deviation of the mean for six fish and are representative of at least two independent experiments.

FIG. 3.

Cumulative mortality for CpG ODN-injected Atlantic salmon challenged with IPNV at 7 days postinjection. The average mortality for duplicate tanks (n = 70 fish) is shown on the ordinate and is plotted against the number of days postchallenge.

FIG. 4.

(A) Effects of CpG ODN and poly(I-C) injection on Mx expression in Atlantic salmon head kidney. PCR was performed as described in Materials and Methods. The level of expression was calculated relative to the actin expression level. Data are the means ± standard deviations of results for three to four fish. (B) Effects of CpG ODN and poly(I-C) injection on Mx expression in Atlantic salmon spleen. PCR was performed as described in Materials and Methods. The level of expression was calculated relative to the actin expression level. Data are the means ± standard deviations of results for four fish. ** and *, statistical significance in comparison to the control at a P value of < 0.01 and < 0.05, respectively.