ANALYSIS OF INHIBITORY EPITOPES IN THE PLASMODIUM-FALCIPARUM RHOPTRY PROTEIN RAP-1 INCLUDING IDENTIFICATION OF A 2ND INHIBITORY EPITOPE

Immune responses to Plasmodium falciparum rhoptry-associated protein I (RAP-1), RAP-2, and RAP-3 appear to contribute to protection against infection by this human malarial parasite, This conclusion is suggeste d by results of monkey immunization trials and of cell culture studies showing antibody dependent inhibition of erythrocyte invasion, In the present study, splenectomized owl monkeys were infected with P. falci parum in order to monitor anti-RAP-l antibody production as antiparasi te immunity developed, The monkeys responded to a primary infection wi th the production of antibodies to a fragment of RAP-l containing amin o acids 1 to 294 (RAP-1(1-294)), After drug cure and reinfection, the monkeys had a prolonged prepatent period, indicating they had already developed partial immunity to the parasite, Sera from these animals sh owed major increases in anti-RAP-1(1-294) antibodies, In contrast, onl y low levels of antibodies to inhibitory B-cell epitope 1 (iB-1), an i nhibitory epitope in RAP-1(1-294) with the sequence N200TLTPLEELYPT211 , was observed after the initial parasite infection, and the anti-iB-1 antibodies were not readily boosted upon reinfection, These results s uggest that iB-1 is an immunogenic but not immunodominant epitope and that anti-iB-1 antibodies do not substantially contribute to early sta ges of naturally acquired immunity in the owl monkey model. To identif y additional epitopes bound by inhibitory antibodies, mouse monoclonal antibodies were produced with a recombinant fusion protein containing RAP-1(1-294), Monoclonal antibody 1D6 inhibited parasite invasion of erythrocytes in vitro, 1D6 did not bind peptide iB-1 but rather bound a second inhibitory epitope called iB-2. iB-2, like iB-1, is found nea r the amino terminus of p67, a RAP-1 processing product thought to be involved in merozoite invasion of erythrocytes, Since anti-iB-1 antibo dies were not readily produced during parasite infection, it may be de sirable to direct antibody responses to particular epitopes in RAP-1, such as iB-1 and iB-2.