Abstract
Hemolysin E (HlyE) is an immunogenic novel pore-forming toxin involved in the pathogenesis of typhoid fever. Thus, mapping of B-cell epitopes of Salmonella enterica serovar Typhi (S. Typhi) is critical to identify key immunogenic regions of HlyE. A random 20-mer peptide library was used for biopanning with enriched anti-HlyE polyclonal antibodies from typhoid patient sera. Bioinformatic tools were used to refine, analyze and map the enriched peptide sequences against the protein to identify the epitopes. The analysis identified both linear and conformational epitopes on the HlyE protein. The predicted linear GAAAGIVAG and conformational epitope PYSQESVLSADSQNQK were further validated against the pooled sera. The identified epitopes were then used to isolate epitope specific monoclonal antibodies by antibody phage display. Monoclonal scFv antibodies were enriched for both linear and conformational epitopes. Molecular docking was performed to elucidate the antigen-antibody interaction of the monoclonal antibodies against the epitopes on the HlyE monomer and oligomer structure. An in-depth view of the mechanistic and positional characteristics of the antibodies and epitope for HlyE was successfully accomplished by a combination of phage display and bioinformatic analysis. The predicted function and structure of the antibodies highlights the possibility of utilizing the antibodies as neutralizing agents for typhoid fever.
Highlights
To date, there is no crystal structure available for Salmonella enterica serovar Typhi
Immunoblotting was performed to evaluate the antigenicity of the rHlyE against pooled acute human typhoid (Pty) sera with pooled human healty (Phty) as control
There is no report on the antigenic regions or epitopes of the Hemolysin E (HlyE) to date but previous reports suggested there are both linear and conformational epitopes present in S
Summary
There is no crystal structure available for Salmonella enterica serovar Typhi To analyse a diverse collection of sequences, bioinformatic tools such as Pepitope and EpiSearch web servers are used to map the peptides on 3D antigenic protein structures to clarify specific epitope regions[10, 11]. Such an approach was applied successfully to identify the epitope sequences against RNAIII-activating protein (RAP) of Staphylococcus aureus through biopanning with polyclonal anti-TRAP antibodies using a random peptide library[12].
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