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
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.
] 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.
] 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
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
] 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
]. 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
]. 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.
] 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.
] 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.