EPITOPE MAPPING OF MONOCLONAL-ANTIBODIES DIRECTED AGAINST LIPOPHOSPHOGLYCAN OF LEISHMANIA-MAJOR PROMASTIGOTES

Monoclonal antibodies (MAbs) were generated against Leishmania major p romastigote lipophosphoglycan (LPG) to use as tools in defining functi onal epitopes of this major cell surface glycoconjugate. Epitope mappi ng of four MAbs, designated 4A2-A2, 2G11-A3, 5E6-D10 and 5E10-F2, reve aled that the phosphorylated oligosaccharide repeat unit PO4-6[Gal(bet a 1-3)]Gal(beta 1-4)Man alpha 1-, P3, is a highly immunogenic epitope which has previously been demonstrated, by chemical analyses, to be a repeat unit specific to L. major. Two antibodies, 4A2-A2 and 5E10-F2, also recognised the repeat unit PO4-6[Ara(beta 1-2)Gal(beta 1-3)]Gal(b eta 1-4)Man alpha 1-, P4a, with less affinity than P3, while 2G11-A3 r ecognised P4a with greater affinity than for P3. The L. major metacycl ic-specific antibody 3F12 only recognised repeat units terminating wit h arabinose residues. In particular, 3F12 recognised P4a, which is upr egulated in metacyclic LPG compared to the procyclic form of the molec ule. The oligosaccharides P3, P4a and P5a are specific to L. major LPG . The epitopes of 4A2-A2, 2G11-A3, 5E6-D10 and 5E10-F2 were found on t he cell surface and in the flagellar pocket of both procyclic and meta cyclic V121 promastigotes, but were only detected at very low levels o n amastigotes. The repeat unit P3 is able to inhibit attachment of pro cyclic promastigotes to the midgut of the sandfly vector, but neither Fab fragments of the four antibodies nor purified P3 could inhibit att achment of metacyclic promastigotes to the macrophage cell line J774. It was also shown that human sera from patients with cutaneous leishma niasis recognised purified P3. The data suggests that while P3 is an i mmunogen in the natural course of infection of the human host, P3 play s no role in attachment and internalisation of promastigotes into the macrophages of the mammalian host.