Phase 1 clinical trial of apical membrane antigen 1: an asexual blood-stage vaccine for Plasmodium falciparum malaria

Abstract

Apical membrane antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. A phase 1 trial was conducted with 30 malaria-naive volunteers to assess the safety and immunogenicity of the AMA1-C1 malaria vaccine. AMA1-C1 contains an equal mixture of recombinant proteins based on sequences from the FVO and 3D7 clones of Plasmodium falciparum. The proteins were expressed in Pichia pastoris and adsorbed on Alhydrogel. Ten volunteers in each of three dose groups (5 mug, 20 mug, and 80 mug) were vaccinated in an open-label study at 0, 28, and 180 days. The vaccine was well tolerated, with pain at the injection site being the most commonly observed reaction. Anti-AMA1 immunoglobulin G (IgG) was detected by enzyme-linked immunosorbent assay (ELISA) in 15/28 (54%) volunteers after the second immunization and in 23/25 (92%) after the third immunization, with equal reactivity to both AMA1-FVO and AMA1-3D7 vaccine components. A significant dose-response relationship between antigen dose and antibody response by ELISA was observed, and the antibodies were predominantly of the IgG1 isotype. Confocal microscopic evaluation of sera from vaccinated volunteers demonstrated reactivity with P. falciparum schizonts in a pattern similar to native parasite AMA1. Antigen-specific in vitro inhibition of both FVO and 3D7 parasites was achieved with IgG purified from sera of vaccinees, demonstrating biological activity of the antibodies. To our knowledge, this is the first AMA1 vaccine candidate to elicit functional immune responses in malaria-naive humans, and our results support the further development of this vaccine.

Figures

FIG. 1.

Local reactogenicity in recipients of the AMA1-C1 vaccine. The percentages of volunteers who experienced injection site erythema (A), induration (B), or tenderness (C) after the first (black bar), second (gray bar), and third (white bar) immunizations with 5 μg, 20 μg, or 80 μg of AMA1-C1 are presented.

FIG. 2.

Immunological responses to AMA1 allelic variants in recipients of the AMA1-C1 vaccine. Antibody units against AMA1-3D7 (A) and AMA1-FVO (B) measured by ELISA assays in sera collected on day 0 (day of vaccination 1; *), day 42 (14 days postvaccination 2; white bars), day 180 (day of vaccination 3; gray bars), and day 194 (14 days postvaccination 3; black bars). Samples with ELISA unit values below the limit of detection were assigned values of zero. * indicates that the mean for the cohort was zero. The bars represent the arithmetic mean antibody units against AMA1-3D7 (A) and AMA1-FVO (B) for the dose cohort: 5 μg/vaccination (n = 10), 20 μg/vaccination (n = 9), and 80 μg/vaccination (n = 7). The circles represent the individual unit values of responders.

FIG. 3.

AMA1-C1 vaccine induces antibodies that recognize the native AMA1 protein on parasites. Representative confocal micrographs illustrating indirect immunofluorescent antibody detection and localization on P. falciparum FVO parasites with anti-AMA1 monoclonal antibody 1C8. Sera collected on days 0 (A to D) and 194 (E to H) were from an individual with anti-AMA1-FVO ELISA titers of 2,816 U on day 194. The sera were tested at a dilution of 1:400, and anti-AMA1 monoclononal antibody 1C8 was diluted 1:1,600. Shown are green fluorescence, representing the vaccinee's antibody against AMA1 (A, E); the corresponding red fluorescence, representing the mouse monoclonal anti-AMA1 antibody (B, F); and the merged images (C, G) and bright-field micrographs (D, H) of the test sera. A 10-μm scale bar is included in panel E.

FIG. 4.

Anti-AMA1-3D7 and -FVO ELISA values correlate with growth inhibition by immune sera on day 194. Total IgG was purified from each serum collected on day 194 (2 weeks postvaccination) as described in Materials and Methods. Antibody levels against AMA1-3D7 (A) and AMA1-FVO (B) in purified IgG were plotted against the corresponding in vitro growth inhibition. Samples were tested at two different final concentrations of total IgG, 5 μg/ml (open symbols) and 10 μg/ml (black symbols). A hyperbolic function was used to derive the best-fit curve shown for each antigen.