Naturally acquired antibodies specific for Plasmodium falciparum reticulocyte-binding protein homologue 5 inhibit parasite growth and predict protection from malaria

Abstract

Background: Plasmodium falciparum reticulocyte-binding protein homologue 5 (PfRH5) is a blood-stage parasite protein essential for host erythrocyte invasion. PfRH5-specific antibodies raised in animals inhibit parasite growth in vitro, but the relevance of naturally acquired PfRH5-specific antibodies in humans is unclear.

Methods: We assessed pre-malaria season PfRH5-specific immunoglobulin G (IgG) levels in 357 Malian children and adults who were uninfected with Plasmodium. Subsequent P. falciparum infections were detected by polymerase chain reaction every 2 weeks and malaria episodes by weekly physical examination and self-referral for 7 months. The primary outcome was time between the first P. falciparum infection and the first febrile malaria episode. PfRH5-specific IgG was assayed for parasite growth-inhibitory activity.

Results: The presence of PfRH5-specific IgG at enrollment was associated with a longer time between the first blood-stage infection and the first malaria episode (PfRH5-seropositive median: 71 days, PfRH5-seronegative median: 18 days; P = .001). This association remained significant after adjustment for age and other factors associated with malaria risk/exposure (hazard ratio, .62; P = .02). Concentrated PfRH5-specific IgG purified from Malians inhibited P. falciparum growth in vitro.

Conclusions: Naturally acquired PfRH5-specific IgG inhibits parasite growth in vitro and predicts protection from malaria. These findings strongly support efforts to develop PfRH5 as an urgently needed blood-stage malaria vaccine.

Clinical trials registration: NCT01322581.

Keywords: Plasmodium falciparum; RH5; blood-stage immunity; endemic population; malaria; prospective cohort study.

Figure 1.

Study participants and sampling flowchart. Abbreviations: IgG, immunoglobulin G; PCR, polymerase chain reaction; PfAMA1, Plasmodium falciparum apical membrane protein 1; PfRH5, Plasmodium falciparum reticulocyte-binding protein homologue 5.

Figure 2.

Naturally acquired immunoglobulin G (IgG) specific for PfRH5 and PfAMA1 increases with age and is boosted by Plasmodium falciparum transmission. Semiquantitative levels of immunoglobulin G (IgG) specific for PfRH5 (A) and PfAMA1 (B) in plasma samples from 357 Malians across age groups, as indicated by background-subtracted optical density (OD 450 nm) in enzyme-linked immunosorbent assay. Boxes enclose interquartile range, central lines represent medians, whiskers indicate the 5th–95th percentile, and dots are outliers. Between-group differences were assessed by the Mann–Whitney test. C, Seroprevalence of IgG with arbitrary units (AU) >1 specific for PfRH5 or PfAMA1 across the indicated age groups, where an AU of 1 is defined as the mean OD value plus 3 standard deviations for 20 malaria-naive US donors. D, Fold-change in PfRH5-specific and PfAMA1-specific IgG levels from before to after a single malaria transmission season in 50 individuals who began the season uninfected and had ≥1 P. falciparum infection between May 2011 and January 2012. Each individual was tested for both antigens. Fold-change relative to a baseline of 1 was compared by the Wilcoxon signed-rank test. Fold-change differences between PfRH5 and PfAMA1 were compared by the Mann–Whitney test. E, PfRH5-specific IgG subclass responses in AU among PfRH5 responders (n = 48) and PfAMA1 responders (n = 64) with seroprevalence for each antigen-specific IgG subclass shown below. Central lines represent medians, and error bars indicate the interquartile range. **P < .01; ***P < .001; ****P < .0001. Abbreviations: AU, arbitrary units; IgG, immunoglobulin G; NS, not significant; OD, optical density; PfAMA1, Plasmodium falciparum apical membrane protein 1; PfRH5, Plasmodium falciparum reticulocyte-binding protein homologue 5.

Figure 3.

PfRH5-specific immunoglobulin G (IgG) predicts protection from febrile malaria. Time to first febrile malaria episode using enrollment date (A) or first polymerase chain reaction (PCR)–confirmed Plasmodium falciparum blood-stage infection (B) as the start time in individuals with and without detectable PfRH5-specific IgG, as determined by Kaplan-Meier survival analysis. C, A Cox model was used to evaluate the effect of different covariates on the risk of the first febrile malaria episode using time of first PCR-detectable P. falciparum blood-stage infection as the start time for malaria risk analysis. Hazards ratios and 95% confidence intervals are represented by boxes and horizontal bars, respectively. Abbreviations: AU, arbitrary units; IgG, immunoglobulin G; PfAMA1, Plasmodium falciparum apical membrane protein 1; PfRH5, Plasmodium falciparum reticulocyte-binding protein homologue 5.

Figure 4.

Affinity-purified, human PfRH5-specific IgG inhibits growth of Plasmodium falciparum parasites (3D7) in vitro. Growth inhibitory activity was assessed on P. falciparum 3D7 parasites using PfRH5-specific IgG affinity purified from uninfected, Malian donors; the IgG flow-through fraction; or IgG purified from US donors at IgG concentrations from 2.34 to 150 µg/mL. Data points represent the mean of duplicate wells from 1 experiment. Error bars represent standard deviations. Some error bars are not visible on this scale due to small standard deviations. Abbreviations: IgG, immunoglobulin G; PfRH5, Plasmodium falciparum reticulocyte-binding protein homologue 5.