A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray

Abstract

Abs are central to malaria immunity, which is only acquired after years of exposure to Plasmodium falciparum (Pf). Despite the enormous worldwide burden of malaria, the targets of protective Abs and the basis of their inefficient acquisition are unknown. Addressing these knowledge gaps could accelerate malaria vaccine development. To this end, we developed a protein microarray containing approximately 23% of the Pf 5,400-protein proteome and used this array to probe plasma from 220 individuals between the ages of 2-10 years and 18-25 years in Mali before and after the 6-month malaria season. Episodes of malaria were detected by passive surveillance over the 8-month study period. Ab reactivity to Pf proteins rose dramatically in children during the malaria season; however, most of this response appeared to be short-lived based on cross-sectional analysis before the malaria season, which revealed only modest incremental increases in Ab reactivity with age. Ab reactivities to 49 Pf proteins measured before the malaria season were significantly higher in 8-10-year-old children who were infected with Pf during the malaria season but did not experience malaria (n = 12) vs. those who experienced malaria (n = 29). This analysis also provided insight into patterns of Ab reactivity against Pf proteins based on the life cycle stage at which proteins are expressed, subcellular location, and other proteomic features. This approach, if validated in larger studies and in other epidemiological settings, could prove to be a useful strategy for better understanding fundamental properties of the human immune response to Pf and for identifying previously undescribed vaccine targets.

Fig. 1.

Kaplan–Meier estimates of the cumulative probability of malaria during the 6-month malaria season, according to age category. The number of individuals at risk in each age category is shown. Arrows indicate the points at which plasma was collected for protein microarray analysis (before and after the 6-month malaria season). The P value was obtained using the log-rank test.

Fig. 2.

Impact of age and Pf transmission on Pf-specific Ab profiles. Heat maps of proteins analyzed for immunoreactivity across plasma samples collected before (A) and after (B) the malaria season show that the breadth and intensity of Ab reactivity increases with age and in response to Pf transmission. The 491 immunogenic proteins are represented in rows in descending order of immunoreactivity. Individual plasma samples are in columns and grouped by age in years (2–4 yrs, 5–7 yrs, 8–10 yrs, and 18–25 yrs). Within each age group, samples are sorted by increasing average immunoreactivity. Red indicates positive immunoreactivity, black indicates intermediate immunoreactivity, and green indicates no immunoreactivity. (C) Of the 491 immunogenic proteins, the average number recognized by Pf-exposed individuals increased with age both before (P < 0.0001) and after (P < 0.0001) the malaria season. Significant increases in the number of proteins recognized from before to after the malaria season within age groups are indicated by an asterisk (P < 0.0001 for all significant changes). (D) Number of proteins recognized by at least 90% of Pf-exposed individuals increased with age and Pf transmission. (E) Average level of Ab reactivity to the 491 immunogenic Pf proteins (reactivity to the negative control subtracted) measured before the malaria season increased with age. In both children and adults, the level of Ab reactivity increased from before to after the malaria season. Statistically significant (P < 0.05) increases are indicated by an asterisk.

Fig. 3.

Pf-specific Ab profiles associated with protection from malaria. Heat map showing the difference in immunoreactivity measured before the malaria season against 49 proteins (in rows) in malaria-susceptible (Left, n = 29) and malaria-protected (Right, n = 12) children aged 8–10 years. (Right) Bar graph shows that the mean Ab reactivity against each of these proteins is higher in protected (red bars) vs. susceptible (blue bars) children (α = 0.05, false discovery rate-corrected). (Upper) For comparison, Ab reactivity against 5 proteins expressed in the rapid translation system that did not discriminate between protected and susceptible children is shown. These 5 proteins correspond to the malaria vaccine candidates CSP, LSA-3, MSP-1, MSP-2, and AMA-1. For the heat map, red indicates positive immunoreactivity, black indicates intermediate immunoreactivity, and green indicates no immunoreactivity. For the bar graph, data are mean ± SEM.