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. 2004 Apr 15;92(3):237-43.
doi: 10.1016/j.imlet.2004.01.001.

Induction of SARS-nucleoprotein-specific immune response by use of DNA vaccine

Min-Sheng Zhu  1 , Ying Pan, Hua-Qun Chen, Yue Shen, Xiao-Chun Wang, Yong-Jun Sun, Kai-Hua Tao
Affiliations

Induction of SARS-nucleoprotein-specific immune response by use of DNA vaccine

Min-Sheng Zhu et al. Immunol Lett. .

Abstract

Induction of effective cytotoxic T lymphocyte (CTL) and/or a specific antibody against conserved viral proteins may be essential to the development of a safe and effective severe acute respiratory syndrome coronavirus (SARS-Cov) vaccine. DNA vaccination represents a new strategy for induction of humoral and cellular immune response. To determine the ability of SARS-Cov nucleoprotein (N protein) to induce antiviral immunity, in this report, we established a stable C2C12 line expressing SARS-Cov N protein, which was used as a target for specific CTL assay. We also expressed recombinant N proteins in Escherichia coli and prepared N protein-specific polyclonal antibodies. C3H/He mice were immunized with N protein-expressible pcDN-fn vector by intramuscular injections. We found that the DNA vaccination induced both N protein-specific antibody and specific CTL activity to the target. When C3H/He mice were immunized by three separate injections, high antibody titre (1:3200-1:6400, average titre is 1:4580) and high CTL activity (67.4+/-8.4% (E:T = 25:1), 69.6+/-6.7% (E:T = 50:1) and 71.8+/-6.2% (E:T = 100:1)) were observed. In the case of two vaccine injections, CTL activity was also high (56.6+/-12.7% (E:T = 25:1), 57.4+/-11.7% (E:T = 50:1) and 63.0+/-6.3% (E:T = 100:1)) However, antibody titres were much lower (1:200-1:3200, average titre is 1:980). Our results suggest that SARS-Cov nucleocapsid gene might be a candidate gene for SARS DNA vaccination.

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Figures

Fig. 1
Fig. 1
Schematic diagram of plasmid constructs expressing SARS-Cov N protein in mammalian expression vector and prokaryotic expression vector. The vehicle vector for DNA vaccination, pcDNA3.1 contains a CMV promoter and BGH polyadenylation sequence. Full length of n gene (labeled as SARS-n in this figure) released from pGEM-fn by EcoRI/BamHI digestion was inserted into pcDNA3.1, the resultant plasmid was indicated as pcDN-fn. For expressing protein in E. coli, EcoRI site in 5′ end of n gene was mutated to NdeI site and thereafter inserted NdeI/BamHI fragment into pET24a vector. The resultant plasmid is indicated as pET-fn. pET-pn indicates a truncated mutant of pET-fn in which only 1–120 aa region (ΔSARS-n) is remained as described in Section 2. TAA indicates stop codon. The number is the amino acid position of N protein sequence.
Fig. 2
Fig. 2
Expression and purification of SARS-Cov N protein in E. coli. Full length of n gene (1–422 aa) (pET-fn) and partial n gene (1–120 aa) (pET-pn) were inserted into pET24a vector and transformed into BL21 (DE3) E. coli. After induction with IPTG, cells were harvested and lysed by 8 M urea and ultrasonic. Cell lysate was centrifuged. Recombinant proteins in supernatants were purified by DEAE-A50 absorption and gel extraction method . A: expression of full length N protein in E. coli. Lane M: marker; lane 1: without IPTG induction; lane 2: with IPTG induction; lane 3: purified protein. B: expression of partial N protein in E. coli. Lane M: marker; lane 1: with IPTG induction; lane 2: without IPTG induction; lane 3: purified protein. C: Western blot assay. Cell lysates were resolved on SDS-PAGE gel and transferred to nitrocellulose membrane. The membrane was then incubated with 1:1000 serum of C3H/He mice which had received pcDN-fn vaccination and HRP labeled secondary antibody, sequentially. Positive bands were visualized by ECL reagent. Lane M: marker, lane 1: cell lysate of pET-pny lane 2: cell lysate of pET-fn; lane 3: BL21 (DE3) control cell lysate.
Fig. 3
Fig. 3
Expression of SARS-Cov nucleocapsid protein in C2C12 cells. 2×106 C2C12 cells were harvested and washed twice with cold HBS buffer (21 mM HEPES pH7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM glucose) and re-suspended with 0.5 ml HBS buffer mixed with 10 μg pcDN-fn DNA. The cell suspension was electroporated (60 μF, 200 V). The transfected cells were selected with G418 (250 μg/ml) and stable N protein-expressing clones were screened by Western blot assay. Lane 1:C2C12 cell control; lane 2: N protein-expressible line 1; lane 3: N protein-expressible line 2; lane 4: no N protein expression line.
Fig. 4
Fig. 4
Induction of SARS-Cov N protein-specific IgG antibody in mice immunized with pcDNA3.1 and pcDN-fn DNA. C3H/He mice given two or three intramuscular injections of the different DNA vaccines at 100 μg per mouse were bled at 5th to 6th week post-immunization, and ELISA was used to measure IgG antibody titers in individual mice.
Fig. 5
Fig. 5
Induction of SARS-Cov N protein-specific CTLs in mice immunized with pcDN-fn DNA. Splenocytes of the mice receiving pcDN-fn or pcDNA3.1 vaccination were re-stimulated in vitro with N protein-expressible C2C12 cells and tested for cytolytic activity by LDH-release assay. Cytolytic activity was defined as specific lysis percentage and detected at various E:T ratio. The values of each experimental point represent the mean of triplicates. ** Represents P<0.01.
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References

    1. Marra M.A., Jones S.J., Astell C.R., Holt R.A., Brooks-Wilson A., Butterfield Y.S. The Genome sequence of the SARS-associated coronavirus. Science. 2003;300:1399–1404. - PubMed
    1. Rota P.A., Oberste M.S., Monroe S.S., Nix W.A., Campagnoli R., Icenogle J.P. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science. 2003;300:1394–1399. - PubMed
    1. Ruan Y.J., Wei C.L., Ee A.L., Vega V.B., Thoreau H., Su S.T. Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection. Lancet. 2003;361:1779–1785. - PMC - PubMed
    1. Wilson J.A., Hart M.K. Protection from Ebola virus mediated by cytotoxic T lymphocytes specific for the viral nucleoprotein. J. Virol. 2001;75:2660–2664. - PMC - PubMed
    1. Vanderzanden L., Bray M., Fuller D., Roberts T., Custer D., Spik K. DNA vaccines expressing either the GP or NP genes of Ebola virus protect mice from lethal challenge. Virology. 1998;246:134–144. - PubMed

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