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. 2008 Jun 15;118(1):65-71.
doi: 10.1016/j.imlet.2008.03.003. Epub 2008 Apr 8.

Search for potential target site of nucleocapsid gene for the design of an epitope-based SARS DNA vaccine

Noton Kumar Dutta  1 , Kaushiki Mazumdar, Byoung-Hee Lee, Min-Won Baek, Dong-Jae Kim, Yi-Rang Na, Sung-Hoon Park, Hyun-Kyoung Lee, Hiroaki Kariwa, Le Quynh Mai, Jae-Hak Park
Affiliations

Search for potential target site of nucleocapsid gene for the design of an epitope-based SARS DNA vaccine

Noton Kumar Dutta et al. Immunol Lett. .

Abstract

It is believed today that nucleocapsid protein (N) of severe acute respiratory syndrome (SARS)-CoV is one of the most promising antigen candidates for vaccine design. In this study, three fragments [N1 (residues: 1-422); N2 (residues: 1-109); N3 (residues: 110-422)] of N protein of SARS-CoV were expressed in Escherichia coli and analyzed by pooled sera of convalescence phase of SARS patients. Three gene fragments [N1 (1-1269 nt), N2 (1-327 nt) and N3 (328-1269 nt)-expressing the same proteins of N1, N2 and N3, respectively] of SARS-N were cloned into pVAX-1 and used to immunize BALB/c mice by electroporation. Humoral (by enzyme-linked immunosorbent assay, ELISA) and cellular (by cell proliferation and CD4(+):CD8(+) assay) immunity was detected by using recombinant N1 and N3 specific antigen. Results showed that N1 and N3 fragments of N protein expressed by E. coli were able to react with sera of SARS patients but N2 could not. Specific humoral and cellular immunity in mice could be induced significantly by inoculating SARS-CoV N1 and N3 DNA vaccine. In addition, the immune response levels in N3 were significantly higher for antibody responses (IgG and IgG1 but not IgG2a) and cell proliferation but not in CD4(+):CD8(+) assay compared to N1 vaccine. The identification of antigenic N protein fragments has implications to provide basic information for the design of DNA vaccine against SARS-CoV. The present results not only suggest that DNA immunization with pVax-N3 could be used as potential DNA vaccination approaches to induce antibody in BALB/c mice, but also illustrates that gene immunization with these SARS DNA vaccines can generate different immune responses.

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Figures

Fig. 1
Fig. 1
Insert preparation, expression, purification and detection of N (N1/N2/N3) protein in E. coli Origami™ B (DE3). (a) Schematic representation of plasmid constructs expressing SARS-CoV N protein in prokaryotic expression vector and mammalian expression vector. Full-length N gene labeled as N1 (nucleotides: 1–1269) and truncated as N2 (nucleotides: 1–327) and N3 (nucleotides: 328–1296). The number is the amino acid position of N protein sequence. (b) PCR products of N1 (1269 bp), N2 (327 bp) and N3 (942 bp), M: DNA marker. (c) Prokaryotic expression vector (pET21a) cloning (BamH1 and Sal1 digestion). (d) Expression of N (N1/N2/N3) protein in E. coli by 12% SDS-PAGE. M: protein marker, C: control [protein from normal E. coli Origami ™ B (DE3)], I: 4 h after induction with 1 mM IPTG, P: N protein purification with His Band affinity Ni-IDA resin column. (e) SDS-PAGE gels were transferred onto cellulose nitrate membranes for western blotting for antigen confirmation. (f) Analysis of antigen. Coomassie blue stained from SDS-PAGE [M, protein marker and lane 1: positive control (purified protein)], western blotting with purified protein (lane 2), serum from convalescent SARS patient (lane 3) and serum from a healthy volunteer (lane 4).
Fig. 2
Fig. 2
Insert preparation, expression and confirmation of N (N1/N2/N3) protein in mammalian cells (ATCC 293T). (a) PCR products of N1 (1269 bp), N2 (327 bp) and N3 (942 bp), M: DNA marker. (b) Eukaryotic expression vector (pVAX-1) cloning (BamH1 and Pst1 digestion). (c) Expression of the constructs, determined by western blot analysis with antisera reactive with SARS CoV N was evaluated after transfection of the indicated plasmid expression vectors in ATCC 293T cells. C: control (whole cell lysate transfected with vector), lane 1: vector inserted gene (pVAX-N1/pVAX-N2/pVAX-N3), lane 2: Recombinant SARS N (N1/N3) protein as positive control.
Fig. 3
Fig. 3
Detection of humoral immune response in the immunized BALB/c mice. Mouse sera were collected 10 days after the final immunization. SARS-CoV N (N1/N3)-specific IgG, IgG1 and IgG2a were assessed. Data are presented as means ± S.D. *p > 0.001 and **p > 0.01.
Fig. 4
Fig. 4
N-specific lymphocyte proliferation assay. Pooled splenocytes were obtained from mice (five mice per group) immunized with the DNA vaccine on day 10 post-immunization. Splenocytes were stimulated in vitro with N protein (N1/N3-test groups), Con A (positive controls), and BSA (irrelevant antigen controls). Splenocytes from the control groups (pVAX-1 or PBS) were stimulated with N protein, and served as negative controls and sham controls. The stimulation index (SI) was calculated using the following formula: SI = (mean OD of Con A or antigen-stimulated proliferation)/(mean OD of non-stimulated proliferation). Each bar represents the mean SI ± S.D. of five mice. *p > 0.01 and **p > 0.05.
Fig. 5
Fig. 5
Changes in CD4+CD8+ ratio of BALB/c mice in different groups. The CD4+ and CD8+ T subtypes were counted on day 10 post-immunization. Compared with the control group, the CD8+ T cells increased and the ratio of CD4+/CD8+ decreased in the experimental group, indicating that CTL activity was induced by the recombinant plasmid. *p < 0.05.
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