Complement and Antibody-mediated Enhancement of Red Blood Cell Invasion and Growth of Malaria Parasites

Abstract

Plasmodium falciparum malaria is a deadly pathogen. The invasion of red blood cells (RBCs) by merozoites is a target for vaccine development. Although anti-merozoite antibodies can block invasion in vitro, there is no efficacy in vivo. To explain this discrepancy we hypothesized that complement activation could enhance RBC invasion by binding to the complement receptor 1 (CR1). Here we show that a monoclonal antibody directed against the merozoite and human polyclonal IgG from merozoite vaccine recipients enhanced RBC invasion in a complement-dependent manner and that soluble CR1 inhibited this enhancement. Sialic acid-independent strains, that presumably are able to bind to CR1 via a native ligand, showed less complement-dependent enhancement of RBC invasion than sialic acid-dependent strains that do not utilize native CR1 ligands. Confocal fluorescent microscopy revealed that complement-dependent invasion resulted in aggregation of CR1 at the RBC surface in contact with the merozoite. Finally, total anti-P. berghei IgG enhanced parasite growth and C3 deficiency decreased parasite growth in mice. These results demonstrate, contrary to current views, that complement activation in conjunction with antibodies can paradoxically aid parasites invade RBCs and should be considered in future design and testing of merozoite vaccines.

Keywords: CR1; Complement; Malaria; Merozoites; Red blood cells.

Graphical abstract

Fig. 1

FS and anti-merozoite monoclonal antibody mAb5.2 enhance P. falciparum invasion of RBCs relative to HIS. a) Two SA-independent strains of P. falciparum, 7G8 and 3D7, were tested in the presence of 10% FS, 3 min HIS, 5 min HIS, or 30 min HIS. Invasion of RBCs decreased with progressively longer heat inactivation times. b) Invasion of RBCs in the same experiment as panel A expressed as percent enhancement relative to 30 min HIS. See Fig. S3 for additional replicates. c) Invasion of P. falciparum strain 7G8 was tested in the presence of monoclonal anti-merozoite surface protein 1(MSP1) mAb5.2 (40 μg/ml), an isotype control antibody (IgG2b), or PBS in 10% FS or 30 min HIS. Invasion of RBCs is expressed as percent enhancement in FS relative to 30 min HIS. Each panel depicts a representative experiment. See Fig. S5 for absolute parasitemia and one additional replicate. Error bars represent standard deviations for triplicate wells. P values were obtained by one-way ANOVA with post-hoc comparisons.

Fig. 2

Enhancement of RBC invasion by anti-merozoite monoclonal antibody mAb5.2 is complement dependent. a) Addition of C2 and fB rescued RBC invasion enhancement properties of mAb5.2 in 3 min HIS. Invasion of P. falciparum strain 7G8 was tested in the presence of 40 μg/ml anti-MSP1 mAb5.2, 40 μg/ml IgG2b isotype control, or PBS in the presence of 10% FS, 3 min HIS, or 30 min HIS. C2 and fB were added to a final concentration of 25 μg/ml and 200 μg/ml respectively. The glycoprotein fetuin was used as a control at 225 μg/ml. Fig. S6A shows the absolute parasitemia. Additional replicates are shown in Fig. S7. b) Enhancement of RBC invasion by anti-MSP1 mAb5.2 was eliminated by the peptide complement inhibitor compstatin but not by control peptide. Conditions were the same as panel A. Invasion of RBCs is expressed as percent enhancement relative to 30 min HIS. Fig. S9A shows the absolute parasitemia. Additional replicates are shown in Fig. S9B and S9C·Error bars represent standard deviations of triplicate wells. P is based on two-sample t-test for the comparison between mAb5.2 and IgG2b or PBS.

Fig. 3

Enhancement of RBC invasion by mAb5.2 is abolished in the presence of sCR1. Invasion of P. falciparum strain 7G8 was tested in the presence of 40 μg/ml anti-MSP1 mAb5.2 or 40 μg/ml IgG2b isotype control in the presence of 10% FS or 30 min HIS. The final concentration of sCR1 or fetuin was 80 μg/ml. Fig. S10 shows the absolute parasitemia and additional replicates. Error bars represent standard deviations of triplicate wells. P is based on two-sample t-test.

Fig. 4

SA-dependent strains of P. falciparum are more reliant on complement to invade NA-treated RBCs than SA-independent strains. a) SA-dependent strains show greater enhancement of invasion into NA-treated RBC (median 54.8%) in the presence of FS relative to 30 min HIS than SA-independent strains (median 31.5%), P < 0.01 by two-sample t-test. Fig. S11 shows absolute parasitemias. Fig. S12 shows one additional replicate. b) Enhancement of invasion into NA-treated RBCs in the presence of C3/C4-reconstituted serum relative to C3/C4-inactivated serum. Fig. S11C shows absolute parasitemias. Error bars are standard deviations of triplicate wells. P values are based on two-sample t-test.

Fig. 5

Antibody and complement-mediated attachment of merozoites to RBC CR1. Attachment of P. falciparum strain 3D7 was tested in the presence of anti-MSP1 mAb5.2 or IgG2b isotype control, in the presence of a) C3/C4-inactivated or C3/C4-reconstituted serum and b) C3-depleted or C3-reconstituted serum. Fig. S13 shows additional replicates. P is based on two-sample t-test. Error bars represent standard deviations for triplicate wells. c) In the presence of C3 and anti-MSP1 mAb5.2 merozoites attach to RBCs via CR1 (yellow) which shows intense aggregation at the site of merozoite contact. This area is also positive for C3 (cyan) and shows lipid accumulation by DID staining (red). Hoechst 33,342 was used to stain the DNA (blue) and MSP1 staining shows green. On the other hand, use of C3-depleted serum resulted in absence of CR1 aggregation, C3 deposition, or lipid accumulation. Scale bar is 2 μm. d) Zoomed view of C3 deposition (i and iii) and CR1 co-localization (ii and iv) in C3-reconstituted and depleted serum. e) Quantitation of the merozoites with C3 deposition and CR1 co-localization in C3-reconstituted and depleted serum in the presence of anti-MSP1 mAb5.2. *P < 0.001, by chi-square test with Fisher's exact test, under the null hypothesis that the CR1 and C3b co-localization are equal in complement sufficient and deficient serum. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Complement decreases inhibitory activity of polyclonal IgG from MSP1₄₂ vaccine recipients. a) RBC invasion of P. falciparum strain FVO in the presence of purified total IgG (2.5 mg/ml) from MSP1₄₂ vaccine recipients and either C3/C4-inactivated or C3/C4-reconstituted serum, N = 13. b) RBC invasion in the presence of total IgG from malaria naïve non-vaccinee controls, N = 14. Horizontal markers represent medians. P is by paired t-test.

Fig. 7

Anti-P. berghei antibodies and complement enhance parasite growth in mice. a) Time course of parasite growth in mice injected with anti-P. berghei antibody (Anti-Pb), control antibody (Cont Ab), or PBS. b) Day 7 enhancement of parasitemia from panel A relative to mice that received PBS. P value is based on analysis of variance. c) Time course of P. berghei parasitemia in C3^–/− (C3 KO) and wild type mice that received 250 μg of Anti-Pb or Cont Ab, or equivalent volume of PBS. d) Day 7 enhancement of parasitemia from panel c. P is based on two-sample t-test. Additional replicates are shown in Fig. S14. Error bars represent standard errors for each group of 4–5 mice.