Chicken single-chain variable fragments against the SARS-CoV spike protein

Abstract

The major concern for severe acute respiratory syndrome (SARS), caused by the SARS-associated coronavirus (SARS-CoV), is the lack of diagnostic and therapeutic agents. Using a phage display technology in a chicken system, high-affinity monoclonal antibody fragments against the SARS-CoV spike protein were characterized. Ten truncated spike protein gene fragments were expressed in Escherichia coli cells. Following the immunization of chickens with these recombinant spike proteins, two single-chain variable fragment (scFv) antibody libraries were established with short or long linkers to contain 5x10(7) and 9x10(6) transformants, respectively. After four rounds of panning selection, the scFv antibodies of randomly chosen clones were demonstrated by Coomassie blue staining, and verified by western blot analysis. In a comparison of nucleotide sequences with the chicken germline gene, we found that all clones varied in the complementarity-determining regions, that two scFv antibodies reacted significantly with SARS-CoV-infected Vero cells, and that those two specific scFv antibodies recognized the same region of the spike protein spanning amino acid residues 750-1000. In conclusion, the results suggest that the chicken scFv phage display system can be a potential model for mass production of high-affinity antibodies against the SARS-CoV spike protein.

Fig. 1

Total cellular lysates seen using Coomassie blue dye (A), while single-chain variable fragment (scFv) antibodies were shown by the western blotting technique (B). The recombinant scFv.S (with short linker) and scFv.L (with long linker) antibodies were identified as 24 and 28 kDa bands, respectively. Total cellular lysates from XL-1 bacteria were loaded into lane L. Lane M denotes prestained protein markers.

Fig. 2

Binding activity of randomly selected clones analyzed by ELISA. Cellular lysates containing single-chain variable fragment (scFv) antibodies from various Ssc (A) and Lsc (B) library clones were examined for their binding to SARS-CoV-infected cell lysates using a commercially available kit. Negative control (N) is a cellular lysate lacking scFv expression. Bound scFv was detected using anti-chicken light-chain antibodies and was measured at 450 nm. The ELISA data were represented as mean of the duplicated wells ± S.E.M. using SigmaPlot Statistical Analysis software.

Fig. 3

Sequence analysis of selected single-chain variable fragment (scFv) genes. Light-chain (V_L) (A) and heavy-chain variable (V_H) (B) region gene sequences were compared with that of the chicken germline gene showing the similarity. Sequence gaps were introduced to maximize alignment as indicated by blank spaces. Framework region (FR) and complementarity-determining region (CDR) boundaries are indicated above each germline sequence.

Fig. 4

Specific binding of recombinant single-chain variable fragment (scFv) antibodies to SARS-CoV-infected Vero E6 cells. The method of detection was described in Section 2. Biochips precoated with SARS-CoV-infected cells were immunostained with convalescent serum (A), normal serum (B), and Ssc22 (C), Ssc35 (D), Lsc18 (E), and Lsc22 (F) scFv antibodies. Infected cells probed with bacterial cell lysate lacking scFv (G). Panels (H–N) show the morphology and distribution of SARS-CoV-infected cells under a light microscope before treatment.

Fig. 5

Epitopes mapped with specific single-chain variable fragment (scFv) antibodies using western blot analysis. Panel (A) shows 10 truncated S fragments on a Coomassie blue-stained polyacrylamide gel. Both Ssc35 (B) and Lsc18 (C) scFv antibodies but not cellular lysate (not shown) recognized a truncated S6 fragment spanning amino acid residues 750–1000.

Fig. 6

Competitive inhibition assay of two representative single-chain variable fragment (scFv) antibodies against the S6 fragment. The amount of bound Ssc35 and Ssc18 scFv in the presence of free S6 inhibitor was measured and expressed as a percentage of the binding of scFv in the absence of an inhibitor. B and B0 are the amounts of bound scFv in the presence and absence of the inhibitor, respectively.