Background
Despite all efforts to curb
Plasmodium falciparum malaria infections it still is an important cause of morbidity and mortality in many developing countries, with an estimated 700,000 deaths annually [
1]. The burden of disease is highest in children below five years of age where much of the mortality is attributable to severe malaria. Except for RTS,S, which is currently in clinical phase 3 trial, no vaccine is available to date, but vaccine development is encouraged by the fact that children living in endemic areas attain conditional immunity to severe malaria after a relatively few number of episodes during childhood [
2]. Genetic diversity of different parasites and antigenic variation of surface antigens pose an obstacle for vaccine development.
Severe malaria, the most life threatening form of the disease is believed to be mediated by cytoadhesion of
P. falciparum-infected erythrocytes to a variety of receptors on the endothelial lining of the host’s blood capillaries. This post-capillary sequestration severely affects vital organs such as the brain, kidneys, lungs or placenta [
3]. Cytoadherence is conferred by
P. falciparum derived proteins on the surface of infected erythrocytes that play a key role as both virulence factors and as targets of naturally acquired immunity [
4,
5]. Of these, the major contributor to pathology of
P. falciparum is the
P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 is a large protein of approx. 200–400 kDa, it is a highly polymorphic antigen which is encoded by a family of ~60
var genes per haploid genome [
6].
Var genes present with a two-exon structure encoding a semi-conserved C-terminus that contains a predicted transmembrane region, and a highly polymorphic extracellular N-terminus. This part has a modular structure containing various numbers of Duffy-binding-like (DBL) domains and cysteine-rich domains that have been shown to be involved in sequestration of the infected erthrocytes [
7‐
9]. The most N-terminal sequence including the DBLα domain is the most conserved domain within the
var gene domains also conferring cytoadherence [
10,
11]. A range of host receptors have been shown to interact with PfEMP1 thus determining the binding to various tissues [
10,
12,
13]. Rosetting and sequestration conferred by expression of different PfEMP1 molecules has been implicated in severe disease [
14].
var genes have been classified into three major groups (A, B and C) and two intermediate groups (B/A and B/C) based on the presence of one of the 5′ upstream sequences (upsA, B or C), and the position and orientation of the gene within a genomic context [
15,
16]. It has been speculated that severe malaria is determined by the expression of a restricted and antigenically semi-conserved subset of PfEMP1 [
17,
18]. The best understood host-parasite interaction concerning PfEMP1 is pregnancy-associated malaria (PAM) in which PfEMP1 molecules binding to CSA are involved [
19].
var genes have further been sub-classified by the distribution of cysteines throughout the head structures and positions of limited variation (PoLV) [
20].
Few studies have investigated the expression of
var genes in field isolates representing different forms of severe malaria [
21‐
27]. These studies suggested that the transcription patterns of
var genes vary between different malaria manifestations. Differences in epidemiology, severe disease classification, and
var classification have also made comparison between studies difficult. Using quantitative real time reverse transcription PCR (qRT-PCR), we have previously shown that group A and B
var transcripts were up-regulated in children from Tanzania with severe malaria as opposed to asymptomatic infections [
25]. Although qRT-PCR is a standard method for detection and quantification of gene expression levels [
28], without subsequent sequencing this technique is not informative to study diversity of genes. Given the importance of immunity against PfEMP1 and its possible association with protection against malaria, it is essential to gain amore detailed understanding of diversity of these molecules at sequence level. Only this allows the determination of how such diversity influences the development of protective immunity. In this study, the genetic diversity of expressed PfEMP1 molecules in parasite populations directly isolated from children with severe malaria was examined.
Discussion
Studies on
var gene diversity are important in understanding malaria pathogenesis and in the design of disease interventions such as a vaccine or chemotherapies. In the present study, we examined
var gene expression from clinical isolates of children with severe malaria and asymptomatic infections from Tanzania. In each isolate dominant expression of one particular
var gene was found, together with less abundant variant transcripts and unique sequences. However, the dominant sequences differed between isolates. This suggests that each parasite contains its own set of
var gene variants. This has the consequences that exposure to multiple infections and hence
var gene products do not necessarily confer immunity to future malaria infections [
46,
47].
By analysing the expressed
var gene repertoires in severe malaria cases versus asymptomatic controls, we showed that the diversity within the
var gene family is enormous with a minimal degree of overlaps between isolates. Kyriacou
et al.[
21] have found a minimal overlap in
var gene repertoires after analysing the expressed sequence tags from Malian children with malaria infections. A recent study on molecular epidemiology of
var genes in Africa has also shown a minimal overlap in
var repertoires among parasite genomes [
48]. In contrast, Albrecht
et al.[
49] reported a large overlap of the
var gene repertoire in Western Amazon isolates.
var repertoires of natural parasite populations found within specific geographical regions showed a degree of overlapping, suggesting the circulation of a similar
var gene repertoire. This has important implications for the acquisition of long-term immunity by the exposed individuals [
47].
The diversity of
var genes within a natural
P.
falciparum population in a particular geographical region is difficult to define, and to assess whether the diversity is constant due to functional constrain on this molecule, fluctuating or constantly turning over, and how fast the turnover rate of the PfEMP1 repertoires could be. Changes in the
var repertoire are believed to be due to high allelic and ectopic recombination rates of
var genes in field isolates [
37,
50,
51] which are influenced by transmission intensity. The diversity of the PfEMP1 repertoire of parasites in a given geographical area is a key factor in the development of clinical immunity. The vast antigenic diversity and complexity of
var gene repertoires in parasite populations may explain why individuals are repeatedly susceptible to
P. falciparum infections and never develop sterilizing immunity. The antigenic variation and high switching rate of
var gene expression are effective mechanisms adopted by
P. falciparum to evade the host’s immune system, for survival, and effective transmissions.
In this study, several sequences were observed more frequently than others within individual patients. This is consistent with previous studies of
var gene diversity [
21,
22,
46,
50,
51]. The variability of the DBL-1α and upstream sequences within an isolate was found to be similar to different isolates in both the groups (SM & AM). AM isolates were more diverse as reflected by the presence of more singletons suggesting that
var genes associated with asymptomatic infection have an enormous repertoire which could explain the difficulty of acquiring immunity to mild or asymptomatic malaria.
Isolates from children with severe malaria were predominantly found to transcribe
var genes with a DBL-1α domain that had a reduced number of cysteine residues which is the characteristic of
var group A. Similar results have been reported previously from other research groups in Kenya, Mali, and Brazil [
20‐
22]. This supports the notion that severe malaria might be caused by a restricted subset of
var genes and confirms that group A
var genes are involved in severe disease similarly as we had shown in a previous study that group A
var genes were up regulated in children with cerebral malaria [
25]. However, most studies on
var gene diversity have been relying on the use of DBL-1α fragments [
50]. DBL-1α primers amplify only a small fragment of the
var gene that is more conserved than other
var domains and that is found in most of PfEMP1 proteins. Due to the complex nature of
var genes, only recently complete
var genes could be cloned routinely [
52] and could provide in future additional information on understanding
var gene transcription and its association to disease phenotype.
Cluster analysis revealed several ‘unique sequences’ of
var genes which were transcribed only in isolates from patients with severe malaria. Expression of these ‘unique sequences’ in a patient who lacks a pre-existing antibody response against this variant might trigger the development of severe malaria. Once exposed to these potentially virulent
var genes individuals living in endemic areas may acquire immunity to severe malaria. In areas of high endemicity this might happen early in life after only a few clinical episodes. The distribution of PoLV motifs showed 8 motifs which were highly associated with severe disease. Based on the MOTIFF algorithm, Normark
et al.[
53] identified 15 DBL-1α degenerate sequence motifs pertinent to severe disease and three motifs associated with the high rosetting phenotype after analysing 93 patients with well-characterized disease. Once again pointing in the direction that disease phenotypes are correlated with the expression of certain PfEMP1 variants and motifs. This is highly relevant information for vaccine development and understanding disease pathogenesis.
The distribution of PF11_0008, a group A
var gene, which previously has been identified in the 3D7 genome and the isogenic isolate NF54 [
42], was found in three SM isolates (ISM11, ISM33, ISM48) and in one AM sample (IAM17), although in low frequencies. This, and the observation that PFD0020c also has been more frequently found in SM cases suggests that the
var genes of laboratory strains are shared among the field isolates. The recent report by Claessens
et al.[
54] showing that up-regulation of the group A
var gene 3D7_PFD0020c is associated with adhesion to human brain endothelial cells further supports this notion.
Competing interests
The authors declare no competing interests.
Authors’ contributions
JM, SR, MR, HPB conceived and designed the study, JM and SR conducted the laboratory analysis, WQ and JM analysed the data. JM and HPB wrote the manuscript with contributions from all other authors. All authors read and approved the final manuscript.