Antibodies to reticulocyte binding protein-like homologue 4 inhibit invasion of Plasmodium falciparum into human erythrocytes

Abstract

Plasmodium falciparum invasion into human erythrocytes relies on the interaction between multiple parasite ligands and their respective erythrocyte receptors. The sialic acid-independent invasion pathway is dependent on the expression of P. falciparum reticulocyte binding protein-like homologue 4 (PfRh4), as disruption of the gene abolishes the ability of parasites to switch to this pathway. We show that PfRh4 is present as an invasion ligand in culture supernatants as a 160-kDa proteolytic fragment. We confirm that PfRh4 binds to the surfaces of erythrocytes through recognition of an erythrocyte receptor that is neuraminidase resistant but trypsin and chymotrypsin sensitive. Serum antibodies from malaria-exposed individuals show reactivity against the binding domain of PfRh4. Purified immunoglobulin G raised in rabbits against the binding domain of PfRh4 blocked the binding of native PfRh4 to the surfaces of erythrocytes and inhibited erythrocyte invasion of parasites using sialic acid-independent invasion pathways and grown in neuraminidase-treated erythrocytes. Our results suggest PfRh4 is a potential vaccine candidate.

FIG. 1.

PfRh4 is expressed as a 160-kDa fragment in the invasion supernatant and binds to the surfaces of erythrocytes in an enzyme-dependent manner. (A) Western blots of saponin-treated schizont pellets (left) and invasion supernatants (right) were probed with an anti-Rh4 (αRh4) antibody. 3D7, HB3, and W2mefΔ175 express PfRh4, which is absent from W2mefΔRh4. The asterisk, white arrowhead, and black arrowhead highlight bands running at 190 kDa, 180 kDa, and 160 kDa, respectively. (B) (Top) Schematic representation of the various domains of PfRh4 against which rabbit polyclonal antibodies were raised. The black bar above each antibody name (R922, R206, and R936) highlights the region of the fusion protein used. C denotes cysteine residues, and the black bar within the schematic represents the transmembrane domain of PfRh4. (Bottom) Western blots of saponin-treated schizont pellets (lanes P) and culture supernatants (lanes S) were probed with three separate anti-Rh4 antibodies. (C) Immunodetection of parasite proteins with anti-RH4 and anti-EBA-175 antibodies after binding and elution from untreated and enzyme-treated erythrocytes. Lanes: I, input lane; P, proteins eluted from PBS control; U, untreated erythrocytes; N, neuraminidase; T_L, low trypsin; T_H, high trypsin; and C, chymotrypsin-treated erythrocytes. Low trypsin and high tryspin are trypsin treatments with 0.1 and 1.5 mg/ml of enzyme, respectively. Molecular masses are indicated on the left (in kDa) for all panels.

FIG. 2.

PfRh4 binds to the erythrocyte surface through its N-terminal region. (A) Schematic representation of the various six-His-tagged PfRh4 recombinant proteins. The C denotes cysteine residues, and the black bar represents the transmembrane domain of PfRh4. The number below each fusion protein indicates the amino acid sequence that it encompasses. (B) rRh4.9 binds erythrocytes in a manner similar to that of native PfRh4. Immunodetection of the recombinant fusion protein with anti-Rh4 (αRh4) antibodies after binding and elution from untreated and enzyme-treated erythrocytes is shown. Lanes: P, proteins eluted from PBS control; U, untreated erythrocytes; Nm, neuraminidase; T_L, low trypsin; T_H, high trypsin; and C, chymotrypsin-treated erythrocytes. Low trypsin and high tryspin refer to trypsin treatments with 0.1 and 1.5 mg/ml of enzyme, respectively. Molecular masses are indicated on the left (in kDa). (C) Minimal binding domain of PfRh4. Binding of six-His-tagged recombinant PfRh4 proteins (Rh4.10, Rh4.11, Rh4.12, and Rh4.13) to untreated erythrocytes was detected using mouse monoclonal anti-His₅ (αHis) antibodies. Molecular masses are indicated on the left (in kDa).

FIG. 3.

Reactivities of human antibodies to rRh4.9. The reactivity of human IgG to rRh4.9 was measured by ELISA using purified rRh4.9 protein. All samples were tested in duplicate and adjusted for background reactivity. The error bars represent the ranges of two duplicates. The samples tested were from malaria-exposed residents of Madang, Papua New Guinea (numbered samples), and nonexposed Melbourne residents (M1 to M7). OD 405, OD at 405 nm.

FIG. 4.

Antibodies raised to the Rh4.9 binding domain inhibit PfRh4 binding to the surfaces of erythrocytes. (A) Anti-Rh4 (αRh4) IgG at final concentrations of 0.0008 to 0.024 mg/ml (left) and 0 to 1.5 mg/ml (right) was incubated with 250 μl of 3D7 invasion supernatants prior to the erythrocyte binding assay. Immunodetection of parasite proteins with anti-Rh4 antibodies after binding and elution from untreated erythrocytes is shown. (B) Anti-Rh4 IgG at final concentrations of 0.0008 to 0.024 mg/ml (left) and 0 to 1.5 mg/ml (right) was incubated with 250 μl of 3D7 invasion supernatants prior to the erythrocyte binding assay. Immunodetection of parasite proteins with anti-EBA-175 antibodies after binding and elution from untreated erythrocytes is shown. (C) Nonimmune IgG from normal rabbit serum (NRS) at final concentrations of 0.0008 to 0.024 mg/ml (left) and 0 to 1.5 mg/ml (right) was incubated with 250 μl of 3D7 invasion supernatants prior to the erythrocyte binding assay. Immunodetection of parasite proteins with anti-Rh4 antibodies after binding and elution from untreated erythrocytes is shown. The concentrations mentioned above refer to the total IgG purified using protein G columns.

FIG. 5.

Strain-specific invasion inhibition of parasite growth using PfRh4 antibodies. (A) Parasite growth for the 3D7, W2mef, W2mefΔ175, and W2mefΔRh4 parasite lines grown in the presence of purified IgG from rabbit prebleed serum (NRS) or anti-Rh4 purified IgG antibodies (αRh4) in normal erythrocytes. (B) Growth of the 3D7 and W2mefΔ175 lines in neuraminidase-treated erythrocytes in the presence of purified IgG from rabbit preimmune serum, purified IgG from second-bleed serum, and purified IgG from third-bleed serum from rabbits immunized with PfRh4. (C) Growth of the 3D7 parasite line in the presence of a dilution series for purified nonspecific IgG from rabbit prebleed serum (NRS) and purified anti-Rh4 IgG antibodies (αRh4) in neuraminidase-treated erythrocytes shows that Rh4 antibody inhibition of parasite growth is concentration dependent. The final concentrations of IgG antibodies ranged from 0 to 2 mg/ml in each invasion assay. For all panels, parasite growth was measured as a percentage of the mean parasite growth for four wells, with nonspecific IgG from rabbit prebleed serum control added in each experiment. The error bars represent the standard error of the mean for duplicate wells in two independent experiments. The concentrations mentioned above refer to the total IgG purified using protein G columns.