Fig. 1.
Immunization with AMA1-RON2L complex but not AMA1 alone protects mice against lethal PyYM challenge. (A) Five mice per group were immunized with AMA1, RON2L-KLH, or AMA1-RON2L complex and then challenged with 104 iRBCs i.v. Five mice immunized with buffer in Freund’s adjuvant were used as controls. Error bars indicate mean ± SEM. (B) Kaplan-Meir curve of the overall survival of animals in A. (C) ELISA titers of antibody response against AMA1 and RON2L from sera of mice from A. Sera were used at 1:8,000 and 1:2,000 dilutions for AMA1 and RON2L, respectively. Error bars indicate mean ± SEM at OD405. (D) In silico homology model of PyAMA1-PyRON2L complex based on PfAMA1-PfRON2 complex structure. Arrows indicate the two cysteines in the PyRON2L peptide. (E) Mutation of Cys1856 and 1868 to Ala abolishes RON2L binding to PyAMA1. (F) Mutating the two cysteines to alanines (c/a) in the RON2L peptide required for binding to AMA1 abrogates complex-driven protection in mice. Five mice per group were challenged with 105 iRBCs. Error bars indicate mean ± SEM. (G) Protection requires vaccination with the AMA1-RON2L complex, because immunizing animals with the two antigens in separate sites (AMA1 + RON2L) does not confer protection. Five mice per group for the control and AMA1-RON2L groups and four mice for the AMA1+RON2L group were challenged with 105 iRBCs. Error bars indicate mean ± SEM. (H) Passive transfer of IgG from mice immunized with the AMA1-RON2L complex controls parasitemia. In this experiment, 400 µg of IgG from either control (PBS-Freund’s adjuvant–immunized mice) or AMA1-RON2L–immunized mice were passively transferred on days −1, 0, and +1 and challenged on day 0 with 105 iRBCs using five mice per group. Error bars indicate mean ± SEM. (I) Kaplan–Meier curve of the overall survival of animals in H.