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Infection and Immunity, December 2006, p. 6778-6784, Vol. 74, No. 12
0019-9567/06/$08.00+0     doi:10.1128/IAI.01187-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Limited Cross-Reactivity among Domains of the Plasmodium falciparum Clone 3D7 Erythrocyte Membrane Protein 1 Family

Louise Joergensen,¹ Louise Turner,¹ Pamela Magistrado,¹ Madeleine A. Dahlbäck,¹ Lasse S. Vestergaard,¹ John P. Lusingu,^1,2 Martha Lemnge,² Ali Salanti,¹ Thor G. Theander,¹ and Anja T. R. Jensen^1*

Centre for Medical Parasitology at Department of Medical Microbiology and Immunology, University of Copenhagen and Copenhagen University Hospital, 2200 Copenhagen, Denmark,¹ National Institute for Medical Research, NIMR Tanga Centre, P.O. Box 5004, Tanga, Tanzania²

Received 28 July 2006/ Returned for modification 5 September 2006/ Accepted 20 September 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
The var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family is responsible for antigenic variation and sequestration of infected erythrocytes during malaria. We have previously grouped the 60 PfEMP1 variants of P. falciparum clone 3D7 into groups A and B/A (category A) and groups B, B/C, and C (category non-A). Expression of category A molecules is associated with severe malaria, and that of category non-A molecules is associated with uncomplicated malaria and asymptomatic infection. Here we assessed cross-reactivity among 60 different recombinant PfEMP1 domains derived from clone 3D7 by using a competition enzyme-linked immunosorbent assay and a pool of plasma from 63 malaria-exposed Tanzanian individuals. We conclude that naturally acquired antibodies are largely directed toward epitopes varying between different domains with a few, mainly category A, domains sharing cross-reactive antibody epitopes. Identification of groups of serological cross-reacting molecules is pivotal for the development of vaccines based on PfEMP1.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The Plasmodium falciparum variant surface antigens (VSA), including the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, play an important role in the pathogenesis of malaria (6, 12, 13, 16, 24, 25) by mediating adherence of infected erythrocytes to receptors on the vascular lining (33). This adherence enables the parasite to escape clearance in the spleen and can be detrimental to the human host by facilitating high parasite growth rates and unchecked and harmful inflammation (8). Individuals living in areas where P. falciparum is endemic acquire natural protective immunity from malaria over a period of several years by a gradual acquisition of specific immunoglobulin G (IgG) against different VSA (reviewed in references 7 and 12). PfEMP1 is the best-characterized VSA (40), and antibodies to these molecules have been associated with protection against malaria in both children (20) and pregnant women (29). PfEMP1 molecules are encoded by the var gene family, comprising 40 to 60 highly diverse genes per haploid genome (3, 35, 40). The genome of the clone 3D7 encodes 59 full-length var genes, which can be grouped into three major groups (A, B, and C) and two intermediate groups (B/A and B/C) on the basis of chromosomal location, direction of transcription with respect to chromosome telomeres, domain structure of the encoded proteins, and sequence similarities in coding and noncoding regions (15, 18). The extracellular and variable sequence of PfEMP1 comprises four different domain types: the N-terminal segment, the C2, the cysteine-rich interdomain region (CIDR), and the Duffy binding-like (DBL) domains (9, 40). The CIDR domains group as three (, ß, and ) and the DBL domains as seven (, ß, , , , , and x) distinct sequence classes (3, 36, 40). Groups A and B/A make up the largest PfEMP1s, with a 7- to 10-domain structure, which is different from the 4-domain-type structure predominant of groups B, B/C, and C (18).

Several studies have demonstrated that parasites causing severe P. falciparum malaria in young children who have little or no protective immunity tend to express VSA linked to severe malaria (VSA_SM) that are serologically distinct from those expressed by parasites causing uncomplicated and subclinical infection in older, more immune individuals (6, 24). The VSA_SM appear to be serologically more conserved and cross-reactive than VSA expressed during uncomplicated malaria infections (VSA_UM) (25), consistent with the finding that immunity to severe malaria is acquired more rapidly than immunity to uncomplicated disease (28). We have previously established a link between expression of group A or B/A (here collectively named category A) PfEMP1 and the VSA_SM phenotype and a link between expression of group B, B/C, or C (here collectively named category non-A) PfEMP1 and the VSA_UM phenotype (13). The serological diversity among parasites expressing VSA_SM is lower than that among parasites expressing VSA_UM, possibly because of functional constraints (18), and this suggests that category A PfEMP1 molecules would be more likely to share cross-reactive antibody epitopes than molecules not belonging to this category.

The sequence similarity between different PfEMP1 domains varies, but it is generally limited and amino acid alignments will often be characterized by relatively short runs of conserved residues interrupted by much longer stretches of high sequence diversity (36, 41). Protective immunity against malaria could be acquired either as individuals build a broad repertoire of antibodies against polymorphic PfEMP1 epitopes or by slow acquisition of antibodies against conserved PfEMP1 epitopes. Previously, some reports have indicated that agglutinating VSA antibodies do not seem to be directed against conserved epitopes (22), while others have shown that some cross-reactivity must exist since a single P. falciparum infection is capable of inducing antibodies cross-reacting with VSA expressed on heterologous parasite isolates (10, 23). Additionally, antibodies reacting with linear conserved epitopes have been shown to be acquired by individuals living in areas where malaria is endemic (26, 38).

This is the first study addressing the question of whether groups or distinct sequence classes of PfEMP1 are more likely than others to share cross-reactive antibody epitopes.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Protein expression. A total of 60 (numbered 1 to 60) different recombinant protein domains representing 11 of the 14 group A and B/A; 20 of the 44 group B, C, and B/C PfEMP1; and VAR2CSA (PFL0030c) of clone 3D7 were cloned and expressed for competition enzyme-linked immunosorbent assay (ELISA). Domains and subtypes DBL (, ß, , , ), C2, and CIDR (, ß, ) were defined as described in reference 36, and specific primers for each recombinant protein domain were designed accordingly.

The DBL1 (PFB1055c, PFD1000c, PFD1235w, MAL6P1.316, PF08_0106), DBL3ß (PFD1235w), C2 (PFD1235w), CIDR2ß (PFD1235w), DBL2x (PFL0030c), DBL4 (PFL0030c), DBL5 (PFL0030c), and DBL6 (PFL0030c) domains were PCR amplified from clone 3D7 genomic DNA and subcloned into the pGEX-4T1 vector (GE Healthcare, Hørsholm, Denmark). The NTS (PF11_0008, PF13_0003), DBL1 (PFA0005w, PFA0015c, PFA0765c, PFD0020c, PFD1015c, MAL6P1.4, PF07_0139, PF07_0050, MAL7P1.1, MAL7P1.56, PF08_0141, PF08_0142, PFI0005w, PFI1820w, PFI1830c, PF10_0406, PF11_0007, PF11_0008, PF11_0521, PFL0935c, PFL2665c, PF13_0003, PF13_0364), DBL1x (PFL0030c), CIDR1 (MAL7P1.55), CIDR1 (PF08_0107) DBL2 (PF11_0008), DBL3 (PF13_0003), DBL4 (PF13_0003), CIDR2ß (PF13_0003), C2-1 and C2-2 (PF13_0003) domains were subcloned into the Gateway (Invitrogen, Taastrup, Denmark) pDEST-15 vector.

The recombinant domains were expressed as fusion proteins at the carboxy terminus of glutathione S-transferase (GST) from Schistosoma japonicum (34) in Escherichia coli and purified by affinity chromatography on glutathione Sepharose 4B (GE Healthcare).

The DBL1 (PFD0995c), DBL2ß, and DBL5 (MAL6P1.316) domains were subcloned into the pAcSecG2T baculovirus vector (BD Biosciences, Brøndby, Denmark) for production of GST-tagged proteins. The NTS, DBL2ß-C2, DBL4, DBL4-DBL5, DBL5, CIDR1 (PFD1235w), DBL2 (PF08_0107), DBL3x and CIDR2ß (PF11_0008), and DBL2ß and CIDR1 (PF13_0003) domains were subcloned into the pBAD-TOPO vector (Invitrogen). For production of the carboxy-terminal V5 epitope and histidine-tagged protein, the inserts were excised by EcoRI and PmeI digestion and subsequently subcloned into the EcoRI and blunt-ended BglII sites of baculovirus transfer vector pAcGP67-A (BD Biosciences). Recombinant baculovirus was generated by cotransfection of the pAcSecG2T or pAcGP67-A construct and Bsu36I-linearized Bakpak6 baculovirus DNA (BD Biosciences Clontech) into insect Sf9 cells. Recombinant products were expressed by infection of insect High-Five cells with recombinant baculovirus. GST-tagged protein was purified as described above. His-tagged protein was purified from culture supernatants on a Co²⁺ metal chelate agarose column as previously described (13).

To test for systematic differences between serological recognition of bacterial and baculovirus-expressed polypeptides, antibody recognition of all proteins was compared by using the same pool of plasma as subsequently used for the competition ELISA. The mean optical densities (ODs) did not differ significantly between the proteins expressed in the two systems.

Plasma. Plasma samples from 63 different malaria-asymptomatic Tanzanian donors (mean age, 11.3 years; standard deviation, 4.3 years) living in Mgome village in the Tanga region of Tanzania were pooled and used for the competition ELISA. This lowland village is characterized by an entomological inoculation rate ranging between 91 and 405 infectious bites/person/year (5). Prior to this study, informed consent was obtained from all individuals and/or their parents and ethical clearance was granted by the Ministry of Health and the Ethics Committee of the National Institute for Medical Research in Tanzania.

Competition ELISA. The competition ELISA was carried out with the different protein domains expressed in E. coli or baculovirus. Blocking of the plasma pool was done with 4 µg/ml recombinant protein (x: recombinant proteins 1 to 60) for 2 h at room temperature. The preabsorbed pool was diluted 1:100, and 100 µl was added to duplicate wells of Maxisorp microtiter plates precoated with antigen (1 to 5 µg/ml) or GST as a control antigen (14). In addition to the preabsorbed pool (pPx), a nonabsorbed pool (nP) was included in each plate. Following 1 h of incubation at room temperature, plates were washed four times in phosphate-buffered saline (PBS)-Tx100 (PBS, 0.5 M NaCl, 1% Triton X-100, pH 7.4). Plates were incubated with peroxidase-conjugated rabbit anti-human IgG (Dako, Glostrup, Denmark) diluted 1:3,000 in PBS-Tx100 including 1% bovine serum albumin for 1 h and washed. Antibody reactivity was visualized by adding 100 µl/well o-phenylenediamine substrate (0.6%; Dako) diluted in distilled H₂O with 0.05% (vol/vol) H₂O₂. Color reactions were stopped by the addition of 100 µl of 2.5 M H₂SO₄, and OD was measured at 492 nm. The percent reduction in antibody reactivity was calculated as follows: 100 – [(OD_pPx/OD_nP) x 100]. Most assays were performed twice, and the results were similar. Data from the last experiment or in case the assay only was performed once, the first and only experiment were assigned a white (0 to 50%), gray (>50 to 75%), or black (>75%) color according to the reduction in antibody reactivity and depicted in SigmaPlot 8.0 (Systat Software Inc., Point Richmond, CA). Proteins were defined as sharing cross-reactive antibody epitopes if the percent reduction in OD was greater than 50%.

Indirect ELISA. To analyze whether CIDR domains were more frequently recognized by human antibodies compared to DBL1 domains, we tested the reactivity of plasma from 20 children aged 2 to 4 years and living in one of two villages, Mgome and Ubiri. Ubiri is an intermediate-altitude village with an estimated entomological inoculation rate of 1.8 to 34 infectious bites/person/year (5). DBL1 of PF11_0008, MAL6P1.316, PF08_0106, PF08_0142, PFI1820w, PFD0995c, PFL0935c, and PFB1055c; CIDR1 of PFD1235w; CIDR1 of PF08_0107 and PF13_0003; and CIDR2ß of PF13_0003 were cloned, expressed, and purified as described above. The ELISAs were optimized to give very similar ODs with a positive pool at different dilutions and the samples tested as described above.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Because of the limited availability of whole genome sequences from different P. falciparum genomes, our analysis was directed toward cross-reactivity among clone 3D7-derived PfEMP1 domains.

Protein expression and assay design. We cloned and expressed 60 different recombinant protein domains representing 32 of the 59 full-length var genes in clone 3D7 and used these in a competition ELISA.

Prior to the competition ELISA analysis, a pool of plasma from 63 different Tanzanian donors (mean age, 11.3 ± 4.3 years) living in a village with a high malaria transmission intensity (19) was generated. The IgG reactivity of this pool was tested with each of the 60 recombinant domains as the coating antigen in an indirect ELISA previously optimized to give a high OD reading for each protein. The OD readings of the pool varied from a minimum OD of 0.5 (DBL1 of PF13_0003, PF11_0007, and PF07_0050), a median OD of 1.0 (PF11_0008 DBL3x, PF13_0003 DBL4 and CIDR2ß, and PFD1000c DBL1), and a maximum OD of 4.0 (PFD1235w CIDR1 and PF13_0003 DBL2ß).

Category A and non-A PfEMP1 and antibody cross-reactivity. To detect antibody epitopes shared between PfEMP1 domains, the plasma pool was preincubated with each of the 60 recombinant domains and subsequently tested in a series of indirect ELISAs with the same 60 domains as the coating antigen. As a control experiment, the plasma pool was preincubated with GST and the ELISA reactivity to each of the recombinant domains was compared to the antibody reactivity before preincubation. For all 60 recombinant polypeptides, preincubation with GST exhibited a <25% reduction in OD, except for PF08_0142 DBL1 (32%) and PF13_-0003 CIDR1 (26%). We therefore defined antibody cross-reactivity with a conservative cutoff of a 50% reduction in OD.

The 60 protein domains were sorted according to whether they appeared in category A or non-A PfEMP1 in a checkerboard diagram showing the inhibition of OD reactivity following preincubation with different domains (Fig. 1). Generally, there was only little evidence of serological cross-reactivity between domains, with only 11 (1%) of 1,245 heterologous protein combinations showing inhibition above the cutoff, whereas inhibition was seen in 37 (76%) of 49 assays when the competing and coating antigens were identical. Interestingly, category A domains (0.9%; 10 of 1,245 heterologous combinations) were more likely to inhibit ELISA reactivity than category non-A PfEMP1 domains (0.1%; 1 of 1,245 heterologous combinations) (Fig. 1). Of the 11 protein domains showing serological cross-reactivity, 4 were expressed in baculovirus and 7 were expressed in E. coli. A few domains were produced in both expression systems and shown to behave similarly in the competition ELISA experiments (data not shown).

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FIG. 1. Cross-reactive antibody epitopes shared among 60 PfEMP1 domains of the clone 3D7 genome. Reactivity for category A PfEMP1 domains (A and B/A according to reference 18) are on the left, and category non-A PfEMP1 domains (B, C, and B/C according to reference 18) are on the right. Responses with the same protein as the competition and coating antigen are on the diagonal. The reduction in antibody reactivity was calculated as described in Materials and Methods. White, <50% reduction; gray, 50 to 75% reduction; black, >75% reduction. Blanks with no square are protein combinations not tested.

Antibody cross-reactivity and PfEMP1 domain types. Since the different domain types (DBL, -ß, -, -, and - and CIDR) were not equally distributed between the two categories; we reanalyzed the data according to domain type (Fig. 2 and Table 1). Unexpectedly, DBL1 domains only rarely shared cross-reactive epitopes (1.0%; 3 of 305 heterologous combinations), indicating that antibodies to conserved amino acid sequences in DBL1 (such as LARSFADIG) were less frequent than expected. By contrast, 4 of 21 (19.0%) heterologous CIDR combinations showed inhibition in the competition assays (Table 1).

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FIG. 2. Cross-reactive antibody epitopes shared among 60 PfEMP1 domains of the clone 3D7 genome as described in the legend to Fig. 1. Reactivities are grouped according to domain type and subtype. Blanks with no square are protein combinations not tested.

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TABLE 1. Total numbers of different domain types encoded by the clone 3D7 genome and numbers of domain types tested and found to contain cross-reacting antibody epitopes

With eight different recombinant DBL1 and four different CIDR domains in an indirect ELISA, we found that plasma antibodies from 20 children aged 2 to 4 years more frequently recognized CIDR than DBL1 domains. Both domain types were more frequently recognized by plasma samples from a high malaria transmission intensity area, Mgome, compared to samples from Ubiri, a moderate malaria transmission intensity area of Tanzania (Fig. 3).

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FIG. 3. Percentages of children aged 2 to 4 years recognizing eight recombinant DBL1 and four different CIDR domains of clone 3D7 PfEMP1. Recognition of DBL1 (gray box plot) and CIDR (white box plot) domains was tested with plasma from 20 different children aged 2 to 4 years living in the villages of Mgome (high transmission) and Ubiri (moderate transmission). The 10th and 90th percentiles are shown. Dots show the percentages of samples positive in each individual protein ELISA.

Evidence of serological cross-reactivity was mainly found between similar domain types (CIDR versus CIDR or DBL versus DBL) and could often be explained by sequence similarity (Table 2). In four cases, inhibitory activity was present without any evidence of sequence similarity between the competing and coating antigens, indicating that conformational epitopes might contribute to cross-reactivity.

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TABLE 2. Sequence identities between PfEMP1 domainsa

Top Abstract Introduction Materials and Methods Results Discussion References

 
Results from previous work have shown that VSA associated with severe malaria are serologically more conserved than variants associated with uncomplicated or mild malaria (6, 24) and that expression of group A PfEMP1 confers a VSA_SM phenotype on parasites (13, 39). In this study, except for a few mainly category A domains, we found recombinant polypeptides with the primary sequence of clone 3D7 PfEMP1 domains rarely competing for antibodies present in plasma from individuals naturally exposed to P. falciparum. Among the different domain types, heterologous CIDR combinations showed inhibition more frequently in the competition assay and were more frequently recognized by individual plasma samples from children aged 2 to 4 years compared to DBL1 domains. This could be explained by a higher antigenicity and the presence of cross-reactive antibody epitopes in the CIDR domains. CIDR domains have previously been shown to induce cross-reactive antibodies in mice following immunization with plasmid DNA expressing CIDR domains (2, 11), and such antibodies have been suggested to modify disease progression in vaccine experiments where Aotus nancymai monkeys were challenged with a heterologous parasite line (21).

From a biological point of view, limited cross-reactivity among PfEMP1 molecules expressed from a single parasite genome would make sense. Individual parasites have the ability to swap expression among 50 to 60 different antigenically distinct PfEMP1 variants through a clonal antigen switching process which is not yet fully understood (4, 27, 35). By this process, P. falciparum is able to establish chronic monoclonal infections which are probably essential for parasite survival in areas with limited and highly seasonal transmission (37). If induction of cross-reactive PfEMP1 antibodies were a frequent event, this would limit the parasite's benefit of carrying many different var genes. The same applies to the initial phase of P. falciparum infections. Studies of var gene expression in parasites isolated just after liver release from individuals experimentally infected with strain NF54 (the parental strain of clone 3D7) suggest that different var genes are being transcribed by individual parasites and that many different PfEMP1 variants might be presented by the parasites establishing the asexual blood stage infection (17). This broad PfEMP1 expression could enhance parasite sequestration in hosts with different levels of preexisting immunity and extend the duration of infection. The presence of antibodies capable of cross-reacting with many different PfEMP1 molecules would reduce the chances of parasite survival even in hosts with little preexisting immunity.

Although we could not test for cross-reactivity between PfEMP1 molecules encoded by different genomes, such cross-reactivity clearly exists. By the age of 3 to 4 years, children in areas where malaria is highly endemic have acquired surface-reactive antibodies against most VSA variants (1). In the present study, antibodies could be detected against all of the recombinant protein domains tested despite its being very unlikely that any of the plasma donors had been exposed to parasites expressing PfEMP1 identical to those of clone 3D7.

The extracellular domain of the different PfEMP1 molecules within the clone 3D7 and other parasite genomes shows a high degree of primary sequence polymorphism. However, a number of conserved subfamilies, such as var1-3, have been described (15, 28, 30, 31). These var genes are present in many P. falciparum genomes, and within each subfamily stretches of high amino acid sequence similarity are found. Such PfEMP1 subfamilies are likely to induce cross-reactive antibodies, and it is conceivable that the global PfEMP1 repertoire is structured into distinct serologically cross-reactive types which could share functions such as binding identical receptors (e.g., CSA, CD36, and others) or being expressed at the same stage of the parasite life cycle (32). Thus, we would speculate cross-reactivity to be more frequent at the intergenomic level compared to the intragenomic level. Identification of groups of serologically cross-reacting PfEMP1 molecules is pivotal for the development of vaccines based on variant antigens that should induce broad protection.

 
We thank Lotte Bram and Susanne Pedersen for excellent technical assistance.

This study received financial support from the Danish Council for Health Science Research (grant 271-05-0427), a grant from the Foundation for the National Institute of Health through the Grand Challenges in Global Health initiative, and a grant from the Danish International Development Agency (104.DAN.8.L.312). A.T.R.J. is supported by the Howard Hughes Medical Institute. A.S., J.P.L., and P.M. are supported by the Gates Malaria Partnership.

 
* Corresponding author. Mailing address: University of Copenhagen, Department of Medical Microbiology and Immunology, Panum 24.2.24, Blegdamsvej 3, Copenhagen DK-2200, Denmark. Phone: 4535327682. Fax: 4535327851. E-mail: atrj{at}cmp.dk.

Published ahead of print on 2 October 2006.

Editor: W. A. Petri, Jr.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, December 2006, p. 6778-6784, Vol. 74, No. 12
0019-9567/06/$08.00+0     doi:10.1128/IAI.01187-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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