Synthetic peptide studies on the severe acute respiratory syndrome (SARS) coronavirus spike glycoprotein: perspective for SARS vaccine development

Abstract

Background: The S (spike) protein of the etiologic coronavirus (CoV) agent of severe acute respiratory syndrome (SARS) plays a central role in mediating viral infection via receptor binding and membrane fusion between the virion and the host cell. We focused on using synthetic peptides for developing antibodies against SARS-CoV, which aimed to block viral invasion by eliciting an immune response specific to the native SARS-CoV S protein.

Methods: Six peptide sequences corresponding to the surface regions of SARS-CoV S protein were designed and investigated by use of combined bioinformatics and structural analysis. These synthetic peptides were used to immunize both rabbits and monkeys. Antisera collected 1 week after the second immunization were analyzed by ELISA and tested for antibody specificity against SARS-CoV by immunofluorescent confocal microscopy.

Results: Four of our six synthetic peptides (S2, S3, S5, and S6) elicited SARS-CoV-specific antibodies, of which S5 (residues 788-820) and S6 (residues 1002-1030) exhibited immunogenic responses similar to those found in a parallel investigation using truncated recombinant protein analogs of the SARS-CoV S protein. This suggested that our S5 and S6 peptides may represent two minimum biologically active sequences of the immunogenic regions of the SARS-CoV S protein.

Conclusions: Synthetic peptides can elicit specific antibodies to SARS-CoV. The study provides insights for the future development of SARS vaccine via the synthetic-peptide-based approach.

Figure 1.

Titration of rabbit serum samples diluted from 1:25 in a twofold series against constant antigen concentration of 10 mg/L. Anti-peptide antiserum is indicated by ▴ with the corresponding preimmune serum, indicated by ▵, as negative control. Anti KLH-conjugated peptide antiserum is indicated by ▪, and the corresponding preimmune serum is indicated by □ as negative control. (A), R_S1 antiserum (▴) and R_S1_KLH antiserum (▪); (B), R_S2 antiserum (▴) and R_S2_KLH antiserum (▪); (C), R_S3 antiserum (▴) and R_S3_KLH antiserum (▪); (D), R_S4 antiserum (▴) and R_S4_KLH antiserum (▪); (E), R_S5 antiserum (▴) and R_S5_KLH antiserum (▪); (F), R_S6 antiserum (▴) and R_S6_KLH antiserum (▪); (G), R_MIX antiserum (▴) and R_MIX_KLH antiserum (▪).

Figure 2.

Titration of monkey serum samples diluted from 1:25 in a twofold series against constant antigen concentration of 10 mg/L. Anti-peptide antiserum is indicated by ▴ with the corresponding preimmune serum, indicated by ▵, as negative control. Anti KLH-conjugated peptide antiserum is indicated by ▪, and the corresponding preimmune serum is indicated by □ as negative control. (A), M_S1 antiserum (▴) and M_S1_KLH antiserum (▪); (B), M_S2 antiserum (▴) and M_S2_KLH antiserum (▪); (C), M_S3 antiserum (▴) and M_S3_KLH antiserum (▪); (D), M_S4 antiserum (▴) and M_S4_KLH antiserum (▪); (E), M_S5 antiserum (▴) and M_S5_KLH antiserum (▪); (F), M_S6 antiserum (▴) and M_S6_KLH antiserum (▪); (G), M_MIX antiserum (▴) and M_MIX_KLH antiserum (▪).

Figure 3.

Positive results in immunofluorescent confocal microscopy are indicated by the presence of green fluorescent signals in the cytoplasm of the African green monkey kidney Vero cells. (A), negative control using rabbit R_S1 preimmune serum, observed under light microscope (A-1) and confocal microscope (A-2). Similar results were observed in all other rabbit preimmune sera. (B), negative control using monkey M_S1 preimmune serum, observed under light microscope (B-1) and confocal microscope (B-2). Similar results were observed in all other monkey preimmune sera. (C), negative control using SARS patient serum on noninfected Vero cells, observed by light microscope (C-1) and confocal microscope (C-2). (D), positive control using SARS patient serum. Positive results in confocal microscopy were shown for the following rabbit and monkey antiserum samples: R_S2_KLH antiserum (E); R_S5 antiserum (F); R_S5_KLH antiserum (G); R_S6_KLH antiserum (H); M_S3 antiserum (I); M_S6 antiserum (J); M_S6_KLH antiserum (K); M_MIX antiserum (L); and M_MIX_KLH antiserum (M).