Schistosoma mansoni schistosomula antigens induce Th1/Pro‐inflammatory cytokine responses

Summary Larvae of Schistosoma (schistosomula) are highly susceptible to host immune responses and are attractive prophylactic vaccine targets, although cellular immune responses against schistosomula antigens in endemic human populations are not well characterized. We collected blood and stool from 54 Schistosoma mansoni‐infected Ugandans, isolated peripheral blood mononuclear cells and stimulated them for 24 hours with schistosome adult worm and soluble egg antigens (AWA and SEA), along with schistosomula recombinant proteins rSmKK7, Lymphocyte Antigen 6 isoforms (rSmLy6A and rSmLy6B), tetraspanin isoforms (rSmTSP6 and rSmTSP7). Cytokines, chemokines and growth factors were measured in the culture supernatants using a multiplex luminex assay, and infection intensity was determined before and at 1 year after praziquantel (PZQ) treatment using the Kato‐Katz method. Cellular responses were grouped and the relationship between groups of correlated cellular responses and infection intensity before and after PZQ treatment was investigated. AWA and SEA induced mainly Th2 responses. In contrast, rSmLy6B, rSmTSP6 and rSmTSP7 induced Th1/pro‐inflammatory responses. While recombinant antigens rSmKK7 and rSmLy6A did not induce a Th1/pro‐inflammatory response, they had an association with pre‐treatment infection intensity after adjusting for age and sex. Testing more schistosomula antigens using this approach could provide immune‐epidemiology identifiers necessary for prioritizing next generation schistosomiasis vaccine candidates.


| INTRODUC TI ON
There are currently 206.4 million people suffering from schistosomiasis worldwide 1 with the majority of infected individuals living in Africa. National control programmes in high-risk countries such as Uganda treat infected school-age children and adults through mass drug administration (MDA), using the drug praziquantel (PZQ).
Although the MDA programmes have a high coverage in the targeted areas, national coverage still remains relatively low. For instance, in 2015 just over a third of the estimated 13.2 million people who required treatment in Uganda were reported to have received it. 1 In addition, MDA programmes have no effect on recurrent reinfections in hotspots of transmission. 2 More unsettlingly, there are reports of reduced efficacy of PZQ with multiple rounds of MDA. 3,4 This suggests that MDA alone is insufficient to control morbidity and prevent schistosomiasis transmission. Therefore, an integrated approach with other interventions such as vaccines is required. 5,6 A few schistosomiasis vaccines, such as Sh28GST, Sm-TSP-2 and Sm-14 are currently in early human clinical trials, 7,8 although the trial data are not yet available. Therefore, novel schistosome vaccine antigens are still needed for the vaccine development pipeline.
Schistosome larvae within the vertebrate host (known as schistosomula) are susceptible to host immune responses in animal models, and thus antigens from this stage are thought to be potential vaccine candidates. [9][10][11] The schistosomula develop when infective free-swimming fresh-water larvae (cercariae) burrow through the host skin and lose their bifurcated tails in a process called transformation. 12 Additional structural changes, such as the loss of the cercarial glycocalyx coat during the transformation process, expose the developing schistosomula to host-mediated immune responses.
As the skin-stage schistosomula develop into the lung-stage schistosomula and adult worms, they acquire host antigens 13 masking themselves from host immune effector mechanisms. 14 By targeting antigens from the early schistosomula, it might be possible to attack this stage and prevent infection of the host. Potential vaccine candidates expressed in the newly transformed schistosomula include SmKK7 (Smp_194830), 15,16 SmLy6A (Smp_019350) and SmLy6B (Smp_105220) 13,17 and the tetraspanins SmTSP6 (Smp_059530) and SmTSP7 (Smp_099770). 18,19 SmKK7 is secreted by both the cercariae and the schistosomula 16 ; it has also been reported to be found in the nervous system of adult S. mansoni and to be homologous to a component in scorpion venom, acting as a potassium ion channel blocker. Schistosoma mansoni lymphocyte antigens, rSmLy6A and rSmLy6B (also known as SmCD59a and SmCD59b), are members of the three-finger protein domain (TFPD) superfamily. Although they are homologous to the TFPDcontaining human CD59, which protects human cells from complement fixation, rSmLy6A and rSmLy6B do not inhibit host complement fixation and as such their function remains unknown. 20 rSmLy6A and rSmLy6B are highly expressed by the schistosomula, and as probable GPI-anchored proteins on the schistosome tegument, they likely interact directly with host immune cells. 21 Schistosoma mansoni transmembrane proteins, tetraspanins rSmTSP6 and rSmTSP7, similarly to other tetraspanin family members, are thought to be involved in cell membrane biology. 19,22 As all of the proteins described above are at the interface between the parasite and the host immune system, and thus may be novel vaccine antigens, we assessed the cellular responses to these antigens in individuals residing in an S. mansoni endemic area.

| Ethics statement
Ethical approval for the study was obtained from the Makerere

| Recruitment of study participants
We examined immune responses in peripheral blood mononuclear cells (PBMCs) of participants from TheSchistoVac study (http:// www.theschistovac.eu) which aimed to develop safe candidates for a prophylactic schistosomiasis vaccine. The design of TheSchistoVac study is described elsewhere. 23 Briefly, a cohort of 372 people from an S. mansoni endemic fishing village, Namoni, in Mayuge District, Eastern Uganda, were recruited in September 2011; infected individuals were treated with two doses of PZQ (40 mg/kg) 1 week apart and followed up at 5 weeks and at 1 year after PZQ treatment.
Venous blood was drawn before treatment and peripheral blood mononuclear cells (PBMCs) isolated by density gradient centrifugation using Lymphoprep ™ (STEMCELL Technologies Inc, Cambridge, MA, USA) and cryopreserved in liquid nitrogen. Here, we report pretreatment cellular immune responses in 54 participants from the cohort described above.

| Microscopic examination of stool for ova
Stool samples were collected before treatment (to determine pretreatment infection intensity), 5 weeks after treatment (to examine effectiveness of the treatment) and at 1 year after PZQ treatment (to  (smp_194830), rSmLy6A (smp_019350), rSmLy6B (smp_105220), rSmTSP6 (smp_059530) and rSmTSP7 (smp_099770). 19 The antigens used in the present study are summarized in Table 1. These antigens were identified as highly expressed products in the schistosomula life cycle stage after screening the Schistosoma transcriptome using a DNA microarray, as previously described. 17,19 Specifically, a >5-fold increase in expression when comparing normalized expression averages from snail (egg, miracidia, mother sporocyst and daughter sporocyst) to schistosomula (3-, 24hours, 3-and 6-day schistosomula) life stages was observed. 17 Recombinant antigens were expressed in Escherichia coli and purified using methods as previously described for rSmKK7, 14 rSmLy6A and rSmLy6B. 15 For rSmTSP6 and rSmTSP7, 84 and 78 amino acids were expressed, representing the extracellular loop 2 of these proteins, respectively, as defined by TMHMM2.0 software. 26

| PBMC stimulation
Peripheral blood mononuclear cells were thawed, rested for 6 hours and stimulated for 24 hours with a panel of S. mansoni antigens in RPMI 1640 Medium (Thermo Fischer): AWA (10 μg/mL), SEA (10 μg/ mL), rSmKK7 (2 μg/mL), rSmLy6A (2 μg/mL), rSmLy6B (2 μg/mL), rSmTSP6 (2 μg/mL) and rSmTSP7 (2 μg/mL). Due to a limited number of PBMCs available per individuals and a large number of antigens tested, each antigen was tested in singlicate. After 24 hours, the supernatants were collected and stored at −80°C. Medium without stimulus was used as a negative control. were assigned values corresponding to half of the lowest standard value and those above the highest limit of detection were given the value of the highest standard. Cytokines that were wholly below the lower limit of the assay (IL-4 and IL-7) and chemokines wholly above the upper limit of the assay (MIP1α, MIP1β and RANTES) were excluded from analysis. Samples from unstimulated PBMCs that produced TNF levels >100 pg/mL were also excluded from analysis, as these were considered unreliable 28,29 ; high IL-1β and IL-1ra levels were observed in the same samples.

| Statistical data analysis
Statistical analysis was carried out using STATA version 13 (StataCorp, College Station, TX, USA) and graphs drawn using GraphPad Prism version 6.0g (GraphPad Software Inc., San Diego, CA, USA).
Cytokine, chemokine and growth factor levels were not normally distributed and were, therefore, transformed using Box-Cox transformation. 30,31 The paired Student's t test was used to compare responses between antigen stimulated and unstimulated PBMCs.
Because the mean cytokine/chemokine/growth factor levels of more than two antigen-stimulated PBMCs was compared to that of the unstimulated PBMCs, the error rate due to multiple testing was adjusted by considering a Bonferroni correction, taking into account the number of comparisons being made. In this instance, a P-value of 0.007 (= 0.05/7, the number of antigens in the study) for each cytokine/chemokine/growth factor was taken to be statistically signifi-

| Schistosoma mansoni adult worm and egg antigens induce Th2 responses
Whole parasite preparations of S. mansoni, particularly the egg antigens, are known to induce Th2 responses. [27][28][29] We sought to validate

| Cytokine, chemokine and growth factor responses to schistosomula antigens cluster depending on the antigen
To provide a more global assessment of responses to schistosomula antigens in S. mansoni-infected individuals, cytokine Age, median (range) 12  Females, n (%) 28 (52) Pre-treatment infection intensity Light ( Finally, growth factor responses clustered around responses to rSmKK7 and rSmLy6A (cluster A), responses to rSmLy6B, rSmTSP6 and rSmTSP7 (cluster B) and a single growth factor cluster for PDGFFbb (cluster C) ( Figure S3B).

| Responses to rSmKK7 and rSmLy6A are positively associated with pre-treatment infection intensity
To explore whether clusters of pre-treatment cellular responses were associated with infection intensity before or 1 year posttreatment, the global test was performed. 32 This test examines the association between clusters (eg, groups of correlated immune parameters) and an outcome (eg, pre-treatment or post-treatment infection intensity). After adjusting for age and sex, a significant positive association between pre-treatment infection intensity and responses to rSmKK7 and rSmLy6a was found for the IFNγ cytokine cluster (Table 3, P = 0.026), for the eotaxin chemokine cluster (Table S1, P = 0.020)) and a combination of growth factors (Table S2, P = 0.015). No significant associations were found between clusters of immune response and 1-year post-treatment infection intensity (Table 3, Tables S1 and S2).

| D ISCUSS I ON
The schistosomula are vulnerable to host immune responses after transformation from the cercariae stage. 33 A paired Student's t test was used to test differences between medium and antigens. *P < 0.05, **P < 0.007, ***P < 0.001, ****P < 0.0001 infection. 45 Furthermore, vaccinating mice with a chimera of two recombinant schistosomula antigens SmTSP2 and SmLy6D (Sm29) formulated in CpG-Alum also protects vaccinated mice against infection. 46 Whether the schistosomula antigens used in the present study, SmLy6B, SmTSP6 and SmTSP7, are protective in animal models of schistosomiasis remains to be determined. In a study in Brazil, Sm14-specific Th1 responses were produced by PBMCs of people who were resistant to reinfection with schistosomiasis. 40 This suggests that high antigen-specific Th1 responses may play a protective role in human resistance to reinfection. However, with no clear resistant group in our cohort 1-year post-treatment, we instead looked at the association between cytokine responses and infection intensity before and after treatment with PZQ. We found no association between infection intensity at either pre-or post-treatment and that Th1/pro-inflammatory cytokines to rSmLy6B, rSmTSP6 and rSmTSP7 suggesting that in our cohort these responses may not be protective in humans and other schistosomula antigenic targets should be explored.
Recombinant SmLy6B, rSmTSP6 and rSmTSP7 induced production of regulatory cytokines IL-10 and IL-1ra. This is consistent with work done with another schistosomula tegument antigen, SmLy6D (Sm29), that induced IL-10 production in PBMCs of S. mansoniinfected individuals. 41 In fact, the expression of SmLy6D (Sm29) is similar to that of rSmLy6B 16,19 and rSmTSP6 and rSmTSP7. 19 The con- SmKK7 is a component. 16 This inconsistency suggests that other constituents of cercarial ES material could be the major stimulants of IL-10 production. In fact, glycosylated components of cercarial ES may play a role in the production of IL-10. 47 In addition, the anomaly in IL-10 production could be caused by a difference in the stimulant used.
Turner and colleagues used native cercarial ES material released from transforming cercariae, 47 while the SmKK7 used in the present study is a purified recombinant protein without glycosylated moieties.
Interestingly, rSmTSP7 induced IL-13 production in stimulated PBMCs of infected participants, whereas the other schistosomula antigens did not. In addition, the same rSmTSP7 induced Th1/proinflammatory cytokine production. These findings imply a mixed Th1/Th2 response to recombinant schistosomula antigen SmTSP7.
As much as murine studies have suggested that Th1 responses against migrating larvae may be more host protective than Th2 responses, 46 that blocks potassium channels. 16,53 As potassium channels are vital in the regulation of T cell activation after antigenic stimulation, 54 it is possible that SmKK7 affects potassium channels, thereby modulating T cell activation and suppressing cytokine production by T cells. 16 It remains to be experimentally determined if indeed SmKK7 modulates human T cell activation. Furthermore, Th1/proinflammatory immune responses to rSmKK7 and rSmLy6A were positively associated with pre-treatment infection intensity. However, the suitability of these antigens as vaccine candidates is debatable because they elicited low responses and were not associated with protection against S. mansoni reinfection.
The cytokine responses to the recombinant antigens observed in this study likely reflect the differences in the expression of the gene products of the schistosomula antigens, and their localisation within the S. mansoni organism. SmLy6B, SmTSP6 and SmTSP7 are localized in the tegument and their gene products are highly expressed during the 3-, 24-hour, 3-and 6-day schistosomula as well as adult life stages 17,19 ; SmLy6A, also a tegument antigen, is only highly expressed in the 6-day schistosomula, while SmKK7 is a secreted molecule. 16 Because of the small number of participants in the present analysis, a larger study may be warranted to investigate the association between cytokine responses to the schistosomula antigens In essence, the global test may be useful for studies of cohorts with many independent measurements.
The rate of exposure to the Schistosoma through water contact is associated with resistance to reinfection with schistosomiasis. [55][56][57] It follows that exposure history during follow-up is an important variable in the understanding of reinfection with schistosomiasis.  Figure 4. b Global test P-value. c Adjusted for age and sex. d Positive direction of association.
Bold text represents a significant difference. cercariae) infection to assess safety and optimal dose of such controlled infections (NCT02755324). Based on the findings from this trial, future CHIM for schistosomiasis could investigate human responses to promising Schistosoma antigens including schistosomula antigens in endemic populations.
In conclusion, our findings show that the schistosomula antigens rSmLy6B, rSmTSP6 and rSmTSP7 induce a predominantly Th1/proinflammatory response. The recombinant antigens SmLy6B, SmTSP6 and SmTSP7 might be good targets to investigate further as vaccine antigens against S. mansoni infection.

ACK N OWLED G EM ENTS
We thank the participants of Namoni for taking part in this study.
We appreciate the work done by the field team from the Vector

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.