Longitudinal analysis of naturally acquired PfEMP1 CIDR domain variant antibodies identifies associations with malaria protection

Abstract

BACKGROUNDMalaria pathogenicity is determined, in part, by the adherence of Plasmodium falciparum-infected erythrocytes to the microvasculature mediated via specific interactions between P. falciparum erythrocyte membrane protein (PfEMP1) variant domains and host endothelial receptors. Naturally acquired antibodies against specific PfEMP1 variants can play an important role in clinical protection against malaria.METHODSWe evaluated IgG responses against a repertoire of PfEMP1 CIDR domain variants to determine the rate and order of variant-specific antibody acquisition and their association with protection against febrile malaria in a prospective cohort study conducted in an area of intense, seasonal malaria transmission.RESULTSUsing longitudinal data, we found that IgG antibodies against the pathogenic domain variants CIDRα1.7 and CIDRα1.8 were acquired the earliest. Furthermore, IgG antibodies against CIDRγ3 were associated with reduced prospective risk of febrile malaria and recurrent malaria episodes.CONCLUSIONThis study provides evidence that acquisition of IgG antibodies against PfEMP1 variants is ordered and demonstrates that antibodies against CIDRα1 domains are acquired the earliest in children residing in an area of intense, seasonal malaria transmission. Future studies will need to validate these findings in other transmission settings and determine the functional activity of these naturally acquired CIDR variant-specific antibodies.TRIAL REGISTRATIONClinicalTrials.gov NCT01322581.FUNDINGDivision of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH.

Keywords: Adaptive immunity; Immunoglobulins; Immunology; Infectious disease; Malaria.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Study design.

Participants and time points for used for “healthy baseline” and longitudinal analysis.

Figure 2. IgG antibodies against PfEMP1 variants belonging to the A or B/A groups or having the EPCR-binding phenotype are rapidly acquired during childhood.

(A) Hierarchical clustering heatmap showing IgG reactivity to each of the 35 PfEMP1 variants in 680 subjects at enrollment (May 2011 healthy baseline). Clustering was performed using the Ward.D method and the Pearson distance metric. AU refers to arbitrary concentration units, which were calculated by fitting data to a dilutional standard curve of pooled hyperimmune plasma from malaria-exposed Malian adults. (B) IgG reactivity obtained at May 2011 healthy baseline versus age for each PfEMP1 variant (solid lines) or control antigen (dotted lines) with loess fit curves and 95% confidence intervals by P. falciparum PCR status determined at enrollment.

Figure 3. IgG antibodies against CIDRα2-6 variants are acquired more slowly during childhood.

(A) IgG reactivity obtained at May 2011 healthy baseline versus age for each CIDR domain class (dashed lines) or control antigen (dotted lines) with loess fit curves and 95% confidence intervals by P. falciparum PCR status determined at enrollment. (B) Linear portion of plot in A (age range 3 months to 8 years), with regression lines and 95% confidence intervals (see Supplemental Table 2). For comparison, a regression line for all variants together is represented by the black dotted line.

Figure 4. Longitudinal analysis of PfEMP1 variant–specific IgG over multiple malaria seasons.

IgG reactivity specific to PfEMP1 variants was determined for 60 children ages 6 months to 8 years at 5 cross-sectional surveys before the malaria season. (A) Malaria incidence over 5 malaria seasons for 60 children aged 6 months to 2 years; 3–5 years; and 6–8 years (n = 20 per age group). Plus signs along the left margin indicate subjects with asymptomatic P. falciparum parasitemia at enrollment. The bubble area is proportional to parasite density determined at each visit. (B) The number of malaria episodes per 2-week period by age group over 5 malaria seasons. (C) Longitudinal IgG reactivity at 5 cross-sectional surveys in the same children.

Figure 5. Longitudinal acquisition of IgG antibodies against specific PfEMP1 variants is hierarchical.

Using longitudinal data, seropositivity was determined for each variant within each subject at each time point to determine the year of seroconversion. Seroconversion year was then used to generate a matrix representing the number of times that seroconversion for a variant (rows) precedes another variant (column) across all subjects. Consensus orderings were determined by sorting each matrix to minimize the total count of consensus-violating seroconversions. The color scale represents the number of times that 1 seroconversion is observed preceding another seroconversion. Analysis was performed at the level of (A) individual variants, (B) CIDR domain class, (C) upstream sequence group, and (D) binding phenotype. Note that heatmap diagonal is 0 in A by definition, because seroconversion to a specific antigen cannot precede itself. In B–D, diagonal counts indicate seroconversions to 1 class, group, or phenotype that are followed by another distinct member of the same class, group, or phenotype.