Memory B-cell and antibody responses induced by Plasmodium falciparum sporozoite immunization

Abstract

Background: Immunization of healthy volunteers during receipt of chemoprophylaxis with Plasmodium falciparum sporozoites (CPS-immunization) induces sterile protection from malaria. Antibody responses have long been known to contribute to naturally acquired immunity against malaria, but their association with sterile protection after whole sporozoite immunization is not well established. We therefore studied the induction and kinetics of malaria parasite antigen-specific antibodies and memory B-cells (MBCs) during CPS-immunization and their correlation with protection from challenge infection.

Methods: We assessed humoral reactivity to 9 antigens representing different stages of the life cycle of P. falciparum by performing standardized MBC enzyme-linked immunospot and enzyme-linked immunosorbent assays on peripheral blood mononuclear cells and plasma samples from 38 Dutch volunteers enrolled in 2 randomized controlled clinical trials.

Results: MBCs and antibodies recognizing pre-erythrocytic and cross-stage antigens were gradually acquired during CPS-immunization. The magnitude of these humoral responses did not correlate with protection but directly reflected parasite exposure in CPS-immunization and challenge.

Conclusions: Humoral responses to the malarial antigens circumsporozoite protein, liver-stage antigen-1, apical membrane antigen-1, and merozoite surface protein-1 do not to predict protection from challenge infection but can be used as sensitive marker of recent parasite exposure.

Clinical trials registration: NCT01236612 and NCT01218893.

Keywords: Plasmodium; antibody; immunization; malaria; memory B-cell; protection; sporozoite.

© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.

Figures

Figure 1.

Study design. Samples from 2 clinical chemoprophylaxis and sporozoites (CPS)–immunization trials were analyzed. In study A, volunteers were immunized on 3 occasions, separated by 4-week intervals, with bites of 15 Plasmodium falciparum–infected mosquitoes (3 × 15) while receiving a prophylactic regimen of the antimalarial drug chloroquine (gray box). For challenge infection 21 weeks after the last immunization (17 weeks after the final chloroquine dose), immunized volunteers were split into 2 groups, with one receiving P. falciparum–parasitized erythrocytes and the other exposed to bites from 5 infective mosquitoes. Plasma and peripheral blood mononuclear cell (PBMC) samples were obtained before immunization (D0), 1 day before challenge (C − 1), and 35 days after challenge (C + 35). In study B, 3 different immunization groups were exposed on 3 occasions, separated by 4-week intervals, to bites from 15 (3 × 15), 10 (3 × 10), or 5 (3 × 5) P. falciparum–infected mosquitoes while receiving chloroquine prophylaxis. All groups were challenged 19 weeks after the last immunization (15 weeks after the last chloroquine dose) with 5 P. falciparum–infected mosquito bites. Plasma and PBMC samples were collected before immunization (D0); 28 days after the first (I), second (II), and third (III) immunization; 1 day before challenge (C − 1), and 35 days after challenge (C + 35).

Figure 2.

Antibody responses against antigens representing different life cycle stages of the malaria parasite. Antibody responses were determined before immunization (D0) and 19 to 21 weeks after the last chemoprophylaxis and sporozoites (CPS)–immunization (C − 1) for volunteers (14 from study A and 5 from study B) immunized 3 times with bites from 15 infected mosquitoes. Responses are expressed as arbitrary units (AU) for malaria antigens representing different life cycle stages of Plasmodium falciparum in the human host (schematically represented by the illustration on the left) (A) Circumsporozoite protein (CSP) and liver-stage antigen 1 (LSA-1) are expressed in pre-erythrocytic stages (B) Exported protein 1 (EXP-1), thrombospondin-related anonymous protein (TRAP), merozoite surface protein 1 (MSP-1) and MSP-2, apical membrane antigen 1 (AMA-1) and glutamate-rich protein (GLURP) are present in liver and blood-stage parasites (C) Erythrocyte binding protein 175 (EBA-175) is exclusively expressed in P. falciparum–infected erythrocytes. For study B, responses were assessed for CSP, LSA-1, AMA-1, and MSP-1 only. Data are presented as ladder plots, with each dot representing an individual volunteer and dotted lines connecting values before and after immunization for each volunteer. The black line represents assay background levels (upper 99% confidence interval of the mean of >40 malaria-naive samples tested). Differences between D0 and C − 1 were analyzed using the Wilcoxon matched-pairs signed rank test. *P < .05, **P < .01, and ***P < .001. Abbreviation: NS, not significant.

Figure 3.

Acquisition kinetics of antimalarial antibody and memory B-cells (MBC) responses. Antibody (A; in arbitrary units [AU]) and MBC responses (B; presented as the percentage of antigen-specific MBCs of total immunoglobulin G [IgG]–expressing MBCs) for circumsporozoite protein (CSP) and merozoite surface protein 1 (MSP-1) were analyzed 28 days after the first (I), second (II), and third (III) immunization and 19 weeks after the third immunization (C − 1) for all 24 CPS-immunized volunteers in study B. Data were corrected for the volunteers’ background response before immunization and are presented as ladder plots, with each dot representing an individual volunteer and dotted lines connecting the different time points for each volunteer. Differences between the time points were analyzed by the Friedman test with the Dunn multiple comparison post hoc test. *P < .05, **P < .01, and ***P < .001.

Figure 4.

Influence of parasite exposure during immunization on the magnitude of the antibody response and protection status. A and B, Chemoprophylaxis and sporozoite (CPS)–immunized volunteers from study B protected from mosquito challenge after exposure on 3 occasions, to bites from 15 (3 × 15; orange; n = 4), 10 (3 × 10; yellow; n = 8), or 5 (3 × 5; white; n = 5) Plasmodium falciparum–infected mosquitoes. Circumsporozoite protein (CSP)- and merozoite surface protein 1 (MSP-1)–specific antibody levels (in arbitrary units [AU]; A) and blood-stage parasitemia (expressed as the number of P. falciparum–parasitized erythrocytes per mL of blood, determined by quantitative polymerase chain reaction; B) were analyzed 28 days after the first (I), second (II), and third (III) CPS-immunization. C and D, 3 × 5 CPS-immunization did (white; n = 5) or did not (purple; n = 5) result in protection from subsequent mosquito challenge. C, CSP- and MSP-1–specific antibody (in AU) and (D) erythrocytic parasitemia (in P. falciparum/mL) 28 days after immunizations I, II, and III are displayed. E, Correlation between CSP and MSP-1 antibody responses (in AU) 28 days after immunization III and cumulative parasitemia (in P. falciparum/mL) over the course of all CPS-immunizations for all study B volunteers. All antibody responses were corrected for the volunteers’ background response before immunization and are presented as individual values (dots) and whisker box plots (box, median with 10th–90th percentile; whiskers, minimum to maximum) and individual values (dots). Differences between groups over time were analyzed by repeated-measures, mixed-model 2-way analysis of variance with the Bonferroni post hoc test. Correlation was analyzed by the Spearman coefficient (r). *P < .05, **P < .01, and ***P < .001.

Figure 5.

Boosting of antibody levels in chemoprophylaxis and sporozoite (CPS)–immunized volunteers by challenge infection. Antibody levels (in arbitrary units [AU]), corrected for the volunteers’ background before immunization) for circumsporozoite protein (CSP) and merozoite surface protein 1 (MSP-1) in immunized volunteers were determined prior to (C − 1) and 35 days following (C + 35) challenge infection. Each dot represents an individual volunteer for which values before and after challenge are connected by a dotted line. A, Mosquito challenge (MC) of 22 protected (experienced pre-erythrocytic parasites only) CPS-immunized volunteers (5 from study A and 17 from study B). The white triangle denotes a volunteer with quantitative polymerase chain reaction–detectable blood-stage parasitemia on day 21 after challenge. B, MC of 7 unprotected (experienced both pre-erythrocytic and blood-stage parasites) CPS-immunized volunteers from study B. C, Blood-stage challenge (BC; exposure to erythrocytic parasites only) of 9 CPS-immunized volunteers from study A. Differences between time points were analyzed by the Wilcoxon matched-pairs signed rank test. *P < .05, **P < .01, and ***P < .001. Abbreviation: NS, not significant.