An analysis of the binding characteristics of a panel of recently selected ICAM-1 binding Plasmodium falciparum patient isolates

Abstract

The basis of severe malaria pathogenesis in part includes sequestration of Plasmodium falciparum-infected erythrocytes (IE) from the peripheral circulation. This phenomenon is mediated by the interaction between several endothelial receptors and one of the main parasite-derived variant antigens (PfEMP1) expressed on the surface of the infected erythrocyte membrane. One of the commonly used host receptors is ICAM-1, and it has been suggested that ICAM-1 has a role in cerebral malaria pathology, although the evidence to support this is not conclusive. The current study examined the cytoadherence patterns of lab-adapted patient isolates after selecting on ICAM-1. We investigated the binding phenotypes using variant ICAM-1 proteins including ICAM-1Ref, ICAM-1Kilifi, ICAM-1S22/A, ICAM-1L42/A and ICAM-1L44/A using static assays. The study also examined ICAM-1 blocking by four anti-ICAM-1 monoclonal antibodies (mAb) under static conditions. We also characterised the binding phenotypes using Human Dermal Microvascular Endothelial Cells (HDMEC) under flow conditions. The results show that different isolates have variant-specific binding phenotypes under both static and flow conditions, extending our previous observations that this variation might be due to variable contact residues on ICAM-1 being used by different parasite PfEMP1 variants.

Figure 1. Static adhesion assay, 2 µl spots at 50 µg/ml protein concentration were placed onto 6 cm dishes and standard protein static binding assays carried out with IE suspended in binding buffer at a parasitaemia of 3% and a haematocrit of 1%.

The results show the mean of binding (IE/mm²) (Figure 1A) and %ICAM-1^Ref binding (Figure 1B–E), and the bars represents SE (n≥3).

Figure 2. Static adhesion assay.

The same method as for Fig. 1 but with the addition of 5 µg/ml ofmAbs 15.2 (A), My13 (B), 8.46A (C) and BBIG-I1 (D) prior the IE incubation with ICAM-1^Ref. The results show the percentage of binding in the presence of ICAM-1 mAb compared to the no mAb treatment (A), and the bars represents SE (n>3).

Figure 3. Flow endothelial cell adhesion assay.

HDMEC seeded on channels pre-coated with fibronictin; IE were passed on confluent cells for five minutes followed by washing by binding buffer for two minutes before counting 6 fields in two different channels. The parasitaemia was 3% and a haematocrit of 2%. The results show the mean of binding with no inhibitory mAbs (A) and the % binding on treatment with 5 µg/ml of 15.2 anti-ICAM-1 (B) and 5 µg/ml IV-C7 anti-CD36 (C) mAbs compared to no mAb treatment. The bars represents SE (n≥3).