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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The persistence of parasites in vivo over a long time requires their adaptation to the immune pressure, which to some degree can be accomplished by the expression of variant antigens.5 However, other mechanisms appear to exist enabling the parasite to evade immune pressure, as indicated by experiments in P. falciparum in vitro cultures. A laboratory strain of P. falciparum, grown in the presence of suboptimal inhibitory concentrations of antibodies to conserved epitopes of two asexual blood stage antigens, developed a specific lower sensitivity to inhibition mediated by those antibodies.6 Possibly, a similar mechanism could be invoked to explain the results from a study in Burkina Faso, where parasite field isolates were less sensitive to in vitro growth inhibition mediated by immunoglobulins from the parasite donor than from other donors living in the same area.7
To extend the latter study, P. falciparum field isolates and plasma immunoglobulins were obtained on two occasions 14 days apart from children living in Ouagadougou, Burkina Faso. The isolates were assessed for their sensitivity to the growth inhibitory effects of autologous and heterologous antibodies. Furthermore, the isolates obtained on the two occasions were characterized by polymerase chain reaction (PCR)–based genotyping.
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MATERIALS AND METHODS |
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Study area. The study area is situated in the central region of Burkina Faso, a typical zone of Sudanese savannah, with markedly seasonal malaria transmission associated with the rainy season (July–October). During this period, inhabitants of the area receive on the average more than one infective Anopheles bite per night.8 The population of the study villages belongs to the Mossi ethnic group and lives by subsistence farming. Families living in two villages, Goundry and Bogré, in the vicinity of Ouagadougou consented to participate in the study.
Isolates of P. falciparum. Blood samples were collected in 1999 during the high transmission season (August–October) from 63 randomly selected asymptomatic children (3–7 years old). Five milliliters of venous blood from each child was added to tubes containing EDTA. One milliliter of whole blood from each donor was frozen immediately at -20°C for preparation of DNA for PCR analyses. From the remaining blood sample, plasma was collected after centrifugation at 1,200 rpm for 10 minutes. The leukocyte interface was removed after washing the erythrocytes twice in RPMI 1640 medium (Gibco, Paisley, United Kingdom). To prepare isolates for in vitro inhibition experiments, each isolate was cultured for 18–22 hours in RPMI 1640 medium plus 10% AB human non-immune serum.9
Thick and thin blood films were prepared from all blood samples and 100 high-power fields were examined by microscopy. The number of parasitized erythrocytes per microliter of whole blood was calculated in relation to the number of leukocytes, assuming a fixed white cell count of 8,000/µL. Each film was read twice by two experienced senior laboratory technicians, and a third reading was made when the difference between the readers exceeded 30%. The median value of the three readings was used. The percentage parasitemia was calculated from thick blood films assuming a fixed erythrocyte concentration of 4 x 106/µL of blood.
Fifty-five of the blood samples were positive for P. falciparum by microscopy, with a mean ± SEM parasitemia of 0.31 ± 0.09%. While the majority of these donors showed parasitemias between 0.01% and 0.29%. Thirty-two of the isolates developed into schizonts during in vitro culture and were used in parasite growth inhibition assays. Blood samples were collected on two occasions (days 0 and 14) from 24 of the children to study the dynamics of parasite inhibitory antibody responses. Only eight of these children were positive for P. falciparum on the two occasions as determined by microscopy. On the basis of inquiries to the parents or guardians, none of the children had a fever in the past 24 hours before the day 0 sampling or during the period between the day 0 and day 14 sampling.
Immunoglobulin fractions. The plasma samples were inactivated by incubation for one hour at 56°C. Immunoglobulin fractions were prepared as described by Harboe and Ingild.10 Briefly, 2 mL of plasma were incubated with 1 g of (NH4)2SO4 for four hours at room temperature, centrifuged, washed three times in 1.75 M (NH4)2SO4, and the pellet re-suspended in 2.5 mL of sterile distilled water. The immunoglobulin fractions were transferred into RPMI 1640 medium by passing over a PD10 column (Pharmacia, Uppsala, Sweden) and eluted with 3.5 mL of the medium. The immunoglobulin fractions were kept at -30°C until use.
In vitro invasion inhibition assay. All in vitro invasion inhibition experiments were performed at Laboratoire d’Immuno-Parasitologie, Center National de Recherche et de Formation sur le Paludisme (Ouagadougou, Burkina Faso).
The assay was performed as previously described.11 Briefly, the ABO blood group of each donor was determined by hemagglutination using Serafol-D bedside cards (Biotest AG, Dreieich, Germany). All tests were performed with compatible blood groups. Immunoglobulin from the same donor (autologous) or other donors (heterologous) were added in serial dilutions to those cultures of the P. falciparum isolates that had developed to schizonts after the initial 18–22 hours of incubation. All inhibition experiments were performed in the presence of Swedish human AB serum from a pretested batch. The cultures were set up in duplicate in flat-bottomed, cell culture, 96-well plates (Costar, Corning, NY) and were incubated at 37°C for 18–22 hours in a candle jar. Monolayers of each culture were then prepared in quadruplicate on eight-well multitest slides (ICN Biomedicals, Inc., Aurora, OH) and fixed in 1% glutaraldehyde, followed by air-drying. Parasites were stained with acridine orange and the percentage of newly infected erythrocytes was determined by counting 25 microscopic fields per well in a fluorescence microscope. The parasite isolates were also cultured in the presence of a negative control immunoglobulin (Swedish non-immune donor). The invasion inhibition was determined by counting parasites using fluorescence microscopy and calculation by the formula: (% parasitemia in control wells - % parasitemia in test wells) x 100/(% parasitemia in control wells).
The mean ± SEM parasitemia at the start of the experiment was 0.8 ± 0.04%, and the mean ± SEM parasitemia at the end of the experiment in the control wells was 2.7 ± 0.3%. To enable comparison of the results from different experiments, the 50% inhibition concentration of antibodies to P. falciparum for the immunoglobulin fractions was calculated by linear interpolation.
Preparation of P. falciparum antigen. Strain F32 of P. falciparum was maintained in continuous culture in vitro as described by Trager and Jensen,9 using Albumax II (Gibco-BRL, Grand Island, NY) as an alternative to human serum in the culture medium.12 When the parasitemia levels reached 10%, antigens were isolated on 60% Percoll (Pharmacia) from a late stage-synchronized culture.13
Enzyme-linked immunosorbent assay (ELISA). Determination of the concentration of total anti-malaria antibodies was performed using an extract of mature stages of cultured P. falciparum as antigen.13 Ninety-six-well ELISA plates (Costar) were coated with 50 µL/well of parasite extract (10 µg/mL).14 The immunoglobulin fractions were diluted 1:1,000 and incubated for one hour at 37°C, and IgG was assayed with alkaline phosphatase–conjugated goat-anti-human IgG (Fc-fragment specific) (Mabtech AB, Nacka, Sweden) with p-nitrophenyl phosphate disodium salt (Sigma, St Louis, MO) as substrate. The concentrations of anti-malarial antibodies were calculated from standard curves obtained in a sandwich ELISA with six concentrations of highly purified IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and goat anti-human IgG both as capture antibody (10 µg/mL) and detecting antibody. Cut-off values for seropositive samples were calculated as the mean optical density values at 405 nm plus 2 SD of the values obtained with sera from eight Swedish donors who had not been exposed to malaria.
Preparation of parasite DNA and PCR. DNA was prepared from whole blood as described by Snounou et al.15 Briefly, blood was lysed with saponin and after centrifugation, the parasite-containing pellet was resuspended in lysis buffer (40 mM Tris, pH 8.0, 80 mM EDTA, 2% sodium dodecyl sulfate) and incubated in proteinase K and sarcosyl. The DNA was extracted with phenol, followed by extraction with phenol-chloroform and chloroform. The DNA was then precipitated in 3M sodium acetate (pH 5.0) and absolute ethanol and incubated in a freezer (-20°C) for two hours. After centrifugation, washing with 70% ethanol, and drying, the DNA pellet was resuspended in TE buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA).
A PCR typing technique was used to characterize parasites collected in a study of asymptomatic children carriers of malaria parasites.16 Plasmodium falciparum populations were genotyped by amplification of regions of two single-copy unlinked genes for merozoite surface protein-1 (MSP-1) and MSP-2, which exhibit allelic and length polymorphism.17 Three classes of block 2 of the msp-1 gene denoted K1, MAD20, and RO33 and two classes of block 3 of msp-2, denoted Indochina (IC) and FC27, were examined. In the first reaction (PFG-Nest1), block 2 of the msp-1 gene was amplified by the use of oligonucleotide primer pair M1-OF and M1-OR. Three separate second amplification reactions (PFG-Nest2) were required to complete the analysis of block 2. The olignucleotide pairs M1-2MF and M1-2MR specifically detected the MAD20 allelic family, M1-2KF and M1-2KR detected the K1 allelic family, and M1-2RF and M1-2RR detected the RO33 allelic family. Block 3 of the msp-2 gene was amplified in the first reaction using the oligonucleotide primer pair M2-OF and M2-OR, followed by two separate second amplifications to detect the IC allelic family (M2-ICF and M2-ICR), whereas M2-FCF and M2-FCR were used to detect the FC27 allelic family.
Statistical analysis. Statistical evaluation was done using the unpaired, two-tailed, Student’s t-test and P values < 0.05 were considered significant.
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RESULTS |
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). Similarly, when analyzing the inhibitory activity on one isolate of the autologous immunoglobulin fraction or of several (3–6) heterologous immunoglobulin fractions, lower 50% inhibition concentrations were seen in 70% of the heterologous combinations compared with the autologous one, but in this case the difference did not reach statistical significance (P = 0.12).
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). No clear-cut patterns of inhibitions were obtained. Day 0 immunoglobulin tested against the day 0 and day 14 isolates showed higher inhibition of growth of the latter isolate in four of eight cases. Comparison of the parasite inhibitory capacity of the day 0 and day 14 immunoglobulins with the autologous day 14 isolate showed that the latter immunoglobulin showed higher inhibition in 8 of 14 cases, while the day 0 immunoglobulin was more efficient in four cases (Table 2). Although the difference in inhibitory activity between the day 0 and day 14 immunoglobulins correlated with a difference in reactivity of antibody to P. falciparum in a majority of the cases, there was no significant correlation between inhibitory activity and the concentration of antibodies (P = 0.30).
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). If one considers all bands detected in the different allele families of MSP-1 and MSP-2, the number of different parasite genotypes in individual isolates ranged from one to nine. The mean number of parasite genotypes in the day 0 and day 14 isolates was 3.8 and 5.1, respectively, a difference that was not statistically significant (P = 0.08). In the majority of cases, the PCR patterns obtained from the day 0 and day 14 samples collected from a single individual were distinct, indicating substantial changes in the composition of the parasite populations in the isolates. In all but one individual (7B), at least one new genotype was detected in the day 14 isolate.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In concordance with reports from other high transmission areas,2,19,20 our results show that asymptomatic infections in Burkinabe children are in general very complex, consisting of multiple parasite clones. The previously demonstrated rapid turnover of parasite clones in a natural P. falciparum infection3 is well reflected in our study, where in most of the cases, the day 14 isolates contained new genotypically different parasites compared with the day 0 isolates. This appearance of new genotypes in the day 14 samples may indicate that the individual had acquired a new infection during the two-week period between the sample collections. However, since the isolates analyzed are derived from parasites circulating in the blood, the new parasites observed in the day 14 samples may also have been sequestered in the microvasculature at the time of the day 0 sample. Analyses of the daily dynamics for 14 days of P. falciparum parasites in asymptomatic children in Tanzania showed that the infections consisted of several inherently synchronous, genotypically distinct subpopulations.21 New genotypes were occasionally introduced, while other genotypes reappeared every second day and were present, circulating and sequestered, in the children through all the study period.21
In our study, the presence of sequestered parasites at the occasion of the day 0 sampling is indicated in 50% (4 of 8) of the children, where the day 0 immunoglobulin showed a lower inhibitory activity against the day 0 isolate than against the day 14 isolate. Consistent with this, in these cases the day 14 isolate was inhibited to a similar or somewhat higher level by the day 14 immunoglobulin compared with those of the day 0 immunoglobulin. If the new parasite clones in the day 14 isolates were derived from a new infection and introduced during the period in between the sample collections, they would not be recognized by the day 0 immunoglobulin. Thus, in the latter case the day 0 immunoglobulin would be expected to show higher inhibitory activity with the day 0 isolate than with the day 14 isolate. However, if one takes into consideration the overall inhibitory activity of the day 0 and day 14 immunoglobulins against the autologous parasite isolates, no clear-cut patterns could be discerned.
The selection by immune pressure of parasites with lower sensitivity to antibody-mediated inhibition may involve parasites expressing polymorphic or variant antigens not recognized by the antibodies.5 However, non-polymorphic antigens may also be targets in this context, as indicated by in vitro experiments, where a laboratory strain of P. falciparum was grown in the presence of suboptimal inhibitory concentrations of antibodies to the antigens Pf155/ring-infected erythrocyte surface antigen (RESA) and Pf332.6 The parasites gradually developed a specific decreased sensitivity to inhibition mediated by the antibodies used in the cultures, while the inhibitory effect by other antibodies was unaffected. Thus, the antibody pressure in those in vitro experiments gave rise to parasites with a specific decreased sensitivity to inhibition, which is similar to what we observed in the present study with fresh parasite isolates submitted to an antibody pressure in vivo.
The inhibition of parasite growth in vitro was determined on the basis of the number of newly infected erythrocytes.11 Since the immunoglobulin fractions used contain antibodies to many different P. falciparum antigens, their inhibitory activity is a composite of inhibition of invasion, inhibition of merozoite dispersal, and inhibition of intra-erythrocytic parasite growth.5 Moreover, some antibodies in the immunoglobulin may also promote merozoite invasion/parasite growth,22,23 thus counteracting the inhibitory activity of other antibodies. However, all the immunoglobulin fractions used in the present study inhibited parasite growth efficiently.
In a previous study that analyzed the effects of autologous and heterologous sera on parasite in vitro growth, no clear-cut differences in sensitivity of the isolates to inhibition were seen.24 However, that study was performed with a small number of isolates, and whole serum was used for inhibition, with possible involvement of non-antibody-mediated effects. In a study on antibody responses in Kenyan children to variant parasite-derived antigens on the surface of P. falciparum-infected erythrocytes, isolates taken during episodes of clinical malaria were less likely to be recognized by the corresponding child’s own pre-existing antibody response than by that of other children.25 An almost exclusive appearance during clinical disease was observed of parasite variants corresponding to gaps in each child’s developing repertoire of antibodies. However, in our study, the children were asymptomatic during the two weeks between the samplings, indicating that their pre-existing antibody response could cope with the infection.
In conclusion, in concordance with the results of our previous study, freshly isolated P. falciparum parasites from children living in a malaria-endemic area of Burkina Faso were less sensitive for growth inhibition in vitro by autologous immunoglobulin fractions compared with heterologous ones. Analyses of two consecutive isolates taken 14 days apart, with regard to genotypes and sensitivity to growth inhibition in vitro, did not give any clear-cut indications on possible mechanisms leading to a reduced inhibitory activity in autologous parasite/antibody combinations. Although new parasites with distinct genotypes occurred in the day 14 isolates, this appeared in several cases to be due to persisting parasite clones, which were sequestered at the time of collection of the day 0 sample. The frequent presence of persisting parasite clones in asymptomatic children indicates that the parasite possesses as yet undefined mechanisms to evade neutralizing immune responses.
Received August 22, 2002. Accepted for publication March 3, 2003.
Acknowledgments: We express our appreciation to the blood donors whose participation has made this study possible.
Financial support: This work was supported by grants from the Swedish International Development Cooperation Agency/the Swedish Agency for Research Development with Developing Countries (SIDA/SAREC) and the Swedish Medical Research Council.
Authors’ addresses: Ahmed Bolad and Klavs Berzins, Department of Immunology, The Wenner-Gren Institute, Stockholm University, SE-10691, Stockholm, Sweden, Telephone: 46-8-164170, Fax: 46-8-157356, E-mail: klavs{at}imun.su.se. Issa Nebié and Nadine Cuzin-Ouattara, Centre National de Recherche et de Formation sur le Paludisme, 01 BP 2208, Ouagadougou, Burkina Faso. Alfred Traore, Centre de Recherches en Sciences Biologique, Alimentaires et Nutritionelles-University of Ouagadougou, Boite Postale 7021, Ouagadougou, Burkina Faso. Fulvio Esposito, Department of Molecular, Cell and Animal Biology, University of Camerino, I-62032 via Camerini 2, Camerino (MC), Italy.
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