Abstract
BackgroundImmunological characterization of potential blood-stage malaria antigens would be a valuable strategy in the development of an effective vaccine. Identifying B and CD4+ T cell epitopes will be important in understanding the nature of immune response. A previous study has shown that Plasmodium falciparum apical asparagine-rich protein (PfAARP) stimulates immune response and induces potent invasion-inhibitory antibodies. Antibodies to PfAARP provide synergistic effects in inhibition of parasite invasion when used in combination with antibodies to other antigens. In the present study, an attempt was made to identify B cell and CD4+ T cell epitopes of PfAARP.MethodsBalb/c mice were immunized with recombinant PfAARP and both cellular and humoral responses were analysed at various time points. Computerized databases [immune epitope database (IEDB) and B cell epitope prediction (BCEPred)] were used to predict epitope sequences within PfAARP and predicted peptides were synthesized. In addition, nine 18 amino acid, long-overlapping peptides spanning the entire length of PfAARP were synthesized. Using these peptides, B cell and CD4+ T cell responses in PfAARP immunized mice were measured by ELISA and ELISPOT assays.ResultsHere, it is demonstrated that immunization of mice with PfAARP induced long-lasting, high-titre antibodies (4 months post immunization). Also, the recombinant protein was effective in inducing a pronounced Th1 type of immune response quantified by IFN-γ ELISA and ELISPOT. It was found that the predicted peptides did not represent the immunogenic regions of PfAARP. However, of the nine overlapping peptides, three peptides (peptides 3, 5 and 7) were strongly recognized by PfAARP-immunized sera and represented B cell epitopes. Also, peptide 3 elicited IFN- γ response, suggesting it to be a T-cell epitope.ConclusionsInduction of long-lasting humoral and cellular response on PfAARP immunization in mice underscores its possible use as a blood-stage malaria vaccine candidate. Mapping of immunogenic regions may help in designing fusion chimera containing immunologically relevant regions of other vaccine target antigens and/or for multi-component vaccine candidates.
Highlights
Immunological characterization of potential blood-stage malaria antigens would be a valuable strategy in the development of an effective vaccine
Plasmodium falciparum apical asparaginerich protein (PfAARP) was expressed as a soluble protein in cytosolic fractions and purified to homogeneity by a three-step chromatographic procedure, including immobilized metal affinity chromatography followed by anion exchange chromatography and reverse-phase high performance liquid chromatography (RP-HPLC), to remove endotoxin from the purified protein
Gel clot assay based on Limulus Amoebocyte Lysate (LAL) reagent kit showed that endotoxin level of the IEX purified fraction was high (~5000 EU per 25 μg protein) which was significantly reduced to ~2.5 EU per 25 μg of the protein on RP-HPLC of IEX fractions
Summary
Expression, purification and characterization of recombinant PfAARP PfAARP (amino acid 20–107 with a C-terminal His tag) was cloned from the full length synthetic gene construct with 5′-CACATCATCAcatatgATTCTGCGTAAT AATAAAAGCC-3′ and 5′-TATATActcgagTCAGTGG TG G TG G TG G TG G TG ATC T TC AT TG TC T T CTTCATC-3′ as forward and reverse primers, respectively, between NdeI and XhoI restriction sites. Phenotypic characterization and T cell epitope mapping, two groups of three mice each were immunized with 25 μg of the recombinant antigen in complete Freund’s adjuvant and given a booster dose in incomplete Freund’s adjuvant with same amount of antigen on day 21 before harvesting spleen. Mice injected with adjuvant-formulated PBS were used as the control group Both the control and the test group mice were sacrificed on the same day and isolated splenic cells were analysed for T cell response. Spleen cells from PfAARPimmunized mice were added to the wells at 5 × 105 cells per well in the presence of recombinant protein or respective peptides at concentration of 50 μg/ml, in final volume of 200 μl and incubated for 48 h at 37 °C in a 5 % CO2 incubator. To determine the level of significance, a Student t test was performed and a p < 0.05 was considered significant
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