Typhoid fever is a disease caused by Salmonella Typhi that was implicated in millions of illnesses worldwide annually. Individuals that do not recover fully from typhoid fever can become asymptomatic carriers of the disease. Host antibodies against the S. Typhi antigens, HlyE (for acute typhoid) and YncE (for carriers) were previously reported to be useful biomarkers for the disease. Here, we expressed and purified recombinant HlyE and YncE antigens and tested the IgG, IgA and IgM responses in 422 sera samples retrieved from acute typhoid patients, other febrile, food handlers, and healthy individuals. The results showed that HlyE-IgG, -IgA and -IgM ELISAs have a collective sensitivity of 83% while YncE-IgG and -IgA ELISAs identified 16 possible carriers based on their antibody profiles. The identification of sensitive biomarkers for typhoid carrier detection is crucial for disease eradication.
Antibody phage display is a useful tool for the isolation and identification of monoclonal antibodies. Naive antibody libraries are able to overcome the limitations associated with the traditional hybridoma method for monoclonal antibody generation. Antibody phage display is also a preferred method for antibody generation against toxins as it does not suffer from toxicity mediated complications. Here, we describe a naïve multi ethnic scFv antibody library generated via two-step cloning with an estimated diversity of 2 × 10(9). The antibody library was used to screen for monoclonal antibodies against Hemolysin E antigen, a pore forming toxin produced by Salmonella enterica serovar Typhi. A soluble monoclonal scFv antibody against the HlyE toxin (IgM scFv D7 anti-hlyE) was isolated from the library. This shows the value of the naïve library to generate antibodies against toxin targets in addition to the potential use of the library to isolate antibodies against other immunogenic targets.
The detection and measurement of different antibody isotypes in the serum provide valuable indicators of the different stages of typhoid infection. Here, the ability of S. Typhi recombinant hemolysin E (HlyE) to detect multi-isotype antibody responses in sera of patients with typhoid and paratyphoid A was investigated using an indirect antibody immunoassay. Nanogram amounts of HlyE were found to be sufficient for detection of IgG and IgA isotypes and, in a study of individuals' sera (n = 100), the immunoassay was able to distinguish between typhoid and non-typhoid sera. The overall sensitivity, specificity and efficiency of the ELISA were 70% (39/56), 100% (44/44) and 83% respectively.
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.