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

Abstract

By attaching infected erythrocytes to the vascular lining, Plasmodium falciparum parasites leave blood circulation and avoid splenic clearance. This sequestration is central to pathogenesis. Severe malaria is associated with parasites expressing an antigenically distinct P. falciparum erythrocyte membrane protein 1 (PfEMP1) subset mediating binding to endothelial receptors. Previous studies indicate that PfEMP1 adhesins with so-called CIDR␣1 domains capable of binding endothelial protein C receptor (EPCR) constitute the PfEMP1 subset associated with severe pediatric malaria. To analyze the relative importance of different subtypes of CIDR␣1 domains, we compared Pfemp1 transcript levels in children with severe malaria (including 9 fatal and 114 surviving cases), children hospitalized with uncomplicated malaria (n ϭ 42), children with mild malaria not requiring hospitalization (n ϭ 10), and children with parasitemia and no ongoing fever (n ϭ 12). High levels of transcripts encoding EPCR-binding PfEMP1 were found in patients with symptomatic infections, and the abundance of these transcripts increased with disease severity. The compositions of CIDR␣1 subtype transcripts varied markedly between patients, and none of the subtypes were dominant. Transcript-level analyses targeting other domain types indicated that subtypes of DBL␤ or DBL domains might mediate binding phenomena that, in conjunction with EPCR binding, could contribute to pathogenesis. These observations strengthen the rationale for targeting the PfEMP1-EPCR interaction by vaccines and adjunctive therapies. Interventions should target EPCR binding of all CIDR␣1 subtypes. KEYWORDS Plasmodium falciparum, antigenic variation, gene expression, malaria, PfEMP1 B ased on simple clinical observations, malaria patients can be divided into a smaller group with severe manifestations and a much larger group with uncomplicated disease (1). In areas of Africa with high to moderate rates of malaria transmission, severe malaria is seen almost only in children below 5 years of age (2). At the first entry point for health care, the majority of African children diagnosed with Plasmodium falciparum malaria suffer from uncomplicated febrile disease and are treated as outpatients. Downloaded fromHowever, based on the initial assessment, the examining physician hospitalizes some patients with more manifest symptoms. These children are not well, but based on simple triage, they can be further divided into a large group with uncomplicated disease, who, upon correct treatment, will be very likely to survive the disease, and a smaller group with severe disease, where a proportion will die despite the administration of what is currently considered optimal care (3). It is estimated that around 10 million children suffer from severe malaria every year and that 5 to 10% of these children die (4). Even though most children in areas where malaria is endemic are expected to experience several bouts of malaria during childhood, only one to three of these bouts are likely to ca...

Linking EPCR-Binding PfEMP1 to Brain Swelling in Pediatric Cerebral Malaria

“…Our study extends previous work suggesting higher transcript levels of DC8 and group A EPCR-binding variants in severe pediatric malaria (Jespersen et al, 2016; Mkumbaye et al, 2017) and the presence of both parasite subsets in Ret+ and Ret−CM cases (Abdi et al, 2015). Strikingly, we found that a CIDRα1.7 sequence was the dominant var tag in 50% of swelling cases.…”

“…Transcripts linked to EPCR-binding, including both DC8 and group A EPCR-binding variants, were significantly increased in Ret+CM compared to UM (Figures 2B, 2C, and Table S4). Additionally, a group A head structure linked to rosetting/unknown binding properties was enriched in Ret+CM cases (DBLα1.5/6a, DBLα1.5/6b, and CIDRδ of DC16), as was a C-terminal domain linked to PECAM-1 binding (DBLγ and DBLβ7/9 of DC5) (Figures 2D, 2E, and Table S4) (Berger et al, 2013) The application of second generation primers (Mkumbaye et al, 2017; Petersen et al, 2016) that can distinguish between the six subtypes of EPCR-binding CIDRα1 domains confirmed that both DC8 (CIDRα1.1) and group A (CIDRα1.4–1.7) transcript levels were significantly increased in Ret+CM cases compared to UM, whether considered individually or as the sum of all CIDRα1 transcripts (Figure S2 and Table S4). …”

“…Genomic DNA was removed (TURBO DNA-free, Ambion), and cDNA was synthesized using random hexamers and SuperScript III reverse transcriptase (Invitrogen). Var transcript levels were determined by qRT-PCR using published amplification conditions (Lavstsen et al, 2012; Mkumbaye et al, 2017; Rottmann et al, 2006), SYBR green, and an AB7300 real-time system. Complete removal of DNA using the stated protocol was previously validated by comparing expression levels in conditional var knockdown parasites (DC-J; Deitsch) to 3D7 expression levels in vitro .…”

Cerebral malaria is associated with differential cytoadherence to brain endothelial cells

“…A particularly strong example of this is where parasites expressing var genes encoding PfEMP1 containing EPCR-binding domains have shown a strong association with the development of SM, including CM (Avril et al, 2012;Claessens et al, 2012;Lavstsen et al, 2012;Bengtsson et al, 2013;Bertin et al, 2013;Jespersen et al, 2016;Kessler et al, 2017;Mkumbaye et al, 2017). A particularly strong example of this is where parasites expressing var genes encoding PfEMP1 containing EPCR-binding domains have shown a strong association with the development of SM, including CM (Avril et al, 2012;Claessens et al, 2012;Lavstsen et al, 2012;Bengtsson et al, 2013;Bertin et al, 2013;Jespersen et al, 2016;Kessler et al, 2017;Mkumbaye et al, 2017).…”

In vitro inhibition and reversal of Plasmodium falciparum cytoadherence to endothelium by monoclonal antibodies to ICAM-1 and CD36

“…More recent work has identified EPCR as being associated with severe malaria [2], including the possibility of structural conservation of the binding site on PfEMP1 that might support the development of a vaccine [7, 8]. Therefore, blocking and disrupting pRBC adhesion to host receptors could reduce the probability of developing severe malaria (SM).…”