Introduction Schistosoma mansoni age-infection curves in endemic human populations characteristically show a peak prevalence in children and early adolescence and then a decline beginning in the late teenage years to lower levels of prevalence among adults [1]. This has led many researchers to hypothesize that humans can acquire immunity to S. mansoni, leading to partial resistance against reinfection [2]. Since the natural lifespan of S. mansoni worms is approximately 5–10 years [3],[4], the decline in prevalence coincides with the time at which worms acquired in early childhood would naturally begin to die in persons living in endemic areas. One theory holds that upon worm death, either naturally or as a result of treatment, critical schistosome antigens not normally or appropriately encountered by the host during chronic infection are released. The release of these antigens alters the immune response patterns that result from exposure to intact worms [5],[6], and it is hypothesized that these changes in immune responses are responsible for the increased resistance to reinfection [2]. We previously reported the age-independent development of immunological resistance to reinfection with S. mansoni in a cohort of adult males occupationally exposed, by washing cars in Lake Victoria, undergoing repeated cycles of reinfection and praziquantel-induced cure [7]. Resistance to reinfection by all three of the schistosome spcies that cause most human disease has been associated with both cellular [8],[9],[10] and humoral immune responses, most notably IgE in response to parasite-specific antigens [11]–[16]. In turn, variations in these immune responses have been related to factors such as age, stage of disease, and duration of infection [17]–[24]. More recently, we have expanded our studies to include a second cohort of men who are also exposed to infectious water through their occupation of harvesting sand in Lake Victoria. Upon discovering differences in the two cohorts in the number of treatments and cures needed before increased resistance to reinfection was demonstrated, we explored demographic and immunologic factors that may explain the discrepancies. Methods Study population All participants in this study were adult males occupationally exposed to S. mansoni by washing cars or harvesting sand on the shores of Lake Victoria near Kisumu, Kenya. The car washers stand ankle- to knee-deep in the lake to wash cars that have been driven into the shallow water at the edge of the lake. Enrollment of car washers began in June 1995, and follow-up continued until January 2009. With the exception of the period between January 2000 and September 2003, enrollment of new car washers was continuous throughout the duration of the study, so follow-up time varies for each individual. The sand harvesters stand waist- to chest-deep in the water to shovel sand off the bottom of the lake. After filling their boats with sand, they then transport the sand to shore and stand in the water at the edge of the lake while they unload the sand onto the shore. Recruitment of sand harvesters began in March 2005, and follow-up continued until January 2009. Both groups of men are ethnically homogeneous, with 90% of the car washers and 98% of the sand harvesters belonging to the Luo tribe. The car washing and sand harvesting sites are shown in Figure 1. The carwash is adjacent to the city of Kisumu, and the site is a busy area also populated with fishermen, fish merchants, and various other vendors. Although located only 5.2 km around the lakeshore and 3 km across the lake, the sand harvesting site differs considerably as it is located off the shores of the small fishing village of Usoma, a rural community separated and distinct from the city of Kisumu. The presence of S. mansoni-infected Biomphalaria sudanica snails has been confirmed at both exposure sites [25],[26]. 10.1371/journal.pntd.0000637.g001 Figure 1 Map of area around Lake Victoria near Kisumu, Kenya, showing locations of carwash and sand harvesting sites. All study participants gave written informed consent prior to enrollment. Study procedures were approved by the institutional review boards of the University of Georgia and the Centers for Disease Control and Prevention, the Scientific Steering Committee of the Kenya Medical Research Institute (KEMRI), and the KEMRI/National Ethics Review Committee of Kenya. Patient follow-up Upon enrollment, men were tested for S. mansoni eggs by the modified Kato-Katz method using two slides from each of three consecutive stool samples. Individuals positive for S. mansoni were treated with 40 mg/kg praziquantel (PZQ), and follow-up stool samples were taken 4–6 weeks later to assess for cure. If necessary, men were re-treated with PZQ until cure was demonstrated by three consecutive stool samples that were negative for schistosome eggs. Upon becoming stool negative, men were continually followed and retested for S. mansoni eggs at 4-week intervals. Each time a new infection was found, the study participant was treated with PZQ until he demonstrated cure. Blood samples were taken every six months for subjects enrolled in 2003 or later and approximately yearly for car washers enrolled prior to 2003. Blood was tested for schistosome-specific antibodies, HIV-1 specific antibodies, and the ability of their peripheral blood mononuclear cells (PBMCs) to produce cytokines [7],[27]. The prevalence of malaria and soil-transmitted helminths in these populations was low. In the rare event malaria or soil-transmitted helminths were found the subjects were offered appropriate treatment. Water exposure was measured by the number of cars washed or the number of days worked in the lake harvesting sand. Daily records of the number of cars washed by each car washer or the number of hours worked each day harvesting sand for each sand harvester were kept by on-site members of the carwash and sand harvester consortia who were employed as field workers for the present study. Since the number of hours spent in the water each day for sand harvesters was highly consistent (mean 5.3±0.9 hours), and sand harvesters likely receive most of their exposure to schistosomes as they are standing near the edge of the lake unloading the sand from their boats rather than when they are harvesting sand in waist- to chest-deep water away from the shore, we have chosen to use days worked rather than hours worked in water exposure calculations for the sand harvesters. Sand harvesters were given credit for one day of work for each day that they worked for at least one hour. It is important to note that one car washed is not equivalent to one day of work harvesting sand, thus direct comparisons between the two groups of men are not appropriate, Cytokine production and evaluation Isolation of PBMCs and cell cultures were performed as previously described [28]. Briefly, PBMCs were separated from venous blood using the ficoll-hypaque technique. PBMCs were washed and resuspended in RPMI containing 5% AB+ normal human sera, antibiotics and L-glutamine. The cells were incubated with 10 µg/ml soluble worm antigen preparation (SWAP) or 5 µg/ml soluble egg antigens (SEA) for five days at 37C in 5% CO2 and the supernatant fluids collected. PBMC production of the cytokines interleukin (IL)-5, IL-10, IL-13, and IFN-γ in response to SWAP and SEA was measured by capture ELISA using commercially-available kits (R&D Systems, Minneapolis, MN) according to manufacturer's instructions. Cytokine production was only performed on blood samples obtained after October 2003. Antibody evaluation Anti-SWAP IgE isotype ELISAs were performed on plasma from the venous blood samples as previously described [29],[30]. External positive and negative controls (EC) comprised of pooled samples of high responders and normal human serum (NHS) from non-endemic volunteers, respectively, were run on each plate. Anti-SWAP IgE values for each sample were standardized according to the following formula: IgE-specific ELISAs against the recombinant antigens ‘tegument allergy like’ (TAL)-1 (formerly Sm22.6) and TAL-2 (formerly Sm21.7) [31] were performed on a subset of baseline samples from 23 car washers and 20 sand harvesters. TAL-1 and TAL-2 were cloned and purified as previously described [32],[33]. ELISA plates were coated with recombinant antigen at 2 µg/ml. Following incubation with plasma samples (1∶20 dilution), antigen-specific IgE binding was measured using directly conjugated mouse anti-human IgE (Southern Biotech, Birmingham, AL). Statistical methods Since almost all measurements were non-normally distributed, the Wilcoxon rank sum test was used for group comparisons, and the Wilcoxon sign rank test was used for paired comparisons of the same subjects at different time points. An alpha level of 0.05 was considered statistically significant for all comparisons. All analyses were performed with GraphPad Prism 5 or SAS version 9.1. The number of cars washed or days worked harvesting sand between each cure and reinfection was estimated in an accelerated failure time model with the LIFEREG procedure in SAS [34]. Each infection interval was defined as the time between the documentation of cure and subsequent reinfection. Thus, “interval 1” is the interval between the first cure after study entry and the first reinfection following the first cure, “interval 2” is the interval between the time of the second cure and second reinfection, and so forth. Interval number was entered into the model as a categorical variable with interval 1 as the reference category. Thus the length of each cure-to-reinfection interval was statistically compared to that of the first interval. The LIFEREG procedure can accommodate failure time data that is right- or left-censored. The first interval was considered left-censored for subjects negative at study entry. Intervals during which the subject left the study or follow-up ended before reinfection occurred were considered right-censored. Censored observations accounted for 59 of 570 total intervals (10.4%) among the car washers and 30 of 144 total intervals (20.8%) among the sand harvesters. Intervals during which more than three months elapsed between the last negative stool and a subsequent positive stool were excluded from the analyses, though other intervals from that same subject could be included. Entire subjects were excluded from the analysis if they did not have at least one complete infection interval—i.e. left the study without ever becoming egg-negative or after the initial cure but before the first reinfection. Because daily records of car washing activities are incomplete prior to 1999, subjects whose entire follow-up occurred before February 1999 are not included in this analysis. For subjects enrolled before February 1999 and followed further, the cure-to-reinfection intervals occurring after February 1999 are included, beginning with the numbered interval that the subject had reached at that point. The final study population consisted of 120 car washers with a mean follow-up time of 74.4 months (range: 9.1–165.5) and 53 sand harvesters with a mean follow-up time of 37.9 months (range: 12.6–61.1). The mean number of cure-to-reinfection intervals was 6.5 (range: 1–18) and 3.0 (range: 1–8) for the car washers and sand harvesters, respectively. For each car washer, the number of reinfections per 100 cars washed (RCW) during the at-risk time over the course of follow-up was calculated as an indication of relative resistance to S. mansoni reinfection. For the sand harvesters, this measure was calculated as the number of reinfections per 100 days worked harvesting sand (RDW) during the at-risk time over the course of follow-up. At-risk time is the time between cure and reinfection. Cars washed (or days worked) in the time between infection and cure are not included in the RCW or RDW calculations. As the RCW or RDW is averaged over the entire duration of follow-up, in theory those men who enter the study with a higher level of resistance or develop resistance over the course of the study will have a lower RCW or RDW than men who retain a high degree of susceptibility over the course of follow-up. For some analyses, car washers and sand harvesters are dichotomized based on the mean RCW or RDW of each respective group. For ease of discussion, men with a below-mean number of reinfections are referred to as “more resistant phenotype,” and men with an above-mean number of reinfections are referred to as “more susceptible phenotype.” Factors associated with having a more resistant phenotype were evaluated in a logistic regression model. Results Baseline characteristics Demographics Baseline characteristics of car washers and sand harvesters are given in Table 1. Sand harvesters were significantly older and reported working in the lake significantly more years prior to study entry than did car washers. Essentially all (98%) sand harvesters reported being born in Usoma, the lakeside village where they harvest sand. Conversely, the car washers are mostly from the city of Kisumu or emigrants from other areas of Kenya, and only 11% reported being born in a village near Lake Victoria. Initial mean egg counts were high (914 epg in the car washers and 876 epg in the sand harvesters) and were not significantly different between the two groups. The prevalence of HIV seropositivity was also high and similar in the two groups (18% versus 20%, Table 1). 10.1371/journal.pntd.0000637.t001 Table 1 Demographic characteristics of car washers and sand harvesters at study entry. Car washers Sand harvesters p-value Age in years [mean (std)] 24.5 (9.0) 27.7 (7.9) 0.0010 Years worked in lake [mean (std)] 5.7 (7.4) 11.1 (8.0) 10 yrs 1.5 (0.5, 4.9) 2.6 (0.4, 16.1) 3.7 (1.0, 13.9) 1.7 (0.3, 9.6) Age >25 yrs 1.1 (0.4, 2.9) 0.6 (0.1, 2.7) 4.5 (1.2, 16.9) 4.3 (0.8, 24.5) HIV positive 0.4 (0.1, 1.0) 0.4 (0.1, 1.3) 1.0 (0.2, 4.4) 1.4 (0.3, 7.2) More resistant phenotype is defined as experiencing a below-average number of reinfections ( 450 cars to become reinfected over the duration of the study (labeled “remained susceptible”). In both groups, the baseline bleed was taken at study entry before any treatment was administered. In the group that developed resistance, the later bleed depicted in the figure is the first bleed after the cure for which the length of cure-to-reinfection intervals surpassed 450 cars. In the group that remained susceptible, the later bleed is the final bleed collected for the study. In the men who became more resistant to reinfection after experiencing multiple reinfections and cures, the level of anti-SWAP IgE significantly increased between baseline and the time at which development of resistance was first evidenced. No significant changes in anti-SWAP IgE over the course of follow-up were observed in men who did not show evidence of development of resistance to S. mansoni by the end of the study. While the men who remained susceptible were more likely to be S. mansoni positive at the time of the later bleed than those men who developed resistance (71% vs 13%, p 90% becoming positive for antibodies to schistosomes by age 10 (J. Verani, unpublished data). A similar situation has been reported among children in fishing villages along the Ugandan shoreline of Lake Victoria, where Odogwu and colleagues found S. mansoni infection in 25% and 86% of children aged <3 years in two endemic villages [35]. Thus, men from Usoma likely had exposure to the lake as children long before they began working as sand harvesters, were probably initially infected with S. mansoni at an early age, and had likely experienced the natural death of worms multiple times prior to being treated as part of this study. In contrast, S. mansoni infections present at study entry in car washers likely represent more recent infections, and they had likely experienced the death of no or few worms prior to treatment with praziquantel. These two groups of occupationally exposed adult males also differed considerably in their immune response patterns to schistosome antigens, and these differences are also likely explained by their different histories of exposure to S. mansoni. The baseline immune responses are suggestive of more recent infections in car washers. PBMC cytokine production in response to SEA at the time of enrollment was higher in car washers than amongst sand harvesters by all four measured cytokines. High responses to SEA have been associated with early S. mansoni infection, and these responses then decrease as infection becomes more chronic and exposure to constantly released egg antigens leads to development of immunoregulatory mechanisms [19],[20],[22],[23]. Similar to other researchers who have shown no difference in humoral responses to crude worm antigens in patients with early and chronic schistosomiasis [19], baseline anti-SWAP IgE responses did not differ between our cohorts. However, in both car washers and sand harvesters, older men had significantly higher levels of anti-SWAP IgE than did younger men, independent of exposure history. While increases in parasite-specific IgE with increased age are usually attributed to longer exposure to infection, Naus et al also reported increased IgE responses against schistosome worm antigens in older age groups in an immunologically naïve immigrant population recently arrived to an S. mansoni-endemic area of Kenya, suggesting that the increase may be innately age-related and not dependent on duration of schistosome infection [18]. Although baseline differences between car washers and sand harvesters were not seen in IgE responses to the heterogeneous worm antigens present in SWAP, differential IgE responses to the recombinant S. mansoni antigens TAL-1 and TAL-2 were observed between the two groups. Fitzsimmons et al have shown that TAL-1 expression is concentrated primarily in the adult worm, while TAL-2 is expressed on all life cycle stages, including miracidia, cercariae, and eggs [33]. Levels of anti-TAL-1 IgE antibodies were increased after treatment of S. mansoni infected individuals in the Fitzsimmons et al study, while anti-TAL-2 IgE antibodies were unchanged by treatment. The authors hypothesized that TAL-1 worm antigens are sequestered during active infection and are only released upon worm death. Conversely, the immune system is continuously exposed to TAL-2 due to the constant release of eggs during S. mansoni infection [32], thus leading to down regulation of responses to TAL-2. The current finding of higher pretreatment levels of anti-TAL-1 IgE in sand harvesters than in car washers and similarly low anti-TAL-2 responses in both groups fits this hypothesis. As the natural lifespan of an adult S. mansoni worm is approximately 5–10 years [3],[4], the car washers had likely not been exposed to any or many dying worms before receiving PZQ treatment as part of the current study, while the sand harvesters had likely already experienced multiple episodes of naturally dying worms, based on exposure since early childhood. Car washers who were HIV positive at study entry were less likely to develop resistance over the course of follow-up than were men who were HIV negative. We previously reported that patients with schistosomiasis and HIV coinfection had significantly lower production of the cytokines IL-4 and IL-10 than schistosome-infected persons who were HIV negative [28]. Other researchers have reported an association between IL-4 production in response to schistosome antigens and increased resistance to reinfection with S. mansoni [8], Schistosoma haematobium [9], and Schistosoma japonicum [10]. HIV infection was not related to the ability to develop resistance in the sand harvesters, most probably because they had already been infected with and developed protective immune mechanisms against schistosomes prior to becoming infected with HIV as adults. While neither age nor number of years worked in Lake Victoria prior to study entry were associated with resistance among the car washers, only age was independently predictive of a resistant phenotype among the sand harvesters. As most sand harvesters likely had lake exposure since childhood before they began working harvesting sand, length of time worked in the lake became insignificant in the analysis after adjustment for age, as age is a better predictor for duration of water exposure in this group. Many previous studies have shown various immune responses to be correlated with resistance to reinfection with all three species of schistosomes, most commonly the production of parasite-specific IgE [11], [14]–[16]. While we did not find any baseline antibody or cytokine responses to be predictive of the ability to develop resistance among the car washers, this was not unexpected given that a change in resistance did not become apparent until the men had experienced on average seven previous cures. However, among those car washers that did eventually demonstrate an increase in resistance against reinfection, we have documented increases in anti-SWAP IgE production that parallel the development of resistance. Increases in anti-SWAP IgE production did not occur in those who remained susceptible. We did not see a similar increase in anti-SWAP IgE as the interval between cure and reinfection increased in sand harvesters. In conclusion, we have again demonstrated that resistance to reinfection with S. mansoni can be acquired or augmented by adults after multiple rounds of reinfection and PZQ-induced cure. However, we now also show that the ability to acquire this resistance and the rate at which resistance is acquired is markedly different in two populations within close geographic proximity to one another that share high levels of occupational exposure to S. mansoni infested water. These differences are likely attributable to differences in history of exposure to S. mansoni infection and their resulting immunologic status at baseline. As many conflicting results have been reported in the literature regarding immunologic parameters associated with the development of resistance to schistosome infection, these factors should be considered in the design of future immuno-epidemiologic studies and eventual vaccine study design.
