Immune responses in pigs induced by recombinant DNA vaccine co-expressing swine IL-18 and membrane protein of porcine reproductive and respiratory syndrome virus

Abstract

In this study, two DNA vaccines, which express the membrane (M) protein of porcine respiratory and reproductive syndrome virus (PRRSV) (pEGFP-M) and co-express both M and swine IL-18 (pEGFP-IL18-M), were constructed and their abilities to induce humoral and cellular responses in piglets were comparatively evaluated. Experimental results showed that both recombinant DNA vaccines could not elicit neutralizing antibodies in the immunized piglets. However, both DNA vaccines elicited Th1-biased cellular immune responses. Notably, pigs immunized with the plasmid pEGFP-IL18-M developed significantly higher levels of IFN-γ and IL-2 production response and stronger specific T-lymphocyte proliferation response than the pigs inoculated with the plasmids pEGFP-M and pEGFP-IL18 (P < 0.05). These results illustrated that co-expression of M and IL-18 proteins could significantly improve the potency of DNA vaccination on the activation of vaccine-induced virus-specific cell-mediated immune responses in pigs, which may be used as a strategy to develop a new generation of vaccines against highly pathogenic PRRSV.

Keywords: DNA vaccine; IL-18; M protein; PRRS; immune response.

Figure 1

Schematic diagrams of recombinant plasmids—pEGFP-M, pEGFP-IL18 and pEGFP-IL18-M. pEGFP-N1 was used for the construction of the recombinant plasmids.

Figure 2

Fluorescent photos of the transfected MARC-145 cells. (A) The picture of negative control; (B) Green fluorescence images of cells transfected with pEGFP-N1; (C) Green fluorescence images of cells transfected with pEGFP-M; and (D) Green fluorescence images of cells transfected with pEGFP-IL18-M.

Figure 3

Western blot analysis of lysates of recombinant eukaryon expression plasmids transfected cells, pEGFP-IL18-M (lane 1), pEGFP-M (lane 2), pEGFP-N1 (lane 3), and negative control (lane 4). MARC-145 cells lysates without transfection were used as the control. Protein marker is shown on the right side.

Figure 4

M-specific enzyme-linked immunosorbent assay (ELISA) antibody responses in piglets immunized with different recombinant plasmids. Serum samples (n = 5) were collected at various time points and the specific antibodies to porcine respiratory and reproductive syndrome virus (PRRSV) were measured by indirect ELISA. Data are presented as the mean ± S.D. Different letters (a, b) above the columns at the same time point indicate significant difference (P < 0.05) between the treatments.

Figure 5

Lymphocyte proliferation responses of piglets immunized with different recombinant plasmids. Blood samples (n = 5) were collected at various time-points and were stimulated with PRRSV protein in triplicate. Data are presented as the mean ± S.D. Different letters (a, b, c) above the columns at the same time point indicate significant difference (P < 0.05) between the treatments.

Figure 6

(A) IFN-γ level in peripheral blood from piglets inoculated with the recombinant plasmids (B) IL-2 level in peripheral blood from piglets inoculated with the recombinant plasmids. Different letters (a, b, c) above the columns at the same time point indicate significant difference (P < 0.05) between the treatments.