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Infection and Immunity, December 2002, p. 7013-7021, Vol. 70, No. 12
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.12.7013-7021.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of Parasitology, Institute for Tropical Medicine, University of Tübingen, Germany,1 Medical Research Unit, Albert-Schweitzer Hospital, Lambaréné, Gabon2
Received 29 March 2002/ Returned for modification 7 May 2002/ Accepted 2 August 2002
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ABSTRACT |
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INTRODUCTION |
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To date, P. falciparum erythrocyte membrane protein 1 (PfEMP-1), encoded by ca. 50 var genes, is the best-characterized surface antigen of IEs (3, 31, 33). The proteins expressed in knobs are highly divergent in their amino acid sequences and clonally variant. Antibodies to PfEMP-1 in sera of adults living in endemic areas recognize specific conserved, semivariable and hypervariable regions on the protein and associations between such antibodies and protection from severe disease have been shown (4, 23, 36).
The recent analysis of the P. falciparum genome sequencing project has identified other multicopy gene families unique to Plasmodia, the largest of which belongs to the rif (repetitive interspersed family) gene family (7, 11, 37). This family of genes encoding 27- to 39-kDa membrane-associated proteins is estimated to be present in the order of ca. 200 copies. Rif genes have two exons, the first of which encodes a putative signal peptide and the second of which encodes an extracellular domain made up of a conserved and a variable region, followed by a transmembrane segment and a short intracellular (cytoplasmic) portion. Unlike var genes, only one of which is expressed at any one time, several rif genes are believed to be concomitantly expressed on the surface of IEs, as evidenced by reverse transcription-PCR (RT-PCR) experiments with RNA prepared from different life cycle stages (6, 9, 19, 20). The function of these clonally variant rifins on the surface of IE remains speculative but because of its surface locality and sequence diversity, these proteins may play an essential role in the host-parasite interface during the asexual blood stage.
We have carried out for the first time a large cross-sectional survey of individuals exposed to natural P. falciparum infections to evaluate the presence of specific anti-rifin antibodies in their sera capable of recognizing recombinant forms of rifin proteins (RIF). Two well-defined study cohorts consisting of clinically immune adults and semi-immune children living in an area of Gabon where malaria is endemic were used for this analysis.
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MATERIALS AND METHODS |
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Study cohort. Before carrying out the study protocol, ethical clearance was obtained from the Ethics Committee of the International Foundation of the Albert Schweitzer Hospital in Lambaréné. All study participants or their parents or guardians gave informed consent. Human sera were collected from 99 clinically immune malaria-exposed individuals who were between the ages of 15 and 64 (median age, 32 ± 16.5 years) participating in a cross-sectional study. Such an individual is described as one living in an area of high malaria endemicity who is at least 15 years old and is protected from disease, showing no clinical symptoms of malaria, regardless of detectable parasitemia. The second group was composed of 90 children with uncomplicated P. falciparum malaria infection that were between 1.3 and 6.5 years old living in the study area (median age, 3.9 ± 1.7 years). The criteria for uncomplicated malaria included a blood smear count of 1,000 to 200,000 parasites/µl, a body temperature higher than 37.5°C, or a history of fever up to 24 h prior to examination. Individuals showing signs of severe malaria were excluded from the study cohort. Blood samples were taken before the initiation of malaria treatment.
PCR amplification and cloning. DNA was extracted from a parasite isolate by using the commercially available QIAamp MiniKit (Qiagen, Hilden, Germany), as previously described (16). DNA was amplified by PCR by using Pwo polymerase (Peqlab Biotechnologie GmbH, Erlangen, Germany) with the help of a set of the degenerate primers Rif-F (5'-ATG AAA RTC CAY TRY TAT AAY ATA TTA TTR TTT) and Rif-R (5'-YTT YTT WCG WCG RTA WCG YAA). Given that there are 200 to 500 rifin sequences in the genome and few data are available on expressed rifins, the most straightforward strategy was to first identify random genomic rifin sequences rather than transcribed sequences. Gel-purified PCR products were cloned into the TA cloning vector pCR2.1 (Invitrogen, Groningen, The Netherlands). The presence of a DNA insert in white recombinant colonies obtained after transformation was screened by PCR amplification with vector-specific primers, M13(-21) and M13(+21), as recommended by the manufacturers. Positive clones were confirmed by nucleotide sequencing with the same primer set on an ABI 373 Prism automated sequencer with a Big Dye terminator sequencing kit (Applied Biosystems, Foster City, Calif.).
Rifin-specific sequences were amplified for cloning into pTrcHis Topo TA expression vector (Invitrogen) by using combinations of primers corresponding to individual cloned rif sequences. Primer pairs were constructed, so as to exclude 5' upstream sequences covering signal sequences and 3' downstream sequences encoding the transmembrane segment. Truncated rifin sequences were thus amplified with the first primer pair ExRif-F/29 (5'-TTA TGC GAA TGT GAA CTA) and ExRif-R/29 (5'-GCC TAT ATT CAA TGA TGA); the second and third pairs were ExRif-F/40,44 (5'-AGA TCA TTA TGC GAA TGC) and ExRif-R/40 (5'-CAT TCC AAT GTC ATA ATT) or ExRifR/44 (5'-CCA ATC CTT TTA GAA ATA), and the last pair of primers was ExRif-F/50 (5'-AGA TTA TTA TGC GAA TGC) and ExRif-R/50 (5'-CGA CAT GTC AGT CCA AAA).
Nucleotide sequences. Sequence identities were confirmed by BLAST analysis. A comparison of the deduced amino acid sequences was made by using the GCG pileup programme (EST Cluster Programme, Heidelberg, Germany). Nucleotide sequences were deposited in the GenBank database under the following accession numbers: rif-21 (AF483814), rif-24 (AF483815), rif-25 (AF483816), rif-29 (AF483817), rif-32 (AF483818), rif-38 (AF483819), rif-40 (AF483820), rif-44 (AF483821), rif-50 (AF483822), rif-52 (AF483823), rif-55 (AF483824), rif-57 (AF483825), and rif-58 (AF483826).
Expression and purification of recombinant proteins. Gel-purified PCR products were cloned by using the pTrcHis Topo TA expression kit based on the histidine-tagged system. Escherichia coli TOP10 cell pellet was resuspended in 8 M urea dissolved in phosphate buffer (20 mM phosphate, 10 mM imidazole, 500 mM NaCl [pH 7.4]), and cells were disrupted by sonication. The solubilized fusion protein was loaded onto HisTrap columns for affinity chromatography (Amersham Pharmacia Biotech, Freiburg, Germany) and eluted with phosphate buffer containing urea and increasing concentrations of imidazole. Purified fusion proteins were stored at -20°C in aliquots in the final elution buffer (phosphate buffer containing 8 M urea and 100 mM imidazole). Fractions were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on a 12% polyacrylamide gel to determine purity. The protein yields were determined by performing a protein assay (Bio-Rad, Munich, Germany), according to the supplier's instructions, and calculated to be between 0.2 and 0.5 mg per 100 ml of induced culture.
Antibody production and testing. Affinity-purified fusion protein was used to immunize rats. Each rat received a primary intraperitoneal injection of 50 µg of emulsified with Freund complete adjuvant, followed by three intradermal and subcutaneous booster injections in Freund incomplete adjuvant at 2- to 3-weekly intervals (Pineda, Berlin, Germany). Antiserum was collected 2 weeks after the last boost. The antibody titer of each rat was determined by an enzyme-linked immunosorbent assay (ELISA), as described below. Rat antisera diluted stepwise from 1:103 to 1:106 were tested. An anti-rat immunoglobulin G (IgG) conjugated to horseradish peroxidase (Sigma, St. Louis, Mo.) (diluted 1 in 15,000) served as a secondary antibody.
Analysis of human immune responses to rifin antigens. An ELISA was used to assess IgG antibody reactivity to the purified His6-rif proteins. All ELISAs were repeated in at least two independent experiments after we determined the optimal antibody and antigen concentrations in checkerboard titrations. To control for day-to-day and test-to-test variations, all sera were tested together for each antigen at one time. We coated 96-well microtiter plates (Costar, Corning, N.Y.) with 100 µl of RIF protein at a concentration of 0.3 µg/ml in coating buffer (15 mM Na2CO3, 35 mM NaHCO3; pH 9.5) and left them overnight at 4°C. Control wells contained coating buffer alone. The plates were washed four times with washing buffer (0.5% Tween 20, phosphate-buffered saline [PBS]) and blocked with blocking buffer (4% bovine serum albumin, PBS) for 5 h at room temperature. Then, 100 µl of human serum (diluted 1:250 in 1% bovine serum albumin in washing buffer) per well was added to duplicate wells and was incubated overnight at 4°C. The plates were washed four times. Next, 100 µl of peroxidase-conjugated goat F(ab')2 fragment recognizing the Fc portion of human IgG (diluted 1:15,000; ICN Cappel, Eschwege, Germany) was added to each well. After 1 h, the plates were again washed four times. The reaction was developed for 10 min at room temperature in the presence of commercially available TMB peroxidase substrate (KPL Europe, Guildford, United Kingdom). The reaction was stopped with 1 M H3PO4, and the absorbance measured at 450 nm with a 550-nm reference filter in a Hitech Digiscan Photometer (Asys, Eugendorf, Austria). The optical density at 450 nm (OD450) values specific for antibody reactivity to the RIF proteins were obtained by subtracting average OD450 values for blank wells from average OD450 values for RIF proteins. The cutoff value was calculated by determining the mean OD450 value plus three standard deviations that was obtained from 28 German blood donors with no known exposure to malaria. Since the cutoff ultimately discriminates positivity from negativity, determinations of these values were repeated in parallel and in independent assays, and there were no significant variations between the values obtained for each experiment.
Competition ELISA. To confirm the presence of rifin-specific antibodies in adult sera, competition ELISAs were performed with all RIF antigens. Two different serum samples that were highly reactive with the fusion proteins were chosen for testing. The final concentrations of fusion proteins used were 0.1, 0.3, 1, and 5 µg/ml. Microtiter plates with 96 wells were coated as described above. The test sera were diluted 1:250 and preincubated in the presence of different concentrations of recombinant proteins for 2 h at room temperature. The serum-protein mixture was then added to the antigen-coated plates. All further steps were carried out as described above.
Affinity purification of anti-rifin antibodies from human immune sera. Whole E. coli cell lysates containing expressed recombinant rif-29 were prepared as described above, and the fusion protein was solubilized for 30 min at 4°C in 2% SDS. Insoluble material was removed by centrifugation at 4,000 x g at 4°C for 30 min. After affinity purification of rif-29 on a nickel-chelate column, 50 µg of the purified material was used for coupling to 200 µl of cyanogen bromide-activated Sepharose 4B beads (Sigma). Then, 1 ml of a pool of highly reactive human immune sera, diluted 1:5 in PBS, was incubated with the rif-29-tagged Sepharose for 4 h at 4°C. The resin was washed with a total of 10 ml of PBS, and bound antibodies were eluted with 100 mM glycine (pH 2) and neutralized immediately with 1 M Tris (pH 10). Antibody-containing fractions were determined by spectrophotometry at 280 nm, pooled, and tested by ELISA for reactivity to recombinant rif-29. To test the purity of the eluted antibodies in Western blot analysis, P. falciparum FCR3 was grown to the late trophozoite stage and IEs were enriched by MACS (Miltenyi Biotec) (35) to 90%. To facilitate handling of the sample, hemoglobin was removed by osmotic lysis of the erythrocytes, and free parasites as well as erythrocyte ghosts were collected by centrifugation. The resulting pellet was analyzed by Western blotting with the eluted affinity-purified anti-rif-29 antibodies. Rat anti-rif-29 antibodies diluted 1:2,000 were analyzed in parallel as a control.
Indirect immunofluorescence microscopy of P. falciparum. Immunofluorescence microscopy was performed essentially as described previously (30). Briefly, eight-well slides were treated for 30 min with NaN3. P. falciparum cultures with a parasitemia of 10% were washed in PBS and resuspended to a hematocrit of 1% in PBS. Cells were left on the slides for 30 min and briefly fixed in 1% glutaraldehyde; this step was followed by a 10-min incubation in methanol prior to air drying. Uninfected erythrocytes were tested in parallel. The wells were then incubated in the presence of 20 µl of the affinity purified anti-rif-29 antiserum at 4°C overnight. The pools of untreated semi-immune sera or European sera (each diluted at 1:5) served as positive or negative controls, respectively. Reactivity to rat immune sera was also examined. After three washes with PBS, slides were incubated with 1:250 dilutions of goat anti-human or anti-rat antibody and 1:25 dilution of fluorescein isothiocyanate (FITC)-labeled rabbit anti-goat antibody. Parasite DNA was counterstained with DAPI (4',6'-diamidino-2-phenylindole). Slides were analyzed on a Zeiss Axioplan microscope.
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RESULTS |
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1,000 bp were cloned into the TA cloning vector pCR2.1, and sequences of 25 independently isolated clones were generated. Except for one clone, all deduced amino acid sequences were confirmed as being rifin specific, and a closer analysis of the sequences revealed 13 different rif sequences. The overall rif genomic organization displayed structural similarities to RIF-1 repetitive elements previously described for other laboratory P. falciparum strains (11). Several of these characteristics were also manifested in our sequences and included the presence of two exons separated by an intron. The presumed intron sequences were defined as being 81 to 234 bp in length and were determined by the presence of consensus sequence elements at the putative 5' and 3' intron splice sites (18, 32). Subsequent excision of the intron sequences (at the position as indicated by the arrowhead in Fig. 1) yielded long open reading frames, the longest having 369 amino acids and the shortest having 303.
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Expression of recombinant RIF proteins. First attempts to express full-length proteins from our novel rifin sequences were unsuccessful, presumably because of the presence of hydrophobic signal sequences and transmembrane regions often known to render recombinant proteins toxic to the cells. Consequently, we sought to express truncated RIF proteins covering the central portion of the protein. With the help of specially designed primers (indicated by arrows on Fig. 1), amplification of partial RIF proteins 173 to 186 amino acids in length was carried out, the result of which was the successful expression in E. coli of 4 of the 13 sequences as His6 fusion proteins. These bacterially synthesized products, termed rif-29, rif-40, rif-44, and rif-50, were ca. 27 to 31 kDa, and included 5 kDa of nonrifin sequences, which are accounted for by the presence of the carrier His6 tag and amino acids specified by vector sequences. The recombinant proteins were purified in milligram quantities by affinity chromatography on HisTrap columns and analyzed on SDS gels (Fig. 2). Proteins purified to 95% homogeneity were used to immunize rats and in ELISA assays to assess humoral responses in individuals from an area where malaria is endemic.
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Age-dependent acquisition of antibodies. Since variant-specific immunity (as measured by the presence of agglutinating antibodies that are reactive to surface antigens of IEs) (4, 10, 14) is acquired in an age-dependent manner, we also investigated the correlation between antibody recognition of rifins and age of the individual child by logistic regression analysis, considering only positive responses from the malaria-exposed group of children. No significant increase of anti-rifin antibody responses with age to the recombinant proteins was observed. Because of the overall low reactivity of children's sera, only very small numbers were available for our analysis, and it therefore remains to be seen whether analysis of a larger data set would yield other results. In addition, these children were of preschool age, and therefore have not yet acquired substantial immunity.
Purity of rif-29 affinity-purified antibodies. Rif-29 was successfully prepared in large quantities in E. coli in soluble form and was chosen for coupling to Sepharose beads for affinity purification from the pool of highly reactive human immune sera. The purity of the affinity-purified anti-rif-29 antibodies was demonstrated in a Western blot against whole parasite lysates (Fig. 6). The antibodies recognized a single band ca. 27 kDa, a size predicted for rifins. Anti-rif-29 antibodies raised in a rat served as a positive control.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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recombinant proteins. The results obtained in the present study are highly distinguishable from the previous one in that seroreactivity and prevalence of antibodies to the recombinant rifins showed a much broader specificity range than the DBL- fusion proteins (26). Recognition of rifins by the malaria-exposed children was generally lower, except for rif-44, which appeared to be better recognized than the others by almost half the sera tested. The reason for this result is unclear. An obvious factor contributing to the reactiveness of rif-44 is its low cutoff value in comparison to other RIF fusions. Clearly, a low cutoff value would result in more reactivities not only from the semi-immune samples but also from the immune samples. This could also explain the high reactivity of 99% of rif-44 in the latter group. An alternative explanation is that rif-44 belongs to a subset of stably expressed dominant and commonly recognized RIF proteins, which are present on the infected red blood cell surface. From our competition ELISA results, one possible conclusion is that there are both commonly recognized and cross-reactive epitopes; presumably, this is also as a result of exposure to many different rifins during the course of a single or more infections.
We have addressed the difference in cutoff values by looking at the correlation between the OD values for rif-44 and the other three proteins by dot plot analyses. Our finding of a strong correlation between responses to the different antigens indicates that the frequency distributions of the antibody responses are similar for all of the proteins so that similar cutoff values can be expected for these proteins. However, this was not the case, but since the proteins under study are not in their native forms, conformational and other differences between the constructs could presumably account for part of the variability in the level of antibody recognition. Higher cutoff values could possibly arise from nonspecific binding of European sera to these recombinant proteins. For example, an immunoreactive epitope present on these proteins but not on rif-44 may cross-react with Europeans but not Gabonese subjects.
So far, the large repertoire of variant-specific PfEMP-1 antibodies acquired by older individuals living in areas with high-intensity transmission is believed to form the basis of immunity and is associated with protection against malaria. Our data provide evidence for the existence of a second family of erythrocyte surface antigens recognized by human antibodies induced during infection. The ELISA results suggest that rifin epitopes, such as PfEMP-1 epitopes, are also efficiently and frequently responded to by both the adults and the children in our study, although the serum samples from acutely infected children have a much smaller antibody repertoire. The high proportion of cross-reactive responses in the adult hosts against RIF proteins is probably due to the presence of certain parasite isolates expressing dominant subsets of these variants on the surface, a situation analogous to that described for different PfEMP-1 epitopes found on "common" isolates (5).
In a previous study, Fernandez et al. (9) described the immunoprecipitation of both rifins and PfEMP-1 by using immune sera from areas where malaria is endemic. After removal of PfEMP-1 molecules from the surface of erythrocytes with trypsin, agglutination of red blood cells by immune sera was still observed, a phenomenon attributed to the presence of rifin-targeted PfEMP-1-independent antibodies. With the help of an affinity-purified rifin-specific antibody, we were able to show the presence of rifins only on the membrane of IEs. These findings are in agreement with a third study in which immune sera from Kenya or the Gambia also showed membrane-associated fluorescence (19). However, further immunoprecipitation and trypsinization experiments from this group did confirm the exposure of rifins on the surface of the infected red blood cell.
Even though our study is in agreement with RIF proteins being naturally antigenic, we have been unable to show surface exposure of our rifin molecules analyzed here, despite the availability of rifin-specific antibodies. Although our results point to considerable overlap in the repertoire of rifin epitopes between different parasites, we do expect to find several other not-yet-characterized surface-specific domains that are both non-cross reactive and diverse in nature. The failure to surface label unfixed live erythrocytes may also be explained by the fact that the affinity-purified human antibodies recognize predominantly linear epitopes rather than conformational and functional surface epitopes. The same arguments for the lack of reactivity with hyperimmune rat sera also hold true. The production of native rifins is under way, and antibodies to conformationally intact molecules will be assessed in future experiments for erythrocyte surface interaction.
The functional role of rifins as a multigene family of clonally variant surface proteins has not yet been elucidated. Based on previously described work that shows the expression of multiple rifins on the surface of IEs (9), it remains plausible that anti-rifin antibodies could interfere with some important aspect of life or cell function of the parasite, such as cytoadherence or rosette formation. Further studies will provide us with a broader insight into a potential protective value and the contribution of anti-rifin antibody responses in the acquisition of immunity.
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ACKNOWLEDGMENTS |
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This work received the financial support of the fortüne-Programme (692-0-0/1 and 863-0-0) of the Medical Faculty of the University of Tübingen and the European Commission (QLK2-CT-1999-01293).
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FOOTNOTES |
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) and the semi-immune group (
) carrying antibodies to 0 to all 4 RIF recombinant proteins were determined.
), rif-44 (•), or rif-50 (
)/ml, and the OD reactivities were determined.
