In earlier works we have described that mice immunized with outer
membrane protein OmpC survive the challenge with live Shigella flexnerii 3a. We have also identified conformational
epitope of this
protein, that was recognized by mice
antibodies. The aim of current work was to investigate whether synthetic OmpC
epitope homologs can elicit immunological response sufficient in protecting mice against
shigellosis. Several linear
peptides containing RYDERY motif were synthesized and conjugated to poly-
lysine. These conjugates appeared to be poor immunogens and to boost the immunological response an addition of the adjuvant (MPL) was required. Unfortunately, the MPL alone caused a very high immunological reaction that was masking response to peptidic
epitope. Under those circumstances we used
tetanus toxoid (TT) as the
carrier protein for the
peptides and the agent stimulating immunological response. Series of
cyclic peptides, homologs of the OmpC main
epitope were synthesized and conjugated to TT. The loop size in
cyclic peptides varied by number of
glycine residues, i.e., 1-3 residues added to the GLNRYDERYIGK motif. The linear GLNRYDERYIGC-TT was also prepared as the control. The latter conjugate gave the highest immunological response, followed by the cyclic-GGLNRYDERYIGC-TT and cyclic-GLNRYDERYIGC-TT. The third
peptide, cyclic-GGGLNRYDERYIGC-TT, gave a very low response, although it was the most resistant to proteolysis. However,
antibodies obtained against cyclic-GGLNRYDERYIGC-TT were more potent to recognize both OmpC and Shigella flexnerii 3a cells than the
antibodies against linear GLNRYDERYIGC-TT. Furthermore, the
monoclonal antibodies raised against linear GLNRYDERYIGC-TT showed 20-fold lower dissociation constant (KD) than the naturally occurring polyclonal
antibodies from umbilical cord sera.
Monoclonal antibodies also gave a weaker signal in electron microscope than mice and human polyclonal
antibodies. In overall, our results point to
cyclic peptides as better candidates for a
vaccine development, since they are eliciting production of the higher affinity
antibodies against Shigella cells and OmpC.