Rapid and precise epitope mapping of monoclonal antibodies against Plasmodium falciparum AMA1 by combined phage display of fragments and random peptides

We describe an approach for the rapid mapping of epitopes within a malaria antigen using a combination of phage display techniques. Phage display of a ntigen fragments identifies the location of the epitopes, then random pepti de libraries displayed on phage are employed to identify accurately amino a cids involved in the epitope. Finally, phage display of mutant fragments co nfirms the role of each residue in the epitope. This approach was applied t o the apical membrane antigen-1 (AMA1), which is a leading candidate for in clusion in a vaccine directed against the asexual blood stages of Plasmodiu m falciparum. As part of the effort both to understand the function of AMA1 in the parasite life cycle and to define the specificity of protective imm une responses, a panel of monoclonal antibodies (MAbs) was generated to obt ain binding reagents to the various domains within the molecule. There is a pressing need to determine rapidly the regions recognized by these antibod ies and the structural requirements required within AMA1 for high affinity binding of the MAbs. Using phage displaying random AMA1 fragments, it was s hown that MAb5G8 recognizes a short linear epitope within the prodomain of AMA1 whereas the epitope recognized by MAb 1F9 is reduction sensitive and r esides within a disulphide-bonded 57 amino acid sub-domain of domain-1. Pha ge displaying random peptide libraries and mutant AMA1 fragments were emplo yed for fine mapping of the MAb5G8 core epitope to a three-residue sequence in the AMA1 prodomain.