The immunological characteristics of SARS-CoV spike protein were investigated by administering mice with plasmids encoding various S gene fragments. We showed that the secreting forms of S1, S2 subunits and the N-terminus of S I subunit (residues 18-495) were capable of eliciting SARS-CoV specific antibodies and the region immediate to N-terminus of matured S 1 protein contained an important immunogenic determinant for elicitation of SARS-CoV specific antibodies. In addition, mice immunized with plasmids encoding S I fragment developed a Th1-mediated antibody isotype switching. Another interesting finding was that mouse antibodies elicited separately by plasmids encoding S I and S2 subunits cooperatively neutralized SARS-CoV but neither the S I nor S2 specific antibodies did, suggesting the possible role of both S I and S2 subunits in host cell docking and entry. These results provide insights into understanding the immunological characteristics of spike protein and the development of subunit vaccines against SARS-CoV. (C) 2004 Elsevier Inc. All rights reserved.