A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage

Abstract

Severe acute respiratory syndrome (SARS) is a newly emergent human disease, which requires rapid diagnosis and effective therapy. Among antibody sources, immunoglobulin Y (IgY) is the major antibody found in chicken eggs and can be used as an alternative to mammalian antibodies normally used in research and immunotherapy. In this study, phage-expressing chicken monoclonal scFv antibody was chosen and characterized with phage display antibody technology. Truncated fragments of SARS-CoV spike protein were cloned in pET-21 vector and expressed in BL-21 Escherichia coli (E. coli) cells. After purification, the purity of these recombinant spike proteins was examined on SDS-PAGE and their identity verified with Western blot analysis using anti-his antibodies and sera from convalescent stage SARS-CoV-infected patients. Using these bacteria-derived proteins to immunize chickens, it was found that polyclonal IgY antibodies in the egg yolk and sera were highly reactive to the immunogens, as shown by Western blot and immunocytochemical staining analysis. A phage displaying scFv library was also established from spleen B cells of immunized chicken with 5 x 10(7) clones. After four panning cycles, the eluted phage titer showed a 10-fold increase. In sequence analysis with chicken germline gene, five phage clones reacted, with large dissimilarities of between 31 and 62%, in the complementarity-determining regions, one dominant phage 4S1 had strong binding to fragment Se-e, located between amino acid residues 456-650 of the spike protein and this particular phage had significantly strong binding to SARS-CoV-infected Vero E6 cells. Based on the results, we conclude that generating specific scFv-expressing phage binders with the phage display system can be successfully achieved and that this knowledge can be applied in clinical or academic research.

Fig. 1

(A) Schematic drawing of cloning strategy for 10 truncated S genes. (B) Amino acid residual locations and predicted molecular weights of these truncated proteins expressed in E. coli cells.

Fig. 2

Coomassie blue staining and Western blotting of various his-fused S fragments. Ten recombinant his-fused S fragments (Sb-b to Sk-i) were purified from E. coli cellular lysates using Ni²⁺-charged sepharose and visualized on SDS–PAGE (A). The molecular weights of the recombinant S fragments were 12–29 kDa, as expected. The proteins were transferred onto nitrocellular paper and analyzed by anti-his antibodies conjugated with horseradish peroxidase (B).

Fig. 3

Western blotting of truncated S fragments with patients’ sera. Sera of one patient who recovered from SARS-CoV infection (A) and an un-infected individual (B) were tested for the presence of specific anti-SARS-CoV antibodies. Secondary antibodies were anti-human Ig antibodies conjugated with horseradish peroxidase.

Fig. 4

Binding activity of randomly chosen phages from library 4S after the fourth panning cycle. Fifteen phage clones (4S1 to 4S15) were amplified and prepared to test their binding to SARS-CoV-infected Vero E6 cell lysates in an ELISA test. Wild-type M13 phage and one irrelevant phage expressing anti-Eno1 scFv were used as negative controls. Binding phages were detected by anti-M13 phage antibodies conjugated with horseradish peroxidase. The color was developed and absorbance was measured at 450–650 nm.

Fig. 5

Sequence analysis of scFv genes of five potentially positive clones. The amino acid sequences of light and heavy chain variable regions (V_L and V_H) of scFv fragments were compared to those of the chicken germline gene. Sequence gaps were introduced to maximize alignment and indicated by blank spaces. FR and CDR boundaries were indicated above germline sequence.

Fig. 6

Binding activity of 4S1 phage binder to native S protein. Commercially available Biochips, precoated with SARS-CoV-infected Vero E6 cells were immunostained, respectively, with sera from a convalescent-stage patient (A), un-infected person (B), post- (C) and pre-immunized (D) chickens, 4S1 phage (E) and wild-type M13 phage (F).

Fig. 7

Epitope mapped by 4S1 phage binder. 4S1 phage (A) and an anti-ENO1 phage (B) were examined for their binding activity to 10 purified and truncated S protein fragments coated on ELISA plates.