1 Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E3
2 Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada L8N 3Z5
3 St Joseph's Hospital, Hamilton, ON, Canada L8N 4A6
Correspondence
Alexander N. Zakhartchouk
zakhartchouk{at}sask.usask.ca
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ABSTRACT |
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MAIN TEXT |
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The causative agent of SARS was identified as a new type of coronavirus, the SARS coronavirus (SARS-CoV). SARS-CoV is an enveloped virus with a positive-sense, single-stranded RNA genome of 29 727 nt. The genome is composed of a region encoding an RNA-dependent RNA polymerase, a region representing four coding sequences for viral structural proteins (S, E, M and N) and several putative uncharacterized proteins (Marra et al., 2003; Rota et al., 2003
).
The SARS-CoV nucleocapsid (N) gene encodes a 50 kDa protein harbouring a putative nuclear localization signal (Marra et al., 2003). However, the N protein is distributed predominantly in the cytoplasm of SARS-CoV-infected and N gene-transfected cells (Chang et al., 2004
). The SARS-CoV N protein is a highly charged, basic protein that can self-associate to form dimers (He et al., 2004
; Surjit et al., 2004
). The three-dimensional structure of the N-terminal portion of the protein is similar to those of other RNA-binding proteins (Huang et al., 2004
). The coronavirus N protein is thought to participate in the replication and transcription of viral RNA and to interfere with cell-cycle processes of host cells (Parker & Masters, 1990
; Kuo & Masters, 2002
; He et al., 2003
). In addition, the N proteins of many coronaviruses are highly immunogenic and expressed abundantly during infection (Liu et al., 2001
; Narayanan et al., 2003
). Indeed, high levels of IgG antibodies against N have been found in sera from SARS patients (Leung et al., 2004
).
For some coronaviruses, there is evidence that the N protein can prime the protective response or, in some cases, induce protective immunity on its own (Seo et al., 1997; Liu et al., 2001
). We therefore focused our studies on characterization of the N protein of SARS-CoV as a target antigen for vaccine development. We chose an adenoviral vector for SARS-CoV N protein expression, because adenovirus-vectored vaccines have been used to express antigens in vivo for the generation of both humoral and cellular immune responses and they can elicit potent and protective immune responses in a variety of animal models (Sullivan et al., 2000
, 2003
; Shiver et al., 2002
).
To generate an E1/partially E3-deleted, replication-defective human adenovirus 5 (Ad5) vector expressing the N protein, cDNA of the SARS-CoV N gene (strain Tor-2) was cloned into the expression cassette of plasmid pH5L. The pH5L plasmid contains the left portion of the Ad5 genome (nt 16100) with deletion of E1 and insertion of transcriptional control elements [the human cytomegalovirus immediate-early promoter and intron A, and the bovine growth hormone poly(A) signal]. The resulting plasmid, pH5L-N, was linearized and transfected into 293 cells together with a plasmid containing the right portion of the Ad5 genome with the 1878 bp deletion in the E3 region, using the ProFection mammalian transfection system (Promega). Homologous recombination led to generation of the recombinant virus, named Ad5-N-V. Adenovirus in crude lysate was plaque-purified by limiting dilution and agar overlay, and Ad5 vector clones were analysed by digestion of the viral genome with restriction enzymes and detection of N protein expression.
To corroborate the insertion of the N gene into the E1 region of Ad5, the viral genome was extracted from 293 cells at 24 h post-infection (p.i.) and digested with restriction enzymes. Due to the insertion of the N gene into the expression cassette, the genome of the recombinant virus Ad5-N-V contained a ClaI site and two BamHI sites (Fig. 1a). Digestion with BamHI resulted in three fragments of 18 208, 12 498 and 3553 bp, whilst digestion with ClaI produced two fragments of 30 737 and 3522 bp (Fig. 1b
). This suggested that recombinant Ad5-N-V contained the SARS-CoV gene N in the E1 region and that the direction of gene transcription was the same as that of E1.
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To examine the product expressed by Ad5-N-V, 7080 % confluent 293 cell monolayers in a six-well plate were infected with 10 TCID50 recombinant or wild-type Ad5 per cell. After virus adsorption for 1 h, cells were incubated in modified Eagle's medium (MEM) containing 10 % fetal bovine serum (FBS). At 24 h p.i., the medium was changed to methionine/cysteine-free MEM (for labelling with 35S) (Sigma) or phosphate-free MEM (for labelling with 32P) (Sigma), supplemented with 5 % FBS. After 1 h incubation, [35S]methionine/cysteine [100 µCi (3·7 MBq) per well; Perkin Elmer] or trisodium [32P]orthophosphate [300 µCi (11·1 MBq) per well; Amersham Biosciences] was added. After 4 h labelling, cells were harvested. Proteins were immunoprecipitated from cell lysates in radioimmunoprecipitation buffer by using rabbit serum 03-52 and samples were analysed by 12 % SDS-PAGE.
Immunoprecipitation of recombinant Ad5-N-V-infected cells labelled with [35S]methionine/cysteine revealed a band of 50 kDa (Fig. 2a, lane 1). No similar band was observed in uninfected cells or in cells infected with Ad5 (Fig. 2a
, lanes 2 and 3).
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These data suggested that SARS-CoV N protein was expressed and phosphorylated in Ad5-N-V-infected 293 cells. Phosphorylation of the N protein is a well-documented phenomenon in many coronaviruses (Stohlman & Lai, 1979; Wilbur et al., 1986
; Lomniczi & Morser, 1981
; King & Brian, 1982
). Although the functional significance of N protein phosphorylation is still not clear, in the case of mouse hepatitis virus, phosphorylated N protein has been suggested to have a higher RNA-binding capacity than the unphosphorylated protein, and its dephosphorylation was found to be connected with initiation of infection (Wilson et al., 1990
; Mohandas & Dales, 1991
). Interestingly, Ying et al. (2004)
tried to confirm SARS-CoV N protein phosphorylation by several methods, but failed. Moreover, a review of SARS-CoV protein mass spectrometry data suggested that there was no phosphorylation of the N protein. Our data, showing metabolic labelling of Ad5-infected cells expressing SARS-CoV N, indicated clearly that the N protein is phosphorylated. A possible explanation for the ability of our laboratory to detect phosphorylation whereas others could not is that metabolic labelling using 32P may be a more sensitive method. To our knowledge, this is the first experimental evidence of SARS-CoV N protein phosphorylation.
To determine the ability of Ad5-N-V to induce SARS-CoV-specific immune responses, five 68-week-old female C57BL/6 mice were immunized intraperitoneally with 2x1010 Ad5-N-V particles in 0·1 ml sterile PBS on days 0 and 28. Mice in the control group (n=5) were each immunized with 0·1 ml sterile PBS, using the same route of injection.
On day 45 after the first immunization, sera were analysed for the presence of SARS-CoV-specific IgG by ELISA. For ELISA, each well of a 96-well plate was coated with 0·1 ml purified inactivated SARS-CoV (1 µg ml1). Ad5-N-V induced a SARS-CoV-specific IgG antibody response (Fig. 3a). Both IgG1 and IgG2a antibody subclasses were found in the sera of immunized mice. The presence of both IgG antibody types (IgG1 and IgG2a) in equal levels in sera indicated that the immune response was a balanced Th1/Th2 response.
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As shown in Fig. 3(b), mice immunized with Ad5-N-V developed SARS-CoV-specific IFN-
ELISPOT responses. The presence of IFN-
indicated antigen-specific Th1-type CD4+- and CD8+-cell activation. There was no response in control-immunized mice or in splenocytes stimulated by cell-culture medium alone.
SARS-CoV cellular proliferative responses were assessed by a [methyl-3H]thymidine incorporation assay. Mice splenocytes were resuspended at a concentration of 3x106 cells ml1 in RPMI 1640 containing 10 % FBS. A 100 µl aliquot was added to each well of a 96-well plate together with 0·5 µg purified inactivated SARS-CoV per well or medium alone. After 3 days culture, 0·4 µCi [methyl-3H]thymidine (Amersham Biosciences) was added to each well. After incubation for 16 h, cells were harvested and thymidine uptake was measured by scintillation counting. Proliferative responses were calculated as mean values of triplicate cultures and were expressed as a stimulation index (counts min1 in the presence of antigen/counts min1 in the absence of antigen). As shown in Fig. 3(c), mice immunized with Ad5-N-V developed a significant SARS-CoV-specific proliferative response, whereas control mice did not.
These results showed that Ad5-N-V is capable of generating strong SARS-CoV-specific humoral and cellular immunity and may potentially be used as a SARS-CoV vaccine. Kim et al. (2004) and Zhu et al. (2004)
have also used SARS-CoV N protein for induction of immune responses; however, they used a DNA-based vaccine strategy.
Gao et al. (2003) used adenoviral vectors that expressed the SARS-CoV spike protein S1 fragment and the M and N proteins. Monkeys immunized with all three adenovirus vectors had an antibody response against the S1 protein and a T-cell response against the N protein, as measured by IFN-
ELISPOT analysis of peripheral blood mononuclear cells. Our data are in agreement with those of Gao et al. (2003)
; however, we used only one recombinant adenovirus for mouse immunizations and the immune responses were assessed by using murine splenocytes.
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ACKNOWLEDGEMENTS |
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Received 16 August 2004;
accepted 23 September 2004.