Anti-merozoite antibodies induce natural killer cell effector function and are associated with immunity against malaria

Abstract

Natural killer (NK) cells are potent immune effectors that can be activated via antibody-mediated Fc receptor engagement. Using multiparameter flow cytometry, we found that NK cells degranulate and release IFN-γ upon stimulation with antibody-opsonized Plasmodium falciparum merozoites. Antibody-dependent NK (Ab-NK) activity was largely strain transcending and enhanced invasion inhibition into erythrocytes. Ab-NK was associated with the successful control of parasitemia after experimental malaria challenge in African adults. In an independent cohort study in children, Ab-NK increased with age, was boosted by concurrent P. falciparum infections, and was associated with a lower risk of clinical episodes of malaria. Nine of the 14 vaccine candidates tested induced Ab-NK, including some less well-characterized antigens: P41, P113, MSP11, RHOPH3, and Pf_11363200. These data highlight an important role of Ab-NK activity in immunity against malaria and provide a potential mechanism for evaluating vaccine candidates.

Fig. 1. Anti-merozoite antibodies activate NK cells.

(A) NK cell degranulation and IFN-γ production were measured by CD107a surface and intracellular staining, respectively. Merozoites were opsonized with a pool of malaria-exposed (n = 4) or naïve plasma (n = 4), with PHA/ionomycin included as a positive control. Malaria-exposed plasma samples from adults in Junju county were used to optimize the assay. Data are from four independent experiments and show the median with a 95% confidence interval (95% CI). Differences between groups are compared using the Kruskal-Wallis test. (B) NK cell degranulation upon stimulation with merozoites opsonized with individual plasma samples from malaria-exposed Junju adults (n = 30). PBMCs were collected on three separate days. The graph shows the pairwise correlation of the percentage (%) of NK cells degranulating after coincubation with merozoites opsonized with malaria-exposed plasma between independent experiments conducted on 3 separate days. The red dots show the correlation between experiments conducted on day 1 versus day 2 (experiment 2). In contrast, the blue dots show the correlation between experiments on day 1 versus day 3 (experiment 3). (C and D) Representative flow plots of NK cells incubated with merozoites in the presence of malaria-exposed or malaria-naïve plasma are shown. P. falciparum merozoites opsonized with plasma from individual malaria-exposed adults (red circles, n = 20) or malaria-naïve plasma (blue circles, n = 4) were coincubated with donor NK cells. An additional pool of hyperimmune sera (PHIS) was included as a positive control (brown circles, n = 4). This pool was prepared from Junju adults with high ELISA responses against parasite schizont lysate from the 3D7 strain of P. falciparum. Error bars represent 95% CI of the median values; Kruskal-Wallis test. (E) Pairwise correlation between the proportion of IFN-γ–secreting or IFN-γ–degranulating (CD107a⁺) NK cells activated by opsonized P. falciparum 3D7 merozoites and merozoite ELISA quantifying total IgG responses (OD; n = 142). (F) Spearman’s correlation heatmap between the proportion of NK cells degranulating upon activation by opsonized merozoites from five P. falciparum strains of different geographical origins (n = 20). (G) Viable merozoites were coincubated with uninfected erythrocytes and test immunoglobulins (1.5 ng per well) in the presence or absence of donor NK cells. Data represent the median with 95% CIs of three independent experiments.

Fig. 2. Antibody-mediated NK cell activity is associated with in vivo parasite growth.

Comparison of Ab-NK cell degranulation (A) and IFN-γ production (B) in treated (n = 56) versus nontreated (n = 86) volunteers. Bars represent 95% CI of the median values; Mann-Whitney test. Subgroup analysis of Ab-NK cell degranulation (C) and IFN-γ production (D) for treated volunteers who either developed fever (febrile, n = 26) or did not (nonfebrile, n = 30) and for untreated volunteers in whom parasites were either detected by PCR (PCR⁺, n = 53) or remained negative (PCR⁻, n = 33). Bars represent 95% CI of the median values; Kruskal-Wallis with Dunn’s multiple comparisons test. Kaplan-Meier curves for the time to treatment for volunteers with a high (red) versus low (blue) Ab-NK cell degranulation (E) and IFN-γ production (F); log-rank test, P < 0.0001, n = 142.

Fig. 3. Potential targets of antibodies that mediate Ab-NK.

(A) Antigen-specific Ab-NK cell degranulation from a subset of CHMI individuals (n = 10) against 14 unique recombinant P. falciparum merozoite antigens. The dashed line represents the cutoff value based on the mean plus 3 SDs of the tagged fragment (CD4 tag). Error bars represent 95% CI of the median (red line). (B) Heatmap of individual level antigen-specific Ab-NK cell degranulation from (A). Each row represents a single CHMI participant, and each column represents responses to a single recombinant merozoite surface–associated antigen. (C) Ab-NK cell degranulation against whole merozoite extract was associated with the breadth of antigen-specific recognition. Error bars represent 95% CI of the median (red line) n = 142; Kruskal-Wallis test. (D) The sum of antigen-specific Ab-NK cell degranulation against 14 unique recombinant merozoite antigens was correlated with NK cell degranulation against merozoites; Spearman’s correlation R = 0.80.

Fig. 4. Antibody-mediated NK cell degranulation is associated with clinical protection in a prospective cohort study in Kenyan children.

(A) Comparison of Ab-NK cell degranulation (left) and IFN-γ production (right) in Junju children over or under 5 years of age. (B) Ab-NK response in children who had an active P. falciparum infection compared with those without. Error bars represent 95% CI of the median values; Mann-Whitney test (n = 293). (C) Antibody-mediated NK cell degranulation (left) and IFN-γ production (right) were correlated with the breadth of total IgG responses against recombinant MSP2, MSP3, and/or AMA-1. Error bars represent 95% CI of the median values; Kruskal-Wallis test with Dunn’s multiple comparisons test (n = 293). (D) Ab-NK cell degranulation and IFN-γ production were associated with clinical protection against symptomatic malaria in children. Ab-NK responses were categorized as high or low (blue) on the basis of a threshold (28); log-rank test, P = 0.001 (n = 293). Each dot represents a technical replicate.