Characterization of humoral responses in mice immunized with plasmid DNAs encoding SARS-CoV spike gene fragments - PubMed (original) (raw)

Characterization of humoral responses in mice immunized with plasmid DNAs encoding SARS-CoV spike gene fragments

Fanya Zeng et al. Biochem Biophys Res Commun. 2004.

Abstract

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 S1 subunit (residues 18-495) were capable of eliciting SARS-CoV specific antibodies and the region immediate to N-terminus of matured S1 protein contained an important immunogenic determinant for elicitation of SARS-CoV specific antibodies. In addition, mice immunized with plasmids encoding S1 fragment developed a Th1-mediated antibody isotype switching. Another interesting finding was that mouse antibodies elicited separately by plasmids encoding S1 and S2 subunits cooperatively neutralized SARS-CoV but neither the S1 nor S2 specific antibodies did, suggesting the possible role of both S1 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.

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Figures

Fig. 1

Schematic diagram of full-length SARS-CoV S protein and its derived fragments used in the study. The full-length S gene is shown immediately under the hydrophobicity profile (upper). Signal sequence of S gene is marked as a solid black box and the putative highly hydrophobic regions of S1 are shown in solid box in light grey color. Heptad repeats (HR1 and HR1) and membrane spanning domain (MSD) are indicated as described elsewhere , . All the fragments including the recombinant fragments were cloned into mammalian expression vector pCI-SPpGH, except for SLa and SNa which were cloned in pCI. Fragments, SNb, SLb, SC, SF, SR1, SR2, and SR3 were inserted into pcDNA3.1 in addition. All the constructed plasmids were used for mice experiments. SR1–3 are different recombinants representing the putative hydrophilic regions.

Fig. 2

The expressed S protein in Western blot. Expressed S protein by pcDNA 3.1-6×His-IRES-GFP was detected with monoclonal mouse anti-his-tag antibody. Arrow indicates the protein band with expected size, the lane with protein collected from cells transfected with control plasmid has no detectable band (not shown). Size marker is shown on the left (kDa).

Fig. 3

Mouse anti-S protein IgG profile. Mice (_n_=5 per group) were immunized with different spike gene fragments inserted in pCI-SPpGH at day 1, 15, and 43. Serum samples were collected and grouped for ELISA assay at a dilution of 1:50.

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Characterization of humoral responses in mice immunized with plasmid DNAs encoding SARS-CoV spike gene fragments - PubMed (original) (raw)