Mapping of the region of complement receptor (CR) 1 required for Plasmodium falciparum rosetting and demonstration of the importance of CR1 in rosetting in field isolates

The malaria parasite Plasmodium falciparum induces a number of novel adhesion properties in the erythrocytes that it infects. One of these properties, the ability of infected erythrocytes to bind uninfected erythrocytes to form rosettes, is associated with severe malaria and may play a direct role in the pathogenesis of disease. Previous work has shown that erythrocytes deficient in complement receptor (CR) 1 (CR1, CD35; C3b/C4b receptor) have greatly reduced rosetting capacity, indicating an essential role for CR1 in rosette formation. Using deletion mutants and mAbs, we have localized the region of CR1 required for the formation of P. falciparum rosettes to the area of long homologous repeat regions B and C that also acts as the binding site for the activated complement component C3b. This result raises the possibility that C3b could be an intermediary in rosetting, bridging between the infected erythrocyte and CR1. We were able to exclude this hypothesis, however, as parasites grown in C3-deficient human serum formed rosettes normally. We have also shown in this report that rosettes can be reversed by mAb J3B11 that recognizes the C3b binding site of CR1. This rosette-reversing activity was demonstrated in a range of laboratory-adapted parasite strains and field isolates from Kenya and Malawi. Thus, we have mapped the region of CR1 required for rosetting and demonstrated that the CR1-dependent rosetting mechanism occurs commonly in P. falciparum isolates, and could therefore be a potential target for future therapeutic interventions to treat severe malaria.     

Human complement receptor type 1 (CR1) binds to a major malarial adhesin

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a major adhesin molecule expressed on Plasmodium-falciparum-infected erythrocytes, interacts with several receptors on endothelial cells and uninfected erythrocytes. This 'stickiness', known as rosetting, is a strategy used by the parasite to remain sequestered in the microvasculature to avoid destruction in the spleen and liver. Erythrocyte rosetting causes obstruction of the blood flow in microcapillaries. Recent data suggest a direct interaction between PfEMP1 and a functional site of complement receptor type 1 (CR1; CD35) on uninfected erythrocytes. Consistent with the hypothesis that CR1 is important in malaria pathogenesis is a 40-70-fold increase in the frequency of two CR1 blood-group antigens (at least one of which might rosette less efficiently) in malaria-exposed African populations. Furthermore, structural differences in erythrocyte CR1 between human and non-human primates are probably explained by the selective pressure of malaria.     

C3b binding, but not its breakdown, is affected by the structure of the O-antigen polysaccharide in lipopolysaccharide from Salmonellae

Bacteria whose lipopolysaccharide contains O-antigen side chains activate complement via the alternative pathway. We have shown previously that three strains of Salmonella, differing in the chemical structure of their O-antigens, consumed C3 to different extents when incubated in C4-deficient guinea pig serum. Moreover, sheep erythrocytes coated with lipopolysaccharide purified from these strains mimicked whole cells in C3 consumption, proving that lipopolysaccharide alone could account for these results. We have now measured the deposition of 125I-C3 in this system, and found that C3 deposition parallels C3 consumption in rate and extent, and differs for surfaces bearing different O-antigens, whether tested with bacteria or with erythrocytes coated with purified lipopolysaccharide. We have also examined the fate of C3 on these Salmonellae by measuring the size and quantity of 125I-C3 breakdown fragments by SDS-PAGE, and have determined the kinetics of conversion of C3b to iC3b by using conglutinin, a molecule that binds specifically to iC3b. There is no difference in breakdown of C3b deposited on cells with different O-antigens: all show partial conversion to iC3b and C3dg as indicated by 68,000, 44,000, and 41,000 m.w. bands on reduced SDS gels. Furthermore, for all strains, the Ka of conglutinin binding to iC3b is similar (0.49 to 0.69 X 10(8) M-1), as is the rate of generation of iC3b and the final ratio of iC3b:C3b + iC3b (0.62 to 0.72). We therefore postulate that the fine structure of the O-antigen in lipopolysaccharide determines the magnitude of alternative pathway activation on the bacterial surface by affecting the rate and extent of C3b deposition, but not the rate and extent of breakdown of C3b.     

Invasion by P. falciparum merozoites suggests a hierarchy of molecular interactions

Central to the pathology of malaria disease are the repeated cycles of parasite invasion and destruction of human erythrocytes. In Plasmodium falciparum, the most virulent species causing malaria, erythrocyte invasion involves several specific receptor-ligand interactions that direct the pathway used to invade the host cell, with parasites varying in their dependency on these different pathways. Gene disruption of a key invasion ligand in the 3D7 parasite strain, the P. falciparum reticulocyte binding-like homolog 2b (PfRh2b), resulted in the parasite invading via a novel pathway. Here, we show results that suggest the molecular basis for this novel pathway is not due to a molecular switch but is instead mediated by the redeployment of machinery already present in the parent parasite but masked by the dominant role of PfRh2b. This would suggest that interactions directing invasion are organized hierarchically, where silencing of dominant invasion ligands reveal underlying alternative pathways. This provides wild parasites with the ability to adapt to immune-mediated selection or polymorphism in erythrocyte receptors and has implications for the use of invasion-related molecules in candidate vaccines.     

C3b2-IgG complexes retain dimeric C3 fragments at all levels of inactivation

C3b2-IgG complexes are formed during complement activation in serum by attachment of two C3b molecules (the proteolytically activated form of C3) to one IgG heavy chain (IgG HC) via ester bonds. Because of the presence of two C3b molecules, these complexes are very efficient activators of the alternative complement pathway. Likewise, dimeric C3b is known to enhance complement receptor 1-dependent phagocytosis, and dimeric C3d (the smallest thioester-containing fragment of C3) linked to a protein antigen facilitates CR2-dependent B-cell proliferation. Because the efficiency of all these interactions depends on the number of C3 fragments, we investigated whether C3b2-IgG complexes retained dimeric structure upon physiological inactivation. We used two-dimensional SDS-PAGE and Western blot to study the arrangement of the C3b molecules by analyzing the fragmentation pattern after cleavage of the ester bonds. Upon inactivation with factors H and I, a 185-kDa band was generated under reducing conditions. It released IgG HC and the 65-kDa fragment of C3b alpha' chain after hydrolysis of the ester bonds with hydroxylamine. The two C3b molecules were not 65-kDa-to-40-kDa linked, because neither ester-bonded 65 kDa HC nor 65 kDa-40 kDa fragments were observed, nor was a 40-kDa peptide released after hydroxylamine cleavage. Factor I and CR1 cleaved the C3b2-IgG molecule to its final physiological product, C3dg2-IgG, which migrated as a 133-kDa fragment in reduced form. This fragment released exclusively C3dg (the final physiological product of C3b inactivation by factor I) and IgG HC. C3dg2-HC appeared as a double band on SDS-PAGE only at low gel porosity, suggesting the presence of two conformers of the same composition. Our results suggest that, upon physiological inactivation, C3b2-IgG complexes retain dimeric inactivated C3b and C3dg, which allows bivalent binding to the corresponding complement receptors.     

Reduced immune complex binding capacity and increased complement susceptibility of red cells from children with severe malaria-associated anemia

Plasmodium falciparum malaria causes 1-2 million deaths per year. Most deaths occur as a result of complications such as severe anemia and cerebral malaria (CM) (coma). Red cells of children with severe malaria-associated anemia (SMA) have acquired deficiencies in the complement regulatory proteins complement receptor 1 (CR1, CD35) and decay accelerating factor (DAF, CD55). We investigated whether these deficiencies affect the ability of erythrocytes to bind immune complexes (ICs) and regulate complement activation. We recruited 75 children with SMA (Hb < or = 6 g/dL) from the holoendemic malaria region of the Lake Victoria basin, western Kenya, and 74 age- and gender-matched uncomplicated malaria controls. In addition, we recruited 32 children with CM and 52 age- and gender-matched controls. Deficiencies in red cell CR1 and CD55 in children with SMA were accompanied by a marked decline in IC binding capacity and increased C3b deposition in vivo and ex vivo. Importantly, these changes were specific because they were not seen in red cells of children with CM or their controls. These data suggest that the declines in red cell CR1 and CD55 seen in children with SMA are of physiologic significance and may predispose erythrocytes to complement-mediated damage and phagocytosis in vivo.     

CR2 ligands modulate human B cell activation

A considerable body of evidence from this and other laboratories indicates that complement receptor type 2 (CR2) modulates B cell activation and growth. In the present studies we have examined the effects of three different types of CR2 ligands, i.e., monomeric, aggregated, and latex-bound C3dg; mAb to different CR2 epitopes; and UV-inactivated, non-transforming EBV (EBVUV) for their actions on highly purified, high density resting tonsil B cells. Although none of these ligands induced B cells to enter the cell cycle or synergized with either anti-mu or low m.w. B cell growth factor in triggering B cell mitogenesis, aggregated C3dg, latex-bound C3dg, the OKB7 anti-CR2 mAb, and EBVUV-enhanced thymidine incorporation by phorbol ester-activated tonsil B cells. Such enhancement was not T cell or monocyte dependent. The major action of the CR2 ligands thus seems to be to enhance the transition of B cells activated by certain stimuli from the G1 to the S phase of the cell cycle. In contrast to the action of aggregated and latex-bound C3dg, monomeric C3dg was inhibitory for phorbol ester and aggregated C3dg-induced B cell activation. The HB-5 anti-CR2 mAb, which reacts with a different epitope on CR2 from that of OKB7, did not synergize with PMA in B cell activation. These data provide additional evidence for a role for the CR2 in the control of B cell growth and provide a useful model for studying the CR2-mediated signals that affect the growth of B cells.     

Using Mutagenesis and Structural Biology to Map the Binding Site for the Plasmodium falciparum Merozoite Protein PfRh4 on the Human Immune Adherence Receptor

To survive and replicate within the human host, malaria parasites must invade erythrocytes. Invasion can be mediated by the P. falciparum reticulocyte-binding homologue protein 4 (PfRh4) on the merozoite surface interacting with complement receptor type 1 (CR1, CD35) on the erythrocyte membrane. The PfRh4 attachment site lies within the three N-terminal complement control protein modules (CCPs 1–3) of CR1, which intriguingly also accommodate binding and regulatory sites for the key complement activation-specific proteolytic products, C3b and C4b. One of these regulatory activities is decay-accelerating activity. Although PfRh4 does not impact C3b/C4b binding, it does inhibit this convertase disassociating capability. Here, we have employed ELISA, co-immunoprecipitation, and surface plasmon resonance to demonstrate that CCP 1 contains all the critical residues for PfRh4 interaction. We fine mapped by homologous substitution mutagenesis the PfRh4-binding site on CCP 1 and visualized it with a solution structure of CCPs 1–3 derived by NMR and small angle x-ray scattering. We cross-validated these results by creating an artificial PfRh4-binding site through substitution of putative PfRh4-interacting residues from CCP 1 into their homologous positions within CCP 8; strikingly, this engineered binding site had an ∼30-fold higher affinity for PfRh4 than the native one in CCP 1. These experiments define a candidate site on CR1 by which P. falciparum merozoites gain access to human erythrocytes in a non-sialic acid-dependent pathway of merozoite invasion.     

Plasmodium falciparum erythrocyte invasion through glycophorin C and selection for Gerbich negativity in human populations

Geographic overlap between malaria and the occurrence of mutant hemoglobin and erythrocyte surface proteins has indicated that polymorphisms in human genes have been selected by severe malaria. Deletion of exon 3 in the glycophorin C gene (called GYPCDeltaex3 here) has been found in Melanesians; this alteration changes the serologic phenotype of the Gerbich (Ge) blood group system, resulting in Ge negativity. The GYPCDeltaex3 allele reaches a high frequency (46.5%) in coastal areas of Papua New Guinea where malaria is hyperendemic. The Plasmodium falciparum erythrocyte-binding antigen 140 (EBA140, also known as BAEBL) binds with high affinity to the surface of human erythrocytes. Here we show that the receptor for EBA140 is glycophorin C (GYPC) and that this interaction mediates a principal P. falciparum invasion pathway into human erythrocytes. EBA140 does not bind to GYPC in Ge-negative erythrocytes, nor can P. falciparum invade such cells using this invasion pathway. This provides compelling evidence that Ge negativity has arisen in Melanesian populations through natural selection by severe malaria.     

CR2 is a complement activator and the covalent binding site for C3 during alternative pathway activation by Raji cells

Antibody-independent activation of the alternative C pathway by human lymphoblastoid cell lines latently infected with EBV has been recognized for some time, although the mechanisms involved and the specific cell surface molecule(s) recognized by the C system have not been identified. The present studies, carried out with the purified proteins of the alternative pathway have addressed these questions. Activation of the purified proteins of the alternative pathway by Raji lymphoblastoid cells was found to be antibody independent, confirming earlier findings with serum. Surprisingly, activation was highly dependent on properdin. In other models properdin has been found to augment alternative pathway activation and to be required for lysis of virus infected cells. Molecules which activate the alternative pathway provide binding sites on which C3 breakdown by regulatory proteins is impeded; therefore intact C3b accumulates on the activator. Immunoprecipitation studies with either anti-CR2 or anti-C3 have identified CR2, the R for C3d,g and EBV, as a major covalent and noncovalent binding site for C3 deposition on Raji cells during alternative pathway activation. Covalently bound C3b was dissociated from CR2 by hydroxylamine, indicating attachment via an ester bond. C3b binding after activation was not reduced by an anti-CR2 mAb which blocks CR2 R function, indicating that it was probably not mediated by C3d,g R epitopes on CR2. Direct confirmation of the ability of CR2 to trigger the alternative pathway came from studies with purified CR2 which was found to activate the alternative C pathway in serum or in mixtures of the purified proteins of the pathway. This work provides conclusive evidence that CR2 is a C activator and functions in this capacity on Raji cells.     

Disease-associated loss of erythrocyte complement receptors (CR1, C3b receptors) in patients with systemic lupus erythematosus and other diseases involving autoantibodies and/or complement activation

Although surface membrane density of complement receptor type one (CR1) on erythrocytes (E) is probably an inherited trait among normal individuals, recent evidence from our laboratories suggests that the reduced number of CR1 per E observed in patients with systemic lupus erythematosus (SLE) results from acquired as well as genetic factors. In the present investigation, the number of CR1 per E was quantitated with 125I-monoclonal anti-CR1 and was found to vary inversely with disease activity in patients with SLE who were followed serially for as long as 14 mo. Although evidence for E surface-bound immune complexes or fixed C3b/iC3b was not obtained, periods of disease activity and low amounts of CR1 per E correlated with the presence of 100 to 800 molecules per E of fixed C3dg fragments (less than 100 C3dg per E in normal subjects). Reduced CR1 and excess fixed C3dg on E also were observed in patients with other disorders associated with complement activation, including chronic cold agglutinin disease, autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria (PNH), Sj?gren's syndrome, and mycoplasma pneumonia. A significant negative correlation (r = -0.498) between CR1/E and fixed C3dg/E was demonstrable in 255 individual assays evaluated by regression analysis. CR1 decreased and fixed C3dg increased during active disease; the converse was obtained during remission. In patients with active SLE, both serum complement activity and E CR1 decreased, whereas fixed C3dg fragments increased. By piecewise linear regression analysis, the appearance of 100 to 400 C3dg molecules on patients' E corresponded to a 27 to 60%, reduction in the number of CR1 per E (p less than 0.0002), confirming that fixation of C3 to E was correlated with a loss of CR1. In patients with PNH, low values for CR1 were observed on moderately complement-sensitive PNH type II E in association with increased fixed C3 fragments; however, the markedly complement-sensitive PNH type III E had essentially normal amounts of CR1 and bore little fixed C3. The addition of soluble DNA/anti-DNA immune complexes to normal blood generated levels of fixed C3dg fragments on E comparable to those observed on E from patients with SLE. Kinetic experiments indicated that C3b was fixed to E during the process of immune complex binding and release from E CR1, and that this fixed C3b was subsequently degraded rapidly to fixed iC3b and more slowly to fixed C3dg without the loss of CR1 that occurs in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complement-regulatory proteins in severe malaria: too little or too much of a good thing?

Data from several laboratories suggest that erythrocyte complement-regulatory proteins, in particular complement receptor 1 (CR1), are important in the pathogenesis of severe malaria. Additional studies suggest that the levels of expression of CR1 and the complement regulator CD55 on erythrocytes vary with age, being low in young children and increasing with age. It is proposed that the interplay between the rate at which immunity develops during malaria exposure and the changes in levels of erythrocyte complement-regulatory proteins that occur with age might contribute to the differences in epidemiology of severe malaria-associated anaemia and cerebral malaria.     

Loss of complement receptor type 1 (CR1) on ageing of erythrocytes. Studies of proteolytic release of the receptor.

Complement receptor type 1 (CR1) is a glycoprotein of Mr about 250 000 present on erythrocytes and other cell types. CR1 acts as a cofactor in the factor I-mediated breakdown of complement fragment C3b to form iC3b. Using an assay of cofactor activity, a wide variation in mean CR1 levels between erythrocytes from individual donors is observed. CR1 levels also decrease on ageing of erythrocytes in vivo, and again the rate of loss is widely variable between individuals. However, variable loss of CR1 during ageing of erythrocytes is likely to make only a minor contribution to the observed variation in mean CR1 levels. CR1 is very sensitive to proteolysis, and random proteolytic removal of CR1 from erythrocytes is likely to be an important factor in loss of CR1 on ageing of red cells in vivo. In vitro, mild trypsin treatment, plasmin or thrombin digestion of erythrocytes results in the loss of the factor I cofactor activity from the cell surface, and appearance of this activity in the supernatant. We conclude that an active fragment of CR1 is released from the cell surface on proteolysis. Subsequent prolonged trypsin treatment destroys most of the activity of this fragment. Proteolytic removal of CR1 from red cells may account not only for loss on ageing of cells, but also for the acquired CR1 deficiencies observed by others in systemic lupus erythematosus.     

Structure-guided identification of C3d residues essential for its binding to complement receptor 2 (CD21)

A vital role for complement in adaptive humoral immunity is now beyond dispute. The crucial interaction is that between B cell and follicular dendritic cell-resident complement receptor 2 (CR2, CD21) and its Ag-associated ligands iC3b and C3dg, where the latter have been deposited as a result of classical pathway activation. Despite the obvious importance of this interaction, the location of a CR2 binding site within C3d, a proteolytic limit fragment of C3dg retaining CR2 binding activity, has not been firmly established. The recently determined x-ray structure of human C3d suggested a candidate site that was remote from the site of covalent attachment to Ag and consisted of an acidic residue-lined depression, which accordingly displays a significant electronegative surface potential. These attributes were consistent with the known ionic strength dependence of the CR2-C3d interaction and with the fact that a significant electropositive surface was apparent in a modeled structure of the C3d-binding domains of CR2. Therefore, we have performed an alanine scan of all of the residues within and immediately adjacent to the acidic pocket in C3d. By testing the mutant iC3b molecules for their ability to bind CR2, we have identified two separate clusters of residues on opposite sides of the acidic pocket, specifically E37/E39 and E160/D163/I164/E166, as being important CR2-contacting residues in C3d. Within the second cluster even single mutations cause near total loss of CR2 binding activity. Consistent with the proposed oppositely charged nature of the interface, we have also found that removal of a positive charge immediately adjacent to the acidic pocket (mutant K162A) results in a 2-fold enhancement in CR2 binding activity.     

Avidin-induced lysis of biotinylated erythrocytes by homologous complement via the alternative pathway depends on avidin's ability of multipoint binding with biotinylated membrane.

It was reported that avidin and streptavidin induce lysis of prebiotinylated red blood cells via the alternative pathway of both homologous and heterologous complement. Both of these proteins have four biotin-binding sites, providing a polyvalent interaction with biotinylated components of the erythrocyte membrane. We have compared the effects of mono- and multipoint avidin attachment on the sensitivity of biotinylated erythrocytes to lysis by the complement system. In the presence of anti-avidin antibody, avidin-bearing biotinylated erythrocytes were rapidly lysed by heterologous serum. This lysis was independent from the mode of avidin attachment, implying that complement activation by the classical pathway triggered by interaction between C1 and avidin-bound antibody on the erythrocyte surface is independent from the avidin's ability of polyvalent (multipoint) binding with biotinylated membrane components. In the absence of anti-avidin antibody, biotinylated erythrocytes bearing polyvalently attached avidin were lysed by homologous complement better than cells bearing avidin, which possesses reduced ability for multipoint binding with biotinylated erythrocyte. Two independent approaches to reduce avidin's ability of multipoint binding were used: decrease in surface density of biotin on the erythrocyte membrane and blockage of biotin-binding sites of avidin. Both methods result in reduced lysis of avidin-bearing erythrocytes as compared with erythrocytes bearing an equal amount of polyvalent-bound avidin. Thus the activation of homologous complement via the alternative pathway depends on avidin's ability to 'cross-link' to the biotinylated components of the erythrocyte membrane.     

Endogenous association of decay-accelerating factor (DAF) with C4b and C3b on cell membranes

Decay-accelerating factor (DAF) is a membrane glycoprotein found on various cells that are in contact with complement. It inhibits the formation of the C3 convertases of the complement system, both the classic (C4b2a) and alternative (C3bBb) pathways. In this investigation, we used a homobifunctional cross-linking reagent to search for a DAF ligand on the surface of cells subjected to complement attack. We found that DAF forms complexes with C4b and C3b deposited on the same erythrocytes, but not with the physiologic degradation products of these complement fragments, that is, C4d or C3dg. Taken together with prior observations that DAF action is reversible, and DAF does not affect the structure of C4b or C3b, these findings suggest that DAF functions by competitively inhibiting the uptake of C2 or factor B, and preventing the assembly of the C3 convertases.     

Effect of hybrid complement regulatory proteins on xenogeneic cells

To suppress C3 fragment deposition in the classical pathway complement activation on xenogeneic membranes, decay accelerating factor (DAF) was the most effective molecule among the complement regulatory proteins (CRPs) used in the present study. C3 fragment deposition was closely related to subsequent xenogeneic cell lysis. However, other molecules were also very effective in different ways and include phosphatidylinositol (PI)-anchored short consensus repeat (SCR) 2-4 of membrane cofactor protein (MCP-PI), PI-anchored C1 esterase inhibitor (C1-INH-PI), and PI-anchored SCR8-11 of complement receptor type 1 (CR1-PI). On the other hand, regarding a strategy for downregulating C4 fragment deposition, the use of only C1-INH-PI and PI-anchored SCR1-3 of the C4b-binding protein (C4bp-PI) was found to be effective.     

Regulation of the alternative pathway of complement by pH

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia. The abnormal PNH erythrocytes are highly susceptible to complement-mediated lysis in vitro, especially at pH 6.4. Lysis has been shown to be due to alternative pathway activation. The purpose of this study was to determine why lysis of PNH erythrocytes is increased at acidic pH. The results presented demonstrate that at pH 6.4: binding of C5 and Factor B to C3b deposited on human erythrocytes is markedly enhanced; generation of the two C3 convertases, C3(H2O), Bb and C3b,Bb is increased; and control of C3b on human erythrocytes by CR1 and Factor I is diminished. In addition, it was found that rabbit erythrocytes, which activate the human alternative pathway, are also lysed much better at pH 6.4 than at pH 7.4. These results indicate that the optimal pH for the initiation and amplification of the alternative complement pathway, and probably also for the activation of the membrane attack complex, is 6.4.     

Plasmodium falciparum histidine-rich protein 1 associates with the band 3 binding domain of ankyrin in the infected red cell membrane

Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm. Changes occur in the infected erythrocyte due to altered phosphorylation of proteins and to novel interactions between host and parasite proteins, particularly at the membrane skeleton. In erythrocytes, the spectrin based red cell membrane skeleton is linked to the erythrocyte plasma membrane through interactions of ankyrin with spectrin and band 3. Here we report an association between the P. falciparum histidine-rich protein (PfHRP1) and phosphorylated proteolytic fragments of red cell ankyrin. Immunochemical, biochemical and biophysical studies indicate that the 89 kDa band 3 binding domain and the 62 kDa spectrin-binding domain of ankyrin are co-precipitated by mAb 89 against PfHRP1, and that native and recombinant ankyrin fragments bind to the 5' repeat region of PfHRP1. PfHRP1 is responsible for anchoring the parasite cytoadherence ligand to the erythrocyte membrane skeleton, and this additional interaction with ankyrin would strengthen the ability of PfEMP1 to resist shear stress.     

Chondroitin sulfate A-adhering Plasmodium falciparum-infected erythrocytes express functionally important antibody epitopes shared by multiple variants

Acquired protection from Plasmodium falciparum placental malaria, a major cause of maternal, fetal, and infant morbidity, is mediated by IgG specific for the P. falciparum erythrocyte membrane protein 1 variant VAR2CSA. This protein enables adhesion of P. falciparum-infected erythrocytes to chondroitin sulfate A in the intervillous space. Although interclonal variation of the var2csa gene is lower than that among var genes in general, VAR2CSA-specific Abs appear to target mainly polymorphic epitopes. This has raised doubts about the feasibility of VAR2CSA-based vaccines. We used eight human monoclonal IgG Abs from affinity-matured memory B cells of P. falciparum-exposed women to study interclonal variation and functional importance of Ab epitopes among placental and peripheral parasites from East and West Africa. Most placental P. falciparum isolates were labeled by several mAbs, whereas peripheral isolates from children were essentially nonreactive. The mAb reactivity of peripheral isolates from pregnant women indicated that some were placental, whereas others had alternative sequestration foci. Most of the mAbs were comparable in their reactivity with bound infected erythrocytes (IEs) and recombinant VAR2CSA and interfered with IE and/or VAR2CSA binding to chondroitin sulfate A. Pair-wise mAb combinations were more inhibitory than single mAbs, and all of the mAbs together was the most efficient combination. Each mAb could opsonize IEs for phagocytosis, and a combination of the eight mAbs caused phagocytosis similar to that of plasma IgG-opsonized IEs. We conclude that functionally important Ab epitopes are shared by the majority of polymorphic VAR2CSA variants, which supports the feasibility of VAR2CSA-based vaccines against placental malaria.