The study shows that N protein of SARS-CoV not only is an important B cell immunogen, but also can elicit broad-based cellular immune responses

The study shows that N protein of SARS-CoV not only is an important B cell immunogen, but also can elicit broad-based cellular immune responses. murine model. Serum anti-N immunoglobulins and splenocytes proliferative responses against N Indole-3-carbinol protein were observed in immunized BALB/c mice. The major immunoglobulin G subclass recognizing N protein was immunoglobulin G2a, and stimulated splenocytes secreted high levels of gamma interferon and IL-2 in response to N protein. More importantly, the immunized mice produced strong delayed-type hypersensitivity (DTH) and CD8+ CTL responses to N protein. The study shows that N protein of SARS-CoV not only is an important B cell immunogen, but also can elicit broad-based cellular immune responses. The results indicate that the N protein may be of potential value in vaccine development for specific prophylaxis and treatment against SARS. Keywords: Severe acute respiratory syndrome-coronavirus, Nucleocapsid protein, Immune response, Cell-mediated immunity, DNA vaccine A worldwide outbreak of severe acute respiratory syndrome (SARS) has been associated with a novel coronavirus, termed with SARS-Coronavirus (SARS-CoV), which has strong infectivity and pathogenicity (Drosten et al., 2003, Falsey et al., 2003, Ksiazek et al., 2003). Recently, several new cases have been confirmed in Taiwan and mainland of China, Singapore, and the Philippines. The coronaviruses are large, enveloped positive-stranded RNA viruses. The spike glycoprotein protein (S) forms conspicuous peplomer structures on the surface of the virus particle and is responsible for many of the biological properties of the virus, such as attachment to cell receptors, penetration, and spread by virus-induced cell to cell fusion (Gagneten et al., 1995, Popova and Zhang, 2002, Sanchez et al., 1999, Taguchi and Shimazaki, 2000). Antibodies to this protein not only neutralize the virus in vitro, but also provide protection against lethal virus challenge (Daniel and Talbot, 1990, Taguchi et al., 1995, Torres et al., 1995). Although the S protein is the immunodominant structural Indole-3-carbinol component, coronavirus infection also induces immune responses to other structural proteins, such as the membrane glycoprotein (M) and the nucleocapsid protein (N). The predicted N protein of SARS-CoV is a highly charged, basic protein of 422 amino acids with seven successive hydrophobic residues near the middle of the protein (Marra et al., 2003, Rota et al., 2003). The cellular immune response against N protein of some animal coronaviruses can enhance the recovery from the virus infection (Glansbeek et al., 2002, Wasmoen et al., 1995, Wesseling et al., 1993). Immunization with plasmid DNA encoding microbial antigens has provided protective immunity in some animal models and is therefore considered a potentially useful vaccine strategy (Fuller et al., 2002, Kamili et al., 2004, Padua et al., 2003). This technique involves the transfer of a target antigen gene into muscle cells by a plasmid vector with subsequent endogenous production and presentation for the induction of systemic immune responses. In this aspect, DNA immunization appeared to mimic natural viral infection. Hence, DNA Indole-3-carbinol vaccines may provide a useful tool to dissect the immunogenicity of a certain viral protein and define its roles in viral immunity. In the present study, the intact N protein of SARS-CoV was expressed in and its antigenicity was analyzed with the pooled sera from convalescence phase of SARS patients. Furthermore, a candidate Indole-3-carbinol DNA vaccine containing the N gene was constructed and its immunogenicity was evaluated in mice. Results Expression of the N protein of SARS-CoV in and identification of its antigenicity The plasmid pHT-N encodes a fusion protein about 50 kDa with 6 histidines and other 25 amino acid residues at its N-terminal. The bands of recombinant bacteria-expressed N proteins could be visualized in SDS-PAGE as predicted. There were two other proteins of smaller molecular mass following purification with Ni-NTA affinity resin in addition to the protein band corresponding to the complete fusion protein (Fig. 1 ). Western blot analysis of the total bacterial protein was carried out to Rabbit Polyclonal to CDCA7 determine the antigenicity of the N protein. As shown in Fig. 1, all of the three proteins could react with the pooled serum of SARS patients. Open in a separate window Fig. 1 Expression of SARS-CoV N protein in DH5. DH5 was transformed with the recombinant plasmid pHT-N and cultured with 0.6 mmol/L IPTG for induction of the SARS-CoV N protein expression, and.