Moderately-neutralizing epitopes in non-functional regions dominate the antibody response to Plasmodium falciparum EBA-140.

Plasmodium falciparum Erythrocyte Binding Antigen-140 plays a role in tight junction formation during parasite invasion of red blood cells and is a potential vaccine candidate for malaria. Individuals in malaria-endemic areas possess Erythrocyte Binding Antigen-140-specific antibodies, and individuals with high antibody titers to this protein have a lower rate of re-infection by parasites. The red cell binding segment of Erythrocyte Binding Antigen-140 is comprised of two Duffy-binding-like domains called F1 and F2 that together create region II. The sialic acid-binding pocket of F1 is essential for binding whereas the sialic acid-binding pocket in F2 appears dispensable. Here, we show that immunization of mice with the complete region II results in poorly neutralizing antibodies. In contrast, immunization of mice with the functionally relevant F1 domain of region II results in antibodies that confer a two-fold increase in parasite neutralization compared to the F2 domain. Epitope mapping of diverse F1 and F2 monoclonal antibodies revealed that the functionally relevant F1 sialic acid-binding pocket is a privileged site inaccessible to antibodies, that the F2 sialic acid-binding pocket contains a non-neutralizing epitope, and that two additional epitopes reside in F1 on the opposite face from the sialic acid-binding pocket. These studies indicate that focusing the immune response to the functionally important F1 sialic acid binding pocket may improve the protective immune response of Erythrocyte Binding Antigen-140. These results have implications for improving future vaccine designs and emphasize the importance of structural vaccinology for malaria.