Induction of specific immune responses by severe acute respiratory syndrome coronavirus spike DNA vaccine with or without interleukin-2 immunization using different vaccination routes in mice

Abstract

DNA vaccines induce humoral and cellular immune responses in animal models and humans. To analyze the immunogenicity of the severe acute respiratory syndrome (SARS) coronavirus (CoV), SARS-CoV, spike DNA vaccine and the immunoregulatory activity of interleukin-2 (IL-2), DNA vaccine plasmids pcDNA-S and pcDNA-IL-2 were constructed and inoculated into BALB/c mice with or without pcDNA-IL-2 by using three different immunization routes (the intramuscular route, electroporation, or the oral route with live attenuated Salmonella enterica serovar Typhimurium). The cellular and humoral immune responses were assessed by enzyme-linked immunosorbent assays, lymphocyte proliferation assays, enzyme-linked immunospot assays, and fluorescence-activated cell sorter analyses. The results showed that specific humoral and cellular immunities could be induced in mice by inoculating them with SARS-CoV spike DNA vaccine alone or by coinoculation with IL-2-expressing plasmids. In addition, the immune response levels in the coinoculation groups were significantly higher than those in groups receiving the spike DNA vaccine alone. The comparison between the three vaccination routes indicated that oral vaccination evoked a vigorous T-cell response and a weak response predominantly with subclass immunoglobulin G2a (IgG2a) antibody. However, intramuscular immunization evoked a vigorous antibody response and a weak T-cell response, and vaccination by electroporation evoked a vigorous response with a predominant subclass IgG1 antibody response and a moderate T-cell response. Our findings show that the spike DNA vaccine has good immunogenicity and can induce specific humoral and cellular immunities in BALB/c mice, while IL-2 plays an immunoadjuvant role and enhances the humoral and cellular immune responses. Different vaccination routes also evoke distinct immune responses. This study provides basic information for the design of DNA vaccines against SARS-CoV.

FIG. 1.

Antibody responses to SARS-CoV induced by vaccinations with or without pcDNA-IL-2 by different immunization routes in mice. Serum samples (eight per group) were taken 10 days after the final immunization. Data are presented as means ± SDs. Values of ≥2.1 were considered positive by taking into account the absolute ratio of the responses in postimmunization serum/naïve serum.

FIG. 2.

Detection of SARS-CoV S-protein-specific IgG and other subclasses in vaccinated mice. Mouse sera (eight per group) were collected 10 days after the final immunization and were assayed for IgG1 and IgG2a antibodies against the S protein of SARS-CoV. Data are presented as means ± SDs.

FIG. 3.

S-protein-specific LPA. Pooled splenocytes were obtained from mice (three mice per group) immunized with the DNA vaccine on day 10 postimmunization with pcDNA-IL-2. Splenocytes were stimulated in vitro with S protein (test groups), ConA (positive controls), and bovine serum albumin (irrelevant antigen controls). Splenocytes from the control groups (immunized with pcDNA-IL-2, pcDNA3.1, or PBS) were stimulated with the S protein and served as negative controls and sham controls. The SI was calculated by use of the following formula: (mean OD of ConA- or antigen-stimulated proliferation)/(mean OD of nonstimulated proliferation). Each bar represents the mean SI ± SD for three mice.

FIG. 4.

SARS-CoV S protein-specific IFN-γ (A) and IL-4 (B) ELISPOT results. The numbers of IFN-γ-secreting cells in the spleens of mice harvested 10 days after the final immunization and stimulated in vitro with the S protein are shown. The results represent the averages for triplicate wells for three mice and are expressed as means ± SDs.

FIG. 5.

Analysis of CD8⁺ and CD4⁺ lymphocytes by flow cytometry. Peripheral blood mononuclear cells were isolated from vaccinated mice (n = 5) 10 days after the final immunization. CD4⁺ and CD8⁺ T cells from healthy and immunized BALB/c mice were counted. Values are expressed as means of the ratio of CD8⁺/CD4⁺ ± SDs.