Immunization with VAR2CSA-DBL5 Recombinant Protein Elicits Broadly Cross-Reactive Antibodies to Placental Plasmodium falciparum-Infected Erythrocytes

Design of DBL synthetic genes.Synthetic genes were constructed by GenScript Corporation (Piscataway, NJ), and codons were optimized for P. pastoris expression. N-Glycosylation sites were removed by converting asparagine to glutamine, and DNA sequences carrying more than five adenine nucleotides in a row were mutated to avoid any premature termination without changing coding features.

Recombinant protein expression in Pichia pastoris.Cloning of recombinant proteins was done as previously described (3). In brief, VAR2CSA constructs with a His₆ tag on the C terminus were amplified from synthetic genes and cloned into SnaBI and NotI restriction sites in the pPIC9K vector (Invitrogen). Constructs were digested with SacI and electroporated into P. pastoris strain GS115. VAR2CSA construct boundaries are indicated in Fig. 1. The IT4var22-DBL3 (GenBank accession number EF158076) recombinant protein went from amino acids G1179 to N1487, numbering from the first methionine in the protein. For protein production, P. pastoris was grown overnight at 20°C with shaking at 250 rpm in 0.9 liter buffered complex medium (BM; 1% yeast extract, 2% peptone, 1% yeast nitrogen base, 1 M potassium phosphate buffer [pH 6.0]) plus 2% glycerol (BMG) and shifted to 0.3 liter BM plus 0.5% methanol for protein induction. His-tagged recombinant proteins were harvested from supernatants on day 4 or 5 by using nickel resin or cobalt-nitrilotriacetic acid-agarose (Sigma-Aldrich). Recombinant proteins were analyzed in 4 to 20% SDS-PAGE gels under reduced or nonreduced conditions. Gels were stained with GelCode blue reagent or transferred to a nitrocellulose membrane and detected by Western blotting using anti-His tag antibodies (Invitrogen). Protein concentrations were determined by Bradford assay (500-0205; Bio-Rad). The identities of recombinant proteins were confirmed by mass spectrometry analyses. Purified proteins were stored at −80°C in 1× phosphate-buffered saline (PBS).

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FIG. 1.

Expression of VAR2CSA-DBL5 recombinant proteins in P. pastoris. (A) Protein schematic of VAR2CSA. The original DBL domain boundaries are numbered and indicated by black rectangles. In gray are new, longer domain boundaries predicted by X-ray crystallographic analysis of DBL disulfide bonds (27, 30). The first and last amino acids in VAR2CSA constructs are indicated below the protein schematic. The arrowhead indicates that the 3D7-DBL5 protein was truncated in the N terminus at the amino acid position 95, which resulted in a loss of ∼10 kDa of the protein. One microgram of recombinant proteins was analyzed under nonreducing conditions in a 4 to 20% SDS-PAGE gel and stained by GelCode blue reagent (B) or detected by Western blotting using anti-His tag antibodies (C). MW, molecular weight.

Animal immunization.Immunizations were performed at R&R Rabbitry (Washington) according to animal immunization guidelines. In brief, rabbits received 25 to 100 μg recombinant protein in complete Freund's adjuvant for the first immunization and were boosted four times with the same amount of recombinant protein in incomplete Freund's adjuvant. Preimmune and immune rabbit plasma samples were heat inactivated for 45 min at 57°C and stored at −20°C.

Parasite lines. Plasmodium falciparum parasites were grown in O⁺ erythrocytes and 10% human plasma. CSA-binding laboratory isolates FCR3/IT4-CSA (47), CS2-CSA (41), 7G8-CSA (3), HB3-CSA (3), 3D7-CSA and NF54-CSA (3), and Pf2004-CSA (18) and Pf2006-CSA (18) are described in Table 1 and were maintained by periodic selection on CSA. The HB3 parasite encodes two var2csa gene copies (31). Parasite lines predominantly expressing the HB3 var2csa allele A or the HB3 var2csa B allele were cloned from a mixed HB3-CSA parental line by flow cytometric sorting. For non-CSA binding controls, A4ultra and ItG-ICAM-1 were employed. A4ultra expresses the IT4var14 gene, which was maintained by selection with monoclonal antibody (MAb) BC6 (gift from Chris Newbold, Oxford, United Kingdom). ItG-ICAM-1 expresses IT4var16, which was maintained by selection on intercellular adhesion molecule 1. The A4ultra and ItG-ICAM-1 lines are isogenic with FCR3 and CS2. Genotyping of parasites in the reference CSA-binding panel was done with MSP1/MSP2 primers according to previously published approaches (51). The recently adapted clinical isolates 736, 755, and 711 were isolated in Tanzania from the peripheral blood of pregnant women who had placental malaria. Samples were collected at the time of delivery, frozen, and used within a few cycles of in vitro culture without CSA selection, with the exception that the 736 and 711 isolates were selected once by panning on CSA (1xpcsa).

qRT-PCR and flow cytometry assay on IEs.Quantitative real-time PCR (qRT-PCR) and flow cytometry were performed as previously described (3). In brief, var gene transcription was assessed by qRT-PCR by using primers to the DBL4 domain in VAR2CSA (58) or gene-specific primers to ITvar14 and ITvar16 (61). Results are expressed as fold differences in expression relative to that of the housekeeping Pf adenylosuccinate lyase (Pfasl) (PFB0295w) (3). For flow cytometry, mature-stage IEs were grown in O⁺ blood and incubated with rabbit and human plasma that had been preabsorbed twice on uninfected O⁺ erythrocytes. For each assay, 10 million erythrocytes infected with between 5 and 8% trophozoites were incubated with a 1/25 dilution of rabbit plasma or a 1/10 dilution of human plasma. Bound antibodies were detected by adding Alexafluor 488-conjugated goat anti-rabbit IgG (A-11034; Molecular Probes) or fluorescein isothiocyanate (FITC)-conjugated goat F(ab′)₂ anti-human IgG (PN IM1627; Beckman). Samples were analyzed in an LSR II flow cytometer (Becton Dickinson) and analyzed using FLOWJO 8.1 software (Tree Star Inc.). Binding is presented as the mean of the adjusted geometric mean of fluorescence intensity (MFI) for plasma samples run in duplicate. The adjusted MFI was as follows: (IE_i − UE_i) − (IE_p − UE_p), where IE_i is the MFI of IEs following incubation in immune plasma, UE_i is the MFI of uninfected erythrocytes following incubation in immune plasma, IE_p is the MFI of IEs following incubation in preimmune plasma, and UE_p is the MFI of uninfected erythrocytes following incubation in preimmune plasma.

Plasma and clinical parasite samples.The human plasma and clinical parasite samples used in these studies were collected from African donors, under protocols approved by the relevant ethics review committees. Study participants gave a written, informed consent before donating samples, and samples included those from adult multigravid women from Tanzania (n = 10) (24) and adult men (n = 10) and multigravid women from Cameroon (n = 10) (gift from Isabella Quakyi). Nonimmune adult plasma samples are from donors in Seattle.

Depletion of anti-plasma antibodies on recombinant proteins.For the depletion assay, 200 μg of His-tagged P. pastoris-expressed recombinant protein 7G8-DBL5 or 7G8-DBL1 was incubated with 0.3 ml of nickel-nitrilotriacetic acid-agarose resin (Ni-NTA; Qiagen) overnight at 4°C with rotation. Unbound protein was removed by centrifugation, and the recombinant protein-Ni-NTA mixture was then incubated 1 h at 4°C with 0.1 ml of preimmune or immune plasma. The supernatant corresponding to the depleted anti-DBL plasma was collected by centrifugation and tested by flow cytometry as described above.

IE binding and antibody binding inhibition assays.IE binding was performed as previously described (3). In brief, IEs were tested for binding to 10-μl spots of 0.1 mg/ml bovine CSA (Fluka Biochemika) or 0.05 mg/ml rCD36 (R&D). Binding assays were performed with 10 μl of 1 × 10⁷ IEs/ml enriched via MACS purification (43). As a control, CSA spots were pretreated with chondroitinase ABC (Sigma-Aldrich). For antibody inhibition assays, IgG antibodies were purified from 0.2 ml of preimmune or immune rabbit plasma, using the Pierce Nab spin kit (89949; Pierce). The purified IgG concentration was determined by a bicinchoninic acid (BCA) assay (23225; Pierce). Antibody binding inhibition assays were performed as described above, except that binding inhibitions were compared at two concentrations of CSA (0.01 mg/ml or 0.1 mg/ml) and in the presence of purified IgGs at a final concentration of 0.5 mg/ml or 1 mg/ml. Binding inhibition was calculated as the percentage of binding in the presence of immune or preimmune IgGs.

Sequence analysis.Phylogenetic analyses were done using PAUP*4.0b10 to generate neighbor-joining trees with 1,000 bootstrap replicates. Gap opening and gap extension penalties were 5.0 and 0.05, respectively.

Expression of P. pastoris recombinant proteins.It has previously been shown that immunization with the VAR2CSA-DBL1, -DBL3, or -DBL6 recombinant proteins produced in P. pastoris elicited variant-specific antibodies to CSA-binding parasite lines, but we were unable to produce the VAR2CSA-DBL5 in that study (3). One reason why the DBL5 domain may not have been expressed previously is that the construct ended shortly after the 10th cysteine residue (C10), and recent structural information indicates that PfEMP1 DBL domains have two additional cysteine residues that are involved in domain disulfide bonding (30, 48), similar to the erythrocyte invasion binding ligands (49, 55). The final two cysteine residues in DBL domains frequently have a CX_1-2C motif or CX_1-2CX_1-2C motif (30). By extending the domain boundaries of DBL5 recombinant proteins to include a CX_1-2CX_1-2C motif we were able to secrete this domain from P. pastoris and improve the expression of other poorly expressed domains (2). In this study, we investigated the immunogenicity of three VAR2CSA-DBL5 recombinants and a VAR2CSA-DBL1 recombinant protein by using the new construct boundaries that included two additional cysteine residues believed to be important for domain folding (30, 48) (Fig. 1). As a vaccination control, a DBL domain from a non-VAR2CSA protein, IT4var22-DBL3, was also expressed. Overall, 7G8-DBL1 and 7G8-DBL5 were the best-expressed proteins (from 1 to 1.5 mg/liter), while 3D7-DBL5, IT4-DBL5, and IT4var22-DBL3 recombinant proteins were produced at between 0.1 mg/liter and 0.5 mg/liter. Under SDS-PAGE nonreducing conditions, all of the proteins ran at the expected size, except 3D7-DBL5, which was ∼10 kDa smaller than expected (Fig. 1 and data not shown). Edman degradation confirmed that 3D7-DBL5 had undergone N-terminal truncation at amino acid position 95, accounting for the loss in size and giving it nearly the same domain boundaries as those of the other two DBL5 recombinant proteins.

Development of a panel of CSA-binding parasite lines.An obstacle to developing a PAM vaccine is the lack of a standard panel of CSA-binding parasite lines to evaluate antibody breadth. Most analyses have focused on four lab-adapted parasite lines, IT4/FCR3, 7G8, HB3, and NF54/3D7. The first three parasites have been cloned and represent single genotypes, while NF54 is the parent to the 3D7 genome reference isolate and also appears to contain another minor parasite genotype (22). A single var2CSA gene copy is present in all of the lines except for HB3, which has two distinct gene copies (31).

For this study, two new CSA-binding lines were derived from West African isolates (Pf2004 and Pf2006) and a mixed HB3-CSA parasite line was cloned by flow cytometry to generate parasite lines that transcribed either the HB3 A or B alleles to develop a panel of seven CSA-binding parasite lines. By the use of allele-specific qRT-PCR primers, one of the cloned HB3 lines predominantly expressed the HB3 A allele, while the other strongly transcribed the HB3 B allele but also transcribed a low level of the HB3 A allele (data not shown). Altogether, the parasites in the panel included isolates from Central America, South America, and Africa (Table 1), and the two parasite lines from West Africa were isolated several decades after the other parasites in the panel. The geographic origins of IT4/FCR3 and 3D7 are more ambiguous (Table 1), but the IT4/FCR3 genotype is most similar to that of the Southeast Asian isolates based on chromosome-wide single-nucleotide polymorphism (SNP) haplotype (35).

As expected, all of the CSA-binding parasite lines strongly bound CSA but had limited binding to the blood microvasculature receptor CD36 (Table 2). In contrast, the negative-control A4ultra and ItG-ICAM-1 parasite lines had the opposite phenotype. Compared to other parasites in the panel, the NF54-CSA and 3D7-CSA parasite lines (data not shown) were weak CSA binders, even after repeated selection on CSA for more than eight times, and quickly became a mixture of CSA and CD36 phenotypes despite repeated CSA selection. In contrast, the other CSA-binding lines were highly stable for more than 10 parasite cycles (data not shown) and bound CSA to a much greater extent (Table 2).

To confirm that parasites in the panel had a phenotype similar to that of placental isolates, they were tested by qRT-PCR for var2csa transcription and with plasma samples from malaria-exposed donors. All CSA-binding lines highly transcribed var2csa compared to the housekeeping reference control gene Pf adenylosuccinate lyase (Asl). In contrast, var2csa was weakly transcribed in the negative-control A4ultra and ItG-ICAM-1 parasite lines, which express var genes different from var2csa (Table 2). Furthermore, all CSA-binding parasite lines, except for Pf2006-CSA, were preferentially recognized by comparing a pool of plasma samples from multigravid Cameroonian women to those from immune men, a characteristic of placental isolates (Table 3). By comparison, the non-CSA-binding A4ultra and ItG-ICAM lines were recognized equally well or better by male plasma samples than by female plasma samples. The non-gender-biased recognition of Pf2006-CSA may represent only a gap in the Cameroon plasma pool, because Pf2006-CSA was highly recognized by a pool of samples from multigravid women from Kenya and highly transcribed var2csa (Table 2). In addition, individual plasma samples from primigravid and multigravid women in Malawi had significantly higher levels of IgG to all of the CSA-binding parasite lines in the panel than did those from the men (M. Hommel and J. Beeson, unpublished observations). Taken together, this analysis confirms that var2CSA transcription is strongly linked to CSA binding and that parasites in the panel have the binding and antigenic phenotype of placental isolates.

Conservation and diversity of VAR2CSA sequences in the CSA-binding panel.To investigate whether parasites in the CSA binding panel differed in var2csa sequences, the DBL1 and DBL5 domains were cloned and sequenced and compared to other full-length VAR2CSA sequences available in GenBank. In every parasite line, except for HB3, only a single var2csa DBL1 or DBL5 sequence was amplified. This includes the Pf2006-CSA and Pf2004-CSA parasite lines that have not been cloned but have been selected multiple times on CSA in the laboratory and appear to now contain a single or major parasite genotype by MSP-1/MSP-2 genotyping (data not shown). We also confirmed that CS2-CSA and FCR3-CSA parasites, which are isogenic, had identical DBL5 sequences and that the DBL5 sequence from NF54-CSA was identical to the 3D7 progeny line.

Based on previous studies of full-length var2csa genes, DBL5 is the second most conserved VAR2CSA domain after DBL4 (11). In that study, VAR2CSA domains ranged from a low of ∼61% amino acid identity (DBL6) to a high of ∼88% identity (DBL4). In the present study, no two parasites in the panel expressed the same DBL1 or DBL5 sequence, except the HB3 A and B var2csa alleles, which encoded identical DBL5 domains (Fig. 2). Therefore, there is extensive diversity of VAR2CSA sequences in the parasite population. Overall, the DBL1 domain in the parasite panel averaged 80% amino acid identity (range, 72 to 89%), and the DBL5 domain averaged 86% amino acid identity (range, 83 to 99%), similar to that of a larger sampling of 17 VAR2CSA sequences from around the world (for DBL1, 79% identity, range of 71 to 89%; for DBL5, 87% identity, range of 83 to 99%) (Fig. 2). Although parasites in the panel differed in DBL1 and DBL5 sequences, there was a significant overlap of polymorphism between globally dispersed isolates and relatively restricted polymorphism at variable regions (see Fig. S1 and S2 in the supplemental material). The most divergent sequence was the DBL1 domain from WR80, which had unique polymorphism in the C terminus or subdomain 3 (see Fig. S1 in the supplemental material). The WR80 parasite was derived from Southeast Asia (35) but is not in the CSA panel. Except for the unique variation in the C terminus of DBL1, the WR80 sequence shares other variable blocks with other P. falciparum isolates. These findings are consistent with earlier work demonstrating extensive gene mosaicism between var2csa alleles (11, 56) and suggest that VAR2CSA polymorphism has an ancient origin that is likely maintained in the parasite population by gene recombination. Together, this analysis confirms that the parasite panel is comprised of different VAR2CSA sequences, which reflects diversity in the parasite population.

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FIG. 2.

Sequence comparison of VAR2CSA-DBL5. Neighbor-joining trees comparing DBL1 (A) and DBL5 (B) sequences from CSA-binding parasites in the panel to global isolates. Bootstrap values above 80% are shown. The geographic origin of parasites is indicated by color: yellow, Southeast Asia; light brown, Africa; metallic brown, Central or South America; gray, unknown. Parasites in the reference panel are indicated by oval-shaped or rectangular shading. DBL5 recombinant proteins were made from parasites with rectangular shading.

Immunization with DBL5 recombinant proteins elicits broadly strain-transcendent antibodies to diverse CSA-binding parasite lines.To investigate the immunogenicity of recombinant proteins, rabbits were immunized with each of the three VAR2CSA-DBL5 allelic forms, as well as the 7G8 VAR2CSA-DBL1 recombinant protein and the negative-control IT4var22-DBL3 recombinant protein. Antibodies raised against the three DBL5 proteins had nearly identical endpoint titers against the 7G8-DBL5 recombinant protein (ranged between 3 × 10⁵ and 5 × 10⁵), showing that the recombinant proteins contained cross-reactive epitopes. To investigate whether cross-reactive epitopes were exposed in the native protein, plasma antibodies were tested against CSA-binding IEs. By flow cytometry, each of the three VAR2CSA-DBL5 immunogens elicited broad antibody responses to all or most of the CSA-binding parasites in the panel but not with the non-CSA-binding control A4ultra or ItG-ICAM-1 parasite lines (Table 3). As expected, the FCR3-CSA and CS2-CSA lines, which have been used interchangeably in previous studies, gave similar reactivity profiles (data not shown).

In contrast to DBL5 plasma, rabbits immunized with the negative-control var22-DBL3 or A4var-CIDR1 (3) did not react with the CSA-binding parasite lines. In addition, anti-VAR2CSA-DBL1 plasma had lower reactivity, and the three rabbit plasma reacted with only three or four of the seven CSA-binding parasite lines, including the homologous 7G8-CSA parasite. Whereas DBL5 plasma had greater breadth than did DBL1 plasma, there were differences in serological profiles between the three DBL5 immunogens. For instance, the Pf2006-CSA line was weakly recognized by the IT4-DBL5 plasma but was not reactive with 7G8-DBL5 plasma. Conversely, the 7G8-CSA parasite was well recognized by 7G8-DBL5 and 3D7-DBL5 plasma but was only weakly reactive with one of the three rabbits immunized with IT4-DBL5. Differences were also observed between rabbits immunized with the same DBL5 immunogen.

DBL5 plasma samples were also tested against three fresh clinical isolates collected from the blood of pregnant women. These parasite isolates were not fully adapted to continuous in vitro cultivation, but two of them were selected once in vitro for adhesion to CSA to increase VAR2CSA expression. At the time of DBL5 plasma assessment, all three clinical isolates were confirmed to bind to CSA and not CD36 (see Table S1 in the supplemental material) and reacted in a gender-specific manner with malaria-exposed plasma (Table 3). Of the three clinical isolates, isolate 736 reacted with all three DBL5 plasma samples, and all three of the clinical isolates were recognized by at least one DBL5 plasma sample. Thus, immunization with VAR2CSA-DBL5 recombinant proteins elicited broad antibodies to diverse CSA-binding parasite lines.

To confirm that anti-DBL5 antibodies were responsible for pan-reactivity, plasmas were preadsorbed against recombinant proteins. Preabsorption of rabbit polyclonal DBL5 plasma on 7G8-DBL5 recombinant protein depleted antibody recognition of both homologous and heterologous CSA-binding parasite lines, while preabsorption with the negative-control 7G8-DBL1 recombinant protein had no effect on serological recognition (Table 4). To investigate adhesion blocking of DBL5 plasma, IgG antibodies were purified from two of the rabbits that displayed the broadest serological cross-reactivity and tested for inhibitory activity against three different CSA-binding parasite lines. While purified IgG antibodies from immune rabbits bound in a specific manner to CSA-binding parasite lines by flow cytometry, the level of CSA-binding inhibition was weak and variable (between 3 and 26%) and was not significantly above that observed with a negative-control ItG-ICAM-1 parasite binding to CD36 (see Table S2 in the supplemental material; data not shown). Thus, VAR2CSA-DBL5 immunogens elicited pan-reactive antibody responses to diverse CSA-binding parasite lines but limited or no inhibitory activity under this immunization protocol.