Phase I malaria vaccine trial with a long synthetic peptide derived from the merozoite surface protein 3 antigen

Abstract

The C-terminal conserved region of Plasmodium falciparum merozoite surface protein 3 (MSP3) is the trigger antigen of a protective immune response mediated by cytophilic antibodies. In an open, randomized, two-adjuvant (Montanide ISA 720, aluminum hydroxide) phase I clinical trial we evaluated the safety and immunogenicity of increasing doses of a long synthetic peptide construct spanning the conserved region of MSP3 targeted by biologically active antibodies (MSP3-LSP). Thirty-five healthy volunteers were randomized to receive three subcutaneous injections on days 0, 30, and 120. Of the 100 injections given, 10 caused severe local reactions, 62 caused transient mild to moderate local reactions, and 28 caused no reaction. On the basis of preestablished exclusion criteria, use of the Montanide formulation led to withdrawal of five volunteers after the second injection. This led to a reduction in the subsequent vaccine doses in four of the groups. No vaccine-related serious adverse events occurred throughout the trial. After the third injection, volunteers displayed a marked specific anti-MSP3-LSP antibody response (23/30 individuals, compared with 29/34 individuals for plasma from an area where malaria is endemic), an anti-native MSP3 antibody response (19/30 individuals), a T-cell-antigen-specific proliferative response (26/30 individuals), and gamma interferon production (25/30 individuals). In conclusion, the MSP3-LSP vaccine was immunogenic with both adjuvants, although it was unacceptably reactogenic when it was combined with Montanide. The potential usefulness of the candidate vaccine is supported by the induction of a strong cytophilic response (i.e., the type of anti-MSP3 antibodies involved in antibody-dependent, monocyte-mediated protective mechanisms in areas where malaria is endemic).

FIG. 1.

Time course of anti-MSP3-LSP antibody response after MSP3-LSP immunization. The results are expressed as box plots and whiskers that show the 5th, 25th, 50th, 75th, and 95th percentiles for individual ratio data (optical density for the test/mean optical density for the control +3 standard deviations; plasma dilution, 1/100) for each immunization group. The arrows indicate the times of immunization. The prevalence of responders (ratio, >1) is indicated above each box.

FIG. 2.

Anti-MSP3-LSP antibody isotypic responses. The results are expressed as described in the legend to Fig. 1. The responses were calculated for each isotype (IgG1 [G1], left y axis; IgG2 [G2], IgG3 [G3], IgG4 [G4], and IgM [M], right y axis) by determining the ratio of the optical density after the second injection to the preimmune optical density (open boxes) and the ratio of the optical density after the third injection to the preimmune optical density (shaded boxes) (plasma dilution, 1/100).

FIG. 3.

Anti-P. falciparum recognition by immunoblotting. Plasma samples from five representative volunteers were incubated with P. falciparum mature schizont extract (3D7 clone) on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. The lane labeled with a star contained a positive control obtained by immunizing mice with the recombinant MSP3 C-terminal protein in Montanide. Lanes 1 to 5 show representative results obtained using postimmunization samples from volunteers (month 5). They show various degrees of reactivity with the 48-kDa MSP3 protein of P. falciparum. Molecular weights (mw) (10³) are indicated on the right.

FIG. 4.

Time course of anti-MSP3-LSP proliferative T-cell response. The box plots show the 5th, 25th, 50th, 75th, and 95th percentiles for the distribution of stimulation indices (S.I.) for each immunization group. SI were calculated by determining the ratio of MSP3-LSP-stimulated PBMC cultures to unstimulated PBMC cultures in cpm. Each culture was performed with six replicates. The arrows indicate the times of immunization. The prevalence of responders (SI, >3) is indicated above each box.

FIG. 5.

Proliferative response to overlapping peptides MSP3-a, -b, -c, and -d. The box plots show the 5th, 25th, 50th, 75th, and 95th percentiles for the distribution of stimulation indices (S.I.) induced by the various peptides in each immunization group. SI were calculated by determining the ratio of peptide-stimulated PBMC to unstimulated PBMC in cpm after the second injection. Each culture was performed with six replicates. Stimulation with peptides MSP3-a, -b, -c, and -d was performed parallel to MSP3-LSP stimulation for comparison (Fig. 4).

FIG. 6.

Time course of IFN-γ production by PBMC in response to MSP3-LSP. PBMC were stimulated for 5 days with 30 μg/ml MSP3-LSP. The results are expressed as box plots and whiskers that show the 5th, 25th, 50th, 75th, and 95th percentiles for individual IFN-γ secretion in the supernatant, expressed in pg/ml for each immunization group. The arrows indicate the times of immunization.