Interaction Of Plasmodium Falciparum Antigens With Blood Group Determinants: Preferences And Link To Disease Severity

ABSTRACT The blood type of malaria patients may determine the outcome of disease. Blood group O associates with protection, while non-O blood groups (A, B or AB) are associated with severe malaria. Rosetting of red blood cells (RBC), which is mediated by blood group A and other red blood cell surface molecules, is also associated with severe disease in the two most virulent species of Plasmodium that infect humans. The interaction of parasite ligands with blood group carbohydrates on RBC and endothelial receptors leads to infected RBC sequestration and persistence of the parasite in the vasculature. The objectives of this work were to study and measure the interactions between parasite-expressed factors on the red cell surface and blood group molecules using standard and novel methods, in both laboratory strains and field isolates of Plasmodium falciparum. Expression levels of var and rif transcripts in laboratory isolates selected for binding to blood group antigens were determined, while antibody reactivity to surface antigens were investigated in these isolates. Venous blood samples were collected from 113 children aged 12 years and below diagnosed with malaria and resident in Hohoe, a high malaria transmission zone in the Volta Region of Ghana. The blood type was determined for all patients, while plasma and RBCs were processed and stored for subsequent work. Long term blood group A and B binding variants of laboratory strains of P. falciparum 3D7, FMG and FUP parasites were successfully produced by regular panning on immobilized blood group oligosaccharides, though the production of binding variants in other strains (FCR3 and HB3) were not as successful. Selected parasites were found to bind to both A and B blood group carbohydrates irrespective of the blood group antigen used in selection. The binding isolates also showed a marked adhesion to aorta and dermal endothelial cell (EC) lines. A novel microtiter-plate based quantitative assay to iii specifically measure binding of parasite infected RBC to plate-bound blood group antigens was developed and used to determine the interaction of both laboratory and field strains to the blood group A and B antigens. All blood group antigen-selected isolates showed a strong propensity to form rosettes in the presence of RBC from donors of blood type A, AB or B, in contrast to unselected isogenic isolates which did not form rosettes under the same conditions. The transcription levels of 58 var genes of ring stage parasites of 3D7 and FMG selected on blood group antigens and detected by quantitative real-time polymerase chain reaction (QPCR) showed no consistent pattern of expression between selected and unselected parasites, though the adhesion linked genes Pf13_0003 and IT4var32b of 3D7 and FMG, respectively, had elevated levels in some selected isolates. In conclusion, a novel plate-based assay to directly measure infected erythrocyte adhesion to blood group antigens has been developed. Selection for long term blood group specific parasite binding isolates was also achieved, but with no clear changes in variant surface antigen transcript levels. Notwithstanding, an increased rosette formation and adhesion to non-O blood antigens of selected parasites which suggest a link to severe disease was observed. It is hoped that future investigations using field isolates with clearer categorization of severe cases and a larger sample size would further validate these findings. Whole genome transcriptional studies and sequencing studies in these parasites may also specifically identify the genes and molecules involved in adhesion. Improved knowledge in all these areas will accelerate the design and development of targeted syndrome specific vaccines which will improve the management of this disease.