| Capsular polysaccharide (CPS) of Streptococcus group B (GBS) is a poor immunogen and functions as T cell independent antigen, eliciting low IgG antibody with deficient immunologic memory. We previously identified a peptide, S9, which mimics CPS of type III GBS. Here we have taken steps to develop the mimetic peptide as a vaccine against GBS group III. We enhanced the immunogenicity of the peptide by presenting it on the coat protein of Cowpea Chlorotic Mottle Virus (CCMV). And we searched for better mimetic peptides by constructing a secondary phage display library. To accomplish the first goal, DNA encoding S9 was cloned into five constructions CCMV coat protein loops using recombinant DNA techniques. The results indicated that inserting the S9 peptide sequence into CCMV coat protein loops disrupted virus and virus-like particle assembly. Therefore the S9 peptide was conjugated to CCMV coat protein using chemical linkers. The CCMV-S9 conjugation products remained intact as monomer virions. Mice were immunized with the CCMV-S9, SubE-S9 (a mutant that does not assemble virions), CPMV-S9 (S9 conjugated to Cowpea Mosaic Virus) and S9 conjugated to the carrier protein KLH, with and without Freund's adjuvant. The CCMV-S9, CPMV-S9 and KLH-S9 induce anti-S9 antibody even without the adjuvant whereas SubE-S9 induced an anti-S9 response only with adjuvant. The CCMV-S9 and CPMV-S9 predominantely induced a Th1 response with antigen-specific IgG2a and IFN-gamma production, whereas the KLH-S9 predominantly induced a Th2 response with antigen-specific IgG1 and IL4/IL10 production. To accomplish the second goal, a DNA sublibrary was designed to have approximately one mutation in each displayed peptide sequence. Peptides with higher affinity to the S9 mAb were identified by affinity selection. ELISA analysis from randomly selected phage clones indicated that amino acid residues 3-5 and 7-10 of S9 peptide are important in specific binding activity to S9 monoclonal antibody. These studies identified peptides with greater affinity for the selecting antibody, i.e. enhanced antigenicity. |
