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Praziquantel treatment of schistosomiasis boosts anti-schistosome responses, with ‘type 2 helper T-cell’ bias that may contribute to immunologically mediated killing and to protection against re-infection. Praziquantel treatment during pregnancy was recommended in 2002 but immunological effects of the treatment had not been investigated.
A cohort of 387 S. mansoni infected women was recruited within a larger trial of de-worming during pregnancy (ISRCTN32849447; http://www.controlled-trials.com/ISRCTN32849447/elliott). Women were randomised to receive either praziquantel or placebo during pregnancy. Six weeks after delivery all women received praziquantel. Whole blood culture cytokine responses to S. mansoni worm and egg antigens were measured before and six weeks after each treatment.
Schistosome specific cytokine responses were suppressed during pregnancy. Praziquantel treatment during pregnancy caused significant boosts in gamma interferon (IFNγ), interleukin (IL)-2, IL-4, IL-5 IL-13 and IL-10 responses to schistosome worm antigen and IFNγ, IL-5 and IL-13 to schistosome egg antigen; but these boosts were not as substantial as those seen for treatment after delivery.
Pregnancy suppresses potentially beneficial boost in cytokine responses associated with praziquantel treatment. Further studies are needed on the long term effect of treating schistosomiasis during pregnancy on morbidity and resistance to reinfection among treated women and their offspring.
Praziquantel, the drug of choice against schistosomiasis, [1, 2] has shown excellent safety and therapeutic effect against schistosomiasis morbidity. Praziquantel became available in 1979, but had never been studied in pregnant or lactating women hence, although presumed safe in pregnancy based on animal studies, was widely withheld from pregnant or lactating women during treatment programmes . In 2002, an informal consultation by the World Health Organisation reviewed experience on use of praziquantel in pregnancy. There was little evidence of adverse effects from case reports, or from inadvertent use during pregnancy in mass treatment programmes and it was recommended that pregnant and lactating women with schistosomiasis should be treated [4, 5]. However, the risks and benefits of treatment during pregnancy, and the immunological effects in particular, had not been studied. Thus, in 2005, a WHO scientific working group called for randomised, placebo-controlled trials of treatment during pregnancy for all species of human schistosomes in both low and high transmission areas . We here report immunological findings from such a trial.
Although the precise mode of action of praziquantel is still not clear there is evidence of synergy between praziquantel and the immune system. First, it has been suggested that praziquantel-induced damage to the worm surface tegument may be supplemented by immunologically mediated killing of the exposed schistosome [7-12]. Second, praziquantel treatment has marked effect on anti-schistosome immune responses [13, 14] and leads to a boost in schistosome antigen-specific cytokine responses , which may contribute to subsequent immunity to re-infection[16, 17].
Several factors including age, sex, previous exposure, co-infections and treatment influence an individual's immune response to schistosomiasis [18, 19]. Of particular interest to this study is the influence of pregnancy. Pregnancy is characterised by depression of cell-mediated immunity [20-22] with relative increase in T helper (Th)2-associated and regulatory responses (increased production of interleukin (IL)-4 and IL-10), and decreased Th1 responses (low gamma interferon (IFNγ) and IL-2 production)  More particularly, it has been shown that schistosome specific proliferative responses decline with advancing pregnancy in women with schistosomiasis .
Thus, within a trial of de-worming during pregnancy, we have explored the effect of pregnancy on immune responses to S. mansoni infection with a hypothesis that praziquantel treatment during pregnancy might be associated with reduced boost in immune responses when compared with treatment of non-pregnant women. Such effect might be important in relation to the efficacy of treatment (reducing the synergy between drug and immunologically mediated killing) and in relation to subsequent resistance to re-infection.
A nested cohort of 387 pregnant women infected with Schistosoma mansoni was enrolled within the larger Entebbe Mother and Baby Study (EMABS) on ‘the impact of helminths on the response to immunisation and on susceptibility to infectious diseases in childhood in Uganda’ (ISRCTN32849447; http://www.controlled-trials.com/ISRCTN32849447/elliott) .
Details of recruitment, baseline findings, interventions, randomisation and allocation procedure for the main study have been described . Briefly, the study was a randomised, double-blind placebo-controlled trial using praziquantel versus placebo and albendazole versus placebo during pregnancy in a 2×2 factorial design. Participants were recruited between April 2003 and November 2005. Women in the second or third trimester of pregnancy were eligible for inclusion if they were resident in the Entebbe peninsula of Lake Victoria, planned to deliver their baby in Entebbe Hospital and were willing to participate. They were excluded if the pregnancy was not normal, if they had any history of adverse reactions to anthelmintics or evidence of helminth-induced disease requiring immediate treatment, or if they had participated in the study during an earlier pregnancy. Demographic and socio-economic details and clinical history were obtained at enrolment. Women were asked to provide stool samples for examination for helminth ova and blood for diagnostic and immunological analysis. They were then randomised to receive single dose praziquantel (40 mg/kg) or placebo and single dose albendazole (400 mg) or placebo, taken under observation. Six weeks after delivery all mothers received praziquantel and albendazole with additional anthelmintic treatment, if indicated by stool results.
The nested cohort study of immune responses to schistosomiasis began in November 2003, seven months after enrolment to the larger cohort had started, and was confined to women who had a positive stool result for S. mansoni ova. Within this nested study, follow-up samples for immunological assays were obtained at six weeks post-enrolment during pregnancy (if the woman was still pregnant), six weeks after delivery but before post-delivery treatment and 12 weeks after delivery (six weeks after the post-delivery treatment). Follow-up stool samples were obtained six weeks post-enrolment during pregnancy, after delivery and 12 weeks after delivery.
At first, assays for responses to S. mansoni antigens were performed for all enrolling women. Later, to conserve limited stocks of schistosome antigens, single-step rapid test kit strips (BV European Veterinary Laboratory EVL, Woerden, Netherlands) for circulating cathodic antigen (CCA) in urine were performed to screen for S. mansoni infection, allowing assays to be selectively done on enrolment samples likely to be from schistosome-infected participants. Out of 121 women enrolled when we used CCA screening, 78 were CCA positive and had assays while 43 were CCA negative and missed the assays. We compared S. mansoni infection intensity (median epg) between women who were CCA negative, Kato-Katz positive and the women who were positive on both CCA and Kato-Katz and there was no significant difference (median epg: 59.9 and 48.0 respectively, p=0.84) confirming no selection bias.
Ethical clearance was obtained from the Science and Ethics Committee of Uganda Virus Research Institute, the Uganda National Council for Science and Technology and London School of Hygiene and Tropical Medicine.
A single stool sample per individual, at each time point, was examined by duplicate Kato-Katz thick smears . Slides were read within 30 minutes for hookworm ova and the next day for S. mansoni ova. S. mansoni infection intensity was expressed in eggs per gram (epg) of stool and categorised as light (1-99epg), moderate (100-399epg) or heavy (≥400epg) .
Soluble S. mansoni adult worm (SWA) and egg (SEA) were prepared as previously described . The endotoxin level in both antigens was below levels that could stimulate detectable production of the cytokines assayed.
Cytokine responses were examined by whole blood culture assays as previously described [28, 29]. Briefly, heparinised blood was diluted to a final concentration of 1 in 4 with serum-free medium (RPMI supplemented with glutamine, penicillin and streptomycin) and 200μl per well added to 96-well, round-bottom plates (TC Microwell, NUNC A/S, Roskelde, Denmark). The blood was stimulated with SWA, SEA or phytohaemagglutinin (PHA- Sigma, UK) at final concentrations of 10μg/ml or left unstimulated. Cultures were incubated at 37°C with 5% CO2, and supernatants harvested after 6 days. Supernatants were virally inactivated with 0.03% tributyl phosphate and 1% Tween 80 (Sigma) at room temperature for one hour and stored at −80°C. Supernatants were analysed for IFN-γ, IL-2, IL-4, IL-5 and IL-10 using OptEIA ELISA Kits (BD Pharmingen, USA) and for IL-13 using antibody pairs (BD PharMingen, USA) with standards obtained from the National Institute for Biological Standards and Controls (NIBSC), UK. Supernatants from all time points for each participant were assayed in duplicate on the same ELISA plate and a positive control was used for all plates to monitor for inter-assay variations. The sensitivity of each assay, and cut-off for positive response, was the lowest standard concentration (7.8pg/ml, except for IFNγ (8.6pg/ml)). Cytokine concentration in unstimulated wells was subtracted from concentrations in antigen-stimulated wells to obtain the antigen-specific response.
The sample size for the nested study was determined by the proportion of women with schistosomiasis within the main study. We expected to enrol 250 schistosome-infected, pregnant women (125 each in praziquantel and placebo arms). Initial analyses were made using qualitative variables but quantitative analyses were found to be more informative and are presented in this paper. No interim analyses were performed and there were no stopping rules, but serious adverse events were reported to the data monitoring committee to allow stopping if excess of events occurred in any group.
The analysis had four objectives:
During recruitment period for the nested cohort, 2208 women were recruited to the main study of whom 387 had stool samples positive for Schistosoma mansoni. Cytokine response data at enrolment was available on 103 women who received praziquantel and 105 women who received placebo for praziquantel. For logistical reasons (missed appointments, omission of schistosome antigens in assays or insufficient samples) numbers analysed for particular time points varied: results available for the praziquantel and placebo groups, respectively, were 80 and 72 at six weeks post-enrolment (while still pregnant), 91 and 95 six weeks after delivery and 68 and 77 six weeks after post-delivery treatment. At baseline, the praziquantel and placebo groups showed similar characteristics of the women, intensity of schistosome infection, prevalence of hookworm and HIV, and profile of cytokine responses to schistosome antigens (Table 1). At baseline, type 2 cytokine responses to SWA showed positive associations with schistosome infection intensity (IL-4 (rho=0.13 p=0.061), IL-5 (rho=0.14 p=0.036), IL-13 (rho=0.16 p=0.021)). Baseline responses to SEA showed negative associations with infection intensity for IFNγ (rho −0.17 p=0.017),and IL-13 (−0.14 p=0.041), and a positive association for IL-4 (rho=0.14 p=0.05). Cytokine responses to SWA and SEA showed no association with maternal age, estimated gestational age or with other co-infections such as hookworm.
Results from the placebo group were examined in order to assess effects of pregnancy on anti-schistosome cytokine responses in the absence of treatment (figures (figures1b1b and and2b).2b). At six weeks post-enrolment, IFNγ, IL-5 and IL-13 responses to SWA and SEA were lower than at enrolment (p=0.008 for IFNγ and IL-5 and p=0.006 for IL-13 to SWA; p<0.001 for IFNγ, p=0.002 for IL-5 and p=0.001 for IL-13 to SEA). At six weeks after delivery IFNγ, IL-5 and IL-13 responses to SWA had increased and were higher than at enrolment (p=0.02 for IFNγ, p=0.007 for IL-5 and p<0.001 for IL-13) and at six weeks post-enrolment (p<0.001 for IFNγ, p=0.003 for IL-5 and p=0.001 for IL-13); similarly, IFNγ and IL-13 responses to SEA were significantly higher than at six weeks post-enrolment (p=0.009 for IFNγ and p=0.002 for IL-13). IL-2, IL-4 and IL-10 responses showed no statistically significant changes.
Thus IFNγ, IL-5 and IL-13 responses to SWA and SEA were suppressed during pregnancy: they declined with progressing pregnancy and were higher, after delivery, than at either time-point during pregnancy.
Results during pregnancy were examined in the praziquantel group, and compared with results in the placebo group, in order to assess the effect of praziquantel treatment during pregnancy. Among the women who received praziquantel at enrolment (figure 1a), cytokine production in response to SWA at six weeks post-enrolment was significantly higher than at enrolment (p=0.01 for IFNγ and IL-2; p<0.001 for IL-4, IL-5, IL-13 and IL-10). At six weeks post-enrolment cytokine responses to SWA were significantly higher among the women in the praziquantel group than among the placebo group (p<0.001 for IFNγ, IL-5, IL-13 and IL-10; p=0.003 for IL-2 and IL-4).
The effects of praziquantel treatment during pregnancy on responses to SEA were more limited (figure 2a). Among women who received praziquantel at enrolment, production of IL-2, IL-5 and IL-13 in response to SEA had increased at six weeks post-enrolment (p=0.02 for IL-2, p=0.01 for IL-5 and 0.002 for IL-13). At six weeks post-enrolment, responses to SEA were significantly higher among the women in the praziquantel group than among the placebo group for IL-5 (p=0.003) and IL-13 (p<0.001).
The magnitude of the praziquantel-induced boost in cytokine responses (between treatment and six weeks after treatment) was compared between the praziquantel group (first-treated at enrolment during pregnancy) and the placebo group (first-treated six weeks after delivery), in order to assess the influence of pregnancy on the boost in responses.
Initial comparisons showed that the boost was lower for treatment during pregnancy than for treatment after delivery for all responses except for IL-5 and IL-13 to SWA (table 2). Several possible sources of bias were considered in relation to this analysis because, at the time of treatment, the group first-treated after delivery (originally the placebo group) differed in several important ways from the group first-treated during pregnancy (originally the praziquantel group). The pre-treatment prevalence of hookworm was lower in the group first-treated after delivery (since 47% had already received albendazole, table 1); and 100% of the group first-treated after delivery received albendazole concurrently with their first praziquantel treatment, compared to 55% of those first-treated during pregnancy. However, analyses during pregnancy showed no effect of hookworm co-infection or of concurrent albendazole on the boost in ant-schistosome responses following praziquantel treatment (data not shown), so these differences were considered unlikely to bias the comparison. Pre-treatment levels of cytokine response were higher in the group first-treated after delivery. Although no major difference in enrolment schistosome infection intensity was noted between the two groups, enrolment schistosome intensity showed positive correlations with post-treatment SWA-specific IL-4 (rho=0.38 p<0.001), IL-5 (rho=0.21 p=0.05), and IL-13 (rho=0.23 p=0.039) responses. Post-treatment responses to SEA showed a tendency to negative correlations with enrolment schistosome intensity which did not attain statistically significant levels. However, after adjustment for pre-treatment cytokine responses and enrolment infection intensity the boost in responses remained markedly higher for women treated after delivery than those treated during pregnancy for all responses except IL-5, IL-13 to SWA and IL-2 to SEA (table 2).
In keeping with the suppression of responses during pregnancy and the reduced boost in response following treatment during pregnancy, all six-week-post-treatment responses were lower following treatment during pregnancy than following treatment after delivery. However, among women first-treated during pregnancy, the responses had increased at six weeks after delivery and were not significantly different from the responses at six weeks post-treatment among those first-treated after delivery; except IL-4 and IL-10 to SWA which were still lower among women first-treated during pregnancy than among women first-treated after delivery (p=0.002 for IL4 and 0.008 for IL-10).
To address the hypothesis that suppression of the boost in immune response during pregnancy is associated with reduced cure rate, infection intensity at six weeks after treatment was compared between the group first-treated during pregnancy and the group first-treated after delivery. Of the 80 women in the group first-treated during pregnancy accessed for cytokine responses at six weeks following treatment, 68 (85.0%) did not have egg detected in their stool, eight (10.0%) had light infection, three (3.8%) had moderate infection and one (1.2%) still had heavy infection. Of the 77 women in the group first-treated after delivery, eggs were not detected in 68/76 (89.5%) of the women and 8/76 (10.5%) still had light infection. One of the women did not submit a stool sample at six weeks after the treatment. The difference in post-treatment infection intensity was not statistically significant (p=0.27).
The aim of this study was to elucidate the influence of pregnancy on the immune response to schistosome antigens in S. mansoni-infected women and, particularly, on the effects of praziquantel treatment on cytokine responses, with a hypothesis that pregnancy would cause reduced boost in the cytokine responses following treatment.
First, the study showed alterations of cytokine responses to schistosome antigens during pregnancy among women who did not receive praziquantel at enrolment. Schistosome-specific IFNγ, IL-5 and IL-13 responses were depressed during pregnancy. Further, although there was no association between stage of gestation and cytokine responses at enrolment (perhaps due to the variability of responses between individuals and difficulty in estimating the stage of gestation accurately), there was a decline in these three responses within individuals over a six-week period within the pregnancy. This evidence of suppression of responses during pregnancy confirmed and extended the earlier findings by Novato-Silva et al  and is consistent with effects reported for other antigens [20, 21]. From the onset of pregnancy, the immunological environment of the uterus is controlled to allow foetal allograft retention  and this is associated with a general depression of cellular responses [31, 32]. Schistosome-specific IL-2, IL-4, and IL-10 remained relatively unaffected during pregnancy. IL-2 and IL-4 responses were detected at low frequency, and lack of sensitivity of the assay may have limited our ability to detect any changes. However, these results accord with reports that Th2-associated responses and regulatory activity are generally sustained during pregnancy [33-35], while Th1 responses are suppressed.
Secondly, despite the immunosuppressive effects of pregnancy, praziquantel treatment of S. mansoni during pregnancy caused detectable increase in all measured cytokine responses to SWA and in all responses to SEA except IL-4 (the latter being perhaps below the level of detection of the assay in many cases); thus praziquantel treatment reversed the otherwise declining cytokine profiles observed during pregnancy. The effects were particularly marked for responses to SWA. Other studies with non-pregnant individuals from S. mansoni endemic areas in Uganda have reported that praziquantel treatment cause boosts in IL-4, IL-5, IL-13, and IL-10 responses to SWA, but not in the IFNγ response to SWA or any response to SEA [15, 36].
Responses to SWA and SEA were boosted to different extents following praziquantel treatment during pregnancy. This could have a bearing on morbidity of schistosomiasis during pregnancy, an aspect that was not examined in this study. In both human and experimental murine studies, schistosomiasis morbidity has been closely linked to the host immune responses, with evidence that excess of either type 1 or type 2 responses can cause severe disease [18, 37-39]. Dominance of IFNγ has been associated with acute and childhood-associated hepatosplenic disease [37, 40, 41] while IL-13 together with low IL-10 has been associated with severe hepatic fibrosis .
The significant boost in IFNγ responses observed in the current study has not been reported by related studies [15, 43] among non-pregnant individuals in areas of high S. mansoni transmission. The current study involved pregnant women and the relatively low prevalence and intensities of infection indicated low S. mansoni transmission. Furthermore post-treatment follow up time was different. The boost in IFNγ responses observed in this study (following treatment either during pregnancy or after delivery), but not in high transmission areas, suggests that the praziquantel-induced boost profile depends on transmission and intensity of infection. In fact, we noted a negative correlation between the IFNγ boost and the pre-treatment infection intensity in this study, although this effect as not statistically significant (data not shown)
Studies elsewhere have reported that praziquantel treatment of schistosomiasis mansoni and haematobium not only resolves some of the schistosomiasis associated morbidities, but can also delay resurgence of these morbidities after subsequent reinfection [44, 45]. Thus, praziquantel treatment can induce immunological changes that favour type 2 responses that are associated with resistance to reinfection [27, 46-48], and also prevent or delay onset of specific morbidities in reinfection [44, 45]. Our study has shown that boost in cytokine responses to schistosome antigens was suppressed following treatment during pregnancy as compared to treatment after delivery. However the responses among women treated during pregnancy increased substantially after delivery. Moreover, in spite the suppressed boost, the cure rate during pregnancy was not significantly different from that after delivery. This suggests that the long term benefits associated with treatment-induced alterations of the immune responses may not be affected by the observed suppression of the boost in cytokine responses following treatment during pregnancy.
This is first study to demonstrate effects of pregnancy on immune response following praziquantel treatment of schistosomiasis. Further studies are needed to examine the likely impact of the treatment-induced cytokine alterations during pregnancy on morbidity in the women as well as the babies born to the treated women; in order to assess morbidity and immune responses in the children and the long term effect of praziquantel treatment of pregnant women.
We thank members of the teams that were involved in the study: the EMaBS women, staff of EMaBS and Entebbe Grade B Hospital maternity ward. We thank the Cambridge Schistosomiasis Immunology Group and in particular Frances Jones for preparation of the schistosome antigens used in this study. We are grateful to the Wellcome Trust, DBL-institute for health Research and Development and Makerere University for funding the study. We thank MRC/UVRI Uganda Research Unit on AIDS and Entebbe Hospital for institutional support.
Sources of funding: The Wellcome Trust, through a senior fellowship held by AME (grant number 064693), Makerere University School of Graduate Studies and DBL-Institute for Health Research and Development.
Conflict of Interest: The authors do not have any commercial or other associations that might pose conflict of interest
Preliminary results of this study have been presented at the following fora: