A plasmid containing CpG ODN as vaccine adjuvant against grass carp reovirus in grass carp Ctenopharyngodon idella

Abstract

CpG oligodeoxynucleotides (ODNs) are proved to have strong immune stimulatory activity. Plasmids containing CpG ODNs could be conveniently and low-costly used as vaccine adjuvant. However, they are different among various plasmids, motif repeats, species, etc. In the present study, plasmid pcDNA3.1 (+) containing five repetitions of CpG ODN 1670A named pcDNA3.1-1670A*5 with strong immunostimulation was screened out from twelve recombinant plasmids and three empty vectors by cell proliferation activity, interferon promoter activities and immune related gene expressions in CIK cells. It works through TLR9-mediated signaling pathway, triggering the immune related genes expression. Furthermore, the potentiality of pcDNA3.1-1670A*5 as adjuvant was tested in vivo. pcDNA3.1-1670A*5 was co-inoculated with inactivated GCRV vaccine on grass carp fingerlings. Immunoglobulins (IgM, IgD, IgZ), TLR9, IFNγ2, IFN1, TNF-α, Mx2 and VP4 were examined. Ultimately, pcDNA3.1-1670A*5 significantly enhanced the expressions of IgM in serum, head kidney and spleen, recognition receptor TLR9 as well as antiviral effector molecule Mx2, and inhibited GCRV proliferation in head kidney and spleen tissues. The present study explored the application and mechanism of plasmid containing CpG ODN as high-efficient adjuvant to promote efficiency of vaccine and control disease in grass carp, which will contribute to the development of new type CpG ODN adjuvant in aquaculture industry.

Keywords: adjuvant; grass carp (Ctenopharyngodon idella); grass carp reovirus; immune protection; plasmid containing CpG ODNs.

Figure 1. Effect of plasmids containing CpG ODNs on CIK cells

(A) CIK cells were treated with PBS (control) or different plasmids and determined the proliferation activity by cell counting at 48 h post-stimulation. (B) CIK cells vaccinated with plasmids for 48 h were challenged with GCRV and determined the proliferation activity by cell counting at 24 h post-infection. Data are presented as means ± SE (n=3). The p value was calculated by student's t-test between pcDNA3.1-1670A*5 stimulation group and other groups (*, p ≤ 0.05, **, p ≤ 0.01). Error bars represent standard errors.

Figure 2. Antiviral activities of plasmids containing CpG ODNs in CIK cells

(A) CIK cells were stimulated with PBS (control) or plasmids containing CpG ODNs and subsequently challenged with GCRV at 48 h post plasmids administration. The mRNA expression levels of VP4 were examined at 0, 24 and 48 h post-challenge. The EF1α gene was used as an internal control to normalize the cDNA template. mRNA levels of the target gene were normalized to those in CIK cells at 0 h. The p value was calculated by student's t-test between experimental and control groups (*, p ≤ 0.05, **, p ≤ 0.01). Error bars indicate SE (n = 3). Blank columns for 24 h and shadowy for 48 h. (B) Crystal violet staining of CIK cells after plasmids containing CpG ODNs stimulation and GCRV infection at 48 h post plasmids stimulation. Control group was treated with PBS and subsequently GCRV while mock group without any treatment.

Figure 3. Immune responses to plasmids containing CpG ODNs in CIK cells

The mRNA expressions of CiTLR9 (A), CiRIG-I (B), CiIL-2 (C), CiIFNγ2 (D), CiNFκB1 (E) and CiNFκB2 (F) were measured at 0, 24 and 48 h post-infection. CIK cells were stimulated with PBS (control) or plasmids containing CpG ODNs and infected with GCRV. The EF1α gene was used as an internal control to normalize the cDNA template. mRNA levels of the target gene were normalized to those in CIK cells at 0 h. The p value was calculated by student's t-test between experimental and control groups at each time point (*, p ≤ 0.05, **, p ≤ 0.01). Error bars indicate SE (n=3).

Figure 4. Immune responses of CiIFN1/3 to plasmids containing CpG ODNs

Left, the mRNA expressions of CiIFN1 (A) and CiIFN3 (B) were measured at 24 and 48 h post-infection. CIK cells were stimulated with PBS (control) or plasmids containing CpG ODNs and infected with GCRV. Other captions were the same as Figure 3. Right, CIK cells were co-transfected with 800 ng of pRL-TK and IFN1pro-luc (A) or IFN3pro-luc (B) in 24-well plates. At 16 h post-transfection, the cells were stimulated with PBS (control) or plasmids and infected with GCRV for 16 h or uninfected. Dual luciferase reporter assays were conducted at 12 h after GCRV infection. Treatment durations in this assay was far shorter than those in qRT-PCR which caused different results. Error bars indicate standard deviation (n = 4). Asterisks indicate significant difference from control (*, P ≤ 0.05).

Figure 5. Serum IgM expression detection by western blot

(A) Grass carp serum was obtained from grass carp vaccinated with NS, vaccine, vaccine-pcCpG or pcCpG and subsequently infected with GCRV at D0, D1, D5, D10, D14, D16 and D20. Grass carp serum of GCRV group was obtained at D16, D20, D25 and D29. (B) Expression difference among 5 groups of grass carp at D10.

Figure 6. Serum IgM detection by ELISA

Sera were collected at different days post immunization or GCRV infection from the fish vaccinated with PBS (control), vaccine, vaccine-pcCpG or pcCpG at D0, D1, D5, D10, D14, D16 and D20. The data of GCRV group were integrated to control group being showed as D16 and D20 in control group. ELISA index was normalized to D0. Data are presented as means ± SD (n = 5).

Figure 7. Immunohistochemistry analysis of IgM in spleen tissue of grass carp at D0, D1, D5, D10, D14, D16 and D20 post vaccination or subsequent infection

The data of GCRV group were integrated to control group being showed as D16 and D20 in control group. Original magnification ×400.

Figure 8. mRNA expressions of three immunoglobulins

The mRNA expressions of CiIgM (A), CiIgD (B) and CiIgZ (C) were measured at D0, D1, D5, D10, D14, D16 and D20 post immunization or subsequent infection in spleen (Left) or head kidney (Right). Grass carp were vaccinated with normal saline (NS), vaccine, vaccine-pcCpG and pcCpG respectively, and spleen and head kidney tissues were obtained from 5 random grass carp from each group at each time point. Different columns represent different time points. The 18S rRNA gene was used as an internal control to normalize the cDNA template. mRNA levels of the target gene were normalized to those in spleen tissue at 0 h. Asterisks (*) mark the significant difference (P ≤ 0.05) between experimental and control groups and double asterisks (**) mark the extremely significant difference (P ≤ 0.01). Error bars indicate SE (n = 3).

Figure 9. mRNA expressions of immune related genes individuals

The mRNA expressions of CiTLR9 (A), CiIFNγ2 (B), CiIFN1 (C), CiTNF-α (D) and CiMx2 (E) were measured at D0, D1, D10 and D16 post immunization in spleen (Left) or head kidney (Right). Grass carp were vaccinated with normal saline (NS), vaccine and vaccine-pcCpG respectively, and tissues were obtained from 5 random grass carp from each group at each time point. Other captions were the same as Figure 8.

Figure 10. Antiviral activities of pcCpG in vivo

The mRNA expression of VP4 was measured at 0, 1 and 5 d post infection in spleen (A) and head kidney (B). Grass carp were vaccinated with normal saline (NS), vaccine and vaccine-pcCpG and infected with GCRV at D15 respectively, then spleen and head kidney tissues were obtained from 5 random grass carp from each group at each time point. Other captions were the same as Figure 8.