- Open Access
Convalescent Plasmodium falciparum-specific seroreactivity does not correlate with paediatric malaria severity or Plasmodium antigen exposure
Malaria Journal volume 17, Article number: 178 (2018)
Antibody immunity is thought to be essential to prevent severe Plasmodium falciparum infection, but the exact correlates of protection are unknown. Over time, children in endemic areas acquire non-sterile immunity to malaria that correlates with development of antibodies to merozoite invasion proteins and parasite proteins expressed on the surface of infected erythrocytes.
A 1000 feature P. falciparum 3D7 protein microarray was used to compare P. falciparum-specific seroreactivity during acute infection and 30 days after infection in 23 children with uncomplicated malaria (UM) and 25 children with retinopathy-positive cerebral malaria (CM). All children had broad P. falciparum antibody reactivity during acute disease. IgM reactivity decreased and IgG reactivity increased in convalescence. Antibody reactivity to CIDR domains of “virulent” PfEMP1 proteins was low with robust reactivity to the highly conserved, intracellular ATS domain of PfEMP1 in both groups. Although children with UM and CM differed markedly in parasite burden and PfEMP1 exposure during acute disease, neither acute nor convalescent PfEMP1 seroreactivity differed between groups. Greater seroprevalence to a conserved Group A-associated ICAM binding extracellular domain was observed relative to linked extracellular CIDRα1 domains in both case groups. Pooled immune IgG from Malawian adults revealed greater reactivity to PfEMP1 than observed in children.
Children with uncomplicated and cerebral malaria have similar breadth and magnitude of P. falciparum antibody reactivity. The utility of protein microarrays to measure serological recognition of polymorphic PfEMP1 antigens needs to be studied further, but the study findings support the hypothesis that conserved domains of PfEMP1 are more prominent targets of cross reactive antibodies than variable domains in children with symptomatic malaria. Protein microarrays represent an additional tool to identify cross-reactive Plasmodium antigens including PfEMP1 domains that can be investigated as strain-transcendent vaccine candidates.
Antibody immunity to Plasmodium falciparum malaria is central to the prevention and clearance of symptomatic disease , but is acquired only after years of exposure . Immunity to malaria is non-sterile, and older children and adults in malaria-endemic areas are frequently parasitaemic, but asymptomatic. Children in sub-Saharan Africa carry the burden of morbidity and mortality associated with P. falciparum infection . It is still unclear why some parasitaemic children develop severe malarial syndromes, including cerebral malaria (CM).
Although not all studies agree , prior studies have reported ‘protective’ antibody responses that differentiate asymptomatic cases from symptomatic malaria [5,6,7]. Most studies have found few differences in antibody profiles along the symptomatic disease spectrum [8,9,10], but tested a limited number of antigens. Newer technologies, including protein microarrays, provide an alternative platform for large-scale evaluation of seroreactivity to thousands of antigens . An initial large P. falciparum proteome-wide array encompassed approximately a quarter of the inferred proteome (2320 proteins encoded by 1204 genes) and identified 491 immunoreactive proteins recognized by sera from a cohort in Mali . A subsequent 824 feature array representing 699 Pf genes was down-selected on the basis of initial array data .
In CM, P. falciparum erythrocyte membrane protein 1 (PfEMP1), a parasite variant surface antigen (VSA) encoded by ~ 60 var genes, is critical for cerebral sequestration [13, 14]. Since antibodies to VSA correlate to protection from symptomatic malaria , children who progress to CM may lack antibodies targeting antigens essential for microvasculature sequestration. Each PfEMP1 protein is comprised of multiple extracellular Duffy-binding like (DBL) and cysteine-rich interdomain region (CIDR) domains that are classified into sequence types (e.g. α, β, γ) and subtypes (e.g. CIDRα1) [15, 16]. The N-terminal head structure (DBL-CIDR) of each PfEMP1 protein is a determinant of PfEMP1-host binding specificity. CIDRα2–6 domains encode CD36 binding properties, and CIDRα1 domains encode endothelial protein C receptor (EPCR) binding properties. CIDRβ/γ/δ domains have unknown binding properties but have been associated with rosetting, which occurs when parasitized red blood cells (pRBCs) bind and aggregate uninfected RBCs . Studies of paediatric malaria implicate EPCR-binding parasites in severe disease [18,19,20,21,22,23,24,25]. Breadth and magnitude of PfEMP1 seroreactivity correlates with age and exposure [26, 27], and antibodies to EPCR-binding CIDRα domains are more abundant than antibodies to other CIDR domains and are likely acquired early in life .
Here, partial proteome microarrays were used to identify and characterize differences in global antibody level, breadth and magnitude of P. falciparum antibody responses, and magnitude of PfEMP1 specific antibody responses between children with uncomplicated malaria (UM) and children with stringently defined CM (Ret + CM; WHO definition + malarial retinopathy) in acute infection and at 30 days of convalescence. A new ~ 1000 feature partial proteome array based upon the 3D7 P. falciparum reference genome that prioritized antigens with relevance to diagnostics and human anti-malarial immunity was used. The antigens represented on this array include vaccine candidates as well as proteins that have been associated with either exposure to P. falciparum or protection from clinical disease. The study results indicate that children with UM and CM have broad seroreactivity to the panel of ~ 1000 P. falciparum antigens and have lowest seroreactivity during acute disease to EPCR-binding “virulent” PfEMP1 antigens represented on the array [17, 25]. Despite exposure to “virulent” PfEMP1 during acute disease, antibody responses to the corresponding antigen in convalescence were not detected using this platform that was generated based upon the reference 3D7 genome.
Patient recruitment and sample collection
During the 2015–2016 malaria seasons, children with malaria at Queen Elizabeth Central Hospital (QECH) in Blantyre, Malawi were recruited to the study (see Additional file 1). UM cases were recruited from the Accident and Emergency Department and included children ages 1–12 years with fever, Blantyre coma score of 5, peripheral P. falciparum parasitaemia, and no overt signs of chronic disease, malnutrition, or progression to severe malaria; all were treated as outpatients. Children with CM were admitted to the Paediatric Research Ward at QECH with clinical, WHO-defined cerebral malaria (parasitaemia, Blantyre coma score ≤ 2, no other identifiable causes of coma) and were 6 months–12 years in age. Only children with CM with malarial retinal abnormalities (Ret + CM) were included. HIV + children were excluded. Cases reported in this study are a subset of those previously reported . Of 38 UM and 57 ret + CM cases, cases were selected for inclusion if they met all of the following criteria: (1) attended 30 day follow-up appointment, (2) HIV non-reactive, (3) successful var qRT-PCR typing from acute infection, and (4) sufficient acute and convalescent plasma for the study. Comparable numbers of cases from each group were compared. Age and sex distribution were comparable to the larger group  and were not statistically different between the CM and UM group (Table 1).
Quantification of plasma PfHRP2 levels
PfHRP2 was quantified in patient plasma by ELISA . Samples were diluted using phosphate-buffered saline and plated in duplicate onto plates coated with anti-HRP2 antibody (Cellabs, Brookvale, Australia). Recombinant PfHRP2 was used to generate a standard curve for quantification. Patient samples with PfHRP2 outside of the linear range of the standard curve were diluted further and reanalysed.
Direct quantification of parasite DNA in plasma
Cell-free, parasite DNA was measured directly from acute patient plasma by qPCR using a primer–probe set targeting Plasmodium 18S rRNA . Amplifications were performed in duplicate on an Applied Biosystems 7300 PCR system. Standard curves were generated from harvested parasite cultures of known parasitaemia and used to calculate parasite genomes in plasma samples.
Determination of total Immunoglobulin plasma concentrations
Total plasma IgG and IgM were measured in acute and follow-up (FU) samples using human isotype-specific radial immunodiffusion kits (The Binding Site Ltd.). Plasma was heat-killed prior to use, and all samples were assayed in duplicate.
Protein microarray development and chip design
A partial proteome microarray with 1000 P. falciparum protein features (Pf1000, 3rd generation) was developed at Antigen Discovery, Inc. (ADI, Irvine, CA, USA). Proteins were selected based on internal R&D using P. falciparum full proteome microarrays probed with samples collected from subjects during naturally and experimentally acquired immunity (unpublished). Briefly, proteins were selected based on a prioritization scheme as follows: (1) a curated list of P. falciparum proteins prominently featured in the scientific literature and established targets of P. falciparum candidate vaccines and diagnostics, plus the full repertoire of 3D7 PfEMP1, rifin and stevor proteins, (2) top hits from previous studies with protein arrays that identify correlates of protection or exposure, (3) a ranking of proteins based on ADI internal full proteome microarray data. The criteria for ranking proteins with empirical full proteome data employed a scoring system that prioritized: seroprevalence in either naturally immune populations (> 60%) or pre-erythrocytic experimental immunity (> 20%); predicted transmembrane domains; predicted signal peptides; gene ontology (GO) terms for Function, Process and Component that were broadly related to parasite and host cell surfaces, host-parasite interactions and host immune system. Proteins were selected according to the prioritization scheme until a total of 1000 targets were selected, accounting for fragmented proteins. The 1000 full-length or partial P. falciparum proteins represent 762 genes from P. falciparum reference strain 3D7, including 61 PfEMP1s, vaccine candidate proteins, and 176 conserved Plasmodium proteins of unknown function. Protein microarrays were fabricated as described in Additional file 2.
Protein microarray sample probing
Antibody reactivity (log2 scale) was determined relative to signal of expression system controls (i.e. “Background”), with signal intensity greater than or equal to 2× the background signal considered seropositive  (i.e. R ≥ 1). Antibody breadth, represented as breadth scores, was calculated as the sum of seropositive responses per individual. Antibody magnitude (levels) was defined as the log2-transformed ratio of target signal to background signal, represented by the normalized signal intensity. Antibody “Deltas” were calculated by subtracting normalized antibody levels at 30-day convalescence from acute antibody levels. Samples were probed and analyzed as described in Additional file 2.
PfEMP1 specific annotation and analysis
Pf1000 protein microarray PfEMP1 antigen spots were annotated using VarDom 1.0 Server  to predict domain and total protein architecture. Each PfEMP1 on the array was annotated for gene group, encoded domains and domain cassettes, the presence of the ATS (acidic terminal sequence), and binding phenotype (EPCR, CD36, ICAM-1, or rosetting) [17, 18, 32, 33]. For PfEMP1 proteins, the head structure, C terminal extracellular domains, and ATS were arrayed in separate spots for each PfEMP1, enabling independent assessment of antibody reactivity. PfEMP1 domain annotations for Pf1000 antigens (e.g. spots) on the array are outlined in Additional file 3. All annotations correspond to the DBLα-CIDRα head structure domains except for the ICAM-1 binding domains. var gene expression was determined by qRT-PCR from the peripheral blood using a panel of PfEMP1 specific primers  with an expression Tu value > 8 indicating positive expression .
Hyperimmune IgG and naïve sera were probed on the array as controls. Hyperimmune IgG was pooled from 834 HIV and HBsAg seronegative Malawian adults , and the naïve control was pooled from 30 malaria-naïve adults from the United States. Both controls were probed at 1:100 (20% Escherichia coli lysate; GenScript). Following dilution, the antibody concentration of the hyperimmune serum was 100 μg/ml.
Statistical analysis was performed using Stata 12.1 or Prism 7.0 for Macintosh. Statistical differences were determined by Wilcoxon rank sum (continuous, unpaired variables), Wilcoxon signed rank sum (continuous, paired variables) or Chi squared test (categorical variables). Analysis of P. falciparum antibodies was performed in R version 3.1.1  and Graphpad Prism (GraphPad Software, La Jolla California, USA), as detailed in Additional file 2. Statistical significance was assessed using an α level < 0.05. P values from multiple comparisons were corrected by the false discovery rate approach (FDR) .
Children with CM have high parasite burden and clinical measures reflecting severe disease
A total of 25 children with Ret + CM and 23 with UM were included in the study (Table 1). Both groups had similar age and sex distributions. Children with Ret + CM had significantly elevated levels of PfHRP2  and cell-free plasma parasite DNA (‘cfPf DNA’)  (both P < 0.001), two biomarkers of parasite burden. PfHRP2 predicts Ret + CM and progression to severe disease [29, 38], and the combination of PfHRP2 and cell-free DNA was the best correlate of severe disease  (Fig. 1a). Children with Ret + CM had lower hemoglobin levels (Median: 7.4 vs. 9.6 g/dL, P < 0.001) and lower platelet counts (P = 0.03).
Children with CM were exposed to a more diverse/parasite population during acute infection than children with UM
As reported previously , the PfEMP1 peripheral blood transcript profile data was available for all paediatric malaria cases described here. Children with Ret + CM were exposed to greater parasite diversity (more variant domains) than children with UM (Median number of domains amplified: 21 vs. 11, P < 0.001) (Table 1).
Total IgG is reduced in acute Ret + CM, but not in UM, and normalizes by 30 days post infection
Ret + CM cases had significantly reduced levels of total IgG relative to children with UM during acute infection (P < 0.001), which normalized in convalescence (Fig. 1b). UM IgG levels were similar to healthy sub-Saharan African children  and did not differ across time points. At both time points, total IgG levels correlated significantly with age (acute, rho = 0.50, P < 0.001; convalescent, rho = 0.47, P < 0.001) (see Additional file 4A).
Children with Ret + CM and UM have similar malaria-specific antibody profiles
Both Ret + CM and UM cases had a broad repertoire of P. falciparum-specific antibody during acute infection (Fig. 2a) with no significant differences in the magnitude of IgG reactivity to any of the P. falciparum antigens (Fig. 2b). There were no significant differences in antibody breadth (Fig. 2a) by case severity in convalescence. Age and sex had minimal impact on P. falciparum-specific antibody levels (see Additional file 4B, C). It should be noted that the children in this study were younger (Table 1) than most studies that have examined P. falciparum-specific antibody profiles of cohorts of symptomatic and asymptomatic children. Children with mild symptomatic malaria were compared to children with clinical CM, a syndrome most common in children under 5 years of age.
Helb et al. reported antigens predictive of recent malaria exposure in Ugandan children using protein microarray . Eight of the 10 antigens included in their exposure model were present on the Pf1000 microarray. For six of the eight antigens, high seroprevalence and reactivity in acute infection with a significant increase in reactivity at 30 days of convalescence was observed (Table 2; For controls see Additional file 5).
Reactivity to P. falciparum vaccine candidates and candidate protective antigens was also examined [5, 42]. In acute samples, high seroprevalence and reactivity was observed with significantly greater reactivity at 30 days of convalescence for MSP1, EBA175, and LSA3 (P < 0.05, < 0.01, < 0.01, respectively) (Table 2). Seroprevalence to CSP, the target of the RTS, S vaccine, was just below 50%, with moderate reactivity. When seroreactivity to antigens previously identified as potential correlates of protection from symptomatic disease using protein microarrays  was compared, no differences were observed by case severity.
PfEMP1 (± ATS) specific reactivity in acute infection is similar between UM and CM cases
Next, the relationship/correlation between PfEMP1 antibody reactivity in acute infection and differences in parasite burden or clinical status was assessed. The ATS portion of each PfEMP1 was expressed on a different spot on the array than the extracellular domains, and each 3D7 PfEMP1 was represented by 2 or 3 different spots that encompassed non-overlapping segments of the coding region. Unlike extracellular domains, the intracellular acidic terminal sequence (ATS) portion of each PfEMP1 protein is highly conserved. Therefore, all analyses examining antibody reactivity to PfEMP1 proteins were performed with and without the ATS.
By case severity, no significant differences in the breadth and magnitude of IgG and IgM responses to PfEMP1 specific antigens were observed in acute infection (Figs. 3b, c). After excluding reactivity to the ATS, the median breadth score to PfEMP1 antigens in acute infection for both Ret + CM and UM cases was ~ 30/60 (Fig. 3b).
All children (UM and Ret + CM) seroreact to PfEMP1 domains with apparent high reactivity to ‘non-virulent’ PfEMP1 domains and low reactivity to ‘virulent’ domains in acute infection
As no significant differences in reactivity by case severity (UM vs. Ret + CM) were observed, all cases were pooled for subsequent PfEMP1 specific analyses. PfEMP1 antigens with the highest reactivity in acute infection were highly conserved ATS regions or domains associated with CD36 binding (Fig. 3d). The ATS region is intracellular, and reactivity to this conserved region is a measure of exposure and not protection. The least reactive antigens during acute infection were DC8 and group A antigens associated with EPCR-binding or rosetting phenotypes, both associated with severe disease in paediatric malaria (Fig. 3d). Reactivity across all binding groups was observed in acute infection but to a lesser extent than observed in control sera pooled from hyperimmune (Malawian) IgG (see Additional file 6).
Children exposed to specific PfEMP1 during acute infection do not mount specific IgG responses in convalescence that are detectable by protein microarray
Upon comparing longitudinal changes in Ig reactivity to all Pf and PfEMP1-specific antigens, both overall Pf and PfEMP1 (± ATS) specific IgM responses decline significantly in convalescence for all cases (Fig. 4a). While overall Pf IgG responses increase significantly in convalescence, PfEMP1 (± ATS) specific IgG responses do not (Fig. 4a).
When the PfEMP1 antigens were classified on the basis of their known or predicted binding phenotype [15, 43], 5 EPCR-binding antigens, 2 rosetting antigens, 45 predicted CD36 binding antigens were represented on the array. Additional file 7 shows total responders and responders by case severity during acute infection. The highest breadth scores were observed for rosetting antigens. UM and Ret + CM children had a lower magnitude of response to EPCR-binding antigens than to either CD36-binding or rosetting antigens (Fig. 4b). A Group A ICAM-1 binding motif is often linked to EPCR binding , and there was a similar magnitude of antibody response to these domains (Fig. 4b) between groups despite a significant difference in exposure to the corresponding antigen/motif during acute infection (Percent exposed: 52.6% vs. 21.7%, P = 0.04) (Table 1). The total number of responders (seroprevalence) to each CIDR antigen was variable but not significantly different across binding phenotype group (Additional file 6). Of note, seroprevalence to the Group A-ICAM-1 binding domain was greater than to linked CIDRα1 domains suggesting greater cross reactivity of antibodies to ICAM-1-binding domains (Table 3 and Additional file 8).
Exposure to PfEMP1 domains during acute infection was determined using relative qRT-PCR profiling of var transcripts in the peripheral blood of each case (both UM and Ret + CM) as previously described [19, 25, 32, 44]. As no significant differences in seroreactivity to Pf1000 antigens by case severity (UM vs. Ret + CM) was detected, PfEMP1 exposed cases were assessed altogether for antibody reactivity to PfEMP1 expressed during acute infection focusing upon CIDR domains across three major binding phenotypes. A substantial antibody response at follow-up in children exposed to a given PfEMP1 antigen was not detected (Table 3; see unexposed individuals in Additional file 8). A single exposure model was generated for each PfEMP1 domain with PfEMP1 expression data available. Exposure to a PfEMP1 antigen in acute infection had no correlation with the antibody level to that antigen in convalescence (Fig. 4c). Age and sex were the only variables that significantly affected convalescent (or delta) antibody levels, and both variables affected IgM but not IgG levels (see Additional file 9). Control adult immune IgG seroreactivity was greater than that observed in children (see Additional files 3, 5).
Differences in antibody repertoire between UM and CM cases could explain why some children with P. falciparum infection progress to severe disease. Despite evidence of general suppression of antibody response in acute Ret + CM (Fig. 1), similar robust reactivity to a broad range of P. falciparum antigens was seen in UM and CM cases alike (Fig. 2). Reactivity to unfractionated malaria antigen and specific antigens of interest (e.g. MSP-1, CSP) by EIA or ELISA in children from the Gambia showed that evidence of prior exposure did not prevent progression to severe disease . Here, a large proportion of the P. falciparum proteome was assayed and no differences in the breadth or magnitude of P. falciparum reactivity by case severity were found (UM vs. Ret + CM; Fig. 2). The antigens on the array were selected based upon their interest as vaccine or diagnostic candidates as well as seroreactivity in both natural and experimental infection and enabled measurement of antibody responses during acute disease and in convalescence. While it cannot be definitively ascertained whether antibody present during acute disease reflects current infection or prior infection, a similar pattern in IgM reactivity that is classically indicative of acute infection was observed in the UM and CM groups across both time points, suggesting that the level of prior exposure and generation of antibody to P. falciparum antigens in Ret + CM cases parallels that of children with UM.
In a study employing an 824 feature P. falciparum protein microarray, antibody to a subset of antigens (including MSP1, MSP2, LSA1, LSA3, Pf70, and PfEMP1; Table 2) was associated with protection from symptomatic disease in sub-Saharan African children . Many of these antigens are current or previous vaccine candidates [42, 46,47,48] and have inconsistently been reported as protective in the literature [4, 7, 49, 50]. Independent of disease severity, high seroreactivity to this group of antigens was identified in children with symptomatic malaria (Table 2). Thus, high seroreactivity is common across children living in endemic regions, such as Malawi, and may be a marker of prior (or repeated) P. falciparum exposure. Dent et al. followed a cohort longitudinally, monitoring seroreactivity pre- and post- infection , as did Crompton et al. . Longitudinal studies suggest that the antibody response of younger children to malaria antigens including PfEMP1 is short-lived [12, 26]. The antibody response to P. falciparum appears to increase with age, and it is not yet clear what factors promote antibody stability and which antibodies are important for the prevention of symptomatic disease versus for prevention of progression to severe manifestations such as CM. Taken together with the results presented here, the data suggests that antibody against some of these targets may be more important for the prevention of symptomatic disease than for the progression to severe manifestations, such as CM.
With regard to PfEMP1 specific antigens, and in agreement with previous studies  , magnitude of reactivity to the highly conserved ATS proteins was greatest. As the ATS is an intracellular domain of PfEMP1, reactivity to this region should be regarded as a surrogate of exposure rather than protection. The CIDR domains are the most hypervariant of all PfEMP1 domains/regions, and seroreactivity to CIDR domains in the patient population described here was highly variable across sequence type (i.e. α/β/γ) and subtype (e.g. CIDRα1) (Fig. 3 and Additional file 7). Within the subset of CIDR domains, reactivity to PfEMP1 domains associated with EPCR-binding was lowest and reactivity to those predicted to bind CD36 was highest . The breadth and magnitude of response to all EPCR-binding CIDR domains on the microarray was lower in acute infection than that to CD36-binding or rosetting CIDR domains (Fig. 3d). It is important to note that while the strongest seroreactivity signal observed corresponded to CD36-binding CIDR domains, there are more probes that encode this binding property on the array (45 vs. 5 EPCR-binding and 2 rosetting) yielding a higher likelihood of measuring a maximum response as a central measure of IgG reactivity. Abundance of features does not fully explain our results, as there are over twice as many EPCR-binding antigens on the array as rosetting antigens, yet the seroreactivity to EPCR-binding antigens remains lowest. The PfEMP1 antigens on the Pf1000 array that were annotated for this study contain two adjacent domains: DBL-CIDR head structure (N-terminus) domains or in the case of ICAM-1 binding PfEMP1 antigens, DBL–DBL domains (see Additional file 3). Further studies of the specificity of seroreactivity observed for individual binding domains or to variants from other sequenced strains can be pursued readily using the same platform, and the consistent annotation schema provide an initial view of antibody responses to different classes of PfEMP1.
Seroreactivity to the newly identified var A ICAM-1 binding motif (2 spots on the array)  was found to parallel seroreactivity to EPCR-binding head structures (Fig. 4b). EPCR-binding PfEMP1 often also encode ICAM-binding motifs (Table 3 and Additional file 7). While the magnitude of seroreactivity to this ICAM-1 binding motif was lower than that to CD36-binding and rosetting PfEMP1, a greater fraction of children had detectable antibodies to this motif compared with PfEMP1 extracellular domains associated with EPCR-binding. Exposed children and adults in malaria endemic regions have reactive, inhibitory IgG to this conserved ICAM-1 binding motif, possibly reflecting the greater conservation of the ICAM-1 binding motif than the EPCR-binding CIDR . Here, the data demonstrating greater seroprevalence in children to this motif than to linked CIDR domains is consistent with those results. In adult hyperimmune sera, seroreactivity to ICAM-1 domains on the array was also greater than seroreactivity to EPCR domains (see Additional file 6).
Neither a consistent response to the corresponding antigen in children exposed to a specific PfEMP1 variant in acute infection or a general significant increase in PfEMP1 IgG antibody at 30 days convalescence were detected. Prior studies employing in vitro functional assays demonstrated that children seroconvert against the VSA of the isolate seen in acute infection following disease resolution  and subsequent studies suggest that the major VSA recognized by immune sera is PfEMP1 . An array study performed in Papua New Guinea showed that the anti-PfEMP1 antibody response was more pronounced in children with detectable parasitaemia . It is possible that the study subjects made PfEMP1-specific antibody in response to acute infection, but this antibody was not maintained in convalescence.
The array was developed using the 3D7 proteome, which lacks some EPCR-binding proteins. 3D7 PfEMP1 proteins may lack sufficient homology to Malawian PfEMP1 to be recognized by specific antibody. The PfEMP1 family is hypervariable with less than 50% sequence identity found in homologous domains of homologous genes [15, 16]. Strain-specific polymorphism of MSP1 and AMA1, vaccine antigens that are more conserved than PfEMP1, is proposed to be a major limiting factor in development of protective antibodies . Additionally, incomplete or inaccurate protein folding for certain PfEMP1 antigens on the array may also contribute to the observed differences across studies, especially since many P. falciparum-specific antibodies recognize conformational epitopes. The cell free in vitro expression system and direct printing protocol used in production of the microarray employed here involves minimal manipulation of expressed proteins and no denaturation steps, but correct conformation of recombinant protein is not readily verifiable.
Seroreactivity in both acute infection and convalescence to antigens from all PfEMP1 binding phenotype groups was identified in UM and CM cases. Seroreactivity to EPCR domains is acquired relatively early in life  and may explain why older children are less likely to develop CM. Children with symptomatic malaria had a lower level of seroreactivity to EPCR-binding P. falciparum antigens than to other PfEMP1 antigens. The median age of our subjects was 3–4 years, whereas peak EPCR-binding domain antibody prevalence was detected in children aged 8–10 years . Although the rate of antibody acquisition is dependent upon a number of factors including exposure to Plasmodium, studies that have examined acquisition of “protective” antibody responses to VSA or Plasmodium antigens, significant protection begins to appear in children 8–10 years of age [12, 52]. Children appear to be at risk for symptomatic malaria until they are older than the children described here. Pooled immune IgG from Malawian adults  (see Additional file 6) was seroreactive across all PfEMP1 binding groups. Multiple exposures are likely required to develop significant cross-reactive antibody to hypervariable extracellular PfEMP1 domains. An array that encompasses Malawian PfEMP1 sequences, as reported for the Papua New Guinea study , may be useful to determine when children with malaria develop antibodies to local PfEMP1 variants.
Antibody reactivity to PfEMP1 binding groups should be compared with caution and the findings would be strengthened by supplemental functional assays and comparison with other platforms . The high throughput microarray provides a cost-effective platform to evaluate reactivity to a large number of P. falciparum antigens using small volumes of sample and is readily amenable to simultaneous querying of reactivity of sera to proteins from strains of disparate genetic backgrounds, as recently demonstrated for AMA1 and MSP1 . Due to recent advances in HIV vaccine efforts, there has been renewed interest in vaccine development of a number of infectious diseases associated with chronic antigen exposure. For many of these diseases, including malaria, there is often a robust host antibody response but very slow acquisition of disease-modifying immunity. Given progress in HIV vaccine research, there has been renewed interest in identifying strain-transcendent protective epitopes.
Despite differences in clinical presentation and parasite burden, no significant differences in breadth or magnitude of P. falciparum seroreactivity between children with Ret + CM and children with UM were found using a 1000 feature P. falciparum protein microarray. This suggests that all children had similar levels of prior exposure and supports the notion that children experience uncomplicated malaria episodes in between severe malaria episodes . Children presenting with acute malaria have significant breadth and magnitude of P. falciparum antibody, indicating these antibodies do not preclude progression to symptomatic malaria or more severe disease. Conserved domains of PfEMP1 are more prominent targets of cross reactive antibodies than variable domains in children with symptomatic malaria. The key determinants required for formation of protective antibodies and surrogates of antibody protection require further investigation. Studies of P. falciparum-specific antibody repertoires that incorporate a broader range of strain-specific PfEMP1 antigens with functional assays to characterize protective antibody function will improve the currently incomplete understanding of the role of antibody immunity in prevention of symptomatic paediatric malaria.
Cohen S, McGregor IA, Carrington S. Gamma-globulin and acquired immunity to human malaria. Nature. 1961;192:733–7.
McGregor IA. Mechanisms of acquired immunity and epidemiological patterns of antibody responses in malaria in man. Bull World Health Organ. 1974;50:259–66.
WHO. World malaria report 2016. Geneva: World Health Organization; 2016.
Dodoo D, Theander TG, Kurtzhals JA, Koram K, Riley E, Akanmori BD, et al. Levels of antibody to conserved parts of Plasmodium falciparum merozoite surface protein 1 in Ghanaian children are not associated with protection from clinical malaria. Infect Immun. 1999;67:2131–7.
Dent AE, Nakajima R, Liang L, Baum E, Moormann AM, Sumba PO, et al. Plasmodium falciparum protein microarray antibody profiles correlate with protection from symptomatic malaria in Kenya. J Infect Dis. 2015;212:1429–38.
Nixon CP, Friedman J, Treanor K, Knopf PM, Duffy PE, Kurtis JD. Antibodies to rhoptry-associated membrane antigen predict resistance to Plasmodium falciparum. J Infect Dis. 2005;192:861–9.
Polley SD, Conway DJ, Cavanagh DR, McBride JS, Lowe BS, Williams TN, et al. High levels of serum antibodies to merozoite surface protein 2 of Plasmodium falciparum are associated with reduced risk of clinical malaria in coastal Kenya. Vaccine. 2006;24:4233–46.
Okech B, Mujuzi G, Ogwal A, Shirai H, Horii T, Egwang TG. High titers of IgG antibodies against Plasmodium falciparum serine repeat antigen 5 (SERA5) are associated with protection against severe malaria in Ugandan children. Am J Trop Med Hyg. 2006;74:191–7.
Dobano C, Rogerson SJ, Mackinnon MJ, Cavanagh DR, Taylor TE, Molyneux ME, et al. Differential antibody responses to Plasmodium falciparum merozoite proteins in Malawian children with severe malaria. J Infect Dis. 2008;197:766–74.
Tebo AE, Kremsner PG, Piper KP, Luty AJ. Low antibody responses to variant surface antigens of Plasmodium falciparum are associated with severe malaria and increased susceptibility to malaria attacks in Gabonese children. Am J Trop Med Hyg. 2002;67:597–603.
Doolan DL, Aguiar JC, Weiss WR, Sette A, Felgner PL, Regis DP, et al. Utilization of genomic sequence information to develop malaria vaccines. J Exp Biol. 2003;206:3789–802.
Crompton PD, Kayala MA, Traore B, Kayentao K, Ongoiba A, Weiss GE, et al. A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray. Proc Natl Acad Sci USA. 2010;107:6958–63.
David PH, Hommel M, Miller LH, Udeinya IJ, Oligino LD. Parasite sequestration in Plasmodium falciparum malaria: spleen and antibody modulation of cytoadherence of infected erythrocytes. Proc Natl Acad Sci USA. 1983;80:5075–9.
Howard RJ, Barnwell JW. Roles of surface antigens on malaria-infected red blood cells in evasion of immunity. Contemp Top Immunobiol. 1984;12:127–200.
Rask TS, Hansen DA, Theander TG, Gorm Pedersen A, Lavstsen T. Plasmodium falciparum erythrocyte membrane protein 1 diversity in seven genomes–divide and conquer. PLoS Comput Biol. 2010;6:e1000933.
Smith JD, Subramanian G, Gamain B, Baruch DI, Miller LH. Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family. Mol Bioch Parasitol. 2000;110:293–310.
Rowe JA, Moulds JM, Newbold CI, Miller LHP. falciparum rosetting mediated by a parasite-variant erythrocyte membrane protein and complement-receptor 1. Nature. 1997;388:292–5.
Turner L, Lavstsen T, Berger SS, Wang CW, Petersen JE, Avril M, et al. Severe malaria is associated with parasite binding to endothelial protein C receptor. Nature. 2013;498:502–5.
Lavstsen T, Turner L, Saguti F, Magistrado P, Rask TS, Jespersen JS, et al. Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children. Proc Natl Acad Sci USA. 2012;109:E1791–800.
Abdi AI, Kariuki SM, Muthui MK, Kivisi CA, Fegan G, Gitau E, et al. Differential Plasmodium falciparum surface antigen expression among children with malarial retinopathy. Sci Rep. 2015;5:18034.
Rottmann M, Lavstsen T, Mugasa JP, Kaestli M, Jensen AT, Muller D, et al. Differential expression of var gene groups is associated with morbidity caused by Plasmodium falciparum infection in Tanzanian children. Infect Immun. 2006;74:3904–11.
Bertin GI, Lavstsen T, Guillonneau F, Doritchamou J, Wang CW, Jespersen JS, et al. Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1 is associated with cerebral malaria in Benin. PLoS ONE. 2013;8:e68368.
Jensen AT, Magistrado P, Sharp S, Joergensen L, Lavstsen T, Chiucchiuini A, et al. Plasmodium falciparum associated with severe childhood malaria preferentially expresses PfEMP1 encoded by group A var genes. J Exp Med. 2004;199:1179–90.
Jespersen JS, Wang CW, Mkumbaye SI, Minja DT, Petersen B, Turner L, et al. Plasmodium falciparum var genes expressed in children with severe malaria encode CIDRalpha1 domains. EMBO Mol Med. 2016;8:839–50.
Kessler A, Dankwa S, Bernabeu M, Harawa V, Danziger SA, Duffy F, et al. Linking EPCR-binding PfEMP1 to brain swelling in pediatric cerebral malaria. Cell Host Microbe. 2017;22:601–14.
Barry AE, Trieu A, Fowkes FJ, Pablo J, Kalantari-Dehaghi M, Jasinskas A, et al. The stability and complexity of antibody responses to the major surface antigen of Plasmodium falciparum are associated with age in a malaria endemic area. Mol Cell Proteomics. 2011;10(M111):008326.
Travassos MA, Niangaly A, Bailey JA, Ouattara A, Coulibaly D, Laurens MB, et al. Seroreactivity to Plasmodium falciparum erythrocyte membrane protein 1 intracellular domain in malaria-exposed children and adults. J Infect Dis. 2013;208:1514–9.
Turner L, Lavstsen T, Mmbando BP, Wang CW, Magistrado PA, Vestergaard LS, et al. IgG antibodies to endothelial protein C receptor-binding cysteine-rich interdomain region domains of Plasmodium falciparum erythrocyte membrane protein 1 are acquired early in life in individuals exposed to malaria. Infect Immun. 2015;83:3096–103.
Fox LL, Taylor TE, Pensulo P, Liomba A, Mpakiza A, Varela A, et al. Histidine-rich protein 2 plasma levels predict progression to cerebral malaria in Malawian children with Plasmodium falciparum infection. J Infect Dis. 2013;208:500–3.
Kamau E, Tolbert LS, Kortepeter L, Pratt M, Nyakoe N, Muringo L, et al. Development of a highly sensitive genus-specific quantitative reverse transcriptase real-time PCR assay for detection and quantitation of Plasmodium by amplifying RNA and DNA of the 18S rRNA genes. J Clin Microbiol. 2011;49:2946–53.
Campo JJ, Aponte JJ, Skinner J, Nakajima R, Molina DM, Liang L, et al. RTS, S vaccination is associated with serologic evidence of decreased exposure to Plasmodium falciparum liver- and blood-stage parasites. Mol Cell Proteomics. 2015;14:519–31.
Lennartz F, Adams Y, Bengtsson A, Olsen RW, Turner L, Ndam NT, et al. Structure-guided identification of a family of dual receptor-binding PfEMP1 that is associated with cerebral malaria. Cell Host Microbe. 2017;21:403–14.
Baruch DI, Ma XC, Singh HB, Bi X, Pasloske BL, Howard RJ. Identification of a region of PfEMP1 that mediates adherence of Plasmodium falciparum infected erythrocytes to CD36: conserved function with variant sequence. Blood. 1997;90:3766–75.
Taylor TE, Molyneux ME, Wirima JJ, Borgstein A, Goldring JD, Hommel M. Intravenous immunoglobulin in the treatment of paediatric cerebral malaria. Clin Exp Immunol. 1992;90:357–62.
Team RC. R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2014.
Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B. 1995;57:289–300.
Dondorp AM, Desakorn V, Pongtavornpinyo W, Sahassananda D, Silamut K, Chotivanich K, et al. Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2. PLoS Med. 2005;2:e204.
Seydel KB, Fox LL, Glover SJ, Reeves MJ, Pensulo P, Muiruri A, et al. Plasma concentrations of parasite histidine-rich protein 2 distinguish between retinopathy-positive and retinopathy-negative cerebral malaria in Malawian children. J Infect Dis. 2012;206:309–18.
Imwong M, Woodrow CJ, Hendriksen IC, Veenemans J, Verhoef H, Faiz MA, et al. Plasma concentration of parasite DNA as a measure of disease severity in falciparum malaria. J Infect Dis. 2015;211:1128–33.
Obiandu C, Okerengwo AA, Dapper DV. Levels of serum immunoglobulins in apparently healthy children and adults in Port Harcourt, Nigeria. Niger J Physiol Sci. 2013;28:23–7.
Helb DA, Tetteh KK, Felgner PL, Skinner J, Hubbard A, Arinaitwe E, et al. Novel serologic biomarkers provide accurate estimates of recent Plasmodium falciparum exposure for individuals and communities. Proc Natl Acad Sci USA. 2015;112:E4438–47.
Crompton PD, Pierce SK, Miller LH. Advances and challenges in malaria vaccine development. J Clin Invest. 2010;120:4168–78.
Bernabeu M, Danziger SA, Avril M, Vaz M, Babar PH, Brazier AJ, et al. Severe adult malaria is associated with specific PfEMP1 adhesion types and high parasite biomass. Proc Natl Acad Sci USA. 2016;113:E3270–9.
Mkumbaye SI, Wang CW, Lyimo E, Jespersen JS, Manjurano A, Mosha J, et al. The severity of Plasmodium falciparum infection is associated with transcript levels of var genes encoding endothelial protein c receptor-binding P. falciparum erythrocyte membrane protein 1. Infect Immun. 2017;85:e00841–008516.
Erunkulu OA, Hill AV, Kwiatkowski DP, Todd JE, Iqbal J, Berzins K, et al. Severe malaria in Gambian children is not due to lack of previous exposure to malaria. Clin Exp Immunol. 1992;89:296–300.
Cummings JF, Spring MD, Schwenk RJ, Ockenhouse CF, Kester KE, Polhemus ME, et al. Recombinant Liver Stage Antigen-1 (LSA-1) formulated with AS01 or AS02 is safe, elicits high titer antibody and induces IFN-gamma/IL-2 CD4+ T cells but does not protect against experimental Plasmodium falciparum infection. Vaccine. 2010;28:5135–44.
Daubersies P, Thomas AW, Millet P, Brahimi K, Langermans JA, Ollomo B, et al. Protection against Plasmodium falciparum malaria in chimpanzees by immunization with the conserved pre-erythrocytic liver-stage antigen 3. Nat Med. 2000;6:1258–63.
Genton B, Betuela I, Felger I, Al-Yaman F, Anders RF, Saul A, et al. A recombinant blood-stage malaria vaccine reduces Plasmodium falciparum density and exerts selective pressure on parasite populations in a phase 1-2b trial in Papua New Guinea. J Infect Dis. 2002;185:820–7.
Fowkes FJ, Richards JS, Simpson JA, Beeson JG. The relationship between anti-merozoite antibodies and incidence of Plasmodium falciparum malaria: a systematic review and meta-analysis. PLoS Med. 2010;7:e1000218.
Richards JS, Arumugam TU, Reiling L, Healer J, Hodder AN, Fowkes FJ, et al. Identification and prioritization of merozoite antigens as targets of protective human immunity to Plasmodium falciparum malaria for vaccine and biomarker development. J Immunol. 2013;191:795–809.
Marsh K, Howard RJ. Antigens induced on erythrocytes by P. falciparum: expression of diverse and conserved determinants. Science. 1986;231:150–3.
Chan JA, Howell KB, Reiling L, Ataide R, Mackintosh CL, Fowkes FJ, et al. Targets of antibodies against Plasmodium falciparum-infected erythrocytes in malaria immunity. J Clin Invest. 2012;122:3227–38.
Bailey JA, Pablo J, Niangaly A, Travassos MA, Ouattara A, Coulibaly D, et al. Seroreactivity to a large panel of field-derived Plasmodium falciparum apical membrane antigen 1 and merozoite surface protein 1 variants reflects seasonal and lifetime acquired responses to malaria. Am J Trop Med Hyg. 2015;92:9–12.
Goncalves BP, Huang CY, Morrison R, Holte S, Kabyemela E, Prevots DR, et al. Parasite burden and severity of malaria in Tanzanian children. N Engl J Med. 2014;370:1799–808.
AK, JC, and KK conceptualized and designed the study. AK and VH collected and processed patient samples. AK performed the experiments. AK and JC performed the formal data analysis, and WBM supervised the statistical analysis. WLM, SR, and KS oversaw the malaria cohorts. AK, JC, and KK wrote the manuscript with input from all authors. KS and KK supervised all work. All authors read and approved the final manuscript.
We thank all patients and their families that made this study possible. We also acknowledge the Blantyre Malaria Project’s clinical and laboratory staff and the Malawi Liverpool Wellcome Trust clinical staff, particularly Patricia Phula, for invaluable assistance with sample collection/processing and data sharing/management. We would like to acknowledge the staff and management at Antigen Discovery Inc. for their invaluable assistance with the microarray technology and analysis. Specifically, we thank Arlo Randall for technical assistance with the probing plan design and quality control measures. We would like to thank Chris Plowe, Miriam Laufer, Jason Bailey, and Mark Travassos for advice in the initial phases of this project, Alister Craig for immune IgG, Liise-anne Pirofski for advice about antibody assays and assistance with interpretation of data, and Phil Felgner for expert consultation on protein microarray experimental design throughout the study.
The authors report no conflicts of interest. J.J.C. is an employee of Antigen Discovery, Inc., which produces and commercializes proteome microarrays.
Full dataset and array design can be obtained from KK (firstname.lastname@example.org).
Ethics approval and consent to participate
The study was approved by the institutional review boards at the University of Malawi College of Medicine, Michigan State University, and the Albert Einstein College of Medicine. Informed consent was obtained from the parent or guardian of all study participants prior to enrollment.
The study was made possible by the following Grants: Grant 1061993 from the National Health and Medical Research Council of Australia (SR) and U19AI089683 from the NIH (KBS). This research was also supported by the Michigan State University College of Osteopathic Medicine (KBS), the NIH National Center for Advancing Translational Science (NCATS) Einstein-Montefiore CTSA TL1TR001072 (AK), and the Burroughs Wellcome Fund (AK).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Consort diagram of patient recruitment and study design. All cases were recruited from QECH in Blantyre, Malawi. Cases were included in the study if they attended their assigned 30-day follow-up appointment and fit the comparable age and sex distributions (for adjusted comparison) between groups.
Supplemental methods for array design, probing, and analysis.
Annotations of PfEMP1/var genes and domains encoded.
Assessment of P. falciparum and total Ig levels by age and sex. (a) Spearman correlations comparing age vs. acute total IgG level, age vs. 30-day convalescent total IgG level, and total IgG levels across timepoints show significant linear trends. (b) Volcano plots comparing the effect of age (> 5 years/≤ 5 years) on IgG, IgM seroreactivity to P. falciparum and PfEMP1 antigens indicates that for IgG in acute infection, age affects the magnitude of seroreactivity to a few P. falciparum antigens and a general non-significant trend for higher antibodies to all proteins in older children during acute infection (CM + UM total n = 48). (c) Volcano plots of inverse unadjusted P-values (y-axis) and linear regression effect estimates (x-axis) for comparing the effect of sex (male/female) on IgG, IgM seroreactivity to P. falciparum and PfEMP1 antigens indicate that sex does not have a significant effect on seroreactivity in our patient population in acute infection or convalescence, although non-significant trends for higher overall P. falciparum-specific IgG and IgM in males during acute infection and lower PfEMP1-specific IgG in males for both time points could be observed (CM + UM total n = 48). Dashed lines represent unadjusted P-value of 0.05. Points are highlighted in bold, red if significant after adjustment for the false discovery rate.
Reactivity to markers of prior malaria exposure and antigens of interest in control populations.
Seroreactivity to PfEMP1 antigens in pooled control serum vs. paediatric malaria cases.
CIDR IgG responders at time of acute P. falciparum infection.
Reactivity to head structure/CIDR or ICAM-binding domain in unexposed cases.
Additional exposure model covariates: the effect of cerebral malaria, age, and sex on PfEMP1 antibody level. Volcano plots of linear regression effect estimates (x-axis) for the independent variables (cerebral malaria, Ret + CM/UM; age, ≤ 5/> 5; sex, male/female) included in the exposure models generated for PfEMP1 expressed domains as determined by qRT-PCR of infected blood samples obtained during acute disease. (Total n = 48; CM n = 25; > 5 years of age n = 14; male n = 32).
About this article
Cite this article
Kessler, A., Campo, J.J., Harawa, V. et al. Convalescent Plasmodium falciparum-specific seroreactivity does not correlate with paediatric malaria severity or Plasmodium antigen exposure. Malar J 17, 178 (2018). https://doi.org/10.1186/s12936-018-2323-4
- Plasmodium falciparum
- Protein microarray
- Cerebral malaria
- Uncomplicated malaria