Schistosoma mansoni infection induces plasmablast and plasma cell death in the bone marrow and accelerates the decline of host vaccine responses

Schistosomiasis is a potentially lethal parasitic disease that profoundly impacts systemic immune function in chronically infected hosts through mechanisms that remain unknown. Given the immunoregulatory dysregulation experienced in infected individuals, this study examined the impact of chronic schistosomiasis on the sustainability of vaccine-induced immunity in both children living in endemic areas and experimental infections in mice. Data show that chronic Schistosoma mansoni infection impaired the persistence of vaccine specific antibody responses in poliovirus-vaccinated humans and mice. Mechanistically, schistosomiasis primarily fostered plasmablast and plasma cell death in the bone marrow and removal of parasites following praziquantel treatment reversed the observed cell death and partially restored vaccine-induced memory responses associated with increased serum anti-polio antibody responses. Our findings strongly suggest a previously unrecognized mechanism to explain how chronic schistosomiasis interferes with an otherwise effective vaccine regimen and further advocates for therapeutic intervention strategies that reduce schistosomiasis burden in endemic areas prior to vaccination.


Introduction
Vaccines prevent highly infectious and fatal diseases which are potentially resource-demanding in the event of uncontrolled outbreaks [1]. The critical impact of vaccination on improving public health has been hailed as second only to clean water supplies [2]. With an estimated 2-3 million lives saved annually through vaccination [3], vaccines are integral disease prevention tools, particularly in developing resource-limited countries [4].
Supported by the World Health Organization (WHO), the Expanded Programme on Immunization (EPI) has been instrumental in the prevention and control of infectious diseases globally [5]. However, over 1.5 million children from developing countries die from vaccine preventable diseases annually [3]. Increasingly, clinical evidence highlights lower responses to vaccines in children from developing countries when compared to those from developed countries [4,6,7]. Among the many associated factors, parasitic helminths have been shown to jeopardize the protective memory immunity mounted by several vaccines in children [8][9][10][11][12].
Concerningly, the reliance on EPI vaccines in developing countries often overlaps with the endemicity for helminths such as Schistosoma spp parasites [13]. Over 90% of all schistosomiasis disease cases, a highly debilitating disease with an estimated 258 million people infected globally [14], occur in sub-Saharan Africa alone [15]. Children in developing countries are particularly at risk of contracting this fresh water-borne parasite [14][15][16]. Considering the suggested impact of schistosomiasis on vaccine effectiveness [8][9][10][11][12][13], the overlapping endemicity of schistosomiasis and the critical need for vaccinating people could be an impending challenge, one that could be catastrophic without strategic interventions.
Praziquantel (PZQ) chemotherapy is currently the only safe and effective treatment intervention recommended against all species of schistosomiasis infections [17,18]. Efforts to control schistosomiasis infection in endemic regions are commonly centred around mass drug administration (MDA) campaigns, under which preventive PZQ treatment is administered periodically to population groups at risk of schistosomiasis infections [19].
In this present study, we assessed the influence of S. mansoni infection on long-term protective immunity to the anti-poliovirus vaccine, used here as a proxy for antibody-dependent vaccination strategies. We conducted an observational clinical study in school children from an S. mansoni endemic area of Cameroon. We also conducted an experimental study using laboratory mice to uncover the immunological interactions between the vaccine-elicited responses and S. mansoni infection.
Summed up, the goal of this study was to integrate our findings into the current global antiviral vaccination strategy, with the foresight to improve the effectiveness of antiviral vaccination programs, particularly in developing countries where schistosomiasis is endemic.

Infection with S. mansoni impairs poliovirus specific serological memory in poliovirus vaccinated school children from a schistosomiasis endemic area
Schistosomiasis infected children were previously shown to possess lower serum antibody titres following vaccination against the measles virus [8]. Therefore, we conducted this study to investigate whether individuals harbouring S. mansoni infection had altered poliovirus vaccine specific serological memory responses. Children residing in Bokito, an S. mansoni endemic rural region of Cameroon were recruited from Yoro 1 (Y1), a public school located near S. mansoni infested rivers in the Bokito area in 2016.
Interviewer-administered questionnaires were utilized to collect information from informed and consenting school children assisted by parents or legal guardians. Poliovirus vaccination status was confirmed by consultation of vaccination cards and recall from parents and/or guardians. Stool samples were collected and assessed for excreted S. mansoni eggs using the Kato Katz technique. Blood samples were collected from all children for downstream analyses. Exclusion criteria from this study included children infected with Hepatitis B or C (using rapid diagnostic tests), Malaria (using blood smear and microscopy), or co-infection with geohelminths in addition to S. mansoni (using Kato-Katz) and any children with missing infection status and/or interview related data.
A total of 64 children (age range: 9.2-12.7 years old) were recruited and had their blood samples collected for this first pilot study in 2016. The prevalence of S. mansoni among these children was found to be 82.8% (53/64). Their blood plasma anti-poliovirus specific IgG antibody titres were quantified by ELISA. We noted significantly reduced anti-poliovirus specific IgG antibody titres in S. mansoni-infected children when compared to non-infected children (p<0.01) (Fig 1A).
To further confirm the robustness of this observation, a subsequent study encompassing a larger sample size with children from five schools located in the same S. mansoni endemic region of Bokito was conducted. Similar consent processes and exclusion criteria as described for the 2016 study were employed. From a total of 336 consenting school children, 189 children were finally recruited (S1A Fig). The children in this cohort ranged from 7 to 16 years of age, with a modal age range of 10-14 years old per school (S1 Table). Thirty-three children were recruited from the 2016 study site, Y1, and the rest from surrounding schools i.e., 41 children from Yoro 2 (Y2), 48 children from Bongando (BG), 32 from Kedia (KD) and 35 children from Ediolomo (ED). The prevalence of S. mansoni infection was 24.2% (8/33) at Y1, 53.7% (22/41) at Y2, 6.25% (3/48) at BG, 6.25% (2/32) at KD and 2.86% (1/35) at ED (S1 Table). Measurements of blood plasma anti-poliovirus specific IgG antibody titres for this new cohort of children further revealed that S. mansoni was significantly associated with lowered anti-poliovirus specific IgG in older children (>14 years) (p < 0.05) (Fig 1B).
Collectively, our observations suggested that chronic S. mansoni infection may negatively impact serological memory immunity elicited by poliovirus vaccination in school-aged children from endemic sites. antibody titres. To distinguish between these possibilities, we addressed whether PZQ treatment could restore, even partially, the poliovirus vaccine specific serological memory of S. mansoni infected children. Praziquantel is the leading therapeutic agent for the treatment of schistosomiasis, particularly in endemic areas [20]. We conducted a follow-up investigation of children who participated in both studies performed in the years 2016 and 2018 to directly assess whether anti-polio vaccine titres would rebound post-treatment. A total of eight children with all information were identified (S1B Fig).
Six of the eight children initially found infected with S. mansoni in the year 2016 had cleared the infection upon follow up in the year 2018. Two of the eight children who were previously uninfected in the year 2016 were found infected with S. mansoni in the year 2018. Between the years 2016 and 2018, a total of 3 years had elapsed, thus translating to 3 cycles of single annual doses of PZQ (40mg/kg) treatment cycles under the MDA campaign aimed to eliminate schistosomiasis in endemic areas of Cameroon. In addition, considering that all children in our studies were treated with single doses of PZQ (40mg/kg) outside of the MDA campaign, both S. mansoni infected and non-infected children effectively received an additional PZQ dose making it 4 treatment cycles over the 3-year study period.
Blood plasma anti-poliovirus specific IgG antibody titres of the selected six previously infected and now cured children were comparatively evaluated before and after treatment in relation to their parasitological status changes between 2016 and 2018 (assessed by Kato Katz in 2016 and at the end of the follow up period in 2018). We noted that these children who were previously infected with S. mansoni in 2016 had significantly higher anti-poliovirus specific IgG antibody titres in 2018 after having cleared the parasite (p<0.05) (Fig 1C). For the remaining two children, we noted that these children who were previously egg-negative for S. mansoni in 2016 had consistently lower anti-poliovirus specific IgG antibody titres in 2018 after having acquired the parasite (Fig 1D). Overall, these data suggested a positive association between PZQ treatment's S. mansoni worm killing and infection clearance with the partial restoration of poliovirus vaccine induced antibody titres in children who efficiently cleared their worms post treatment.

Establishing a hexavalent (DTPa-hepB-IPV-Hib) vaccination mouse model
Given the ethical limitations of human research studies involving invasive mechanistic experimentation, we next turned to an experimental mouse model of S. mansoni infection to further investigate whether chronic S. mansoni infection actively suppressed anti-polio antibody responses. Similar to the oral poliovirus vaccine (OPV), inoculation of mice with the inactivated poliovirus vaccine (IPV) has been shown to induce robust serum anti-poliovirus IgG serological responses that can be maintained for up to 400 days [21]. We established an IPV mouse vaccination model using age and gender-matched BALB/c mice that were subcutaneously inoculated twice with a commercial hexavalent (DTPa-hepB-IPV-Hib) vaccine (Hexaxim, Sanofi Pasteur, Lyon, France) or mock treatment (1x PBS) at day 0 and day 30 (

Chronic S. mansoni infection reduces poliovirus specific antibody titres in vaccinated mice that are partially reversed by PZQ treatment
We next assessed, in mice, the impact of chronic S. mansoni infection and its subsequent clearance by PZQ treatment on immune responses induced by the hexavalent (DTPa-hepB-IPV-Hib) vaccine. To this end, four groups of age and gender matched BALB/c mice were established (S3 Fig). The first group represented the non-vaccinated naïve mice. The remaining three mice groups were subcutaneously injected twice with the hexavalent vaccine (DTPa-hep-B-IPV-Hib) at day 0 and day 30, as per our established vaccination model (Fig 2A and 2B). One of the three groups was designated the vaccinated control (Vac+). The remaining two groups of vaccinated mice were subsequently percutaneously exposed to low dose infection with 35 S. mansoni cercariae at 5 days post booster vaccine administration to establish a chronic S. mansoni infection (Vac+Sm+). Analysis of weight changes over the course of infection highlighted a decline from 8 weeks post-S. mansoni-infection (p.i), for the two groups of S. mansoni infected mice (Fig 2A). One cohort of Vac+Sm+ mice was administered 400mg/kg of PZQ between week 9 and 10 p.i (Vac +Sm+PZQ+), that is, after the establishment of the chronic phase of the schistosomiasis infection [22]. Upon PZQ treatment, Vac+Sm+PZQ+ mice recovered their weight within 1 week while Vac+Sm+ continued to decline until the experimental endpoint at week 18 p.i.

Schistosomiasis reduces vaccine efficiency
At week 18 p.i, Vac+Sm+PZQ+ mice had significantly lower spleen and liver weights than Vac+Sm+ mice (p<0.001 and p<0.05, respectively) ( Fig 2B and 2C). Additionally, Vac+Sm + mice had significantly increased spleen and liver weights than Vac+ mice (p<0.0001), which on the other hand were within normal limits as demonstrated by comparable spleen and liver weights of naïve mice. Further, there were no notable differences in the spleen weights of Vac +Sm+PZQ+ and Vac+ mice (p>0.05) Fig 2B) while a minimally significant difference was observed for liver weights between the same groups of mice (p<0.05) Fig 2C). Chronic schistosomiasis was confirmed by the recovery of S. mansoni eggs in the gut and the liver of infected mice. By week 18 p.i, PZQ treated mice showed significantly reduced S. mansoni egg burden in the liver and the intestines when compared to non-treated mice, thus demonstrating the efficiency of our PZQ treatment regimen ( Fig 2D).
Next, we investigated the relationship between chronic S. mansoni infection and the parasites' clearance by PZQ treatment on the levels of anti-poliovirus vaccine induced serological responses. Blood serum was obtained from all mice cohorts at weeks 4, 8, and 18 p.i, and serum ELISAs were performed to quantify anti-poliovirus specific IgG antibody titres over the 18 week infection period. The successful vaccination of the mice was confirmed by observed significantly elevated anti-poliovirus specific IgG antibody titres of Vac+ in comparison to naïve (non-vaccinated) mice at week 4, 8 and 18 p.i (p<0.01) ( Fig 2E). Additional comparisons among the vaccinated groups revealed a significant decline in anti-poliovirus specific IgG antibody titres over time in S. mansoni infected mice (Vac+Sm+ and Vac+Sm+PZQ+) ( Fig 2E). Strikingly, by week 18 after S. mansoni infection, anti-poliovirus specific IgG antibody titres in both vehicle treated and PZQ-treated infected mice were significantly lower than that of Vac + mice (p<0.01 and p<0.05, respectively).On the other hand, PZQ treated Vac+Sm+PZQ + mice had significantly higher titres than their non-treated Vac+Sm+ counterparts at week 18 p.i (p<0.05; Fig 2E and 2F). Furthermore, associative analyses between serum anti-parasite levels (directed against Schistosoma Egg Antigen, SEA) and vaccine-elicited anti-polio IgG antibodies showed a progression of a positive association that gradually changed into a nega-

Chronic S. mansoni infection reduces bone marrow CD138+ plasma B cell responses in mice, and anthelminthic PZQ treatment reverses this effect
The reversible inhibition of anti-poliovirus antibody titres in S. mansoni infected mice prompted us to evaluate cellular immune components that could potentially provide a mechanistic explanation to our observations. Given that long-term vaccine specific serological memory immunity is maintained by B cells such as antibody producing CD138+ plasma B cells [23][24][25][26] that niche in the bone marrow, we investigated the influence of chronic S. mansoni infection and PZQ treatment on specific B cell subsets. First, to unambiguously assess the

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Schistosomiasis reduces vaccine efficiency influence of S. mansoni infection on B cell subsets in the course of an infection, BALB/c mice were infected with a low dose chronic-driving amount of cercariae (35 cercariae each percutaneously), and bone marrow and spleen were collected at weeks 4 and 10 p.i for B cell analyses using flow cytometry. We observed that S. mansoni infected mice had depleted CD19+B220 + total B cells, CD138+ plasmablasts and plasma cells in their bone marrow at week 10 but not at week 4 p.i (S5 Fig).
In vaccinated mice, we noted that B cell frequencies and absolute counts were significantly increased in the bone marrow of Vac+ mice when compared to naïve controls at week 18 p.i (Fig 3A and 3B). This elevation was abrogated in Vac+Sm+ mice where B cell reduction was associated with S. mansoni infection. In Vac+Sm+PZQ+ mice, previously reduced B cell levels were restored to levels similar to those of Vac+ mice ( Fig 3B). Strikingly, the spleen B cell compartment, although stimulated by vaccination (Vac+, p<0.1) was not as robustly affected as in the bone marrow following S. mansoni infection ( Fig 3C). Upon quantification of CD138 + plasma B cell populations (here encompassing plasmablasts and plasma cells) by flow cytometry in bone marrow and spleen (Fig 3A), the bone marrow of Vac+ mice showed significantly higher frequencies and absolute numbers of plasma B cells as defined by Live Lymphocyte +CD19+B220+IgD-IgG1-CD138+ staining ( Fig 3A) when compared to those of naïve mice (p<0.001) and Vac+Sm+ mice (p<0.05) ( Fig 3D). In contrast, there were no notable differences between the frequencies of bone marrow plasma B cells of Vac+ and Vac+Sm+PZQ + mice. The latter presented with significantly higher frequencies and absolute numbers of bone marrow plasma B cells when compared to those of Vac+Sm+ mice (p<0.05) ( Fig 3D). Our observations, altogether, preferentially indicated a parasite-mediated reduction of recruitment or maintenance of plasma B cells in the bone marrow of vaccinated mice as worthy of further consideration to explain, at the B cell level, reduced polio vaccine-elicited serological IgG antibody production in S. mansoni infected hosts. In fact, since this effect was ameliorated by PZQ-mediated parasite killing, more support was hereby lent to our serological observations that treatment against the parasite may improve vaccine response in schistosomiasis-diseased hosts. Focusing on plasma cells and to separate the possibility of the defective migration of these plasma cells to the bone marrow from impaired survival within the bone marrow niche, we analysed a C-type Lectin CD93 that has been demonstrated to be critical in the long-term survival of antibody producing CD138+ plasma B cells within the bone marrow niche but not their migration. [23,27]. To do so, we assessed the expression of CD93 on bone marrow plasma B cells for all mice groups. We noted a reduced surface expression of CD93 on plasma B cells of naïve mice in comparison to Vac+ mice, although the difference was not significant. However, Vac+ mice had a markedly increased expression of CD93 on bone marrow plasma B cells in comparison to Vac+Sm + mice (p <0.05) (Fig 3E). In addition, plasma B cells of Vac+Sm+PZQ+ expressed higher levels of CD93 than Vac+Sm+ mice and a non-significant difference versus Vac+ mice. These

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Schistosomiasis reduces vaccine efficiency findings support a bone marrow plasma B cell-survival-associated route for the negative effect of S. mansoni infection on the sustainability of anti-polio vaccination-elicited memory responses in mice.
In the spleen, however, there was no significant difference between CD138+ plasma B cell frequencies of Vac+ and naïve mice ( Fig 3F). However, Vac+ mice had significantly higher CD138 + plasma B cell frequencies when compared to Vac+Sm+ (p<0.05) (Fig 3F), while no significant differences were observed for absolute numbers. In addition, there was no significant difference between splenic CD138+ plasma B cell frequencies of Vac+ and Vac+Sm+PZQ+ mice and splenic CD138+ plasma B cell frequencies of Vac+Sm+ and Vac+Sm+PZQ+ mice (Fig 3F).
To further assess the tripartite association between infection bone marrow plasma cell frequencies/numbers, serum anti-SEA IgG titres and serum anti-polio IgG titers in our study, we performed associative analyses of these variables (Fig 3G and 3H). We noted that increasing serum anti-SEA IgG titers in S. mansoni infected mice negatively associated with bone marrow plasma cell levels ( Fig 3G). On the other hand, a positive association between bone marrow plasma cell levels and serum anti-polio IgG titers in was noted in vaccinated mice (Fig 3H).
These observations demonstrated that S. mansoni could be associated with reduced antibody-producing cells, the CD138+ plasma B cells, in the bone marrow but not in the spleen of mice. Taken together, we, therefore, hypothesized that the observed reduction of CD93 expression on CD138+ plasma B cells in the bone marrow of S. mansoni infected mice might indicate the parasite's driven increased likelihood of bone marrow CD138+ plasma B cell death. We also hypothesized that PZQ treatment and its associated reduction of parasite burden could mitigate the parasite's impact on these antibody producing cells in mice.

Chronic S. mansoni infection is associated with a reduced survival of bone marrow, but not of spleen, plasma B cells of hexavalent (DTPa-hepB-IPV-Hib) vaccinated mice, and anthelminthic chemotherapy PZQ reverses this effect
To further explore the observed reductions of plasma cells in the bone marrow of vaccinated and S. mansoni infected mice, and their restoration in the bone marrow of their PZQ-treated counterparts, we assessed the survival of various immune cells important in the generation, maintenance and expansion of plasma cells in the different mice groups. First, the dead cell staining fixable viability dye (FVD) was used to identify and quantify dead plasmablasts and plasma cells by flow cytometry (Fig 4A). The bone marrow of Vac+ mice showed significantly lower frequencies of FVD+CD138+plasma B cells, and lower but non-significant plasmablasts, when compared to naive mice (p<0.01 and p = 0.07, respectively) (Fig 4B and 4C). These results indicated the better survival of plasma cell populations in vaccinated mice when compared to non-vaccinated mice, consistent with a survival advantage of plasma cell lineage following vaccination. Similarly, the bone marrow of Vac+ mice had significantly lower frequencies of FVD+CD138+ dead plasmablasts and plasma cells when compared to the infected Vac+Sm+ mice (p<0. p<0.05 and p<0001, respectively) (Fig 4B and 4C) indicating a parasite-driven provoked death of these cells. Likewise, bone marrow frequencies and absolute numbers of FVD+CD138+ dead plasmablasts and plasma B cells of Vac+ mice were either not different or minimally higher than those of infected and treated Vac+Sm+PZQ+ mice (p>0.05 and p< 0.05, respectively) ( Fig 4B and 4C

Schistosomiasis-driven death of plasma cells and plasmablasts in the bone marrow is not mediated by apoptosis
Further assessment of the mechanism of cell death in bone marrow plasma cells was performed by dual staining of the cells with FVD live/dead staining and Annexin V staining to evaluate apoptosis (Fig 5A). We failed to note a disproportionate increase of apoptotic plasma cells (Fig 5B-5D) or apoptotic plasmablasts (Fig 5E-5G) in S. mansoni infected Vac+ Sm + mice, arguing against the central contribution of apoptosis in the observed schistosomiasisassociated death of bone marrow plasma cells. However, cell death due to membrane-damage, i.e., Annexin V+FVD+ cells, showed a pattern consistent with the differential reduction of plasma cells and plasmablasts in schistosomiasis-diseased Vac+Sm+ mice with an equally recapitulative restoration by death rate reduction in Vac+Sm+PZQ+ mice. Taken together, a nonapoptotic process appears to be at play here to foster plasmablasts and plasma cell death in the bone marrow of mice following schistosomiasis.

Discussion
Long-term immunity has been demonstrated for poliovirus vaccines [28,29]. Our observations now suggest a negative association between S. mansoni infection and the ability of the host to maintain serological memory against poliovirus in vaccinated hosts. This observation is consistent with our recent report of S. mansoni infection being associated with reduced responses to the measles virus vaccine in school-aged children from Bokito, Cameroon [8,13]. Additionally, weakened immune responses in S. mansoni infected children have been reported for the measles virus vaccine in Uganda [9], and the hepatitis B virus vaccine in Uganda [10] and Egypt [11]. The similarity of these observations for different viral vaccines now suggests a general impairment of long-term serological memory mounted by viral vaccines in S. mansoni infected children living in endemic areas. Specifically in the present study, our findings revealed a heightened impact of S. mansoni infection with increasing age as we noted a more pronounced effect of S. mansoni associated reductions of vaccine-elicited memory responses in older children from five schools combined.
Interestingly, we noted in our study a high prevalence of S. mansoni infection (>54%) in children from Y1 in the year 2016, which was considerably reduced to less than 24.2% in the

Schistosomiasis reduces vaccine efficiency
year 2018. This reduction in the prevalence of S. mansoni could be attributed in part to the effectiveness and efficacy of annual PZQ treatments of these children under the MDA campaign in Cameroon, despite the permanent risk of reinfection [8]. The success of MDA in

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Schistosomiasis reduces vaccine efficiency reducing schistosomiasis prevalence in school children has recently been confirmed for 44 countries in sub-Saharan Africa [30].
We similarly observed that treatment with PZQ could partially reverse anti-viral vaccine specific serological memory in schistosomiasis-diseased children. This was also the case in a recent study in Uganda which demonstrated that PZQ treatment improved responses to the measles catch-up vaccine in S. mansoni infected preschool children [9]. This could either denote an intrinsic repair potential of PZQ, irrespective of the infection status and/or a potential linkage to its antiparasitic effect. Our subsequent observation of the restoration of antipolio vaccine serological responses in children that were confirmed cured of schistosomiasis argues strongly in favour of PZQ's potential ability to reverse serological vaccine memory suppression mediated by killing adult schistosome parasites, an observation further supported by existing reports showing that progressive chronic schistosomiasis is associated with host immunosuppression [31].
We demonstrated that the long-term maintenance of IPV-specific serological memory in DTPa-hepB-IPV-Hib vaccinated mice was consistent with the previous report of long-term maintenance of poliovirus specific serological memory in mice [21]. Utilizing a well-established mouse model to mimic natural human chronic S. mansoni infections [32], we showed that infection with chronic S. mansoni post vaccination lowers the anti-polio specific serological memory responses over time. Similarly, other studies have demonstrated that S. mansoni suppresses mouse immune responses to the diphtheria toxoid [33], the bacille Calmette-Guérin (BCG) vaccine [34] and HIV specific vaccines [35]. In another study, an association between S. mansoni infection and reductions of serologic IgG antibodies mounted by the Human Papillomavirus vaccine in non-human primates was described [36]. Highlighting the immunosuppressive nature of different Schistosoma spp. in a separate study, chronic S. japonicum infection impaired immune responses against the hepatitis B virus vaccine in mice [37]. These observations strengthen the odds that schistosomiasis impairs its host's ability to maintain long-term vaccine memory immunity.
Serological memory immunity is maintained by the continual supply of antibodies produced by plasma cells [23,26,38]. In our study, CD138+ plasma B cells were substantially reduced in the bone marrow and moderately in the spleens of chronic S. mansoni infected mice. These observations suggest that the negative impact of S. mansoni could be rooted in the bone marrow in addition to satellite defects in the secondary lymphoid organ [13]. Noteworthy, the S. mansoni associated plasma cell reduction was well pronounced in bone marrow but poorly in the spleen, thus suggesting the concentrated impact of S. mansoni on bone marrow plasma cells through mechanisms that remain unclear. As the bone marrow is a niche for the long-term survival of antibody-producing plasma cells, we reasoned that S. mansoni could reduce the survival of these cells. Confirming our hypothesis, CD93, a specific marker of bone marrow plasma cell survival, and not defective migration [23,27], was expressed at markedly lower levels in bone marrow plasma cells of S. mansoni infected mice when compared to noninfected counterparts.
Logically, one would have therefore expected the reduced viability of plasma cells expressing lower levels of CD93 in our settings, consistent with compelling reports [27,39]. Measuring the survival of plasma cells in our study, we noted that higher frequencies of the bone marrow CD138+ plasma B cells in chronic S. mansoni infected mice were stained with the amine dye for dying or dead cell populations. As such, we consequently concluded that the reduced expression of surface CD93 on plasma cells during S. mansoni infection robustly translated into increased death or reduced survival of these cells in mice. A rough parallel could be drawn from human studies where clinical evidence has shown the downregulation of B cell proliferation in S. haematobium infected children [40]. Several routes for explanation are envisaged here to explain the loss of plasma B cells in the bone marrow of patently infected S. mansonidiseased mice. A possibility could be the hijacking of the plasma cell generation/differentiation pipeline during schistosomiasis to foster the expansion/accumulation of another B cell subset. We do, however, find great opposition to this possibility in the assessment of spleen and bone marrow levels of other B cell subsets (marginal, memory and follicular) where no indication of accumulation of any of these B cell subsets was apparent as a result of schistosomiasis. Another explanation could have been an erroneous accumulation of plasma cells in the spleen rather than migrating to the bone marrow to constitute long-lived and powerful antibody-producing cells. However, our results on the levels of plasma cells in the spleen of schistosomiasis-diseased mice do not support an accumulation of these cells in the spleen, arguing against the likelihood of the depletion of plasma cells in the bone marrow being a result of an upstream impaired migration from the spleen. Nevertheless, the observation that splenic T cells, particularly in the Foxp3+ and memory T cell compartment, are altered during schistosomiasis in a way that could favour plasma B cell depletion in the bone marrow do raise the clear possibility that plasma cell depletion might not be sufficient to fully explain the observed reduction of antibody production caused by schistosomiasis. Nonetheless, further investigations on the scope of alteration of schistosomiasis on humoral memory response within the T cell compartment are certainly required despite non-opposing indications on the presently robust observation of a preferential depletion of bone marrow plasmablasts and plasma cells as strong correlates of reduced production of vaccine-elicited antibodies in our setting.
Standing out from our analyses was the possibility of diminished survival and heightened cellular death as demonstrated by dual live/dead and Annexin V staining. Our investigation using this cell death staining combo in bone marrow plasma cells in vaccinated and S. mansoni infected mice did not reveal a preferential accumulation of apoptotic plasma cells but rather that of live/dead staining positive dead cells within the plasma cell compartment. These data argue against apoptosis as the central driving force of plasma cell depletion by schistosomiasis. In fact, besides apoptosis, several means of eukaryotic cell death ranging from necrosis/ necroptosis, autophagy, pyroptosis, or ferroptosis [41] could be at play here. However, given that markers of cell death can only be internalized as a result of damaged cell membranes and that cellular inflammation was not apparent in the bone marrow of schistosomiasis-diseased mice (no increase in total B cell and total T cell numbers), compelling support is lent to the implication of a membrane-damaging and poorly inflammatory cell death process such as necroptosis or ferroptosis. Clearly, this needs to be carefully investigated in follow-up studies to conclusively understand the mechanistic bases of schistosomiasis-associated plasma cell death in the bone marrow. As of now, nevertheless, our data suggest that the Schistosoma spp. parasites could lower the survival of plasmablasts and that of CD138+ plasma B cells in the bone marrow of their hosts.
More pathophysiologically, the discriminative influence on bone marrow rather than splenic plasma cells is intriguing and could well be rooted in the heightened physiological toll chronic infections such as schistosomiasis might have on the bone marrow hematopoietic machinery. Whether chronic schistosomiasis deleteriously solicits the bone marrow hematopoietic machinery as the infection progresses [42] and as such interferes with the maintenance of essential survival cues within the bone marrow for plasma cells such as CD93 expression is to be addressed. Certainly, further experimental evaluations are required to decisively elucidate the mechanism behind these deductions and to validate how far upstream and wide around the B cell activation ladder is the host impaired by S. mansoni infection.
Overall, as yet, our mice data suggest that the chronic S. mansoni driven reduced survival or increased death of CD138+ plasma B cells in the bone marrow associates with impaired maintenance of vaccine specific serological memories. What remains puzzling and worthy of future investigations is the selective impairment of polio-specific responses by schistosomiasis whereas SEA-specific humoral responses do persist and are increased over time, indicating a certain level of antigen-specificity in the schistosomiasis-associated impairment of the memory responses.
Whatever the basis for such a sophisticated immunomodulatory ploy of schistosomes, our observations of the impact of S. mansoni on poliovirus vaccination in both children and mice raise an important public health concern, as they indicate that global progress made against viral infections through vaccination is at risk. Clinical data have shown that S. mansoni lowers vaccine induced memory immunity for the measles virus vaccine in Cameroon [8] and Uganda [9], and the hepatitis B virus vaccine in Uganda [10]. This trend of S. mansoni associated impairment of viral vaccines is more concerning, particularly given the global vaccination efforts to control the current SARS-CoV2 pandemic. The control of immunomodulatory infections of the like of S. mansoni infections is therefore critical especially in regions where the parasites are endemic.
Our study further revealed that treatment with PZQ partially restored long-term poliovirus vaccine specific serologic memory responses in S. mansoni-infected children and mice. Additionally, PZQ treatment improved bone marrow plasma cell responses in mice. These findings support further the utilization of PZQ in schistosomiasis endemic areas despite previous conflicting evidence on its usefulness in ameliorating vaccine responses. Indeed, PZQ has also been shown to improve mice immune responses to the HIV vaccines [35,43] and the hepatitis B virus vaccine [37]. In Olive baboons, PZQ treatment in the chronic stage of S. mansoni infection significantly reversed S. mansoni associated reductions of HPV specific IgG antibodies [36]. Together, our present findings point at a beneficial effect of PZQ on vaccine responses in schistosomiasis-diseased animals and children. However, further studies assessing the effect of PZQ on vaccine immunity, for example, the current POPVAC trial studies in Uganda [44], are imperative.
In conclusion, the present study showed that S. mansoni infection could be associated with impaired vaccine responses in both children and mice, by lowering the survival of CD138 + plasma B cells as shown in mice and ultimately suppressing the maintenance of serological memory. This study further demonstrated that PZQ treatment could partially restore vaccine induced humoral memory responses in S. mansoni infected children and cellular and humoral immune responses in mice.
As such, given the remarkable safety profile of PZQ [45], accumulating evidence on the added benefits of repeated MDA with PZQ [46], and the present findings, we support the WHO consideration of intensified PZQ treatments, for populations in schistosomiasisendemic areas. This strategy could potentially improve vaccination responses in these S. mansoni endemic areas and thus could strengthen vaccination benefits in developing countries, particularly regarding the current vaccination efforts against SARS-COV 2 viruses causing the deadly COVID-19 pandemic.

Ethics statement
Ethical approval for human studies was granted by the Cameroon National Ethics committee for Human Health Research (Approval no. 2018/02/976/CE/CNERSH/SP). Authorization to conduct the study was also granted by the Ministries of Basic Education (Approval no. 3/105/ L/MINEDUB/DREB-C/SDAG/IMS) and Public Health of Cameroon (Approval no. 030-571/ L/MINSANTE/SG/DROS/CRC/NPN/TMC/ 631-12.18). The study was conducted with authorization from local, regional and school authorities. Children and parents'/legal guardians were informed of the objectives and the methodology of the study with assistance from PLOS PATHOGENS Schistosomiasis reduces vaccine efficiency schoolteachers. Participation was voluntary and all children and parents/legal guardians gave informed written consent prior to the study. All collected data were de-identified before downstream analyses to ensure the confidentiality of the participating school children. All school children from the enrolled schools, participant or not, were treated with PZQ as recommended by the Cameroon National program for the control of Schistosomiasis and Soil transmitted Helminthiasis regardless of their parasitological status.
Authorization to conduct experiments on animals was granted by the University of Cape Town Animal Ethics Committee (AER Protocol no. 018/029). All animal experiments were performed to minimize animal suffering in accordance with the guidelines of the Animal Research Ethics committee of the Faculty of Health Science of the University of Cape Town, the South African Veterinary Council (SAVC) and the South African National Standard (SANS 10386:2008).

Human observational study design, population, and data collection
A cross-sectional study was conducted in five public schools in vicinity to S. mansoni infested rivers in 5 different villages namely Yoro 1, Yoro 2, Bongando, Ediolomo and Kedia in Bokito, a rural town located about 100 km from Yaoundé, the capital of Cameroon. This study area has been previously described [47].
The study was conducted in two sub-phases. In the first explorative sub-phase of the study, school children were recruited from Yoro 1 in the year 2016. In the second sub-phase, a follow up of the study with a larger sample size from five different public schools, was carried out in the year 2018. A total of 189 children were recruited from the following schools after exclusion of those infected with malaria, hepatitis B/C and other geohelminths: Yoro 1, Yoro 2, Bongando, Ediolomo and Kedia (S1 Fig). In both phases of the study, school children with at least 6 months of residency within the endemic area and consenting to participate to the study were recruited. In addition, both studies were conducted at least 6 months after PZQ treatment of all children under the Mass Drug Administration (MDA) program of Cameroon. The MDA campaign is aimed at controlling schistosomiasis in endemic regions of Cameroon and is based on the WHO recommended tablet dose pole strategy. The dose of PZQ per child was based on their body height and administered based on the dose-pole to achieve an optimal dosage of 40 mg/kg [48,49]. In addition, all children contacted in our study were treated with a single dosage of 40 mg/kg PZQ regardless of their schistosomiasis status. A total of 8 children were followed up with complete data in both the 2016 and 2018 studies. Of these, 6 children who were infected with S. mansoni in 2016 had cleared the parasite in 2018. Further analyses were conducted to assess the impact of parasite clearance by PZQ on vaccination. The remaining 2 children were not infected in 2016 but were found infected in 2018. Due to their small sample size, no further analyses were conducted for these children.

Standardized Interview administered data collection
Each child was interviewed by a member of the research staff assisted by their class teacher and parents/legal guardians after informed consent was attained. The interviews were carried out and recorded on questionnaires designed to collect demographic information such as age and gender as well as self-reported and/or parent/legal guardian confirmed general health and vaccination status. These deidentified data are available upon request.

Stool and urine sample collection and parasitological assays
Each child was given 2 prelabelled 50ml screw-cap vials for collection of fresh morning stool samples collected at 2 separate days with a 5-day gap in-between. The stool samples were evaluated for parasitological analyses using the Kato-Katz technique to identify children infected

PLOS PATHOGENS
Schistosomiasis reduces vaccine efficiency with S. mansoni as previously described [50,51]. Briefly, 41.7mg of stool was prepared for each sample, 2 stools per child, and assessed by microscopy by 2 independent laboratory technologists for the detection and quantification of S. mansoni eggs. Urine samples (10ml) were collected from each child and assessed by filtration method to identify S. haematobium eggs as previously described [52].

Blood sample collection and assays
Whole blood (4 ml) was aseptically drawn from each child by venepuncture into Heparin coated tubes by experienced and authorized phlebotomists. Post-puncture care was provided for each child to minimize risk of infection. Malaria infection status was assessed by thick smears as previously described [53]. Briefly, a spot of well mixed blood was placed on a clean slide. Using the edge of another clean slide, red blood cells were carefully lysed into a thick smear to release the Plasmodium parasite. The smear was then air-dried followed by Giemsa staining and analysed using an optical microscope. Plasma was isolated from the collected blood by centrifugation and stored at -80˚C until use. HBV and HCV infection statuses were assessed using the DiaSpot HBsAg and DiaSpot HCV-Ab test kits (DiaSpot, Jakarta, Indonesia), respectively, following the manufacturer's instructions. The quantification of total antipoliovirus IgGs in plasma samples was carried out by sandwich ELISA using a commercial kit (Human Anti-Poliomyelitis Virus 1-3 IgG ELISA Kit, Alpha Diagnostic International, Tx, USA) following the manufacturer's instructions.

Animal experimental study
Mice, vaccines, parasites, and treatment. Mice on a BALB/c background were used. The mice were maintained under specific pathogen-free animal conditions at the University of Cape Town, in accordance with the guidelines established by the Animal Research Ethics committee of the Faculty of Health Science of the University of Cape Town and the South African Veterinary Council (SAVC). Age and gender matched mice aged 6-8 weeks were injected subcutaneously with a commercial hexavalent vaccine against infectious diseases diphtheria, tetanus, pertussis, hepatitis B, poliovirus myelitis (inactivated) and haemophilus influenzae type b conjugate (DTPa-hepB-IPV-Hib) (Sanofi Pasteur, Lyon, France), or were mock vaccinated, at 2 sites i.e., 150 ul on the neck and 100 ul on the abdomen, at day 0 (primary vaccination) and day 30 (secondary vaccination). At day 5 post-secondary vaccination, vaccinated mice were anesthetized and infected by percutaneous abdomen exposure with a low dose of 35 live S. mansoni cercariae for 30 minutes as previously described [54]. Live cercariae were generated from Biomphalaria glabrata snails (a gift from Adrian Mountford, York, UK), and NMRI female mice were used to maintain and expand the S. mansoni parasite larvae. Between week 9 and week 10 post S. mansoni exposure, infected mice only were treated with a single daily oral dose of 400mg/kg of PZQ solution (Merck KGaA, Darmstadt, Germany) or mock treated for 7 days. The PZQ solution was prepared as previously described [55]. Briefly, PZQ was weighted at quantities sufficient for 400 mg/kg of animal body weight and mixed with 10 parts 70% Tween + 30% Ethanol using a magnetic stirrer. Next, 90 parts of distilled sterile water were slowly added and stirring to obtain a homogeneous suspension. Administration of 200 ul of the homogeneous suspensions were administered within 3 h after preparation.

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Schistosomiasis reduces vaccine efficiency euthanized. Blood was centrifuged in serum separator tubes (BD Bioscience, San Diego, CA) at 8 000 × g for 10 min at 4˚C to retrieve the serum. The upper aqueous serum phase was aliquoted into tubes and stored at -80˚C until further use.
Anti-poliovirus specific total IgG titres were quantified by sandwich ELISA using a commercial kit (Mouse Anti-Poliovirus Virus 1-3 IgG ELISA Kit, Alpha Diagnostic International, Tx, USA) according to the manufacturer's instructions.

Sampling of the bone marrow and spleen, single cell isolation and flow cytometric analyses
Following euthanasia at week 18 p.i, the rear left femur bone and spleen were collected for each mouse. Single cell suspensions of bone marrow and spleen cells were prepared and immediately used for downstream flow cytometry analyses.

Sampling of the liver and gut, and S. mansoni egg burden determination
The liver and gut samples were excised and collected from each mouse at week 18 post S. mansoni infection. The organs were weighed and S. mansoni eggs were counted after tissue digestion using 4% KOH aq for 18 h, as previously described [32]. The intensity of S. mansoni infection intensity was expressed as the number of eggs detected per gram of faeces (EPG).

Statistics
Statistical analyses were conducted using GraphPad Prism 6.0 software (http://www.prismsoftware.com). Data distribution was assessed by the Shapiro-Wilk test. The Chi-square test was used to assess differences between gender distribution of school children in relation to their S. mansoni infection status. All other statistical evaluations were performed using one of the following tests depending on data distribution: unpaired Student's t-test for equal or unequal variances or Mann-Whitney test for comparison between 2 groups, or Kruskal-Wallis test followed by the Dunn's multiple comparisons, or ANOVA followed by Bonferroni's multiple comparisons or FDR corrected multiple comparisons using the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli [56]. A p-value threshold of < 0.05 was considered as statistically significant.

PLOS PATHOGENS
Schistosomiasis reduces vaccine efficiency schools combined. All children completed questionnaires and had 2 stool samples collected on two separate days for S. mansoni egg detection by the Kato-Katz technique. Plasma was collected only for children without missing data nor infections such as other helminths, malaria, and hepatitis B and/or C. A battery of exams and diagnostic tests were performed on consenting participants and only samples from patients with no missing data for stool examination by Kato Katz, rapid diagnostic testing of Hepatitis B and C viruses and microscopical screening of malaria parasite in blood smears were further used in the present study, as previously described. Finally, 189 children had their plasma samples analysed for anti-poliovirus IgG titres. (B) Samples of children with complete questionnaire data showing complete annual PZQ treatment, and complete anti-poliovirus IgG titre analyses from two studies conducted three years apart were selected. Initial data was collected in the first study conducted in the year 2016 followed by data collection for the same children in a second study in the year 2018.

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Schistosomiasis reduces vaccine efficiency