Preclinical vaccine study of Plasmodium vivax circumsporozoite protein derived-synthetic polypeptides formulated in montanide ISA 720 and montanide ISA 51 adjuvants

Abstract

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate previously assessed in animals and humans. Here, combinations of three synthetic polypeptides corresponding to amino (N), central repeat (R), and carboxyl (C) regions of the CS protein formulated in Montanide ISA 720 or Montanide ISA 51 adjuvants were assessed for immunogenicity in rodents and primates. BALB/c mice and Aotus monkeys were divided into test and control groups and were immunized three times with doses of 50 and 100 μg of vaccine or placebo. Antigen-specific antimalarial antibodies were determined by enzyme-linked immunosorbent assay, immunofluorescent antibody test, and IFN-γ responses by enzyme-linked immunosorbent spot (ELIspot). Both vaccine formulations were highly immunogenic in both species. Mice developed better antibody responses against C and R polypeptides, whereas the N polypeptide was more immunogenic in monkeys. Anti-peptide antibodies remained detectable for several months and recognized native proteins on sporozoites. Differences between Montanide ISA 720 and Montanide ISA 51 formulations were not significant.

Figure 1.

Antibody response of sera from BALB/c mice vaccinated with a mixture of N, R, and C peptides of the Plasmodium vivax circumsporozite (CS) protein formulated in Montanide ISA 720 or Montanide ISA 51, respectively. Each line corresponds to sera of mice immunized with a priming dose of 100 μg of peptide N and C and two boosting doses 150 μg of the three peptide (N + R + C) at 3-week intervals. Higher titers were obtained in groups of mice immunized with C-long synthetic peptides (LSP) than in those immunized with N-LSP, independently on the adjuvant used. Control groups are not shown.

Figure 2.

Antibody response of sera from Aotus monkeys vaccinated with a mixture of N, R, and C peptides of the Plasmodium vivax circumsporozite (CS) protein formulated in Montanide ISA 720 or Montanide ISA 51, respectively. Groups of six monkeys were primed with 100 μg of each N and C peptide and two boosting doses of 300 μg of the mixed peptide (N + R + C) at 0, 2 and 4 months. Antibody titers were followed 6 and 8 months post immunization. Enzyme-linked immunosorbent assay (ELISA) titers < 1:100 were considered negative.

Figure 3.

Comparison of in vitro interferon-gamma (IFN-γ) production by Aotus peripheral blood mononuclear cells (PBMCs) measured by ex vivo enzyme-linked immunosorbent spot (ELIspot). Results show the mean values of six monkeys per group immunized with a blend of three PvCS synthetic polypeptides (N, R, and C) formulated in Montanide ISA 720 and Montanide ISA 51, respectively.