Antibodies that inhibit the growth of blood-stage Plasmodium falciparum may play an important role in acquired and vaccine-induced immunity in humans. However, the acquisition and activity of these antibodies is not well understood.
We tested dialysed serum and purified immunoglobulins from Kenyan children and adults for inhibition of P. falciparum blood-stage growth in vitro using different parasite lines. Serum antibodies were measured by ELISA to blood-stage parasite antigens, extracted from P. falciparum schizonts, and to recombinant merozoite surface protein 1 (42 kDa C-terminal fragment, MSP1-42).
Antibodies to blood-stage antigens present in schizont protein extract and to recombinant MSP1-42 significantly increased with age and were highly correlated. In contrast, growth-inhibitory activity was not strongly associated with age and tended to decline marginally with increasing age and exposure, with young children demonstrating the highest inhibitory activity. Comparison of growth-inhibitory activity among samples collected from the same population at different time points suggested that malaria transmission intensity influenced the level of growth-inhibitory antibodies. Antibodies to recombinant MSP1-42 were not associated with growth inhibition and high immunoglobulin G levels were poorly predictive of inhibitory activity. The level of inhibitory activity against different isolates varied.
Children can acquire growth-inhibitory antibodies at a young age, but once they are acquired they do not appear to be boosted by on-going exposure. Inhibitory antibodies may play a role in protection from early childhood malaria.
Citation: McCallum FJ, Persson KEM, Mugyenyi CK, Fowkes FJI, Simpson JA, Richards JS, et al. (2008) Acquisition of Growth-Inhibitory Antibodies against Blood-Stage Plasmodium falciparum. PLoS ONE 3(10): e3571. doi:10.1371/journal.pone.0003571
Editor: Colin J. Sutherland, London School of Hygiene & Tropical Medicine, United Kingdom
Received: July 16, 2008; Accepted: October 7, 2008; Published: October 29, 2008
Copyright: © 2008 McCallum et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: National Health and Medical Research Council of Australia (Project grant and Career Development Award to J. Beeson; Postgraduate Research Fellowships to F. McCallum and J. Richards), the Welcome Trust (Project grant to K. Marsh and J. Beeson; Program grant to K. Marsh; Fellowships to K. Marsh and T. Williams), the Miller Fellowship of the Walter and Eliza Hall Institute (to J. Beeson), the Teggerstiftelsen, Maud and Birger Gustavssons Stiftelse (K. E. M. Persson) and Wenner-Gren Fellowship (K. E. M Persson). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Plasmodium falciparum malaria is a leading cause of childhood mortality, with around 1 million deaths annually . In malaria-endemic areas effective immunity against malaria develops after repeated exposure that limits blood-stage parasitemia and prevents symptomatic illness and severe complications . Antibodies that inhibit blood stage replication of P. falciparum are believed to be important in mediating both acquired immunity and immunity generated by candidate blood-stage vaccines , , . Serum antibodies that inhibit parasite growth in vitro have been isolated from clinically immune individuals, but are rarely detected in malaria-naïve individuals , . Early studies suggest that these “growth-inhibitory” antibodies are acquired in an isolate-specific manner following convalescence from acute infection . However, the role of growth-inhibitory antibodies in protection from clinical disease in humans, or their function in vivo, has not been established. Furthermore, there is limited knowledge of the acquisition of these antibodies and it is unclear whether inhibitory antibodies can be acquired quickly after limited exposure or instead require repeated exposure over an extended period.
Inhibitory antibodies are thought to mainly act by inhibiting erythrocyte invasion through targeting merozoite surface antigens and invasion ligands , . Data from animal studies support an important role for antibodies against merozoite antigens in immunity , . Merozoite surface protein 1 (MSP1), apical membrane antigen 1 (AMA1), erythrocyte binding antigens (EBAs) and P. falciparum reticulocyte-binding homologues (PfRh proteins) are implicated as important targets of acquired human inhibitory antibodies , , , , and polyclonal and monoclonal antibodies produced in experimental animals against these antigens can inhibit erythrocyte invasion in vitro , , , , , . Studies have found variable associations between the level of antibodies to recombinant merozoite antigens and protective immunity , , , , , , , , , . However, measuring antibodies to recombinant merozoite proteins has significant limitations and antibody reactivity in these assays may not reflect functional activity. Epitope specificity and the affinity of antibodies are likely to be essential for inhibitory activity, and are not measured by standard ELISA. Furthermore, antibodies that can block the inhibitory activity of other antibodies have been described , , and antibodies may also act by inhibiting the processing of antigens required for erythrocyte invasion , . These findings further emphasize the need for a greater understanding of growth-inhibitory antibodies, which is likely to be essential for the development and testing of blood-stage vaccines.
In order to understand the acquisition of human inhibitory antibodies and their potential role in immunity, we have evaluated the presence of inhibitory antibodies, and their association with active infection and markers of blood-stage malaria exposure and immunity, across different age groups and between populations with different transmission levels. Furthermore, we evaluated the relationship between inhibitory antibodies and antibodies to recombinant MSP1-42, which is a leading vaccine candidate that has been shown to induce growth-inhibitory antibodies , , , and the C-terminal fragment of which (MSP1-19) has been identified as a target of human inhibitory antibodies , .
Study site and population
The main study site of Ngerenya, Kilifi district, Kenya has been described previously , . Ngerenya experiences seasonal malarial transmission following the “long” rains in May–July and the “short” rains in November, and in 1997/1998 the predicted entomologic innoculation rate (EIR) was 10 infective bites/person/year. Serum samples collected cross-sectionally from two separate Ngerenya cohorts were used. The first set (n = 150) were selected randomly from samples collected in September 1998 from Ngerenya adults and children (n = 354), as part of a longitudinal cohort study involving randomly selected households . After baseline serum sample collection, individuals were followed-up for 12 months by active surveillance for symptomatic illness . Study participants of the Ngerenya 1998 cohort were divided into 3 age groups, in concordance with age-associated incidence of clinical malaria in the area ; 2–5 years, n = 43; 6–14 years, n = 57 and 18–81 year olds, n = 50. The second set of samples was collected from the same village in October 2002 and comprised 237 children aged 1–8 years (1–5 years, n = 129; 6–8 years, n = 108). This subset was derived from a larger cohort of children (<8 years, n = 297) from randomly selected Ngerenya households . We included all serum samples that were available from children ≥1 year of age.
Written informed consent was obtained from all participants, or their guardians, in the study. Ethical approval was obtained from the Ethics Committee of the Kenya Medical Research Institute, Nairobi, Kenya and from the Human Research Ethics Committee, the Walter and Eliza Hall Institute, Melbourne, Australia.
Growth Inhibition Assays (GIAs)
The P. falciparum lines 3D7 and W2mef were cultured in vitro  and synchronised by alternate day re-suspension in 5% D-sorbitol (Sigma, St Louis, MO, USA) in water. Growth assays were performed over two cycles of parasite replication and parasitemia was measured using flow cytometry (FacsCalibur, Becton, Dickinson, Franklin Lakes, NJ) , . Assays were set-up with mature trophozoites and schizonts, at 1% haematocrit and 0.3% starting parasitemia (50 µl/well) in 96 well U-bottom culture plates (Becton Dickinson, Franklin Lakes, USA) with pre-treated serum (5 µl) from cohort members or from non-exposed United Kingdom or Australian donors (n = 7 to 10). On all plates, PBS was included as a non-inhibitory control and 1F9 anti-AMA1 monoclonal antibody (0.5 mg/ml)  was included as an inhibitory control. Parasite growth for each sample is expressed relative to plate PBS controls. All samples were tested in duplicate in two separate assays.
To remove potential growth inhibitors or enhancers, serum samples were dialysed against PBS using 50 kDa MWCO microdialysis tubes (2051, Chemicon, Temecula, CA, USA) and subsequently reconstituted to original volume using centrifugal concentration tubes (100 kDa MWCO; Pall Corp, Ann Arbor, MI, USA) prior to testing in GIAs . Purification of immunoglobulins (Ig) was performed by ammonium sulphate precipitation, as described .
Enzyme-Linked Immunosorbent Assays (ELISAs)
Serum IgG recognising components of schizont protein extract and recombinant MSP1-42 was measured by ELISA . Schizont protein extract was prepared from 3D7 cultures using standard methods. Recombinant MSP1-42 and MSP1-19 (3D7) proteins were kind gifts from Carole Long (NIH, Bethesda) and Paul Gilson (WEHI, Melbourne), respectively. Samples were tested in duplicate together with serum from 7–10 non-exposed United Kingdom or Melbourne donors. Positive and negative controls were included to allow standardisation.
Statistical analysis was performed using Stata Version 9 (StataCorp, College Station, Texas, USA). The association between continuous variables was assessed using Students t-test or one-way ANOVA, or by Wilcoxon rank-sum test or Kruskal-Wallis test where appropriate. The association between categorical variables was assessed using a chi-square test. Pearson and Spearman's rank correlation coefficients were used to assess the association between two continuous variables. For the Ngerenya 1998 samples, the male to female ratio varied across the three age groups so the association between gender and the variables of interest was assessed separately within each age group. Data exclusions were due to insufficient sample for GIAs (Ngerenya 1998; 3D7, n = 7 and W2mef, n = 8), and outliers (Ngerenya 1998; 3D7, n = 3 and W2mef, n = 4). Survival analysis was performed using the Cox proportional hazards method. Malaria was defined as symptoms of fever or observed fever together with a parasitemia of >2500 parasites/µl.
In the Ngerenya 1998 cohort, individuals with antibody levels equal to or greater than the 75th percentile value for ELISA data were defined as ‘high ELISA responders’. Individuals whose sera inhibited growth below the approximate median value for each parasite line (3D7, <45% growth [% of control], n = 66; W2mef, <35% growth, n = 69) were defined as ‘high inhibitors’. To examine breadth and strength of responses, individuals were defined as ‘strong inhibitors’ (highly inhibitory of growth of both parasite lines (i.e. 3D7 <45% growth plus W2mef <35% growth)), and ‘weak inhibitors’ as those samples that were not strongly inhibitory of either 3D7 or W2mef (3D7 ≥45% growth plus W2mef ≥35% growth). The remaining samples were termed ‘intermediate inhibitors’.
Acquisition of antibody to blood-stage antigens and MSP1-42 increases with age
Among the 150 individuals selected from the Ngerenya 1998 cohort, there was an age-related decrease in the prevalence and density of P. falciparum infection, reflecting the acquisition of immunity in the population (Table 1) . Samples from this cohort were tested for IgG to schizont protein extract (a marker of exposure to blood-stage malaria and acquired immunity) and recombinant MSP1-42 (a major merozoite antigen and vaccine candidate) by ELISA. Levels of IgG to schizont extract and MSP1-42 were significantly correlated (Spearman's rho, rs = 0.79, P<0.001). Median IgG levels to schizont extract and MSP1-42 increased with age in both aparasitemic and parasitemic individuals (P<0.02) (Figure 1 A–D). In concordance, the proportion of high responders to both antigens increased significantly with age (Table 1). There was no significant association between parasitemic status or gender and IgG levels against schizont extract or MSP1-42.
A, B: Median absorbance (Optical Density, OD) to schizont protein extract and MSP1-42, respectively, for aparasitemic participants (n = 91). C, D: Median absorbance to schizont protein extract and MSP1-42, respectively, for parasitemic participants (n = 59). Sera were used at a dilution of 1:1000. Error bars represent the inter-quartile range. P values were calculated using a Kruskal-Wallis test. All samples were tested in duplicate. The difference in median IgG levels for aparasitemic compared to parasitemic participants was not significant for schizont extract or MSP1-42 (P = 0.082 and P = 0.14, respectively).
Age and exposure-related pattern of acquisition of growth-inhibitory antibodies
In contrast to the ELISA data, we did not observe a marked increase in growth-inhibitory antibodies with increasing age among the Ngerenya 1998 samples (Figure 2). Age associated profiles of inhibition differed slightly for the two parasite lines despite a significant positive correlation between growth of 3D7 and W2mef (r = 0.62, P<0.001). A small, statistically significant reduction in the level of parasite growth inhibition with increased age was observed for W2mef (P = 0.007), but not for 3D7 (P = 0.365). After stratifying by parasitemic status, a significant association between reduced growth inhibition of W2mef with age was seen for aparasitemic participants only (P = 0.002, Figure 2B). The proportion of highly inhibitory samples was greatest in young children and decreased with age (3D7, P = 0.294 and W2mef, P = 0.006) (Table 1).
A, B: Mean growth of 3D7 and W2mef, respectively, for aparasitemic participants. C, D: Mean growth of 3D7 and W2mef, respectively, for parasitemic participants. Growth is expressed relative to control (PBS). Error bars represent standard deviation. P values were calculated using a one-way ANOVA. All samples were tested in duplicate in two separate assays. For all samples, including parasitemic and aparasitemic individuals together, mean growth (%, ±SD) was: W2mef (n = 138) 31.8±6.5, 2–5 years; 35.1±9.0, 6–14 years; 38.1±10.3, 18–81 years; 3D7 (n = 140) 42.4±8.3, 2–5 years; 44.2±12.4, 6–14 years; 45.4±7.5, 18–81 years.
Examining associations between growth inhibitory antibodies and exposure, a weak positive correlation was seen between greater W2mef growth (i.e. reduced growth inhibition) and IgG to schizont extract (rs = 0.342, P<0.001; Fig. 3). This correlation was stronger for aparasitemic compared to parasitemic individuals (rs = 0.375, P<0.001 and rs = 0.119, P = 0.153, respectively). No correlation was seen between 3D7 parasite growth and IgG to schizont extract (rs = −0.01, P = 0.887). Associations between parasite growth and IgG to MSP1-42 were similar to those seen for responses to schizont extract. A positive correlation between W2mef growth (i.e. reduced growth inhibition) and IgG to MSP1-42 was observed in both aparasitemic and parasitemic individuals (rs = 0.301, P = 0.005 and rs = 0.265, P = 0.056, respectively). No correlation existed between 3D7 growth and IgG to MSP1-42 among samples (rs = −0.055, P = 0.527). Additionally, no correlation was found between antibodies to recombinant MSP1-19 and inhibition of 3D7 growth (data not shown). Similar results were obtained when the analysis was restricted to samples from children (data not shown); there was a weak positive correlation between antibodies to schizont extract or MSP1-42 and parasite growth using W2mef, but not 3D7.
A, B: Correlation between ELISA response to schizont extract and 3D7 or W2mef growth, respectively. C, D: Correlation between ELISA response to MSP1-42 and 3D7 or W2mef growth, respectively. rs values represent Spearman's rank correlation coefficients. Results for aparasitemic and parasitemic individuals are represented by black or grey diamonds, respectively. Samples used in growth-inhibition assays were dialysed serum.
Mean levels of inhibition were not significantly different between parasitemic versus aparasitemic individuals. In the 6 months following sample collection, 41 individuals (40 children, 1 adult) had at least one episode of symptomatic malaria, and survival analysis was performed for children only (2–5 years, n = 43 and 6–14 years, n = 57). As expected, older children had a reduced risk of symptomatic malaria compared with younger children (HR = 0.24, P<0.001). There was no significant association between inhibition of either 3D7 or W2mef growth by samples and reduced risk of subsequent malaria with or without adjustment for age.
The contrasting associations with age and exposure between growth-inhibitory antibodies and total antimalarial antibodies led us to further examine methods used to measure inhibitory antibodies and to validate our findings. Immunoglobulins were purified from an additional 52 sera samples of the Ngerenya 1998 cohort, chosen to achieve a group of children (≤12 years, n = 30) for comparison with adults (>12 years, n = 22), and tested for growth inhibition in single and two-cycle growth-inhibition assays. Again, we found little correlation between 3D7 or W2mef growth and IgG to schizont extract (Fig. 4. A, B); there was a positive correlation between W2mef growth and IgG to schizont extract for aparasitemic individuals (rs = 0.67, n = 9; P = 0.047). Inhibition of growth was not significantly different for adults compared to children (Eg. mean W2mef growth±SD; adults = 70.7±21.8% versus children = 65.6±18.7, P = 0.365) or for high versus low responders to schizont extract (71.3±19.3% versus 66.6±21.1%, respectively, P = 0.461). We typically performed GIAs over two cycles of parasite replication because we have found this increases the sensitivity of the assay . We found a strong correlation between one and two-cycle assays run in parallel for both W2mef and 3D7. The level of growth inhibition was greater for two-cycle compared to one-cycle assays. When samples were tested in single-cycle assays, there were no significant associations between inhibition results and age, parasitemia or ELISA data. Additionally, a subset of 80 samples from the Ngerenya 1998 cohort were independently dialysed and tested in GIAs against 3D7 and W2mef. Similar to our earlier results, little association with age was observed and mean inhibition of W2mef was greater than for 3D7.
A 3D7 parasite line. B W2mef parasite line. Samples used in the assays were immunoglobulins purified using ammonium sulphate precipitation from Ngerenya 1998 serum samples (n = 52). rs represents Spearman's rank correlation coefficients.
Breadth and strength of growth inhibition responses according to age and exposure
To examine the breadth of response against both parasite lines as well as the strength of growth inhibition among samples from Ngerenya 1998 cohort we defined individuals as ‘strong inhibitors’ (n = 45), ‘intermediate inhibitors’ (n = 40) and ‘weak inhibitors’ (n = 51) based on their activity against both parasite lines. The proportion of strong inhibitors decreased with age (P = 0.009)(Table 1). In addition, the median [inter-quartile range, IQR] IgG level to schizont extract was significantly lower for strong inhibitors (0.19 [0.1–0.51]) compared to intermediate (0.19 [0.01–0.53]) or weak inhibitors (0.36 [0.21–0.78], P = 0.037). There was no evidence of association between breadth and strength of a response with IgG towards MSP1-42 (P = 0.178).
Profile of inhibitory antibodies in young children with less malaria exposure
To evaluate the influence of differing levels of malaria transmission on the acquisition of inhibitory antibodies, we obtained 237 samples that were collected in a cross-sectional survey in the same community in October 2002 from children aged 1–8 years. The level of malarial transmission in the region was lower in 2002 compared to 1998 , which is reflected in the lower proportion of parasitemic individuals at the time of sample collection (Ngerenya 2002, 6.9% versus Ngerenya 1998, 39.3%). To compare growth inhibition between the two studies, analysis was restricted to children of the same age in each study (i.e. 2–8 years; median age of 5.1 years (Ngerenya 2002, n = 207) and 5 years (Ngerenya 1998, n = 61)). Growth inhibition of 3D7 was significantly less in Ngerenya 2002 than Ngerenya 1998 (median [IQR] parasite growth as a percent of control was 100.6 [93.4–105.3] for Ngerenya 2002 versus 43.2 [37.1–48.6] for Ngerenya 1998 samples; P<0.001). For all experiments, the level of growth inhibition by cohort sera was significantly greater than that seen for non-exposed control sera (P<0.001).
Because most Ngerenya 2002 samples were non-inhibitory against 3D7, only an “inhibitory subset” comprised of the top 20% (n = 46) of inhibitory samples (growth relative to controls was <92%) was also tested against W2mef. Median W2mef growth in this inhibitory subset of Ngerenya 2002 child samples was higher (equivalent to reduced inhibition) than that observed for all samples from children (aged 2–8 years) of the Ngerenya 1998 cohort (64.2 [55.8–72.4] versus 32.8 [27.9–36.6] respectively; P<0.001). In considering only the 2002 samples, the inhibitory subset of Ngerenya 2002 samples had higher IgG to schizont extract compared with the remaining less-inhibitory (3D7 growth ≥92%, n = 191) Ngerenya 2002 samples (median [IQR] IgG: 0.16 [0.09–0.57], versus 0.13 [0.04–0.42], respectively, P = 0.038). However, there was no difference in age distribution between the most inhibitory and less-inhibitory Ngerenya 2002 samples (P = 0.599). This suggests that exposure to blood-stage malaria, rather than age per se, is the major determinant for the acquisition of inhibitory antibodies in this cohort of children.
Our findings demonstrate that children exposed to P. falciparum can acquire growth-inhibitory antibodies at a young age after little exposure to malaria. It appears that once these antibodies have been acquired they are not boosted by subsequent exposure in the classical manner described for antibodies to blood-stage antigens. In contrast to the age-associated increase in antibodies to schizont extract and MSP1-42, parasite growth-inhibitory antibodies tended to remain stable or decrease with age, despite on-going exposure, and there was no substantial association with active infection. Antibodies to schizont protein extract probably reflect cumulative exposure to malaria and the overall level of blood-stage immunity. Similar to age associations, there was a trend towards declining growth-inhibitory activity as reactivity to blood-stage antigens in the schizont protein extract increased, but the association was not strong. It is important to note that inhibitory antibodies were clearly related to malaria exposure as we found consistently greater inhibition by samples from malaria-exposed individuals compared to non-exposed controls, and inhibition was greater among samples from children collected during a period of higher malaria transmission. Examination of children in the Ngerenya 2002 cohort with lower levels of malaria exposure compared to those children in the Ngerenya 1998 cohort suggested that the acquisition of inhibitory antibodies was ongoing at that time. In the 2002 cohort there was a positive association between antibodies to schizont extract and inhibitory activity and it appeared that inhibitory activity in this group of children had not yet become saturated. Findings among this cohort also confirmed that inhibitory activity can be acquired at an early age and does not require repeated exposure over several years.
The role of growth-inhibitory antibodies against blood-stage P. falciparum in immunity to malaria, and their relevance to the progression of disease in vivo, remains unclear. In our cohort, growth-inhibitory antibodies were not associated with reduced risk of symptomatic malaria. As expected, age was significantly associated with reduced risk of malaria, yet inhibitory activity was not positively associated with age. The acquisition of inhibitory antibodies at an early age and the relatively stable levels of inhibitory antibodies through childhood suggest that a potential role for these responses in protection from early childhood malaria should be considered. Immunity to severe malaria is acquired relatively quickly after little exposure whereas effective immunity to mild symptomatic malaria is acquired significantly later . It is possible that early immune responses, such as the acquisition of growth-inhibitory antibodies, are important for immunity to severe malaria. Further studies are needed to test this hypothesis.
These findings have important implications for development and evaluation of candidate blood-stage vaccines. It is assumed that antibodies to merozoite antigens should induce inhibitory antibodies, but the relevance of this response to immunity in humans remains to be established. Vaccine trials with merozoite antigens are usually first done among older children with prevention of symptomatic malaria as the end-point; this may not be an appropriate strategy to evaluate these responses. Our results suggest that individual inhibitory antibody responses do not continue to increase in effect once they are acquired, which occurs following limited exposure. This may explain recent findings that immunization of malaria-exposed adults with AMA1 boosted total IgG to recombinant AMA1, but did not increase inhibitory activity . An earlier study in The Gambia similarly found that inhibitory antibodies tended to be lower among adults than children , and recent studies by Dent et al. in western Kenya report maximum inhibitory activity among young children, which subsequently declines with increasing age . Two prior studies also found no association between growth inhibition by dialysed serum  or whole serum  and risk of mild malaria episodes, as we found here. On the other hand, some data suggests growth-inhibitory antibodies may contribute to a reduced risk of reinfection. In western Kenya, MSP1-19 specific inhibitory activity of whole serum and total growth-inhibitory activity of dialysed serum was associated with reduced risk of reparasitisation after treatment , . Further studies are needed to focus on possible associations between inhibitory antibodies and protection from symptomatic malaria in early childhood.
The use of untreated serum/plasma by some studies may have affected the measurement of inhibitory antibodies and the associations with clinical parameters. Non-specific growth-inhibitory factors, such as antimalarials, have been reported in untreated samples , ; therefore we dialysis-treated all samples prior to use in our study. Comparable inhibitory effects of dialysed serum versus immunoglobulin isolated from serum have been shown previously . In the present study, similar associations between growth-inhibitory activity of samples and other parameters were found using dialysed serum or purified immunoglobulins suggesting that antibody rather than other serum factors is the major mediator of growth inhibition in our assays. Chloroquine was the drug most likely to have been used for treatment of malaria in the population at that time  and we confirmed that chloroquine was removed by serum dialysis (data not shown). Furthermore, we found that W2mef was resistant to common anti-malarial drugs at concentrations that could be expected in serum following treatment for malaria (McCallum, Richards, Wilson, unpublished data). Therefore the presence of antimalarials in samples is unlikely to be a factor influencing our growth inhibition results.
In our studies, associations between age, exposure, and inhibitory antibodies were established by extensive testing of two different parasite lines. Additionally, no association between age and level of growth inhibitory activity was found using W2mef variants with different invasion phenotypes (Persson, McCallum, Beeson, unpublished data). Interestingly, W2mef was consistently inhibited to a greater degree than 3D7. Differences in the inhibitory activity against different parasite lines may be explained by differences in the level of exposure in the population to key epitopes expressed by these isolates. 3D7 and W2mef are known to differ in allelic variants of several important antigens, such as AMA1, and use different ligands for erythrocyte invasion , . Recent studies suggest that variation in the use of erythrocyte invasion pathways may act as a mechanism that facilitates immune evasion .
Differences in the acquisition and maintenance of ELISA-measured antibodies compared with growth-inhibitory antibodies may be explained by several factors. Human antibody responses to recombinant merozoite antigens have been reported to be short lived among children , , but appear to stabilise in older children and adults; this response needs to be investigated with respect to inhibitory antibodies. Maintenance of inhibitory antibodies at a sufficient concentration for functional activity may require exposure to a threshold parasite density that is reached in young children during infection, but not in adults and older children who more effectively control and suppress parasite density. A further consideration is that the polyclonal antibody response to merozoite antigens includes inhibitory and non-inhibitory antibodies; repeated exposure may lead to non-inhibitory antibodies reaching a greater concentration or higher affinity than inhibitory antibodies, especially if inhibitory epitopes are fewer in number than non-inhibitory epitopes. Others have reported the acquisition of antibodies that can block the activity of inhibitory antibodies , which may impede the development of inhibitory activity with increased exposure; presently, this effect is not well understood. Antibodies to merozoite antigens may also act by antibody-dependent cellular inhibition of parasite growth rather than by directly inhibiting invasion .
The lack of association between high levels of IgG to recombinant MSP1-42 or MSP1-19 and growth-inhibitory activity raises concerns regarding the accurate measurement of naturally-acquired and vaccine-induced immunity. The sum effects of multiple antibodies towards different merozoite antigens in serum samples may confound associations between measurements of growth inhibition and antibodies to a single antigen. However, in our study children with high ELISA antibodies to MSP1-42, or MSP1-19, often lacked inhibitory activity, suggesting that ELISA is a not a reliable measure of inhibitory activity. Vaccination of monkeys with MSP1-42 induced growth-inhibitory antibodies and protected against P. falciparum challenge . However, vaccination of human volunteers with recombinant MSP1-42 induced inhibitory antibodies only in a subset of individuals, despite high rates of seroconversion to the recombinant antigen , . Further studies are clearly needed to understand the relationship between antibody reactivity in standard immunoassays and growth-inhibitory activity.
These findings have important implications for understanding and measuring immunity, and for the development and evaluation of blood-stage vaccines. Our results suggest that the acquisition of functional antibodies differs from that which has been predicted from studies of acquired immunity using standard immunoassays. Antibodies can be acquired at an early age after limited exposure to malaria, but do not demonstrate continued boosting or enhancement with ongoing exposure. Furthermore, antibodies measured by ELISA to potential targets of inhibitory antibodies, such as MSP1-42 or MSP1-19, showed little correlation with growth-inhibitory activity. Results from this work suggest that future investigations on inhibitory antibodies are needed among younger children at the time when they are first experiencing malaria episodes.
Thanks to all participants in the study and to Tabitha Mwangi for co-ordinating the Ngerenya cohort, to Joanne Chesson, Nimmo Gicheru and Moses Mosobo for technical assistance, and Danny Wilson, Brendan Crabb, and Arlene Dent for helpful ideas and discussion. Human erythrocytes and serum for in vitro culture were kindly provided by the Australian Red Cross Blood Service, Melbourne, Australia, and recombinant MSP1-42 was kindly provided by Carole Long. This manuscript is published with the permission of the Director of the Kenya Medical Research Institute.
Conceived and designed the experiments: FJM KEP KM JGB. Performed the experiments: FJM KEP CKM JR. Analyzed the data: FJM CKM FJF JAS TNW JGB. Contributed reagents/materials/analysis tools: FJM KEP CKM JR TNW KM JGB. Wrote the paper: FJM FJF JAS JGB.
- 1. Elliott SR, Beeson JG (2008) Estimating the burden of global mortality in children aged <5 years by pathogen-specific causes. Clin Infect Dis 46: 1794–1795.
- 2. Marsh K, Kinyanjui S (2006) Immune effector mechanisms in malaria. Parasite Immunol 28: 51–60.
- 3. Cohen S, Butcher GA, Crandall RB (1969) Action of malarial antibody in vitro. Nature 223: 368–371.
- 4. Good MF, Stanisic D, Xu H, Elliott S, Wykes M (2004) The immunological challenge to developing a vaccine to the blood stages of malaria parasites. Immunol Rev 201: 254–267.
- 5. Brown GV, Anders RF, Mitchell GF, Heywood PF (1982) Target antigens of purified human immunoglobulins which inhibit growth of Plasmodium falciparum in vitro. Nature 297: 591–593.
- 6. Wilson RJ, Phillips RS (1976) Method to test inhibitory antibodies in human sera to wild populations of Plasmodium falciparum. Nature 263: 132–134.
- 7. Chappel JA, Holder AA (1993) Monoclonal antibodies that inhibit Plasmodium falciparum invasion in vitro recognise the first growth factor-like domain of merozoite surface protein-1. Mol Biochem Parasitol 60: 303–312.
- 8. Sy NE, Oberst RB, Macalagay PS, Fallarme VD, Cruzada SF, et al. (1990) In vitro growth inhibition of Plasmodium falciparum by sera from different regions of the Philippines. Am J Trop Med Hyg 43: 243–247.
- 9. Wipasa J, Elliott S, Xu H, Good MF (2002) Immunity to asexual blood stage malaria and vaccine approaches. Immunol Cell Biol 80: 401–414.
- 10. Rotman HL, Daly TM, Clynes R, Long CA (1998) Fc receptors are not required for antibody-mediated protection against lethal malaria challenge in a mouse model. J Immunol 161: 1908–1912.
- 11. Egan AF, Burghaus P, Druilhe P, Holder AA, Riley EM (1999) Human antibodies to the 19 kDa C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 inhibit parasite growth in vitro. Parasite Immunol 21: 133–139.
- 12. Hodder AN, Crewther PE, Anders RF (2001) Specificity of the protective antibody response to apical membrane antigen 1. Infect Immun 69: 3286–3294.
- 13. Persson KE, McCallum FJ, Reiling L, Lister NA, Stubbs J, et al. (2008) Variation in use of erythrocyte invasion pathways by Plasmodium falciparum mediates evasion of human inhibitory antibodies. J Clin Invest 118: 342–351.
- 14. O'Donnell RA, de Koning-Ward TF, Burt RA, Bockarie M, Reeder JC, et al. (2001) Antibodies against merozoite surface protein (MSP)-1(19) are a major component of the invasion-inhibitory response in individuals immune to malaria. J Exp Med 193: 1403–1412.
- 15. Rayner JC, Vargas-Serrato E, Huber CS, Galinski MR, Barnwell JW (2001) A Plasmodium falciparum homologue of Plasmodium vivax reticulocyte binding protein (PvRBP1) defines a trypsin-resistant erythrocyte invasion pathway. J Exp Med 194: 1571–1581.
- 16. Duraisingh MT, Triglia T, Ralph SA, Rayner JC, Barnwell JW, et al. (2003) Phenotypic variation of Plasmodium falciparum merozoite proteins directs receptor targeting for invasion of human erythrocytes. Embo J 22: 1047–1057.
- 17. Narum DL, Haynes JD, Fuhrmann S, Moch K, Liang H, et al. (2000) Antibodies against the Plasmodium falciparum receptor binding domain of EBA-175 block invasion pathways that do not involve sialic acids. Infect Immun 68: 1964–1966.
- 18. Branch OH, Udhayakumar V, Hightower AW, Oloo AJ, Hawley WA, et al. (1998) A longitudinal investigation of IgG and IgM antibody responses to the merozoite surface protein-1 19-kiloDalton domain of Plasmodium falciparum in pregnant women and infants: associations with febrile illness, parasitemia, and anemia. Am J Trop Med Hyg 58: 211–219.
- 19. Egan AF, Morris J, Barnish G, Allen S, Greenwood BM, et al. (1996) Clinical immunity to Plasmodium falciparum malaria is associated with serum antibodies to the 19-kDa C-terminal fragment of the merozoite surface antigen, PfMSP-1. J Infect Dis 173: 765–769.
- 20. Polley SD, Mwangi T, Kocken CH, Thomas AW, Dutta S, et al. (2004) Human antibodies to recombinant protein constructs of Plasmodium falciparum Apical Membrane Antigen 1 (AMA1) and their associations with protection from malaria. Vaccine 23: 718–728.
- 21. Cavanagh DR, Dodoo D, Hviid L, Kurtzhals JA, Theander TG, et al. (2004) Antibodies to the N-terminal block 2 of Plasmodium falciparum merozoite surface protein 1 are associated with protection against clinical malaria. Infect Immun 72: 6492–6502.
- 22. Polley SD, Conway DJ, Cavanagh DR, McBride JS, Lowe BS, et al. (2006) High levels of serum antibodies to merozoite surface protein 2 of Plasmodium falciparum are associated with reduced risk of clinical malaria in coastal Kenya. Vaccine 24: 4233–4246.
- 23. John CC, O'Donnell RA, Sumba PO, Moormann AM, de Koning-Ward TF, et al. (2004) Evidence that invasion-inhibitory antibodies specific for the 19-kDa fragment of merozoite surface protein-1 (MSP-1 19) can play a protective role against blood-stage Plasmodium falciparum infection in individuals in a malaria endemic area of Africa. J Immunol 173: 666–672.
- 24. Osier FH, Fegan G, Polley SD, Murungi L, Verra F, et al. (2008) Breadth and magnitude of antibody responses to multiple Plasmodium falciparum merozoite antigens are associated with protection from clinical malaria. Infect Immun 76: 2240–2248.
- 25. Perraut R, Marrama L, Diouf B, Sokhna C, Tall A, et al. (2005) Antibodies to the conserved C-terminal domain of the Plasmodium falciparum merozoite surface protein 1 and to the merozoite extract and their relationship with in vitro inhibitory antibodies and protection against clinical malaria in a Senegalese village. J Infect Dis 191: 264–271.
- 26. Riley EM, Allen SJ, Wheeler JG, Blackman MJ, Bennett S, et al. (1992) Naturally acquired cellular and humoral immune responses to the major merozoite surface antigen (PfMSP1) of Plasmodium falciparum are associated with reduced malaria morbidity. Parasite Immunol 14: 321–337.
- 27. Roussilhon C, Oeuvray C, Muller-Graf C, Tall A, Rogier C, et al. (2007) Long-term clinical protection from falciparum malaria is strongly associated with IgG3 antibodies to merozoite surface protein 3. PLoS Med 4: e320.
- 28. Guevara-Patino JA, Holder AA, McBride JS, Blackman MJ (1997) Antibodies that inhibit malaria merozoite surface protein-1 processing and erythrocyte invasion are blocked by naturally acquired human antibodies. J Exp Med 186: 1689–1699.
- 29. Nwuba RI, Sodeinde O, Anumudu CI, Omosun YO, Odaibo AB, et al. (2002) The human immune response to Plasmodium falciparum includes both antibodies that inhibit merozoite surface protein 1 secondary processing and blocking antibodies. Infect Immun 70: 5328–5331.
- 30. Blackman MJ, Scott-Finnigan TJ, Shai S, Holder AA (1994) Antibodies inhibit the protease-mediated processing of a malaria merozoite surface protein. J Exp Med 180: 389–393.
- 31. Malkin E, Long CA, Stowers AW, Zou L, Singh S, et al. (2007) Phase 1 study of two merozoite surface protein 1 (MSP1(42)) vaccines for Plasmodium falciparum malaria. PLoS Clin Trials 2: e12.
- 32. Ockenhouse CF, Angov E, Kester KE, Diggs C, Soisson L, et al. (2006) Phase I safety and immunogenicity trial of FMP1/AS02A, a Plasmodium falciparum MSP-1 asexual blood stage vaccine. Vaccine 24: 3009–3017.
- 33. Singh S, Miura K, Zhou H, Muratova O, Keegan B, et al. (2006) Immunity to recombinant Plasmodium falciparum merozoite surface protein 1 (MSP1): protection in Aotus nancymai monkeys strongly correlates with anti-MSP1 antibody titer and in vitro parasite-inhibitory activity. Infect Immun 74: 4573–4580.
- 34. Mwangi TW, Ross A, Snow RW, Marsh K (2005) Case definitions of clinical malaria under different transmission conditions in Kilifi District, Kenya. J Infect Dis 191: 1932–1939.
- 35. Mbogo CN, Snow RW, Khamala CP, Kabiru EW, Ouma JH, et al. (1995) Relationships between Plasmodium falciparum transmission by vector populations and the incidence of severe disease at nine sites on the Kenyan coast. Am J Trop Med Hyg 52: 201–206.
- 36. Nyakeriga AM, Troye-Blomberg M, Dorfman JR, Alexander ND, Back R, et al. (2004) Iron deficiency and malaria among children living on the coast of Kenya. J Infect Dis 190: 439–447.
- 37. Beeson JG, Brown GV, Molyneux ME, Mhango C, Dzinjalamala F, et al. (1999) Plasmodium falciparum isolates from infected pregnant women and children are associated with distinct adhesive and antigenic properties. J Infect Dis 180: 464–472.
- 38. Persson KE, Lee CT, Marsh K, Beeson JG (2006) Development and optimization of high-throughput methods to measure Plasmodium falciparum-specific growth inhibitory antibodies. J Clin Microbiol 44: 1665–1673.
- 39. Coley AM, Gupta A, Murphy VJ, Bai T, Kim H, et al. (2007) Structure of the malaria antigen AMA1 in complex with a growth-inhibitory antibody. PLoS Pathog 3: 1308–1319.
- 40. O'Meara WP, Mwangi TW, Williams TN, McKenzie FE, Snow RW, et al. (2008) Relationship between exposure, clinical malaria, and age in an area of changing transmission intensity. Am J Trop Med Hyg 79: 185–191.
- 41. Dicko A, Diemert DJ, Sagara I, Sogoba M, Niambele MB, et al. (2007) Impact of a Plasmodium falciparum AMA1 Vaccine on Antibody Responses in Adult Malians. PLoS ONE 2: e1045.
- 42. Marsh K, Otoo L, Hayes RJ, Carson DC, Greenwood BM (1989) Antibodies to blood stage antigens of Plasmodium falciparum in rural Gambians and their relation to protection against infection. Trans R Soc Trop Med Hyg 83: 293–303.
- 43. Dent AE, Bergmann-Leitner ES, Wilson DW, Tisch DJ, Kimmel R, et al. Antibody-mediated Growth Inhibition of Blood Stage Plasmodium falciparum Decreases with Age and Is Associated with Delayed Time to Infection in Persons with Naturally Acquired Immunity to Malaria. PLoS One in press.
- 44. Shi YP, Udhayakumar V, Oloo AJ, Nahlen BL, Lal AA (1999) Differential effect and interaction of monocytes, hyperimmune sera, and immunoglobulin G on the growth of asexual stage Plasmodium falciparum parasites. Am J Trop Med Hyg 60: 135–141.
- 45. Marsh VM, Mutemi WM, Willetts A, Bayah K, Were S, et al. (2004) Improving malaria home treatment by training drug retailers in rural Kenya. Trop Med Int Health 9: 451–460.
- 46. Duraisingh MT, Maier AG, Triglia T, Cowman AF (2003) Erythrocyte-binding antigen 175 mediates invasion in Plasmodium falciparum utilizing sialic acid-dependent and -independent pathways. Proc Natl Acad Sci U S A 100: 4796–4801.
- 47. Cavanagh DR, Elhassan IM, Roper C, Robinson VJ, Giha H, et al. (1998) A longitudinal study of type-specific antibody responses to Plasmodium falciparum merozoite surface protein-1 in an area of unstable malaria in Sudan. J Immunol 161: 347–359.
- 48. Kinyanjui SM, Conway DJ, Lanar DE, Marsh K (2007) IgG antibody responses to Plasmodium falciparum merozoite antigens in Kenyan children have a short half-life. Malar J 6: 82.
- 49. Bouharoun-Tayoun H, Attanath P, Sabchareon A, Chongsuphajaisiddhi T, Druilhe P (1990) Antibodies that protect humans against Plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes. J Exp Med 172: 1633–1641.