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Centre for Medical Parasitology at the Institute for Medical Microbiology and Immunology, University of Copenhagen, and Department of Infectious Diseases and Department of Clinical Microbiology, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark
Department of Biochemistry, University of Khartoum, Khartoum, Sudan
Background.
The acquisition of immunoglobulin (Ig) G to variant surface antigens (VSAs) seems important for the development of protective immunity against malaria. Unlike VSAs expressed by parasite isolates associated with uncomplicated malaria, VSAs expressed by parasite isolates associated with severe malaria (VSASM) are frequently recognized by IgG.
Methods.
We analyzed levels of anti-VSA IgG in 57 individuals in Daraweesh, Sudan, before and after the transmission season. IgG responses to 79 Plasmodium falciparum isolates from children with defined malaria syndromes and exposed to high transmission in a different part of Africa were also analyzed.
Results.
After the transmission season, individuals with malaria had an increase in IgG recognition to 25.8% (95% confidence interval [CI], 19.9%31.7%) and a decrease in IgG recognition to 7.6% (95% CI, 4.4%10.8%) of 79 parasite isolates, and individuals without malaria had an increase in IgG recognition to 8.1% (95% CI, 6.0%10.2%) and a decrease in IgG recognition to 11.9% (95% CI, 7.0%16.8%) of 79 parasite isolates. Most newly acquired IgG responses were against parasite isolates expressing VSAsSM that are frequently recognized by IgG.
Conclusions.
Anti-VSA IgG levels decrease in the absence of infection, and an episode of clinical malaria induces IgG against a range of VSAs, particularly VSAsSM.
Protective immunity to Plasmodium falciparum is acquired in areas of intense transmission after repeated infections during childhood. Protection against the blood stages of the parasite is mainly antibody mediated [1], and acquisition of a broad repertoire of antibodies against parasite-encoded variant surface antigens (VSAs) on infected erythrocytes is a key element of clinical immunity [2, 3]. Protection against severe malaria (SM) is developed before protection against uncomplicated malaria (UM), which precedes protection against parasitemia [4]. Parasites associated with SM tend to express a restricted subset of VSAs (VSAsSM), and IgG directed against this subset is commonly present at high levels in plasma from children living in areas of endemicity [5, 6]. In contrast, parasites associated with UM generally express more diverse VSAs (VSAUM), which induce low levels of and are less frequently recognized by IgG [5, 6]. Parasites infecting young children predominantly express VSAsSM, but, as the VSA antibody repertoire is expanded as a result of repeated infections, there appears to be an IgG-mediated selection against VSASM expression, so that VSAUM expression dominates in older children [5, 6].
In areas of low and seasonal transmission, acquired immunity reduces the number of clinical episodes of malaria, but it is not sufficient to protect against all febrile episodes [7], and plasma levels of anti-VSA IgG are generally low [8]. Whether this is caused by poor immunological memory, combined with low immunogenicity of VSA, or by an insufficient number of infections for acquisition of a broad repertoire of IgG is unknown. The low pre-existing anti-VSA IgG levels in the plasma of individuals living in areas of low transmission would allow parasites to express VSAs similar to those on parasites infecting young children living in areas of intense transmission.
The most extensively studied VSA family is P. falciparum erythrocyte membrane protein 1 (PfEMP1), which enables the infected erythrocyte to adhere to the vascular endothelium [9]. Its high intraclonal variation, combined with its interclonal variation, allow for potentially vast antigenic variation. Nevertheless, the presence of conserved cysteine residues and that relatively conserved regions of amino acids are surrounded by hypervariable regions of amino acids suggest that the 3-dimensional structure must be maintained to facilitate binding [10]. Different host molecules can function as receptors for PfEMP1; among these, CD36, intercellular adhesion molecule 1, complement receptor 1, and chondroitin sulfate A seem to be the most important (reviewed in [11]). Particularly efficacious adhesion of infected erythrocytes to host receptors is likely to allow parasites to have a high effective growth rate. However, conserved tertiary structures in the PfEMP1 molecule will presumably also reduce the diversity allowed within the binding domains of VSA. It has recently been established that changes in the VSA phenotype are associated with marked changes in var gene expression profiles [12, 13] and that VSAsSM seem to be more conserved than VSAsUM [8, 12]. Furthermore, parasite isolates from children with SM express VSAs that have the capacity to bind multiple receptors [14]. Collectively, these observations suggest that VSAsSM are primarily expressed in nonimmune and semi-immune individuals, because parasites expressing these VSAs are likely to obtain high growth rates because they can effectively bind to endothelial cells. Because of the high sequence variation, for technical reasons, it is not feasible to directly quantify var gene expression in field isolates. Therefore, as an indirect measure of VSA expression during infection, we investigated how individuals living in an area of Sudan that has low, unstable, and seasonal P. falciparum transmission acquired IgG against VSAs expressed by parasite isolates from individuals with strictly defined malaria syndromes.
SUBJECTS, MATERIALS, AND METHODS
Parasite isolates.
Seventy-nine parasite isolates were used as a source of VSAs. Seventy of these were isolated from African individuals (age range, 312 years) diagnosed, on the basis of criteria described in detail elsewhere [6], with cerebral malaria (CM) (n = 28), severe malarial anemia (SA) (n = 8), or UM or malaria not fitting into these categories (n = 34). In addition, we used parasite isolates from individuals (age range, 1664 years) admitted to the Department of Infectious Diseases (n = 9), Copenhagen University Hospital (Rigshospitalet). These latter individuals were Danish residents returning from various parts of sub-Saharan Africa who had P. falciparum UM.
From each individual, a sample of parasitized erythrocytes was snap frozen in liquid nitrogen. On thawing, parasite isolates were cultured until a sufficient number of infected erythrocytes was available for analysis by flow cytometry. The culture time needed to obtain enough parasite material for analysis varied between parasite isolates, but there was no significant correlation between time in culture, age or category of parasite donor, and recognition of parasite isolates by IgG (data not shown).
Samples.
Plasma samples were collected from 57 individuals, 731 years old, living in Daraweesh village, Gedaref State, which is 430 km southeast of Khartoum, Sudan. The area is characterized by a rainy season from July to October, and the remainder of the year is hot and dry. The number of malaria cases varies considerably from year to year, but essentially all malaria cases are diagnosed between September and January [7, 8]. Venous blood samples were collected before the transmission season in September 1994 and after the transmission season in January 1995. During the transmission season, all individuals feeling unwell reported to a health team that was present in the village on a daily basis. Blood smears were obtained from all individuals with a body temperature >37.5°C and/or other symptoms suggestive of malaria. Individuals with malaria symptoms and positive blood smear results were classified as having malaria. We used a plasma pool from Ghanaian adults living in an area of high transmission, and 2-fold dilutions thereof, as positive controls, and we used plasma samples from 6 Danes not previously exposed to P. falciparum as negative controls [6]. In addition, we used a plasma pool from Ghanaian children to group parasite isolates into VSASM and VSAUM recognition patterns [6, 15]. The studies received ethical clearance from the relevant ethical committees, and informed consent was always obtained from the donors or their parents before the collection of samples.
Immunostaining and flow cytometry.
Erythrocytes infected by late developmental stages (hemozoin-containing trophozoites and schizonts) of parasites were purified (to >75% parasitemia) from culture material by exposure to a strong magnetic field (Miltenyi BioTec), as described elsewhere [16]. Immunostaining was performed as described elsewhere [17]. Briefly, aliquots of 2 × 105 purified late-stage infected erythrocytes (LSIEs) (labeled with ethidium bromide to allow flow-cytometric exclusion of remaining uninfected erythrocytes) were sequentially incubated with 5 L of plasma, 0.4 L of goat antihuman IgG (Dako), and 4 L of fluorescein isothiocyanate (FITC)conjugated rabbit antigoat IgG (Dako) in a total volume of 100 L. Samples were washed 2 times in PBS and 2% fetal calf serum between each antibody incubation step. All plasma samples were tested simultaneously with each parasite isolate. For each parasite isolateplasma sample combination, 2-color flow-cytometric data from 5000 LSIEs were analyzed on a FACScan analyzer (BD Biosciences), and the mean FITC fluorescence intensity (MFI) of the LSIEs (high levels of ethidium bromide) was recorded. Nonspecific labeling was evaluated by analysis of uninfected (negative for ethidium bromide) erythrocytes.
Data analysis.
MFI values above the cutoff value, which was calculated as the mean + 2 SDs of the MFI value of the negative control plasma samples, were considered to be positive. Each parasite isolateplasma sample combination was ranked by MFI value and was given a score of between 0 and 5, with 5 being the MFI value for the 2-fold dilution of the positive control plasma samples and 0 being the cutoff value. This ranking was performed separately for each parasite isolate. The recognition pattern of the parasite isolates was tested simultaneously with plasma from Ghanaian children living in areas of high transmission [6, 8]. On the basis of these data, the parasite isolates were divided, according to the rank sum, which was calculated as described elsewhere [6], into those expressing VSAs that are rarely recognized by IgG and those expressing VSAs that are frequently recognized by IgG. The mean + SD rank sums for parasite isolates that are rarely and frequently recognized by IgG were 88.6 + 41.7 and 240.8 + 64.5, respectively. We have previously shown that parasite isolates expressing VSAs that are frequently recognized by IgG are associated with SM [6]. Of the parasite isolates expressing VSAs that are frequently recognized by IgG, 64% were from children with SM, whereas 70% of the parasite isolates expressing VSAs that are rarely recognized by IgG were from children with UM. To measure the seasonal change in the frequency of IgG recognition of single parasite isolates, values were calculated as = MFIpostseason - MFIpreseason for values above the cutoff value. SigmaStat (version 3.0.1; SPSS) was used for univariate analysis comparing the 2 clinical groups. We used the Mann-Whitney rank sum test, except for testing sex distributions in the 2 clinical groups, for which we used Fisher's exact test. Logistic regression was performed using Stata SE (version 8.0; StataCorp), and recognition above the cutoff value was used as the dependent variable. To avoid colinearity, regression modeling with regard to clinical picture and recognition pattern was performed separately. All models were stratified by sex and age, and age was used as a continuous variable.
RESULTS
Levels of anti-VSA IgG in an area of low, unstable, and seasonal transmission of malaria, compared with those in an area of high transmission.
Fifty-seven individuals were included in the study. Plasma samples were obtained before the transmission season (preseason samples) and after the transmission season (postseason samples) and were tested against the panel of 79 parasite isolates. Twenty-four individuals had an episode of clinical malaria during the transmission season; the mean age of these individuals was 16.5 years. The remaining 33 individuals did not have clinical symptoms of malaria during follow-up; the mean age of these individuals was 13.8 years. There was no observed difference between the age (P = .37) and sex distributions (P = .39, Fisher's exact test) of the 2 clinical groups (data not shown).
To determine the general level of anti-VSA IgG in plasma samples from individuals living in this area, we compared it with the level in the negative control plasma samples. When we used preseason samples only, 19% of combinations between endemic parasite isolates (n = 79) and plasma samples (n = 57) had responses at levels above the cutoff value (figure 1A). Thirteen percent of the endemic parasite isolatepreseason plasma sample combinations had responses at levels between the cutoff value and that of the 1 : 16 dilution of the positive control plasma samples (figure 1A). There was no difference between the responses in the 24 individuals with malaria and the 33 individuals without malaria (P = .41). However, in the postseason samples, there was a statistically significant increase in the number of responses in the individuals with malaria (figure 1B). In these individuals, 40% of responses were higher than the cutoff value, compared with only 20% of those in individuals without malaria (P < .001). The majority of individuals with malaria had only 1 episode. These data show that, before the transmission season, the majority (80%) of anti-VSA IgG levels were lower than those in the negative control plasma samples, whereas clinical malaria induced anti-VSA IgG levels that ranged between those in the negative control plasma samples and those in the 1 : 8 dilution of the positive control plasma samples.
IgG specific for a high proportion of VSAs.
To show how a single or a few clinical infections induce anti-VSA IgG against single parasite isolates, we compared the proportion of parasite isolates that were recognized by IgG at levels above the cutoff value in preseason and postseason plasma samples from the 2 clinical groups (figure 2A). The medians of the proportions of parasite isolates recognized above the cutoff value in the preseason samples were not statistically different between individuals with or without malaria during the transmission season (P = .43). In contrast, in the postseason samples, individuals with malaria had anti-VSA IgG against a median of 26% of the parasite isolates, and individuals without malaria had anti-VSA IgG against a median of only 8% of the parasite isolates. This difference was highly significant (P < .001) (figure 2A).
To test how these findings were influenced by a decrease or induction of anti-VSA IgG during the study period, we calculated the seasonal change as values for each parasite isolateplasma sample combination (see Subjects, Materials, and Methods). Importantly, increasing and decreasing valuespositive and negative valuesagainst single parasite isolates were seen in both groups (figure 2B). In individuals with malaria, there was an increase in recognition to 25.8% (95% confidence interval [CI], 19.9%31.7%) and a decrease in recognition to 7.6% (95% CI, 4.4%10.8%) of the 79 parasite isolates. In contrast, individuals without malaria had an increase in recognition to 8.1% (95% CI, 6.0%10.2%) and a decrease to 11.9% (95% CI, 7.0%16.8%) of the 79 parasite isolates. This shows that a single or a few infections in semi-immune individuals induce IgG with specificity to a range of VSAs.
Anti-VSASM IgG in semi-immune individuals.
As an indirect measure of VSA expression during malaria in semi-immune individuals, we recorded the positive seasonal changes in levels of anti-VSA IgG. To distinguish between VSASM- and VSAUM-expressing parasite isolates, we divided them into 2 equal groups on the basis of recognition by a plasma pool from Ghanaian children. Of the parasite isolates expressing VSAs that were frequently recognized by IgG in plasma from Ghanaian children, the majority (64%) were from individuals with SM (CM and SA). Of the parasite isolates expressing VSAs that are rarely recognized by IgG in plasma from Ghanaian children, the majority (70%) were from individuals with UM. We found that the proportions of increasing anti-VSA IgG responses during the transmission season were significantly skewed toward parasite isolates expressing VSAs that are frequently recognized by IgG in individuals with and without malaria (P = .008 and P = .042) (figure 3). Taken together, these data show that IgG against a range of VSAs and, in particular, against VSAsSM that are frequently recognized can be induced as a result of an episode of clinical or subclinical malaria.
To test if recognition of parasite isolates was influenced by age and if parasite isolates from individuals with SM induced higher levels of anti-VSA IgG than parasite isolates from individuals with UM, as was found in a study published elsewhere [6], we used logistic regression. IgG recognition of VSA was used as a dependent variable, and the data were stratified by sex and age, and age was used as a continuous variable (table 1). Only a small increase in IgG recognition of VSA was observed with increasing age. Before the transmission season, it appeared that IgG in plasma samples from individuals with malaria recognized fewer parasite isolates than IgG in plasma samples from individuals without malaria, although this was not significant, with an odds ratio (OR) of 0.86 (95% CI, 0.71.05). After the transmission season, plasma samples from individuals with malaria had an OR of 3.02 (95% CI, 2.533.62) of recognition above the cutoff value for a given parasite isolate, compared with individuals without malaria. Additionally, parasite isolates from individuals with SA and CM were more likely to be recognized than parasite isolates from individuals with UM (table 1). In general, these data confirm the findings when plasma samples from different geographical locations are used [6, 8].
DISCUSSION
Sequestration is an important factor in the pathogenesis of P. falciparum infections, and the VSAs mediating adhesion are key targets of protective immunity. However, acquisition of protection is delayed by a parasite isolate's ability to vary VSA expression. Recently, several differences between VSAsSM and VSAsUM have been described. VSAsSM are generally recognized more frequently by and elicit higher levels of IgG in immune serum samples than VSAsUM [5, 6], and VSA expression in SM appears to be confined to a restricted and antigenically relatively conserved subset [8]. Furthermore, parasite isolates from individuals with CM tend to express larger VSA molecules, compared with parasite isolates from individuals with UM [18], and VSASM expression is associated with rosetting and binding to many different human receptors in vitro [14, 19, 20]. Finally, selection for VSASM expression is associated with expression of group A var genes, which code for a type of PfEMP1 that is relatively large and has a complex domain structure [12]. It is hypothesized that VSAsSM are positively selected in infections in nonimmune and semi-immune individuals, possibly because of efficacious adhesion, and, thus, IgG responses against VSAsSM would predominate in such infections. This would be in line with the observed selection against VSAsSM by immunity [5, 6], which is possibly the result of a relatively high degree of conserved B cell epitopes in VSASM molecules.
In the present study, we investigated how anti-VSA IgG was acquired during the transmission season in an area with a long dry season and unstable malaria transmission. Plasma samples from 57 individuals were collected before and after the transmission season, and 24 had malaria during the transmission season. Our data show that an episode of clinical malaria in individuals living in an area of low transmission can induce IgG against a range of VSAs expressed by 79 heterologous parasite isolates. These results could arise both by recognition of shared conserved epitopes by different VSAs and by the expression of a range of VSAs during infection. Importantly, we found that IgG was induced predominantly against the parasite isolates that are frequently recognized by IgG, of which the majority (64%) were from individuals with SM. This finding suggests that different VSAsSM either share conserved epitopes or are preferentially expressed during infections in unprotected individuals. This association was highly significant for individuals with malaria during the transmission season. There was also an association, although it was only borderline significant, for individuals without malaria during the transmission season. This difference could be due to induction of protective IgG during subclinical infections in individuals without malaria, which is consistent with the selection for expression of VSAsSM during infections.
Before the transmission season, the levels of anti-VSA IgG in the 2 clinical groups were essentially identical, and the majority of responses were below the cutoff value. This finding probably reflects a decrease in anti-VSA IgG induced during the previous transmission season. Although we do not have data from the previous transmission season to substantiate this, this hypothesis would be in line with previous findings [21, 22]. The low levels of anti-VSA IgG in preseason plasma samples could also imply that these individuals were not protected, because protection appears to require a large repertoire of IgG because of the vast variability in VSAs. Previous studies from Daraweesh, Sudan, have shown that some protection, albeit not complete protection, is acquired with age [23]. Although this protection could be caused by multiple factors, it appears that anti-VSA IgG plays an important role [24]. The seemingly preferential expression of putatively more conserved VSAsSM in semi-immune individuals living in areas of low transmission may, after a numberof infections, prepare the immune system to control the most virulent infections. This protection could be achieved by the induction of memory responses predominantly against VSAsSM, because these are preferentially expressed during infections in individuals with low levels of anti-VSA IgG at the beginning of the transmission season. Thus, although anti-VSA IgG levels are low after long periods without transmission, an appropriate IgG response, particularly against VSAsSM, would be induced. This hypothesis would be in line with what is seen in areas of intense transmission, where epidemiological studies have shown that immunity is developed sequentially against SM, UM, and, finally, parasitemia [4, 25]. In line with these findings, recent studies have shown that asymptomatic infections in areas of intense transmission can be associated with decreased susceptibility to clinical disease [21, 26, 27]. Even if the memory responses are not sufficient to protect individuals with low anti-VSASM IgG levels from febrile episodes, they could be important in controlling the progression to SM, and it is worth noting that a recent study at the district hospital serving the area in which Daraweesh is located showed that only 110 of 2488 individuals seen during a period of 2 years who had positive blood smear results fulfilled the World Health Organization criteria for SM [28].
In contrast to the results of studies conducted in the Gambia, Ghana, and Kenya [2, 3, 21, 29], but in agreement with the results of studies conducted in Sudan and India [30, 31], we found that an episode of clinical malaria can induce a broad anti-VSA IgG response to an average of 20% of a large panel of parasite isolates. As was proposed elsewhere, the balance between highly variant specific and cross-reactive IgG responses is likely to be caused by the impact of transmission intensity on the acquisition of immunity and, thereby, the ability to control VSA expression [32]. In conclusion, our data suggest that a range of VSAs is expressed during infections in semi-immune individuals. However, the IgG acquired during episodes of clinical malaria is mainly against VSAsSM, and this acquisition could be the result of both selection of expression and a higher degree of conserved B cell epitopes in these molecules.
Acknowledgments
We thank all plasma and parasite donors for their cooperation. We also thank Kirsten Pihl for excellent technical assistance.
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