| Summary: | The aim of this project was to characterise the humoral immune responses to RhD-derived synthetic peptides as a first step towards developing alternative peptide immunogens to replace RBC in immunising donors for anti-D IgG antibody production for clinical use. In the first part, 4 peptides (Dp6, Dp13, Dp17, and Dp28) containing Th-cell epitopes on the RhD protein were used to stimulate PBMC from 2 D-alloimmunised donors in vitro. A mixture of the four peptides was also utilised to boost HLA-DR15 transgenic mice previously immunised with purified RhD protein. The ability of the four peptides to boost anti-D titre significantly varied between the two donors, with a maximum 3-fold increase in anti-D titre induced by Dp17 in donor 2. The mouse antibody response to peptide boosting was also variable, with 4 of the 6 (66.7%) peptide-boosted DR15 mice demonstrating positive changes in anti-RhD antibody levels. The findings from the in vitro and mice studies demonstrated that these four peptides had the potential to boost anti-D titres in D-alloimmunised donors. In the second part of this study, an alternative approach to designing a totally synthetic peptide immunogen was investigated. An RhD mimotope (DM4) was selected from a set of previously identified RhD mimotopes, and its antigenic mimicry for the D antigen was verified. The DM4 peptide was coupled with the Dp6 peptide as a Th-cell epitope, to construct a chimeric T-B peptide immunogen. The immunogenicity of the T-B and DM4 peptides were evaluated in DR15-transgenic mice. Both peptides were able to induce significant DM4-specific IgG antibodies. The immunogenicity of the DM4 was significantly augmented by colinear coupling to the Dp6 peptide. The immunogenic mimicry of the DM4 peptide for the D antigen was examined through the ability of mouse anti-DM4 antibodies to recognise human D antigen on D-positive RBC. However, these antibodies appeared to recognise D antigen non-specifically, reacting with all tested human RBC regardless of their D phenotypes.
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