Acquisition of IgG to ICAM-1-Binding DBLβ Domains in the Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigen Family Varies between Groups A, B, and C

Abstract

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important malaria virulence factor. The protein family can be divided into clinically relevant subfamilies. ICAM-1-binding group A PfEMP1 proteins also bind endothelial protein C receptor and have been associated with cerebral malaria in children. IgG to these PfEMP1 proteins is acquired later in life than that to group A PfEMP1 not binding ICAM-1. The kinetics of acquisition of IgG to group B and C PfEMP1 proteins binding ICAM-1 is unclear and was studied here. Gene sequences encoding group B and C PfEMP1 with DBLβ domains known to bind ICAM-1 were used to identify additional binders. Levels of IgG specific for DBLβ domains from group A, B, and C PfEMP1 binding or not binding ICAM-1 were measured in plasma from Ghanaian children with or without malaria. Seven new ICAM-1-binding DBLβ domains from group B and C PfEMP1 were identified. Healthy children had higher levels of IgG specific for ICAM-1-binding DBLβ domains from group A than from groups B and C. However, the opposite pattern was found in children with malaria, particularly among young patients. Acquisition of IgG specific for DBLβ domains binding ICAM-1 differs between PfEMP1 groups.

Keywords: PfEMP1; Plasmodium falciparum; antibodies; immunity; malaria.

FIG 1

ICAM-1-binding DBLβ domains. (A) ICAM-1 binding (ELISA optical density at 450 nm [OD450 nm]; means ± SD from three independent experiments) of 11 DBLβ domains, seven of which were predicted to bind ICAM-1 (6 group B and 1 group C). PFD1235w_D4 (group A) and PFD1235w_D5 (*) were used as positive and negative controls, respectively. (B) Phylogeny of ICAM-1-binding (red gene names) and nonbinding (blue gene names) DBLβ, shown as a maximum likelihood tree of 62 DBLβ domains (18, 20, 54, 55). The new DBLβ domains tested in this study are indicated by black dots. UKN indicates unknown group identity. ICAM-1 binding DBLβ domains from groups A (orange shading), B (green shading), and C (gray shading) are highlighted. (C) Schematic domain structure of ICAM-1-binding group B and C PfEMP1 proteins, with domain cassettes (DC) indicated by red boxes. ICAM-1 binders identified in this study (dark green shading) and by Janes et al. (20) (light green shading) are also indicated. The classification and nomenclature of domain groups and subgroups follow those of Rask et al. (56). ND, not determined. (D) Surface expression of HB3VAR21 P. falciparum HB3 IEs, visualized by incubation with HB3VAR21-specific antiserum (gray) or without antiserum (black). (E) Ability of HB3VAR21⁺ IEs to bind to recombinant ICAM-1, EPCR, and CD36. Mean adhesion (three independent experiments) is shown, with SD indicated by error bars.

FIG 2

Difference in IgG responses to group A and B DBLβ domains. Blood samples were obtained from 124 Ghanaian children (labeled Mal+) 2 weeks after they were diagnosed with acute P. falciparum malaria and from 91 healthy children at six different time points over a 1-year period (May, September, November, January, March, and May). (A) Mean IgG scores in all donors against five DBLβ domains from group B (white circles; PFL0020w, KOB84711, AAA75396, ERS009963, and KOB63129) and five from group A (black circles; PFD1235w, JF712902, KJ866957, Dd2VAR32, and AFJ66668) are shown. (B) Difference (ΔIgG) in mean IgG scores against the same five group B and five group A recombinant DBLβ domains.

FIG 3

Individual mean IgG score of each child against DBLβ domains at admission (wk0) and two weeks (wk2) and six weeks (wk6) later. (A) Mean plasma IgG score of each individual Ghanaian child (n = 124) against a total of 31 group A, B, and C DBLβ domains. (B) Mean IgG scores in individual children against 14 ICAM-1-binding DBLβ domains from group A. (C) Nine ICAM-1-binding DBLβ domains from groups B and C. (D) Eight non-ICAM-1-binding DBLβ domains from group A. The wk0 data from 2014 in panels B and D were published in reference . Median levels (dots) and their percentiles (25% and 75%; error bars) are indicated in red. The statistical significance (Mann-Whitney rank sum test) of pairwise comparisons is shown along the top of each panel.

FIG 4

Plasma levels of IgG specific for DBLβ domains from group A, group B, and group C. Samples were obtained from 124 Ghanaian children diagnosed with severe (▲) or nonsevere P. falciparum malaria 2 weeks prior to taking the blood sample. (A) Levels of IgG antibodies specific for individual ICAM-1-binding DBLβ domains (columns) from group A (left) and groups B and C (center) or for non-ICAM-1-binding domains from group A (right). Shading indicates the IgG level score: black, 4; dark gray, 3; gray, 2; light gray, 1; white, 0. The domain subtypes are indicated in Table S1. Danish controls (n = 20) did not react with any of the domains (data not shown). (B) Mean week 2 scores of IgG specific for individual ICAM-1-binding DBLβ domains from group A (black) and groups B and C (white). Error bars indicate 95% confidence intervals. (C) Mean IgG scores against all DBLβ domains in individual children, grouped according to age. Medians are indicated by horizontal red lines.

FIG 5

Mean IgG scores of 124 individual Ghanaian children against DBLβ domains from group A (black) and groups B and C (white) according to severity. Medians are indicated by horizontal red lines.