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.

Single chain antibody fragment with serine protease inhibitory property capable of neutralizing toxicity of Trimeresurus mucrosquamatus venom

“…The hyper mutation in CDRs implied antibodies with high affinity could be generated through an antigen-driven humoral response after the immunization [19]. Similar patterns of somatic mutation were also observed in our previous studies with genetic analysis of scFv antibodies [17,18]. Notably, the light chain used by clones 3S10, 4S9 and 4S15 are identical, suggesting that this particular light chain may play an important role on structure stability and the protective effect of these scFv antibodies against RBC hemolysis and lethal in vivo venom challenge.…”

“…However, the cost for the production of such therapeutics is high and alternative choice for cost-effective production is needed. As comparable to the horses, chickens have been demonstrated suitable for eliciting strong humoral response in our previous studies [17,18]. In this study, our data indicated that bivalent anti-TM/TS IgY antibodies were used for neutralization test, they provided an efficient protection comparable to that offered by bivalent anti-TM/TS IgG antibodies (Fig.…”

IgY-Technology Applied to Studies of Toxoplasma gondii Infection

“…Libraries constructed, using mRNA from an immune source, are enriched in affinity-matured sequences. Repertoires focused on a number of defined targets can be constructed using lymphocyte mRNA from chickens immunized with a mixture of several different antigens from Plasmodium falciparum, Trypanosoma sp., or human viruses [47,48].…”

Chicken Monoclonal IgY Antibody: A Novel Antibody Development Strategy

“…This unique Abs will be a useful tool for research with regard to dyslipidemia, atherosclerosis, and ischemic heart diseases. Lee et al (2007) generated an antibody against the S protein of severe acute respiratory syndrome associated coronavirus (SARS-CoV) in chickens with phage display technology, it was found that one scFV (4S1) showed the greatest binding ability in both phage ELISA and Immunocytochemical staining assays when 10 10 -10 11 phage particles were used. It indicates that this 4S1 scFV phage is a suitable candidate for developing a phage-based diagnostic assay for the detection of SARS-CoV.…”