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Characterization of fine specificity of the immune response to a Plasmodium falciparum rhoptry neck protein, PfAARP

View Article: PubMed Central - PubMed

ABSTRACT

Background: Immunological 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.

Methods: Balb/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.

Results: Here, 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.

Conclusions: Induction 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.

No MeSH data available.


Purification and characterization of PfAARP. a Position of PfAARP ectodomain in the protein sequence. b Mobility of recombinant purified protein on SDS-PAGE. c Western blot of recombinant purified protein with anti-His monoclonal antibody. d RP-HPLC profile of purified protein showing the purity of the protein
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Fig1: Purification and characterization of PfAARP. a Position of PfAARP ectodomain in the protein sequence. b Mobility of recombinant purified protein on SDS-PAGE. c Western blot of recombinant purified protein with anti-His monoclonal antibody. d RP-HPLC profile of purified protein showing the purity of the protein

Mentions: PfAARP described in this study comprises amino acid 20–107, devoid of signal sequence, asparagine repeats, polyproline stretch, and transmembrane domain of the protein (Fig. 1a). 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 finally RP-HPLC, to remove endotoxin from the purified protein. Purified PfAARP showed an apparent mobility at ~17 kDa on SDS-PAGE instead of its predicted molecular mass of 11.6 kDa (Fig. 1b). This discrepancy in mobility of the recombinant protein may be due to highly acidic pI (5.06) of the protein and the unusual amino acid composition. Such aberrant migration on SDS-PAGE has been observed with a number of other malaria proteins, such as RESA and also for Toxoplasma proteins (another Apicomplexan parasite) [24, 25]. Identity of the recombinant protein was confirmed by Western blot analysis under reducing conditions with anti-His monoclonal antibody (Fig. 1c). The results from both SDS-PAGE analysis and RP-HPLC showed that the purity of the recombinant protein was greater than 95 % (Fig. 1d). Gel clot assay based on 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. This endotoxin level is well below the permissible limit (30 EU per 25 µg protein) for bacterially expressed proteins used for immunization studies in small animals. Application of RP-HPLC for endotoxin removal from bacterially expressed proteins has been used previously for other proteins, such as human cryptic plasminogen-derived domain Kringle 5 [26]. Purified PfAARP with low, acceptable level of endotoxin content was used for immunization in mice.Fig. 1


Characterization of fine specificity of the immune response to a Plasmodium falciparum rhoptry neck protein, PfAARP
Purification and characterization of PfAARP. a Position of PfAARP ectodomain in the protein sequence. b Mobility of recombinant purified protein on SDS-PAGE. c Western blot of recombinant purified protein with anti-His monoclonal antibody. d RP-HPLC profile of purified protein showing the purity of the protein
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5015194&req=5

Fig1: Purification and characterization of PfAARP. a Position of PfAARP ectodomain in the protein sequence. b Mobility of recombinant purified protein on SDS-PAGE. c Western blot of recombinant purified protein with anti-His monoclonal antibody. d RP-HPLC profile of purified protein showing the purity of the protein
Mentions: PfAARP described in this study comprises amino acid 20–107, devoid of signal sequence, asparagine repeats, polyproline stretch, and transmembrane domain of the protein (Fig. 1a). 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 finally RP-HPLC, to remove endotoxin from the purified protein. Purified PfAARP showed an apparent mobility at ~17 kDa on SDS-PAGE instead of its predicted molecular mass of 11.6 kDa (Fig. 1b). This discrepancy in mobility of the recombinant protein may be due to highly acidic pI (5.06) of the protein and the unusual amino acid composition. Such aberrant migration on SDS-PAGE has been observed with a number of other malaria proteins, such as RESA and also for Toxoplasma proteins (another Apicomplexan parasite) [24, 25]. Identity of the recombinant protein was confirmed by Western blot analysis under reducing conditions with anti-His monoclonal antibody (Fig. 1c). The results from both SDS-PAGE analysis and RP-HPLC showed that the purity of the recombinant protein was greater than 95 % (Fig. 1d). Gel clot assay based on 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. This endotoxin level is well below the permissible limit (30 EU per 25 µg protein) for bacterially expressed proteins used for immunization studies in small animals. Application of RP-HPLC for endotoxin removal from bacterially expressed proteins has been used previously for other proteins, such as human cryptic plasminogen-derived domain Kringle 5 [26]. Purified PfAARP with low, acceptable level of endotoxin content was used for immunization in mice.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Immunological 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.

Methods: Balb/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.

Results: Here, 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.

Conclusions: Induction 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.

No MeSH data available.