Abstract
As an alternative approach toward the development of a cholera vaccine, the potential of peptide mimics of Vibrio cholerae lipopolysaccharide (LPS) to elicit cross-reactive immune responses against LPS was investigated. Two closely related protective monoclonal antibodies, S-20-4 and A-20-6, which are specific for Ogawa O-antigen (O-specific polysaccharide; O-SP) of V. cholerae O1, were used as the target antibodies (Abs) to pan phage display libraries under different elution conditions. Six phage clones identified from S-20-4 panning showed significant binding to both S-20-4 and A-20-6. Thus, it is likely that these phage-displayed peptides mimic an important conformational epitope of Ogawa antigens and are not simply functionally recognized by S-20-4. Each of the six phage clones that could bind to both monoclonal antibodies also competed with LPS for binding to S-20-4, suggesting that the peptides bind close to the paratope of the Ab. In order to predict how these peptide mimics interact with S-20-4 compared with its carbohydrate counterpart, one peptide mimic, 4P-8, which is one of the highest affinity binders and shares motifs with several other peptide mimics, was selected for further studies using computer modeling methods and site-directed mutagenesis. These studies suggest that 4P-8 is recognized as a hairpin structure that mimics some O-SP interactions with S-20-4 and also makes unique ligand interactions with S-20-4. In addition, 4P-8-KLH was able to elicit anti-LPS Abs in mice, but the immune response was not vibriocidal or protective. However, boosting with 4P-8-KLH after immunizing with LPS prolonged the LPS-reactive IgG and IgM Ab responses as well as vibriocidal titers and provided a much greater degree of protection than priming with LPS alone.
Highlights
These areas are difficult to achieve in most cholera-endemic areas
There are over 200 serogroups of V. cholerae that have been identified on the basis of O-SP, only O1 and O139 are known to cause major epidemics
Selection of S-20-4- and A-20-6-specific Phage Clones—X12, X7, and CX7C phage display libraries were biopanned against mAb S-20-4, which is specific for the protective epitope of Ogawa LPS [10], using low pH elution or nonelution
Summary
Antibodies—Murine ascites fluid containing S-20-4 and A-20-6 mAbs were gifts from Dr J. The bound phage were detected with horseradish peroxidase-conjugated anti-M13 Ab. Site-directed Mutagenesis of Gene III Peptide Motif Region— Each mutation was created by inserting DNA with the desired mutation between the EagI and BglII sites of the vector M13KE (New England Biolabs). The remaining four amino-terminal amino acids were idealized by requiring all-trans-peptide bond configurations This structural motif and alternative peptide conformations, arrived at by the threading of the full 12-mer sequence (NHNYPPLSLLTF) onto similar peptides observed in similar Ab-peptide complexes [37], provided a range of similar hairpin peptide configurations. 4 –5-Day-old CD-1 infant mice were orally administered, by gavage, 25 l of virulent V. cholerae O1 Ogawa strain O395 that contained 9 ϫ 107 bacteria or ϳ300 LD50, which had been cultured overnight in LB medium at 37 °C and mixed 1:1 with antisera from the ninth bleed or normal mouse serum. Challenged mice were kept at 30 °C and monitored after challenge until the termination of the assay
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
