[PMC free article] [PubMed] [Google Scholar] 19

[PMC free article] [PubMed] [Google Scholar] 19. 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. Apical membrane antigen BML-275 (Dorsomorphin) 1 (AMA1) is a leading malarial vaccine candidate, since many studies have demonstrated that recombinant AMA1 induces antibodies that protect against plasmodial infection in simian and rodent models of the human disease (6, 22). AMA1 appears to have an essential role in the erythrocyte invasion process, and anti-AMA1 monoclonal, polyclonal, and Fab fragment antibodies have been shown to inhibit this process in vitro (10, 23). AMA1 is a type I integral protein with eight intramolecular disulfide bonds in the ectodomain, which suggests a three-domain substructure (9). The constrained nature of AMA1 was found to be critical, since antibodies generated toward reduced and alkylated (R-A) AMA1 failed to inhibit invasion (2). This evidence strongly suggests that conformation stabilized by disulfide bonds is important for the generation of a protective immune response. The monoclonal antibody (MAb) 4G2dc1, which binds to correctly folded AMA1 but not to the R-A protein, is a useful reagent for monitoring correct disulfide bonding of recombinant AMA1 (12). In previous studies, 4G2dc1 was shown to react with recombinant AMA1 in 10 different isolates of from diverse geographical locations, and it consistently inhibited the invasion of merozoites into erythrocytes in vitro by 60 to 70% (12). 4G2dc1 also inhibited invasion by parasites in vitro by 44% at 1 mg/ml (13). The exact location of the 4G2dc1 conformational epitope is not known, but clearly it has significance with respect to the generation of protective antibodies that can inhibit merozoite invasion (14). In this study, we have used phage display technology to isolate peptide sequences that mimic the conformation of the 4G2dc1 epitope. Immunization of rabbits with the peptide BML-275 (Dorsomorphin) mimotopes induced high titers of peptide antibodies, which were reactive with native AMA1. Human antibodies were affinity purified from plasma samples from individuals living in regions of Papua New Guinea (PNG) where malaria is endemic, using immobilized peptide immunoadsorbents. Both rabbit and affinity-purified human antibodies inhibited the invasion of human erythrocytes BML-275 (Dorsomorphin) by merozoites in vitro. Our data support the use of Rabbit Polyclonal to SRY 4G2dc1 peptide mimics as in vitro correlates of protective immunity in future AMA1 vaccine trials. MATERIALS AND METHODS Phage library and selection. We constructed a linear peptide library of 20 random amino acids displayed as N-terminal fusions to protein III of filamentous phage M13, using the Fuse 5 vector (20). The library of >5 108 random peptides was screened for MAb 4G2dc1-binding peptides using microtiter plates as described previously (1), with the following modifications. 4G2dc1-coated wells were blocked with 5% BLOTTO (milk powder diluted in phosphate-buffered saline [PBS]), and the 20-mer phage peptide library was preincubated in 1% BLOTTO for 15 min to remove any milk-binding phage before being added to the 4G2dc1-coated wells. Phage was amplified and titrated, and DNA sequencing was performed using procedures similar to those described previously (1). Recombinant antigens. The ectodomain of AMA1 from the 3D7 strain of was expressed in with an N-terminal six-His tag to allow purification by Ni chelate chromatography using methods detailed elsewhere (10). The 3D7 strains of merozoite surface antigen 2 (MSP2) and MSP3 were expressed in and prepared using similar methods (25). The recombinant antigen designated Ag1505H, corresponding to 70% of the C terminus of the ring-stage parasite-infected erythrocyte surface antigen (RESA) polypeptide, was also BML-275 (Dorsomorphin) used as a control antigen (25). ELISAs. Phage enzyme-linked immunosorbent assays (ELISAs) were performed by coating a microtiter plate (Nunc Maxisorp) with 5 g of antibody/ml overnight at 4C and subsequently blocking it with 10% skim milk for 2 h. Phage dilutions (100 l) were prepared in PBS, transferred in duplicate to the coated blocked wells, and incubated for 1 h with shaking. The wells were washed BML-275 (Dorsomorphin) five times with PBS containing 0.05% Tween 20, and 100 l of anti-M13 antibody conjugated to horseradish peroxidase (HRP) (Pharmacia) at 1/5,000 dilution was added to each well. After a further 1-h incubation and washing as described above, bound phage was detected with parasites (3D7 line) were cultured in vitro, and invasion-inhibition experiments were performed as explained previously (17). Briefly, late trophozoite or.