Immunogenicity of well-characterized synthetic Plasmodium falciparum multiple antigen peptide conjugates

Abstract

Given the emerging difficulties with malaria drug resistance and vector control, as well as the persistent lack of an effective vaccine, new malaria vaccine development strategies are needed. We used a novel methodology to synthesize and fully characterize multiple antigen peptide (MAP) conjugates containing protective epitopes from Plasmodium falciparum and evaluated their immunogenicity in four different strains of mice. A di-epitope MAP (T3-T1) containing two T-cell epitopes of liver stage antigen-1 (LSA-1), a di-epitope MAP containing T-cell epitopes from LSA-1 and from merozoite surface protein-1, and a tri-epitope MAP (T3-CS-T1) containing T3-T1 and a potent B-cell epitope from the circumsporozoite protein central repeat region were tested in this study. Mice of all four strains produced peptide-specific antibodies; however, the magnitude of the humoral response indicated strong genetic restriction between the different strains of mice. Anti-MAP antibodies recognized stage-specific proteins on the malaria parasites in an immunofluorescence assay. In addition, serum from hybrid BALB/cJ x A/J CAF1 mice that had been immunized with the tri-epitope MAP T3-CS-T1 successfully inhibited the malaria sporozoite invasion of hepatoma cells in vitro. Spleen cells from immunized mice also showed a genetically restricted cellular immune response when stimulated with the immunogen in vitro. This study indicates that well-characterized MAPs combining solid-phase synthesis and conjugation chemistries are potent immunogens and that this approach can be utilized for the development of subunit vaccines.

FIG. 1

MAP conjugates T3-T1 (A), T3-CS-T1 (B), and T3-MSP1 (C). MAPs are synthesized as a base pair of peptides on the lysine-cysteine core and are conjugated with a second pair of peptides on the alpha sulfur.

FIG. 2

Antibody titers to each MAP in mice of different genetic backgrounds. Antibody levels in serum were determined by ELISA. Titers were determined based on the highest dilution of the sample that generated an optical density greater than twice that seen in the adjuvant control group. The T3-CS-T1 (A), T3-T1 (B), and T3-MSP1 (C) vaccination groups are shown.

FIG. 3

Antibody isotype analysis. Sera obtained at 4 weeks after the third immunization were tested for IgG1 by ELISA for each MAP. The T3-CS-T1 (A), T3-T1 (B), and T3-MSP1 (C) vaccination groups are shown.

FIG. 4

Analysis of T-cell proliferation induced by MAP constructs. Mice of different genetic backgrounds (six to eight per group) were immunized with the T3-CS-T1 (A), T3-T1 (B), or T3-MSP1 (C) MAP. Control groups of mice received adjuvant alone. At the end of the immunization protocol spleen cells were restimulated in vitro with the respective MAP at a concentration of 5 μg/ml. The results are shown as means ± standard errors of the means. ∗, significant difference (Student's t test; P < 0.05) compared to adjuvant control group.