Naturally-acquired cellular immune response against Plasmodium vivax merozoite surface protein-1 paralog antigen

Abstract

Background: Plasmodium vivax merozoite surface protein-1 paralog (PvMSP1P) is a glycosylphosphatidylinositol-anchored protein expressed on the merozoite surface. This molecule is a target of natural immunity, as high anti-MSP1P-19 antibody levels were detected during P. vivax infection and the antibody inhibited PvMSP1P-erythrocyte binding. Recombinant PvMSP1P antigen results in production of a significant Th1 cytokine response in immunized mice. The present study was performed to characterize natural cellular immunity against PvMSP1P-19 and PvDBP region II in acute and recovery P. vivax infection.

Methods: Peripheral blood mononuclear cells (PBMCs) from acute and recovery P. vivax infection were obtained for lymphocyte proliferation assay upon PvMSP1P-19 and PvDBP region II antigen stimulation. The culture supernatant was examined for the presence of the cytokines IL-2, TNF, IFN-γ and IL-10 by enzyme-linked immunosorbent assay (ELISA). To determine whether Th1 or Th2 have a memory response against PvMSP1P-19 and PvDBPII protein antigen, PBMCs from subjects who had recovered from P. vivax infection 8-10 weeks prior to the study were obtained for lymphocyte proliferation assay. Cytokine-producing cells were analysed by flow cytometry.

Results: IL-2 was detected at high levels in lymphocyte cultures from acutely infected P. vivax patients upon PvMSP1P-19 stimulation. Analysis of the Th1 or Th2 memory response in PBMC cultures from subjects who had recovered from P. vivax infection showed significantly elevated levels of PvMSP1P-19 and PvDBPII-specific IFN-γ-producing cells (P < 0.05). Interestingly, the response of IFN-γ-producing cells in PvMSP1P stimulation was fourfold greater in recovered subjects than that in acute-infection patients. CD4(+) T cells were the major cell phenotype involved in the response to PvMSP1P-19 and PvDBPII antigen.

Conclusions: PvMSP1P-19 strongly induces a specific cellular immune response for protection against P. vivax compared with PvDBPII as the antigen induces activation of IFN-γ-producing effector cells following natural P. vivax exposure. Upon stimulation, PvMSP1P-19 has the potential to activate the recall response of Th1 effector memory cells that play a role in killing the parasite.

Figure 1

Cytokine production of PvMSP1P-19-stimulated lymphocyte cultures obtained from individuals with acute P. vivax infection (n  =  15). PBMCs were re-stimulated with PvMSP1P-19 antigen for 48 h and culture supernatant was removed for cytokine detection. (a) IL-2 detection after 48 h of in vitro stimulation. (b) TNF, IFN-γ and IL-10 after 96 h of in vitro stimulation.

Figure 2

The recall of a cellular immune response specific to PvMSP1P-19 antigen by induction of IFN-γ and IL-10 cytokine responses. PBMCs from individuals who had recovered from P. vivax infection 8–10 weeks prior to the study were stimulated by PvMSP1P-19 or PvDBPII antigen with negative (media) and positive controls (PHA) for 96 h. IFN-γ and IL-10 levels in the culture supernatant were measured by ELISA. Data show the average cytokine values from P. vivax-recovered subjects (n  =  20). Significance was determined by one-way ANOVA with Dunnett’s test. The level of significance was set at P  <  0.05.

Figure 3

T-cell responses to PvMSP1P-19 antigen using multiparameter flow cytometry. Intracellular cytokine assay demonstrating the T-cell response of P. vivax-recovered subjects to PvMSP1P-19 or PvDBPII with negative (media) and positive controls (PMA/Ionomycin). PBMCs from individuals who had recovered from P. vivax infection 8–10 weeks prior to the study were removed for lymphocyte proliferation assay and intracellular cytokine detection by flow cytometric analysis. The data are shown as the average levels of cytokine-producing cells in individual subjects (n  =  6). This shows the gating strategy to identify IL-2/IFN-γ/IL-10-producing cells.

Figure 4

T-cell responses to PvMSP1P-19 antigen using multiparameter flow cytometry. (a) Overall PvMSP1P-19-specific IL-2-producing cells. (b) IFN-γ-producing cells. Significance was determined by one-way ANOVA with Dunnett’s test. The level of significance was set at P  <  0.05.

Figure 5

T-cell responses to PvMSP1P-19 antigen using multiparameter flow cytometry. IL-10-producing cells. Significance was determined by one-way ANOVA with Dunnett’s test. The level of significance was set at P  <  0.05.