Antibodies to malaria peptide mimics inhibit Plasmodium falciparum invasion of erythrocytes

Abstract

Apical membrane antigen 1 (AMA1) is expressed on the surfaces of Plasmodium falciparum merozoites and is thought to play an important role in the invasion of erythrocytes by malaria parasites. To select for peptides that mimic conformational B-cell epitopes on AMA1, we screened a phage display library of >10(8) individual peptides for peptides bound by a monoclonal anti-AMA1 antibody, 4G2dc1, known to inhibit P. falciparum invasion of erythrocytes. The most reactive peptides, J1, J3, and J7, elicited antibody responses in rabbits that recognized the peptide immunogen and both recombinant and parasite AMA1. Human antibodies in plasma samples from individuals exposed to chronic malaria reacted with J1 and J7 peptides and were isolated using immobilized peptide immunoadsorbents. Both rabbit and human antibodies specific for J1 and J7 peptides were able to inhibit the invasion of erythrocytes by P. falciparum merozoites. This is the first example of phage-derived peptides that mimic an important epitope of a blood-stage malaria vaccine candidate, inducing and isolating functional protective antibodies. Our data support the use of J1 and J7 peptide mimics as in vitro correlates of protective immunity in future AMA1 vaccine trials.

FIG. 1.

Selection and characteristics of phage clones binding to MAb 4G2dc1. (A) Reactivities of selected phages from each round (R) of panning on 4G2dc1 detected by ELISA. (B) Binding of eight selected clones to 4G2dcl and human anti-AMA1 antibodies (Ab) affinity purified from a pool of plasma samples from PNG by ELISA. (C) Sequences of eight selected clones from rounds (R) 4 to 6. Amino acids that were identical in two sequences are in light gray, and those identical in three or more sequences are underlined. (D) Aligned amino acid sequences of J1, J3, and J7 selected phage peptides; the shaded areas indicate identical amino acids. (E) Selected phage clones bound to 4G2dc1, as shown by ELISA, but not to an antibody of the same isotype. Recombinant AMA1 (10 μg/ml) competed with the phage clones for binding to 4G2dc1. (F) Selected phage clones bound to human anti-AMA1 antibodies affinity purified from a pool of plasma samples from PNG but not to purified human IgG from individuals not exposed to malaria. Error bars indicate the ranges of individual values.

FIG. 2.

Specificities of anti-J1 and anti-J7 rabbit antibodies affinity purified on AMA1. (A) ELISA showing antibodies binding to soluble peptide on microtiter plates. (B) Immunoblots showing specific reactivity with recombinant AMA1. (C) ELISA showing that antibodies bind to bacterially expressed recombinant AMA1 from four P. falciparum lines. Error bars indicate the ranges of individual values.

FIG. 3.

Human antibody titer to AMA1 correlates with the titer to J1 and J7 peptides. Plasma samples (P1 to P 24) from individuals living in an area of PNG where malaria is endemic were assayed by ELISA at a dilution of 1:10,000 on AMA1 and 1:100 on J1 (A) and J7 (B). The correlation coefficient was used to compare data sets. Error bars indicate the ranges of individual values.

FIG. 4.

Colocalization of MAb 4G2dc1 and antipeptide antibodies. Immunofluorescence analysis of parasite smears with rabbit anti-J1 (A), rabbit anti-J7 (B), human anti-J1 (C), and human anti-J7 (D) purified antibodies probed with FITC (left), 4G2dc1 probed with anti-rat rhodamine (middle), and merged images (right). Scale bars, 5 μm.

FIG. 5.

Specificities of J1 and J7 affinity-purified human antibodies. ELISAs show human plasma antibodies binding to antigens before (A) and after (B) purification on immobilized J1. AMA1 and J1 (C) or J7 (D) peptide at 10 and 100 μg/ml competed with J1 (C) or J7 (D) affinity-purified human antibodies for binding to AMA1. J1 antibodies (E) and 4G2dc1 (F) bound to bacterially expressed recombinant AMA1 from four P. falciparum lines. Error bars indicate the ranges of individual values.