Immunogenicity of the Plasmodium vivax merozoite surface protein 1 paralog in the induction of naturally acquired antibody and memory B cell responses

Abstract

Background: The Plasmodium vivax merozoite surface protein 1 paralog (PvMSP1P-19) is a glycosylphosphatidylinositol (GPI)-anchored blood-stage protein that is expressed on the merozoite surface. It is proposed as a blood-stage vaccine candidate against P. vivax because of its ability to induce immune responses upon natural P. vivax exposure and in immunized animals. This study aimed to demonstrate the presence of inhibitory antibodies and memory B cell responses to the PvMSP1P-19 antigen during acute P. vivax infection and after recovery from infection.

Methods: To evaluate the antibody responses to PvMSP1P-19 during and after recovery from P. vivax infection, heparinized blood was collected from P. vivax-infected patients and recovered subjects to detect the total IgG response. The seropositive samples were defined into high and low responders, according to their optical density (OD) values obtained from ELISA. High responders were the subjects who had OD values above the OD of antisera from non-exposed controls plus 4× standard deviations, whereas low responders were the subjects who had OD values less than OD of antisera from non-exposed controls plus 4× standard deviations. The plasma from high and low responders were taken for testing the inhibitory activity against PvMSP1P-19-erythrocyte binding by in vitro EBIA. The sustainability of PvMSP1P-19-specific memory B cell responses after recovery from infection was analysed by ELISPOT.

Results: The anti-PvMSP1P-19 antibody levels were significantly higher in acutely infected P. vivax patients compared to healthy controls (P < 0.0001). Monitoring of the anti-PvMSP1P-19 antibody titre showed that the antibody was maintained for up to 9 months after recovery. Almost all high-responder groups strongly inhibited PvMSP1P-19 binding to erythrocytes, whereas no inhibition was shown in most low-responder samples. Interestingly, the inhibitory activity of the antibodies in some individuals from high-responder samples were stable for at least 12 months. The longevity of the antibody response was associated with the presence of PvMSP1P-19-specific memory B cells at 9 months after recovery from infection.

Conclusions: The PvMSP1P-19 antigen has immunogenicity during the induction of the antibody response, in which both the levels and inhibitory activity are maintained after the patient recovered from P. vivax infection. The maintenance of the antibody response was associated with the response of PvMSP1P-19-specific memory B cells. Therefore, the PvMSP1P-19 antigen should also be considered as a reliable vaccine candidate to develop a blood-stage vaccine against P. vivax.

Keywords: Immunogenicity; Merozoite surface protein 1 paralog; Plasmodium vivax.

Fig. 1

Humoral immune response to PvMSP1P-19. The antibody levels were detected in human plasma from acutely P. vivax-infected patients (n = 40), villagers in endemic areas (n = 15) and naïve controls (n = 22). Each symbol represents an individual sample. The dashed line indicates the cut-off value [mean ± 2 standard deviations (SD) of the optical density (OD) value of malaria-naïve individuals]. A nonparametric Mann–Whitney U test was used to assess the significant difference in the antibody titre levels against the PvMSP1P-19 antigen. The level of significance was set at a P value <0.05

Fig. 2

Longevity of the antibody response to the PvMSP1P-19 antigen after recovery from infection. a A survey of antibody responses during the acute phase (n = 40) and after anti-malarial treatment at 3 months (n = 27), 9 months (n = 28) and 12 months (n = 14). The bars represent the mean values. Mann–Whitney U test was used for statistical analysis and P value calculation. The level of significance was set at a P value <0.05. b The stability of the anti-PvMSP1P-19 antibody responses in the same individual (n = 16) during acute phase and at 3-month recovery phase. c A longitudinal cohort analysis of anti-PvMSP1P-19 response persistence in seropositive individuals (n = 5, HR1 = Pv01, HR2 = Pv02, HR3 = Pv03 , LR1 = Pv04, LR2 = Pv05) at their acute phases and by following-up their recovery phases at 3, 9 and 12 months. The dashed line indicates the cut-off value (means ± 2SD of the OD value of malaria-naïve individuals)

Fig. 3

Inhibitory activity of antibodies against PvMSP1P-19 binding to human erythrocytes. Seropositive plasma samples from acutely P. vivax-infected a high responders (n = 16) and b low responders (n = 13) were performed to evaluate the inhibitory activity of antibodies against PvMSP1P-19-erythrocyte binding. c The activity of the anti-PvMSP1P-19 inhibitory antibody against human erythrocyte binding in individuals (n = 5) in their acute phases and their recovery phases at 9 and 12 months after P. vivax infeciton. The transfected HEK293-T cells expressing PvMSP1P-19 were incubated with plasma and with human erythrocytes. The numbers of rosettes were compared between wells of transfected cells that were incubated with plasma of vivax malaria patients relative to the negative control well. The charts show the mean inhibition of each P. vivax subject. Error bars represent ± standard deviation

Fig. 4

Alteration of CD19⁺ B cells and CD10⁻CD19⁺CD27⁺ memory B cells in P. vivax subjects. a The gating strategy for identifying the CD19⁺B cells and CD19⁺CD10⁻ CD27⁺ memory B cells (MBCs) by using flow cytometry. The B cell subsets in the FACS plots are represented for P. vivax-infected patients. The lymphocytes were first gated, and then, 100,000 events using CD19⁺ were collected to identify the B cell populations. These populations were continuously analysed for the expression of CD10⁻ and CD27⁺, which were classified as MBCs. The percentages of CD19⁺ B cells (b) and CD19⁺ CD10⁻CD27⁺ MBCs (c) in individuals during acute phase and at 3 and 9 months recovery after P. vivax infeciton (n = 11) are shown. Each symbol represents the B cell frequencies for one individual. The horizontal line reflects the median value

Fig. 5

Memory B cell response to the PvMSP1P-19 antigen. a The level of the memory B cell (MBC) response to the PvMSP1P-19 antigen and tetanus toxoid in individual PBMCs from 9 months after P. vivax infection were determined by ELISPOT (n = 11). The frequencies of MBCs are expressed per million cultured PBMCs. Each symbol represents the MBC number for one individual. The line reflects the median value. SPM, spots per million. b The correlation between the ELISA and ELISPOT responses to the PvMSP1P-19 antigen. The data are shown for 11 subjects at 9 months after recovery from P. vivax infection. The numbers of subjects who were double-positive (top left), ELISA-positive but ELISPOT-negative (bottom left), ELISA-negative but ELISPOT-positive (top left) or double negative (bottom right) are shown