Effect of four rounds of annual school‐wide mass praziquantel treatment for schistosoma mansoni control on schistosome‐specific immune responses

Summary This study evaluated potential changes in antischistosome immune responses in children from schools that received 4 rounds of annual mass drug administration (MDA) of praziquantel (PZQ). In a repeated cross‐sectional study design, 210 schistosome egg‐positive children were recruited at baseline from schools in western Kenya (baseline group). Another 251 children of the same age range were recruited from the same schools and diagnosed with schistosome infection by microscopy (post‐MDA group). In‐vitro schistosome‐specific cytokines and plasma antibody levels were measured by ELISA and compared between the 2 groups of children. Schistosome soluble egg antigen (SEA) and soluble worm antigen preparation (SWAP) stimulated higher IL‐5 production by egg‐negative children in the post‐MDA group compared to the baseline group. Similarly, anti‐SEA IgE levels were higher in egg‐negative children in the post‐MDA group compared to the baseline group. Anti‐SEA and anti‐SWAP IgG4 levels were lower in egg‐negative children in the post‐MDA group compared to baseline. This resulted in higher anti‐SEA IgE/IgG4 ratios for children in the post‐MDA group compared to baseline. These post‐MDA immunological changes are compatible with the current paradigm that treatment shifts immune responses to higher antischistosome IgE:IgG4 ratios in parallel with a potential increase in resistance to reinfection.

seldom leads to sterile immunity, 6 it could assist in maintaining lower levels of infection.
Several immunologic correlates of protection against reinfection by schistosomes have been reported and are mainly Th2associated. [7][8][9] These include elevated parasite-specific IgG1 and IgE and antitegumental-allergen-like 1 (TAL-1) IgE, higher IgE:IgG4 ratios to schistosome egg or worm antigens and increased number of eosinophils. 7,8,10,11 Elevated IL-4 and IL-5 production and levels following treatment are also associated with resistance to reinfection, while experimental mouse studies have shown that IL-10 can block the development of post-treatment protective responses. 8,9,12 Annual mass drug administration (MDA) of PZQ to school children in schistosomiasis endemic areas is an integral component of the school-based deworming programme in many countries. In 2012, MDA with PZQ was included for the first time in a Kenyan nationwide school-based deworming (SBD) campaign that also included albendazole for soil-transmitted helminths (STH). 13 So far, 5 rounds of annual SBD have been administered since the launch of the programme, with a target of reaching at least 75% of school-age children as recommended by the WHO. 14 While the main intended benefits of MDA are to reduce morbidity and improve a child's developmental potential, the concomitant effects of these treatments on a child's antischistosome immune responses are largely unknown.
Also, unknown are the consequences of these potentially altered immune responses in regard to potential future reinfections or other health-related outcomes. Thus, millions of children are now undergoing annual MDA with PZQ without data regarding its impact on their immune responses to schistosome antigens. Therefore, we have studied the effects of consecutive 4 years of annual schoolbased MDA on antischistosome immune responses previously associated with immunoregulation and resistance to reinfection.

| MATERIAL S AND ME THODS
This repeated cross-sectional study was conducted as among school children in the Asembo area of Rarieda Sub-county in Siaya County of western Kenya. The design was to evaluate the antischistosome immunologic impact of multiple annual school-based MDAs in the entire student population attending the 5 study schools. Treatments during the MDAs were provided by the study team and administered as directly observed therapy (DOT). School attendance in these schools, based on assessments by teachers was >95%. In addition, due to widespread publicity and the provision of porridge and juice, attendance on MDA days surpassed that and often included nonschool attending siblings. The study protocol was reviewed by the Scientific and Ethical Review Unit (SERU) of the Kenya Medical Research Institute (KEMRI) as well as institutional review boards of the University of Georgia and the Centers for Disease Control and Prevention (CDC) and commenced only after necessary approvals had been granted. Parents or guardians of participating children gave informed consent, and children gave assent prior to being enrolled in the study. A total of 210 schistosomiasis egg-positive participants of ages 10-19 year old were recruited from 2 primary and 3 secondary schools in western Kenya, all within 5 km radius of Lake Victoria at baseline in 2012. After 5 years of their schools having received 4 rounds of annual MDA with PZQ and albendazole, 251 participants from across the same age spectrum were recruited from the same schools. Participants were at least in the fifth grade at baseline, and in the post-MDA group, and would therefore have had an opportunity to receive PZQ at least 4 times. The proportion of children drawn from each grade in each of the 5 schools remained the same at both time points, as did the age and sex structure. Prior to baseline and the fifth year's annual MDA, stool samples were collected from each participant on 3 consecutive days and examined by the Kato-Katz faecal microscopy method for diagnosis of schistosome and STH infections, 15 using 2 slides per stool. Infection intensity was expressed in eggs per gram of faeces (epg), and arithmetic means were calculated. About 10 mL of venous blood was collected by venipuncture by a trained phlebotomist into heparin vacutainer tubes from each participant and transported to the immunology laboratory at the Centre for Global Health Research (CGHR) of KEMRI, in Kisian, near Kisumu city, Kenya, for storage and analysis. A portion of the blood was used to prepare blood smears for malaria diagnosis. The rest of the blood was used for whole-blood cultures for cytokine production or centrifuged to obtain plasma for antibody measurements.

| Cytokine production and evaluation
1.75 mL of the heparinized blood was used to set up whole-blood cultures for evaluation of in vitro production of cytokines (IL-5, IL-10, IL-13, IFNγ) as previously described. 16 Briefly, blood was diluted 1:5 in culture media (RPMI 1640, 1% L-glutamine, 1% Penicillinstreptomycin). 1.5 mL per well of this mixture was used for wholeblood cultures in Corning ® Costar ® 24-well tissue culture plates

| Serologic assays for schistosome-specific antibodies of given isotypes
Heparinized whole blood (~5.75 mL) was centrifuged to yield approximately 3 mL of plasma, which was stored in aliquots frozen at −20°C. Assays were performed to detect antischistosome SEA or SWAP-specific total IgG, IgG4 and IgE or TAL-1-, TAL-2-and TAL-5-specific IgE and IgG4 as previously described [18][19][20] with modifications described below. Recombinant TAL antigens were produced at the CDC from plasmids containing the TAL genes kindly provided by Colin Fitzsimmons, Cambridge University. 21 Final concentrations of coating antigens were prepared as follows: 5 μg/mL for SWAP, 2.5 μg/mL of SEA for IgE, 0.625 μg/mL of SEA for IgG and IgG4, 1.5 μg/mL of TAL 1, 4.0 μg/mL of TAL 2 and 8.0 μg/mL of TAL 5.
Samples were added to the plate in duplicate at a dilution of 1:20 for IgE measurement and 1:50 for IgG or IgG4 measurement. A pool made from plasmas of 12 Kenyan car washers known to be high responders to SWAP, and therefore likely to have responsiveness to

| Data processing and analysis
Data were analysed using GraphPad Prism software version 6 (GraphPad Inc., California, USA). Both baseline data and final year measures were non-normally distributed; hence, analyses were mainly performed using nonparametric tests. Mann-Whitney U or Kruskal-Wallis with Dunns's multiple comparison post-test was used to compare medians for 2-sample and multiple-sample comparisons, respectively.

| Demographic characteristics of study participants
A total of 210 Schistosoma mansoni-infected (egg positive) school children were recruited at baseline from 2 primary and 3 secondary schools from within 5 km of Lake Victoria. These schools subsequently received 4 years of annual, school-based MDA, and all students present at the times of the annual MDAs were treated, with annual coverage approaching 95%. Treatment with PZQ (40 mg/kg body weight) and albendazole (400 mg) was directly observed by the study team.    The mean media control values for IL-5, IL-13 and IFN-g did not differ between baseline values and those after the 4 MDAs. The mean media control value for IL-10, however, did differ and is described below in the section on IL-10.  Figure 1. This is due to greater variability and very high responses by a few individuals in the baseline group, but a lower median response compared to the S. mansoni-negative post-treatment group.

| IFNγ
In the post-MDA group, children who were egg-negative produced more IFNγ in response to SEA, than children at baseline (Figure 4).
There was no significant difference in IFNγ production by PHA or SWAP stimulation between children at baseline and those in the post-MDA group.

| Anti-SEA, anti-SWAP and anti-TAL antibodies
Anti-SEA IgE was significantly higher in the egg-negative post-MDA group than the baseline group. While median anti-SWAP IgE was also higher in the post-MDA group, this difference was not statistically significant, as shown in Figure 5. while these ratios for TAL-1 and TAL-2 were not different ( Figure 10).

| D ISCUSS I ON AND CON CLUS I ON
Several studies have reported the beneficial effect of repeated treatment of schistosomiasis with PZQ following multiple reinfections  were egg-negative and those who were egg-positive. Data are presented as dot plots, medians at centreline, and whiskers at 25th and 75th percentile. Comparison was done by Kruskal-Wallis followed by Dunn's multiple comparison test, with *P < .05 and ****P < .0001 post-treatment reduction in antischistosome IgG4 is also associated with protection against reinfection. 7 Others have suggested that it is the balance between IgE and IgG4 that is important in resistance or susceptibility to schistosome infection. 40,41 Consistent with this hypothesis, we found higher anti-SEA, anti-

D I SCL A I M ER
This study is published with the permission of the Director of the Kenya Medical Research Institute.
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

ACK N OWLED G EM ENTS
We wish to acknowledge the staff of the NTD laboratory at Kenya Medical Research Institute's Centre for Global Health Research for their invaluable contribution to the study and the schools and students that participated in the study, and we thank Drs. David Dunne and Colin Fitzsimmons for the kind donation of the plasmids containing the genes for TAL-1, TAL-2 and TAL-5.

D I SCLOS U R E S
None.
F I G U R E 1 0 Anti-TAL-1 (A), anti-TAL-2 (B) and anti-TAL-5 (C) IgE/IgG4 ratios levels at baseline for children who were all Schistosoma mansoni egg-positive, and after 4 mass drug administrations (MDAs) (post-MDA group) in children who were egg-negative and those who were egg-positive. Data are presented as dot plots, medians at centreline and whiskers at 25th and 75th percentile. Comparison was done by Kruskal-Wallis followed by Dunn's multiple comparison test. *P < .05