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. 2006 Apr 12;24(16):3100-8.
doi: 10.1016/j.vaccine.2006.01.058. Epub 2006 Feb 8.

Immunological characterizations of the nucleocapsid protein based SARS vaccine candidates

Shih-Jen Liu  1 , Chih-Hsiang Leng, Shu-Pei Lien, Hsiang-Yun Chi, Chiung-Yi Huang, Chang-Ling Lin, Wei-Cheng Lian, Chi-Ju Chen, Shie-Liang Hsieh, Pele Chong
Affiliations

Immunological characterizations of the nucleocapsid protein based SARS vaccine candidates

Shih-Jen Liu et al. Vaccine. .

Abstract

The recombinant nucleocapsid (rN) protein of the coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS) was cloned and expressed in Escherichia coli, extracted from cell lysates containing 6M urea, then purified by Ni(2+)-affinity chromatography. In animal immunogenicity studies, we found that most anti-rN protein antibodies were IgG2a in BALB/c mice vaccinated with rN emulsified in Montanide ISA-51 containing the synthetic oligodeoxynucleotide, CpG. In contrast, anti-rN protein antibodies of mice immunized with rN protein in PBS were found to mainly be IgG1. These results indicated that ISA-51/CpG-formulated rN protein was dramatically biased toward a Th1 immune response. To identify the B-cell immunodominant epitopes of the rN protein in the mouse and monkey, the reactivities of antisera raised against purified rN proteins formulated in ISA-51/CpG were tested with a panel of overlapping synthetic peptides covering the entire N protein sequence. Three immunodominant linear B-cell epitope regions were mapped to residues 166-180, 356-375, and 396-410 of the rN protein. When the reactivities of these peptides were screened with human sera from five SARS patients, peptides corresponding to residues 156-175 reacted strongly with sera from two of the SARS patients. These results indicated that the region around residues 156-175 of the N protein is immunogenic in the mouse, monkey, and human. We found that peptides corresponding to residues 1-30, 86-100, 306-320, and 351-365 contained murine immunodominant T-cell epitopes. To identify functional CTL epitopes of the N protein, BALB/c mice were immunized with peptides containing the H-2K(d) CTL motif emulsified in adjuvant ISA-51/CpG. Using an IFN-gamma secretion cell assay and analysis by flow cytometry, peptides containing residues 81-95 were found to be capable of stimulating both CD4(+) and CD8(+) cell proliferation in vitro. We also only observed that peptides corresponding to residues 336-350 were capable of stimulating IFN-gamma production in T-cell cultures derived from peripheral blood mononuclear cells (PBMCs) of macaques immunized with the rN protein emulsified in ISA/CpG adjuvant. Our current results together with those of others suggest that some immunodominant B-cell and T-cell epitopes are conserved in the mouse, monkey, and human. This information is very important for the development SARS diagnostic kits and a vaccine.

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Figures

Fig. 1
Fig. 1
Expression and purification of the rN protein using SDS–PAGE and immunoblot analysis. (A) Coomassie blue-stained 10% reduced SDS–PAGE showing the rN protein purification process. Lane 1, cell lysates after IPTG induction; lane 2, cell lysate before IPTG induction; lane 3, flow-through fraction of crude extracts from the inclusion body fraction; lane 4, fraction washed at pH 8.0 with 8 M urea in homogenate buffer (see Section 2); lane 5, fraction washed at pH 6.3; lanes 6 and 7, fractions washed at pH 5.9; lanes 8 and 9, rN protein eluted at pH 4.5 with 8 M urea in homogenate buffer. (B) Lane 1, purified rN protein stained with Coomassie blue; lane 2, purified rN protein detected by blotting with anti-His antibody. The arrows indicate the electrophoretic mobility of the rN protein.
Fig. 2
Fig. 2
Effects of adjuvant on anti-N protein antibody titers. Mice were immunized with 50 μg of the rN protein in PBS, IFA, or ISA/CpG (see Section 2) subcutaneously. Serum of each mouse was collected at 2 weeks after the second boost and measured with ELISA for detection of N protein-specific antibody titers. (A) Total IgG antibody titers and (B) the IgG2a/IgG1 ratio in each group. Pre-immunization titers were subtracted from the post-immunization titers. Data are presented as the mean ± S.D. from seven mice in each group.
Fig. 3
Fig. 3
Immunodominant B-cell epitopes of the SARS CoV N protein in sera from mice, macaques, and SARS patients. (A) Mice; (B) macaques; (C) SARS patients. The eighty 15-mer overlapping peptides that cover the entire sequence of the SARS CoV N protein were used in an ELISA to measure the immunodominant linear B-cell epitopes. The “relative reactivity” of each peptide with the tested sera was calculated using the mean OD of each peptide divided by the mean OD of peptide no. 1. Samples with ≥5 times the “relative reactivity” were considered to be positive.
Fig. 4
Fig. 4
Diagrammatic representation of the immunodominant linear B-cell and T-cell epitopes of the N protein in mice, macaques, and humans.
Fig. 5
Fig. 5
Immunodominant T-cell epitopes of the SARS CoV N protein in mice and macaques. Immunodominant T-cell epitopes were measured by an IFN-γ secretion assay. The isolated splenocytes from vaccinated mice (A) or peripheral blood mononuclear cells from a vaccinated macaque (B) were cultured with 15-mer individual peptides (5 μg/ml) at 37 °C for 5 days. Levels of IFN-γ in the supernatants were determined by an ELISA assay.
Fig. 6
Fig. 6
Peptide-specific IFN-γ release in the peptide mixture of vaccinated BALB/c mice. (A) The isolated splenocytes at 1 × 106 in 24-well plates were stimulated with 5 μg/ml of each peptide. The numbers 9-1 and 9-2 represent the top two ranking nonamer peptides, respectively (see Table 2); the peptide order was based on the predicted score. (B) Positive wells cultured with corresponding peptides (2.5 μg/ml) at 37 °C for 7 days in the presence of 50 U/ml IL-2. Cultured cells were re-stimulated with the corresponding peptide at 37 °C for 4 h. IFN-γ-secreting CD4+ and CD8+ cells were analyzed by flow cytometry.
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