In vitro growth-inhibitory activity and malaria risk in a cohort study in mali

Abstract

Immunity to the asexual blood stage of Plasmodium falciparum is complex and likely involves several effector mechanisms. Antibodies are thought to play a critical role in malaria immunity, and a corresponding in vitro correlate of antibody-mediated immunity has long been sought to facilitate malaria vaccine development. The growth inhibition assay (GIA) measures the capacity of antibodies to limit red blood cell (RBC) invasion and/or growth of P. falciparum in vitro. In humans, naturally acquired and vaccine-induced P. falciparum-specific antibodies have growth-inhibitory activity, but it is unclear if growth-inhibitory activity correlates with protection from clinical disease. In a longitudinal study in Mali, purified IgGs, obtained from plasmas collected before the malaria season from 220 individuals aged 2 to 10 and 18 to 25 years, were assayed for growth-inhibitory activity. Malaria episodes were recorded by passive surveillance over the subsequent 6-month malaria season. Logistic regression showed that greater age (odds ratio [OR], 0.78; 95% confidence interval [95% CI], 0.63 to 0.95; P = 0.02) and growth-inhibitory activity (OR, 0.50; 95% CI, 0.30 to 0.85; P = 0.01) were significantly associated with decreased malaria risk in children. A growth-inhibitory activity level of 40% was determined to be the optimal cutoff for discriminating malaria-immune and susceptible individuals in this cohort, with a sensitivity of 97.0%, but a low specificity of 24.3%, which limited the assay's ability to accurately predict protective immunity and to serve as an in vitro correlate of antibody-mediated immunity. These data suggest that antibodies which block merozoite invasion of RBC and/or inhibit the intra-RBC growth of the parasite contribute to but are not sufficient for the acquisition of malaria immunity.

FIG. 1.

Distribution of growth-inhibitory activities in the study cohort. The histogram shows the growth-inhibitory activities for the 220 study participants measured just prior to the malaria season. Inhibitory activities ranged from −7% to 97.6%, with a median of 26.0%. Only one individual had a negative value for % growth inhibition (−7% [data not shown]).

FIG. 2.

Growth-inhibitory activity by age group and whether individuals were parasitemic with P. falciparum just prior to the malaria season. Box-and-whisker plots represent the smallest and largest (whiskers), the lower and upper quartiles of (top and bottom of box), and the median (horizontal line across box) growth-inhibitory activity. Growth-inhibitory activity in nonparasitemic individuals increased with age (P < 0.001). Within each age group, the median growth-inhibitory activity was higher in parasitemic than in nonparasitemic individuals, but this difference reached statistical significance only in the 2- to 4-year-old (P < 0.01) and 8- to 10-year-old (P = 0.02) age groups, as indicated by an asterisk above the bar. Sample sizes for nonparasitemic individuals were as follows: 2- to 4-year-olds, n = 68; 5- to 7-year-olds, n = 45; 8- to 10-year-olds, n = 45; and 18- to 25-year-olds, n = 46. Those for parasitemic individuals were as follows: 2- to 4-year-olds, n = 4; 5- to 7-year-olds, n = 4; 8- to 10-year-olds, n = 5; and 18- to 25-year-olds, n = 3. The nonparametric Kruskal-Wallis test was used for statistical analysis.

FIG. 3.

Kaplan-Meier estimates of the cumulative probability of malaria in children aged 2 to 10 years during the 6-month malaria season, by whether growth-inhibitory activity was greater or less than 40%. Malaria was defined as an axillary temperature of ≥37.5°C, P. falciparum asexual parasitemia of ≥5,000 parasites/μl, and a nonfocal physical examination by the study physician. The number of individuals at risk for the first malaria episode in the two groups over the course of the study period is shown below the plot. The P value was obtained using the log rank test.

FIG. 4.

Distribution of growth-inhibitory activities in malaria-immune (no malaria episodes) and susceptible (≥1 malaria episode) children aged 2 to 10 years. Malaria was defined as an axillary temperature of ≥37.5°C, P. falciparum asexual parasitemia of ≥5,000 parasites/μl, and a nonfocal physical examination by the study physician.