Identification of two neutralizing regions on the severe acute respiratory syndrome coronavirus spike glycoprotein produced from the mammalian expression system

Abstract

The Spike (S) protein of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) plays important roles in viral pathogenesis and potentially in the development of an effective vaccine against this virulent infectious disease. In this study, the codon-optimized S gene of SARS-CoV was synthesized to construct DNA vaccine plasmids expressing either the full-length or segments of the S protein. High titer S-specific immunoglobulin G antibody responses were elicited in rabbits immunized with DNA against various segments of the S protein. Two neutralizing domains were identified on the S protein, one at the N terminus (Ser12-Thr535) and the other near the C terminus (Arg797-Ile1192).

Figures

FIG. 1.

(A) Designs of SARS-CoV S DNA vaccines. Schematic representation of the entire S protein is shown at the top, including its natural leader and a TM close to the C-terminal tail. Hypothesized N glycosylation sites are marked by asterisks, and the ACE2 receptor (R) binding domain is also noted. DNA vaccines expressing different segments of the S protein are shown in the lower part of the figure, and their amino acid residue numbers are indicated. The nucleotide boundaries for these constructs are 34 to 3,765 bp for S, 34 to 2,394 bp for S1, 34 to 1,605 bp for S1.1, 1,599 to 2,394 bp for S1.2, 2,389 to 3,768 bp for S2, and 2,389 to 3,576 bp for S2.dTM. The tPA leader sequence has replaced the S natural leader in these constructs, and the variant S2.dTM construct has a C-terminal truncation including the TM domain. These codon-optimized S gene segments were individually subcloned into the DNA vaccine vector pSW3891 (22). (B) Western blot with either viron (Urbani strain)-associated S protein (SARS-CoV) or various S segments (S, S1, S1.1, and S2) expressed from transiently transfected 293T cells. Uninfected VeroE6 cells were used as negative controls. Samples were either treated (+) with the PNGaseF or not treated (−), as labeled. Rabbit serum immunized with the full-length S DNA vaccine was used to detect the SARS-CoV proteins. (C) Titers of rabbit anti-S IgG responses after four DNA immunizations as measured by ELISA. Rabbits were immunized with a Helios gene gun (Bio-Rad) on the shaved abdominal skin as previously reported (23). Plasmid DNA (36 μg) was administrated to individual rabbits for each of the immunizations at weeks 0, 2, 4, and 8. Serum samples tested were taken at week 10. Two different S protein antigens, S (left panel) and S1.2 (right panel), expressed from the transiently transfected 293T cells were used as the ELISA coating antigens. Data represent the geometric mean titers of sera from two different rabbits within each group.

FIG. 2.

Western blot analysis to identify the specificity of anti-S rabbit sera elicited by DNA vaccines expressing different S segments. (A) Rabbit serum raised by S DNA vaccine; (B) rabbit serum raised by S1.1 DNA vaccine; (C) rabbit serum raised by S1.2 DNA vaccine; (D) rabbit serum raised by S2 DNA vaccine. Cell lysates from 293T cells transfected with various S-expressing DNA vaccines and the empty vector are labeled above each Western blot. All samples were run under denatured conditions. (E) Western blot analysis to compare the potential of S2 and S2.dTM to form oligomers with rabbit serum immunized with the S2-expressing DNA vaccine. The 293T cell lysates transfected with either of the two S2 DNA constructs were heat (95°C) treated for 5 min prior to their loading onto sodium dodecyl sulfate-polyacrylamide gels; 4M urea was added to half of the samples (lanes labeled +).

FIG. 3.

Western blot analysis of SARS-CoV-associated S proteins by using rabbit sera elicited by DNA vaccines expressing different S segments (S, S1.1, S1.2, and S2). Cell-associated viral samples were collected 4 days postinfection and 100 ng of protein was loaded into each lane for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lanes: SARS-CoV, VeroE6 lysate infected with SARS-CoV; VeroE6, uninfected VeroE6 cells. S, expected monomeric S glycoprotein.

FIG. 4.

CPE assay to examine the neutralizing antibody responses in DNA-immunized rabbit sera. The neutralization assays were performed with triplicate testing wells in a 96-well flat-bottom plate in the biosafety level 3 laboratory. SARS-CoV (400 50% tissue culture infective doses) in 50 μl/well was incubated with 50 μl of serially diluted rabbit sera or tissue culture medium for 1 h. After the incubation, 100 μl of VeroE6 cells (20,000 cells) was added to each well at a multiplicity of infection of 0.02. The assay results were determined by CPE assay on day 4 of infection. (A) Sample pictures of uninfected (left) and SARS-CoV-infected VeroE6 cells in the presence (right) or absence (middle) of neutralizing rabbit serum elicited by DNA vaccine expressing full-length S antigen. (B) Anti-S neutralizing antibody titers of rabbit sera elicited by different S DNA vaccines. The neutralizing antibody titers are presented as the geometric means of two rabbit sera at the highest antibody dilutions that could completely prevent CPE.