Plasmodium vivax promiscuous T-helper epitopes defined and evaluated as linear peptide chimera immunogens

Abstract

Clinical trials of malaria vaccines have confirmed that parasite-derived T-cell epitopes are required to elicit consistent and long-lasting immune responses. We report here the identification and functional characterization of six T-cell epitopes that are present in the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) and bind promiscuously to four different HLA-DRB1* alleles. Each of these peptides induced lymphoproliferative responses in cells from individuals with previous P. vivax infections. Furthermore, linear-peptide chimeras containing the promiscuous PvMSP-1 T-cell epitopes, synthesized in tandem with the Plasmodium falciparum immunodominant circumsporozoite protein (CSP) B-cell epitope, induced high specific antibody titers, cytokine production, long-lasting immune responses, and immunoglobulin G isotype class switching in BALB/c mice. A linear-peptide chimera containing an allele-restricted P. falciparum T-cell epitope with the CSP B-cell epitope was not effective. Two out of the six promiscuous T-cell epitopes exhibiting the highest anti-peptide response also contain B-cell epitopes. Antisera generated against these B-cell epitopes recognize P. vivax merozoites in immunofluorescence assays. Importantly, the anti-peptide antibodies generated to the CSP B-cell epitope inhibited the invasion of P. falciparum sporozoites into human hepatocytes. These data and the simplicity of design of the chimeric constructs highlight the potential of multimeric, multistage, and multispecies linear-peptide chimeras containing parasite promiscuous T-cell epitopes for malaria vaccine development.

FIG. 1.

Relative HLA class II binding profile of synthetic peptides spanning the complete sequence of PvMSP-1. Purified DRB1*0401, DRB1*0301, DRB1*1101, and DRB1*0101 and biotinylated control peptides were used in binding competition assays. The results, depicted as bars, are presented as the percentage of inhibition of binding of the biotinylated indicator peptides obtained with individual PvMSP-1 peptides. Only the positive reactivity, considered to be when 50 μM individual peptides inhibited more than 50% of the binding, are shown; 75% inhibitions are represented by the dotted lines. Relative amino acid positions (RAP) are based on the PvMSP-1 sequence of the Belem isolate of P. vivax (9).

FIG. 2.

(A) Schematic representation of PvMSP-1 and localization of the promiscuous T-cell epitopes described here. The amino acid sequence identity between the Belem and SalI sequences are adapted from the report by Gibson et al. (22). Peptide amino acid sequences are showed with code numbers (Fig. 1) and their relative amino acid positions based on the PvMSP-1 sequence of the Belem isolate (9). Dimorphic and polymorphic residues are shown as boldface letters. The open boxes represent areas of <50% identity, the solid boxes represent regions with identities ranging between 50 and 90%, and the shaded boxes represent conserved regions with >90% identity. (B and C) Peptide-specific lymphoproliferative responses of terminal T-cell lines derived from 33 individuals with antecedents of natural contact (C1 to C33) with P. vivax and living in an area of low endemicity for malaria (B) and 30 healthy individuals (N1 to N30) living in an area where malaria is not endemic (C). The peptides are identified by their original positions in the linear PvMSP-1 sequence (Fig. 1). Each block represents the mean response (SI) in triplicate for an individual donor. Open boxes, <2.0; shaded boxes, 2.0 to 3.9; hatched boxes, 4.0 to 7.9; and solid boxes, >8.0. Significant responses were considered positive when the SI was higher than 2. %R is the percentage of individuals with positive reactivity.

FIG. 3.

Schematic representation of the Cys-T-B-Cys topology used to synthesize LPCs. The hatched bar represents the N-terminal position of the T-cell epitopes. The sequences for individual P. vivax epitopes described here (PvT4, PvT6, PvT8, PvT19, PvT45, and PvT53) and the sequence of the P. falciparum T1 allele-restricted T-cell epitope previously described (PfT1) (48) are shown. These peptides were synthesized in tandem with (NANP)₃, the minimal B-cell epitope from the repeat region of P. falciparum CSP, and flanked by cysteine residues. The sequences of the promiscuous T-cell epitope identified here are presented and coded with their original position numbers (Fig. 1).

FIG. 4.

(A) Antigenicities of the LPCs determined with anti-CSP MAbs. ELISA plates were coated with 1 μg of the individual LPCs/ml and assayed with several dilutions of P. falciparum (2A10) anti-repeat MAbs. The data are presented as ODs obtained at different concentrations of the MAb 2A10 determined by ELISA. (B) Kinetics of the BALB/c mouse antibody responses generated by immunization with PfB3, PfB6, and seven LPCs. ELISAs were conducted using PfB6 as the antigen. PvT4-PfB3 (◊), PvT6-PfB3 (□), PvT8-PfB3 (▾), PvT19-PfB3 (⧫), PvT45-PfB3 (▪), and PvT53-PfB3 (•) are the individual LPCs containing PvMSP-1 promiscuous T-cell epitopes. PfT1-PfB3 (+) represents the LPC containing the P. falciparum genetically restricted T1 epitope. PfB3 (▴) and PfB6 (▿) represent the polymeric peptides containing only the B-cell epitope (NANP)_n with three and six repeats, respectively. Mice immunized with saline emulsified in FA represent the placebo group (✚). The data are presented as geometric mean titers obtained with a pool of sera obtained at different time points. The immunizations were conducted at 0, 30, and 60 days as indicated by the syringes.