Rural-urban differences in oral typhoid vaccine responses in Uganda: contribution of Schistosoma mansoni

Rural-urban differences in oral typhoid vaccine responses in Uganda: contribution of Schistosoma mansoni | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Rural-urban differences in oral typhoid vaccine responses in Uganda: contribution of Schistosoma mansoni Bridgious Walusimbi, Agnes Natukunda, Rebecca Amongin, Victoria Heyraud, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8832138/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Vaccine immunogenicity varies across populations, yet drivers remain unclear. We compared systemic (plasma) and mucosal (coprological) antibody responses to the oral typhoid vaccine Ty21a, quantified by ELISA, among Ugandan adolescents from urban and Schistosoma mansoni-endemic rural settings. Urban participants elicited higher responses. Causal mediation analysis indicated that S. mansoni partly accounted for reduced mucosal, but not systemic, responses in rural participants, highlighting implications for oral vaccine effectiveness in endemic settings. Health sciences/Diseases Biological sciences/Immunology Health sciences/Medical research Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Main Text Oral vaccines exhibit heterogeneity in immunogenicity and efficacy across populations, with impaired responses often reported in tropical low-income countries (LICs) compared with high-income countries (HICs), 1 , 2 and in rural compared to urban LIC settings. 3 Environmental exposures, including immunomodulators such as helminths, may contribute to this variability yet their effects remain incompletely understood. 4 , 5 Using harmonised protocols from the POPulation differences in VACcine responses (POPVAC) programme in Uganda 6 , 7 , we previously reported rural-urban differences in Salmonella enterica serovar Typhi O-lipopolysaccharide (O:LPS)-specific plasma IgG responses following oral typhoid (Ty21a) vaccination, 3 and inverse associations with Schistosoma mansoni ( Sm ) infection. 8 Given the importance of mucosal immunity in protection against S. Typhi , 9 we extended these findings by evaluating systemic and mucosal antibody responses , focusing on S. Typhi O:LPS-specific plasma IgA and copro - IgA and copro-IgG , across contrasting POPVAC settings: Sm -endemic rural islands of Lake Victoria 10 , 11 and urban mainland communities with lower helminth exposure . 12 , 13 We used causal mediation analysis to assess whether Sm infection contributed to observed rural-urban differences in these responses. Fig. 1 and Supplementary Fig. S1a show the study schedule, with additional details provided in the flowchart ( Supplementary Fig. S1b ) and methods section. Of the 778 participants enrolled across the two POPVAC settings, 718 (92%) were seen at the Ty21a vaccination timepoint. Of these, 699 (97%) received at least one Ty21a dose. Baseline (pre-Ty21a vaccination) data were available for 697 participants ( 403 rural – POPVAC A; 294 urban – POPVAC C) for at least one of the following measures: S. Typhi O:LPS-specific plasma IgA, copro-IgA, copro-IgG, or total copro-IgA ( Supplementary Fig. S1b and Table 1 ). Sex distribution was comparable between settings (57% vs 60% male; p=0.560), but rural participants were younger than urban participants (median age 11 vs 15 years; p <0.001). Sm exposure was markedly higher in rural participants, with 71% testing positive by combined plasma circulating anodic antigen (CAA) and/or stool PCR compared with 43% among urban participants ( p 92% for each of the three doses), but lower among rural participants for doses 2 and 3 ( p <0.001; p =0.009, respectively). Characteristics Rural (POPVAC A) Urban (POPVAC C) n/N (%) n/N (%) p value Socio-demographic Age in years, median (range) 11 (9-17) 15 (13-17) <0.001 Male sex 231/403 (57) 175/294 (60) 0.560 Baseline S. mansoni infection Plasma CAA≥30pg/ml 218/402 (54) 58/294 (20) <0.001 PCR positive 237/400 (59) 106/291 (36) <0.001 PCR positive and/or CAA≥30pg/ml 286/403 (71) 126/294 (43) <0.001 Baseline STH infections Necator americanus , PCR positive 87/400 (22) 3/291 (1) <0.001 Strongyloides stercoralis , PCR positive 31/400 (8) 2/291 (1) <0.001 Oral typhoid (Ty21a) vaccination Received Ty21a dose 1 403/403 (100) 292/294 (99) 0.097 Received Ty21a dose 2 367/398 (92) 291/294 (99) <0.001 Received Ty21a dose 3 316/331 (95) 289/292 (99) 0.009 Table 1. Baseline characteristics of study participants. Data are from POPVAC A (rural, N = 403) and POPVAC C (urban, N = 294) participants who received oral typhoid vaccination and who had baseline data available for at least one of the following measures: total copro-IgA, Salmonella Typhi O:LPS-specific copro-IgG, S . Typhi O:LPS-specific plasma IgA, and S . Typhi O:LPS-specific copro-IgA. Denominators vary due to missing data and are therefore lower than 403 (rural) or 294 (urban) for some variables. P values for comparisons between rural and urban participants were obtained using chi-squared (or Fisher’s exact) tests for categorical variables and Wilcoxon rank-sum tests for continuous variables. Abbreviations: PCR , Polymerase Chain Reaction; CAA , Circulating Anodic Antigen, STH , Soil-transmitted Helminths There was no effect of POPVAC trial interventions (rural: intensive versus standard praziquantel treatment; 10 urban: BCG versus no BCG revaccination 12 ) on antibody concentrations ( Supplementary Table S1 ); therefore, data from both trial arms within each setting were combined for subsequent analyses. Analyses of post-vaccination antibody responses were restricted to participants who received at least one Ty21a dose. Four weeks after Ty21a vaccination, systemic and mucosal S. Typhi O:LPS -specific antibody concentrations were substantially higher in urban than rural participants ( Fig. 2 , Supplementary Table S2 ): after adjustment for age, sex, number of Ty21a doses received and corresponding baseline antibody levels, urban residence was associated with higher S . Typhi O:LPS-specific plasma IgA (geometric mean ratio [95% confidence interval]: 1.44 [1.12-1.84], p =0.004), copro-IgA (1.71 [1.09-2.69], p =0.020)and copro-IgG responses (1.26 [1.05-1.51], p =0.011).By contrast, total copro-IgA levels did not differ significantly by setting, indicating that rural-urban differences reflected antigen-specific vaccine responses rather than global alterations in mucosal IgA production. This likely reflects the composite nature of total copro-IgA, dominated by steady-state mucosal antibody production and continuous environmental antigen exposure. In contrast, antigen-specific vaccine responses require coordinated antigen processing and T cell-dependent class switching and this might account for greater sensitivity to environmental immunomodulation. 14 , 15 Consistent with mucosal immunobiology, copro-IgA concentrations were generally higher than copro-IgG, although this observation is interpreted qualitatively owing to assay differences. Given the marked disparity in Sm exposure between the rural and urban settings, we next assessed associations between baseline Sm infection and Ty21a-induced responses using pooled analyses across all participants. In models adjusted for age, sex, geographical setting, number of Ty21a doses, and pre-vaccination plasma IgA responses, no significant associations were observed between baseline Sm infection and Ty21a-induced responses ( Fig. 3a ; Supplementary Table S3a ). However, interaction tests indicated effect modification by setting for post-vaccination S. Typhi O:LPS-specific plasma IgA (interaction p =0.015) and weaker evidence for copro-IgA (interaction p =0.082), suggesting that the immunological impact of Sm infection on Ty21a responses differed between rural and urban environments ( Supplementary Table S3b ). Stratified analyses supported this interpretation. Among rural participants, Sm infection was associated with reduced post-vaccination S. Typhi O:LPS-specific plasma IgA (aGMR [95% CI]: 0.79 [0.65-0.97], p =0.021; Fig. 3b , Supplementary Table S3b ). This finding is consistent with our previous observations of inverse associations between baseline Sm infection and Ty21a-induced plasma IgG in the same rural participants, 8 and with other reports of helminth-associated attenuation of vaccine-specific systemic antibody responses following parenteral immunisation. 5 , 16 However, Sm infection was not associated with post-vaccination copro-IgA responses. In contrast, among urban participants, baseline Sm infection was associated with reduced post-vaccination S. Typhi O:LPS-specific copro-IgA (0.64 [0.44-0.93], p =0.018), but showed no effect on plasma IgA responses ( Fig. 3c , Supplementary Table S3b ). These findings indicate that the immunomodulatory effects of Sm infection on oral vaccine-induced immunity are not uniform but are likely contingent on the broader immuno-epidemiological context. In rural settings, sustained exposure to multiple enteric pathogens and chronic immune stimulation may result in a more highly conditioned mucosal immune environment , reducing the marginal impact of helminth infection on vaccine-induced responses. 17-19 High cumulative schistosome exposure in our rural setting may also obscure true inverse associations with Ty21a-induced copro-antibody responses, particularly given evidence that Sm infection is associated with increased Salmonella intestinal carriage and altered gut ecology. 20 , 21 Notably, despite this background, we still observed inverse associations between Sm infection and plasma Ty21a-induced antibody in rural participants, suggesting that helminth-associated immunomodulation is better detected in the systemic compartment. This may reflect the intravascular lifecycle of Sm , with circulating egg antigens driving systemic regulatory immune responses, whereas mucosal effects may be less readily resolved against a background of high local immune activation. In contrast, among urban participants, who experience lower cumulative exposure to helminths and enteric infections, Sm infection may exert a more pronounced immunological effect at the intestinal mucosa, resulting in a detectable suppression of antigen-specific mucosal IgA responses following oral vaccination. This interpretation is supported by evidence from our recent work in rural Uganda demonstrating that Sm infection is associated with gut inflammation, microbial translocation, and impaired vaccine responses. 22 Previous studies have shown that Sm infection can alter the intestinal microbial community, and mucosal immune function, 23-25 suggesting potential pathways for helminth-mediated modulation of oral vaccine responses. Building on these findings, future studies could evaluate interventions that modulate the gut immune environment such as dietary strategies, microbiome-directed approaches, or mucosal adjuvants to enhance oral vaccine immunogenicity in helminth-endemic populations. To assess whether Sm infection may have a causal role in rural-urban differences in Ty21a vaccine responses, we performed causal mediation analyses ( Table 2 ). We observed that rural-urban differences in Ty21a-induced S. Typhi O:LPS-specific plasma IgA and copro-IgG responses were largely driven by direct effects of setting, with little evidence of a meaningful contribution from Sm infection. However, for S. Typhi O:LPS-specific copro-IgA, mediation analysis showed that lower post-vaccination responses in the rural setting were partially mediated by Sm infection: w hile the natural direct effect did not reach statistical significance (GMR [95%CI]: 1.51 [0.91-2.51], p =0.109), the natural indirect effect via Sm infection was significant (1.15 [1.02-1.30], p =0.024), corresponding to an estimated 25.52% (95%CI: -6.23–57.26) of the total effect mediated. These findings suggest that while rural-urban differences in systemic Ty21a responses are predominantly driven by factors other than Sm infection, helminth-associated effects contribute specifically to variation in antigen-specific mucosal IgA responses. Although Sm infection was significantly associated with reduced plasma IgA in rural participants ( Supplementary Table S3 ), a role for Sm in explaining the observed rural-urban difference was not supported by mediation analyses, which indicated no detectable indirect effect. The finding that a significant mediation effect was observed only for Ty21a-induced copro-IgA is biologically plausible. Copro-IgA represents a proximal measure of mucosal immune function at the gut-luminal interface, where helminth-associated inflammation, epithelial disruption, and microbial translocation are most pronounced. Given that Ty21a is a live attenuated oral vaccine whose immunogenicity relies on effective mucosal antigen processing and IgA class switching, helminth-driven changes of the intestinal immune environment are most likely to manifest as alterations in antigen-specific mucosal IgA responses. Additional environmental exposures, including variation in nutrition, sanitation, enteric infections (including soil-transmitted helminths), microbial diversity, and differences in drinking water quality, are likely to shape mucosal immune conditioning beyond the effects of Sm infection alone. Finally, the absence of differences in post-vaccination responses to Ty21a between intensive and standard praziquantel treatment arms suggests that while baseline Sm infection contributes to heterogeneity in oral vaccine responses as shown in our mediation analysis, praziquantel treatment alone may be insufficient to reverse established helminth-associated immunomodulation in this high-exposure rural setting, and the treatment–vaccination interval may have been inadequate for such reversal. This likely shows the influence of cumulative environmental exposures, including enteric infections, microbial burden and nutritional factors, that are not addressed by anti-helminthic treatment in isolation. A potential study limitation is pooling of trial arms; although no overall intervention effects were observed, residual or subgroup-specific effects, including sex-specific responses to intensive praziquantel treatment, 10 cannot be excluded. We also did not formally assess the contribution of soil-transmitted helminths, because they were very rare in the urban setting (Table 1). Furthermore, we did not assess functional antibody activity; therefore, it is unclear whether rural-urban differences in concentrations recapitulate differences in protection. Future studies should incorporate functional and cellular and mechanistic immune profiling. Together, these data demonstrate pronounced rural-urban differences in immune responses to oral typhoid vaccination, characterised by stronger systemic and mucosal S. Typhi -specific antibody responses in urban participants. Our causal mediation analyses suggest that while rural-urban differences in systemic Ty21a responses are predominantly driven by factors other than Sm infection, the helminth may contribute to differences in antigen-specific mucosal IgA responses. These findings, based on antibody concentrations and mediation analyses, do not establish causality but highlight environmental exposures as key determinants of heterogeneity in oral vaccine responses. Responses, 4 weeks post-vaccination Total Effect GMR (95%CI) Total Effect p- value Natural Direct Effect GMR (95%CI) Natural direct Effect p-value Natural Indirect Effect GMR (95%CI) Natural Indirect Effect p-value Proportion mediated % (95%CI) S. Typhi O:LPS-specific plasma IgA 1.43 (1.15, 1.76) 0.001 1.48 (1.17, 1.87) 0.001 0.96 (0.90, 1.04) 0.317 -10.35% (-30.80, 10.09) S. Typhi O:LPS-specific copro-IgA 1.74 (1.07, 2.85) 0.026 1.51 (0.91, 2.51) 0.109 1.15 (1.02, 1.30) 0.024 25.52% (-6.23, 57.26) S. Typhi O:LPS-specific copro-IgG 1.26 (1.03, 1.55) 0.027 1.26 (1.02, 1.55) 0.029 1.00 (0.95, 1.06) 0.955 0.66% (-22.21, 23.53) Table 2: Estimates comparing schistosomiasis-endemic rural to urban mediated by S. mansoni infection The table shows effects of the urban-rural environment on Ty21a responses. Natural indirect effect is the effect of the urban-rural environment mediated though S. mansoni infection whereas the natural direct effect is the effect through other pathways. The total effect is the sum of the natural direct and indirect effects. All estimates are adjusted for age, sex, number of Ty21a doses received, and corresponding baseline Ty21a responses. GMR: geometric mean ratio, 95%CI: 95% confidence interval Methods Ethics statement Written informed assent was obtained from all participants and written informed consent from their respective parents or guardians. This included consent for future use of samples for additional related research. Ethics approval was provided by the Uganda Virus Research Institute Research Ethics Committee (references: GC/127/18/09/680, GC/127/19/05/664, GC/127/18/09/682), the London School of Hygiene and Tropical Medicine Observational/Interventions Research Ethics Committee (reference: 16032, 16034), the Uganda National Council for Science and Technology (references: HS2486, HS2491), and the Uganda National Drug Authority (reference: CTA0093, CTA0094). Parent study design and population This analysis uses data and samples from the POPulation differences in VACcine responses (POPVAC) programme, a set of randomised controlled trials 3 , 26 among schoolchildren aged 9–17 years in rural 10 and urban 12 Uganda. The rural trial considered here (POPVAC A; July 2019–September 2020; ISRCTN60517191) was conducted in Schistosoma mansoni- endemic fishing communities on the Koome islands of Lake Victoria. The full trial protocol 27 and primary results 10 have been published. In brief, POPVAC A compared intensive versus standard praziquantel treatment for Sm and its impact on vaccine-specific immune responses. Children in the intensive arm received three praziquantel doses (40 mg/kg) before the first vaccination (BCG) followed by quarterly treatment for one year; the standard arm received a single praziquantel dose at the primary endpoint (8 weeks post-BCG), consistent with national school-based deworming guidelines. The POPVAC programme also included a second rural trial (POPVAC B) evaluating intermittent dihydroartemisinin–piperaquine (DP) versus placebo on the same set of vaccine responses. 28 , 29 As the present investigation did not use POPVAC B samples, all references to the “rural setting” relate exclusively to POPVAC A. The urban trial (POPVAC C; August 2020–August 2021; ISRCTN10482904) 12 , 30 was undertaken in Entebbe municipality, an area with lower helminth and malaria prevalence. POPVAC C evaluated the effect of BCG revaccination compared with no revaccination on immune responses to subsequently administered unrelated vaccines among participants of the Entebbe Mother and Baby Study (EMaBS) cohort. 31 POPVAC C served as the urban comparator for both the wider POPVAC programme 3 and for this investigation. Except for the respective trial interventions (praziquantel in POPVAC A and BCG revaccination in POPVAC C), study procedures were harmonised across sites. In both settings, participants received BCG vaccine (Serum Institute of India) at week 0; yellow fever (YF-17D, Sanofi Pasteur, France), oral typhoid (Ty21a, Vivotif, PaxVax, UK; one capsule per day taken on 3 alternate days) and HPV (Gardasil, Merck & Co, USA) at week 4. Current study design and population The present investigation used samples from randomly selected POPVAC A (rural) and POPVAC C (urban) participants and focuses on the Ty21a vaccine. Vaccine immunogenicity ( S . Typhi O:LPS-specific plasma IgA; S . Typhi O:LPS-specific copro-IgG and IgA; procedures detailed below) was assessed at four weeks post-Ty21a vaccination (week 8 of the trials). Total copro-IgA was also measured. Baseline responses were measured at week 0 in the urban trial. In the rural trial, baseline S . Typhi O:LPS-specific plasma antibodies were measured at week 0 and baseline total copro-IgA and S . Typhi O:LPS-specific copro-antibodies at week -6 ( Supplementary Fig. S1 ), reflecting pre-vaccination sample availability within the parent trial design. Schistosoma mansoni infection status was assessed retrospectively at baseline, prior to any intervention (week -6 in the rural setting and week 0 in the urban setting), using previously published methods. 8 , 10 Infection was defined by the presence of detectable plasma circulating anodic antigen (CAA), measured using the up-converting phosphor lateral flow SCAA20 assay with a positivity threshold of 30 pg/ml, 32 and/or detection of S. mansoni DNA in stool by PCR, adapted from established protocols. 33 Infection with other Schistosoma species has not been documented in our study areas. 34 The stool PCR assay also included primers and probes for Necator americanus and Strongyloides stercoralis DNA detection, as previously reported. 10 ELISA measurement of Salmonella Typhi O-lipopolysaccharide (O:LPS)-specific copro-IgG and IgA Salmonella Typhi O:LPS-specific copro-IgG and IgA were quantified using in-house ELISAs. Firstly, stool samples (0.125g) were suspended in 1 ml of 1X phosphate buffered saline (PBS), vortexed for 1 minute and centrifuged at 3000g for 10 minutes. Supernatants were stored at -80°C until analysis. On the first day of the ELISA assay, Immulon 4HX 96-well plates (Thermo Fisher) were coated overnight at 4 °C with 50 µL/well of S. Typhi O:LPS (Sigma L2387) diluted in bicarbonate buffer (0.1 M Na 2 CO 3 /NaHCO 3 , pH 9.6), excluding the first two columns. Plates were coated at 15 µg/ml for IgA assays and 5 µg/ml for IgG assays. Two-fold serial dilutions of IgA or IgG standards, prepared in coating buffer, were added to the first two columns to generate standard curves. Human colostrum IgA (Sigma I2636; top concentration 500 ng/ml) and human serum IgG (Sigma I2511; top concentration 2000 ng/ml) were used as standards. After coating, plates were washed with 0.05% Tween-20 in PBS (PBST) and blocked for 1 hour at room temperature with 5% skimmed milk powder in PBST. Plates were then washed, and 50 µL of stool supernatants were added and incubated for 2 hours at room temperature. For the standard curve wells (first two columns), 50 µL of 1X PBS was added in place of stool supernatant. Following washing, plates were incubated for 1 hour with 50 µL/well of either goat anti-human IgA-HRP (Bio-Rad, STAR141P; 1:6000 dilution) or polyclonal rabbit anti-human IgG-HRP (Agilent Dako; 1:3000 dilution), diluted in assay buffer (1% skimmed milk powder in PBST). After a final wash, plates were developed in the dark with 100 µL/well of 3,3′,5,5′-tetramethylbenzidine (TMB; Sigma-Aldrich, T0440) for IgA assays or o-phenylenediamine (OPD; Sigma-Aldrich, P9187) for IgG assays. Colour development was stopped with 30 µL/well of 2 M sulphuric acid after 12 minutes (IgA) or 17 minutes (IgG). Optical density was read at 450 nm for IgA assays and 490 nm for IgG assays (reference wavelength 630 nm) using a BioTek ELx808 plate reader. Copro-antibody concentrations (ng/ml) were interpolated from standard curves using Gen5 software (BioTek Instruments Inc., USA) with a five-parameter logistic curve fit. ELISA measurement of total copro-IgA Stool samples were processed as described above. Immulon 4HX 96-well plates (Thermo Fisher) were coated overnight at 4°C with 50 µl/well of purified goat anti-human immunoglobulins (polyvalent; 1 µg/mL; Invitrogen) diluted in carbonate/bicarbonate coating buffer (0.1 M Na 2 CO 3 /NaHCO 3 , pH 9.6). Plates were washed with 1X phosphate-buffered saline containing 0.05% Tween-20 (PBST) and blocked for 2 hours at room temperature with 5% skimmed milk in PBST. After washing, 50 µl/well of standards or samples were added. Standards (human colostrum IgA; Sigma I2636) were prepared in assay buffer (1% skimmed milk in PBST), starting at 500 ng/mL with two-fold serial dilutions, and added to columns 1–2. Stool supernatants were added to columns 3–12, and plates were incubated for 2 hours at room temperature. Plates were then washed and incubated for 1 hour at room temperature with 50 µl/well of goat anti-human IgA conjugated to horseradish peroxidase (HRP; Bio-Rad, STAR141P) diluted 1:6000 in assay buffer. After additional washes, plates were developed in the dark with 100 µl/well of o-phenylenediamine substrate (Sigma-Aldrich), and colour development was stopped by addition of 30 µl/well of 2 M sulphuric acid. Optical density was read at 490 nm with a reference wavelength of 630 nm using a BioTek ELx808 microplate reader. Total copro-IgA concentrations (ng/mL) were interpolated from standard curves using Gen5 data collection and analysis software (BioTek Instruments Inc., USA) with a five-parameter logistic curve fit. ELISA measurement of plasma Salmonella Typhi O-lipopolysaccharide (O:LPS)-specific IgA Plasma IgA specific to Salmonella Typhi O-lipopolysaccharide (O:LPS) was also quantified using an in-house ELISA. Nunc Maxisorp 96-well plates (Thermo Fisher) were coated overnight at 4°C with 50 µl/well of S. Typhi O:LPS (Sigma L2387) diluted in bicarbonate buffer (0.1 M Na 2 CO 3 /NaHCO 3 , pH 9.6). Plates were coated at 5 µg/ml. Plates were washed with 0.05% Tween-20 in 1X phosphate-buffered saline (PBST) and blocked for 1 hour at room temperature with 5% skimmed milk powder in PBST. After washing, plates were incubated for 2 hours at room temperature with 50 µl/well of diluted plasma samples and two-fold serial dilutions of standard sera. Plasma samples were diluted 1:83.3 in assay buffer (1% skimmed milk in PBST). Standards were derived from pooled sera with known O:LPS-specific antibody titres (top concentration 20 ELISA units [EU]/ml), provided by the Oxford Vaccine Centre Biobank. These sera were obtained from individuals with high O-antigen responses following S. Typhi challenge in a controlled human infection study. 35 Following sample incubation, plates were washed and antibody binding detected by incubation for 1 hour at room temperature with goat anti-human IgG-HRP (Insight Biotechnology, UK) diluted 1:6000 in assay buffer. Plates were washed and developed with 100 µl/well of o-phenylenediamine (OPD; Sigma-Aldrich). Colour development was stopped with 30 µl/well of 2M sulphuric acid after 10 minutes. Optical density was measured at 490 nm with a reference wavelength of 630 nm using a BioTek ELx808 plate reader. O:LPS-specific plasma IgA titres were expressed as nominal ELISA units (EU/mL) and interpolated from standard curves using Gen5 data collection and analysis software (BioTek Instruments Inc., USA) with a five-parameter logistic curve fit. Statistical analysis Statistical analyses were conducted in Stata v19.5 (College Station, Texas, USA), with data visualisation done in GraphPad Prism v10.0.0 (GraphPad Software, San Diego, CA, USA). All analyses were conducted only in study participants that were vaccinated with the oral typhoid (Ty21a) vaccine, excluding those who received none of the three Ty21a vaccine doses. Baseline characteristics of study participants were summarised by geographical setting. Schistosoma mansoni prevalence and intensity at baseline (week -6, pre-Ty21a vaccination), Ty21a uptake and sociodemographic characteristics were compared between rural (POPVAC A) and urban (POPVAC C) settings using chi-squared (or Fisher’s exact) tests (categorical variables) and Wilcoxon rank sum tests (continuous variables). Analysis of total copro-IgA and Salmonella Typhi O:LPS-specific plasma and copro-antibody concentrations revealed skewed distributions; therefore, log 10 transformations (concentration + 1) were applied to the data. The primary analysis outcomes were Salmonella Typhi O:LPS-specific plasma and copro-antibody concentrations, and total copro-IgA, measured 4 weeks post-Ty21a vaccination, at the week 8 timepoint of the POPVAC trials. For rural-urban comparisons of log 10 transformed plasma and copro-antibody concentrations, linear regression analyses were conducted. Regression coefficients were back transformed to obtain geometric mean ratios (GMRs) and 95% confidence intervals (95%CIs). Both crude (unadjusted) and adjusted GMRs and 95%CIs are presented. Adjusted analyses controlled for age, sex, number of Ty21a doses received, and baseline (pre-vaccination) plasma and copro-antibody concentrations. Associations between baseline Schistosoma mansoni infection and post-vaccination plasma and copro-antibody concentrations were also assessed using linear regression. In the primary model, we adjusted for age, sex, number of Ty21a doses received, geographical setting, and baseline (pre-vaccination) plasma and copro-antibody concentrations. In the second model, we included an interaction term between S. mansoni infection and geographical setting, and used this model to obtain stratum (setting)-specific estimates. To evaluate whether Schistosoma mansoni infection mediated rural-urban differences in Ty21a-induced immune responses, we performed causal mediation analyses within the counterfactual framework using the Stata “mediate” command. Geographical setting (rural vs urban) was the exposure, baseline S. mansoni infection (CAA and/or stool PCR positivity) the mediator, and post-vaccination antigen-specific plasma and copro-antibody concentrations the continuous outcomes. Analyses were adjusted for age, sex, number of Ty21a doses received and baseline antibody concentrations, and natural direct, indirect, and total effects were estimated, with the proportion mediated reported when the effects were in the same direction. This approach assumes no uncontrolled confounding of the exposure-outcome, mediator-outcome, or exposure-mediator relationships. Participants with missing vaccine or mediator data were excluded. The effects on post-vaccination plasma and copro-antibody concentrations of intensive versus standard praziquantel treatment (rural trial) and of BCG versus no BCG revaccination (urban trial) were also assessed using linear regression. These analyses did not adjust for covariates, as POPVAC trials were randomised. However, we controlled for corresponding baseline vaccine responses to increase precision of analyses. A 5% significance level was used for all analyses. Data availability The de-identified individual participant data that underlie the results reported in this article are stored in a non-publicly available repository (LSHTM Data Compass), together with a data dictionary. Data are available on request via https://doi.org/10.17037/DATA.00005144. Researchers who would like to access the data may submit a request through LSHTM Data Compass, detailing the data requested, the intended use for the data, and evidence of relevant experience and other information to support the request. The request will be reviewed by the corresponding authors in consultation with the MRC/UVRI and LSHTM data management committee, with oversight from the UVRI and LSHTM ethics committees. In line with the MRC policy on Data Sharing, there will have to be a good reason for turning down a request. Patient Information Sheets and consent forms specifically referenced making anonymised data available and this has been approved by the relevant ethics committees. Researchers given access to the data will sign data sharing agreements which will restrict the use to answering pre-specified research questions. Declarations Acknowledgements We thank study participants, their parents and communities for participation, and the Koome sub-county local council, Mukono District authorities, Entebbe Hospital, and Entebbe Municipal Council authorities for supporting the POPVAC trial work. We thank Elizabeth Jones, Andrew Pollard, the Oxford Vaccine Centre Biobank and their study participants for kindly providing control sera for the S . Typhi O:LPS-specific antibody assays; Claudia de Dood, Govert van Dam and Paul Corstjens (Leiden University Medical Center) for production of reagents and support in undertaking the CAA assays. The parent POPVAC programme of work was supported primarily by the Medical Research Council (MRC) of the United Kingdom (grant # MR/R02118X/1 and MC_PC_21034). This award was jointly funded by the UK Medical Research Council (MRC) and the UK Foreign Commonwealth and Development Office (FCDO) under the MRC/FCDO Concordat agreement and is also part of the EDCTP2 programme supported by the European Union. GN received support from the EDCTP2 programme supported by the European Union (grant # TMA2019PF-2707) and from the Wellcome Trust (grant # 224263/Z/21/Z). AN, ELW and AME received support from the UK National Institute of Health and Care Research (NIHR) under its "NIHR Global Health Research Group on Vaccines for vulnerable people in Africa (VAnguard)” [grant # NIHR134531] using UK aid from the UK Government to support global health research. JN was also supported by the Makerere University – Uganda Virus Research Institute Centre of Excellence for Infection and Immunity Research and Training (MUII-plus), funded under the DELTAS Africa Initiative (grant # DEL-15-004). The DELTAS Africa Initiative was an independent funding scheme of the African Academy of Sciences, Alliance for Accelerating Excellence in Science in Africa and supported by the New Partnership for Africa’s Development Planning and Coordinating Agency with funding from the Wellcome Trust and the UK Government. ELW also received funding through the International Statistics and Epidemiology Group (ISEG) from the UK MRC grant # MR/R010161/1. BW was supported by GCRF collaborative Grant (R120442) from the Royal Society awarded to AME. The work was conducted at the MRC/UVRI and LSHTM Uganda Research Unit; both ISEG and the MRC/UVRI and LSHTM Uganda Research Unit are jointly funded by the UK MRC part of UK Research and Innovation and the UK Foreign, Commonwealth and Development Office (FCDO) under the MRC/FCDO Concordat agreement and are also part of the EDCTP2 programme supported by the European Union. The views expressed in this publication are those of the authors and not necessarily those of the funders or the UK government. The funders had no role in study design, data collection, data analysis, data interpretation, writing of the report, or in the decision to submit this article for publication. Author contributions G.N. and A.M.E. conceptualised the study; R.A., V.H., C.B. and J.N. conducted the laboratory work under supervision of G.N.; B.W., A.N., R.A., V.H., J.N., G.N. and E.W. curated the data; B.W., A.N. and G.N. undertook the formal analyses; B.W. and G.N. wrote the original draft, and all authors reviewed and edited the manuscript. Competing interests G.N. and A.M.E. report grants from Wellcome Trust. G.N. reports funding from the EDCTP2 programme supported by the European Union. A.M.E. reports funding from Medical Research Council (MRC) of the United Kingdom for conduct of the parent study; A.M.E. reports funding from NIH, Science for Africa Foundation and DELTAS Africa, outside the submitted work. B.W. and A.M.E. report funding from the Royal Society outside the submitted work. A.M.E., B.W. and A.N. report support from UK National Institute of Health and care Research (NIHR). A.M.E. further reports support from the Serum Institute of India, Uganda National Expanded Programme on Immunisation, and Emergent BioSolutions for conduct of the parent study. All other authors declare no competing interests. References Jiang, V., Jiang, B., Tate, J., Parashar, U. D. & Patel, M. M. Performance of rotavirus vaccines in developed and developing countries. Human vaccines 6 , 532-542 (2010). Hallander, H. et al. Calibrated serological techniques demonstrate significant different serum response rates to an oral killed cholera vaccine between Swedish and Nicaraguan children. Vaccine 21 , 138-145 (2002). Natukunda, A. et al. Schistosome and malaria exposure and urban-rural differences in vaccine responses in Uganda: a causal mediation analysis using data from three linked randomised controlled trials. The Lancet Global Health 12 , e1860-e1870 (2024). https://doi.org/10.1016/S2214-109X(24)00340-1 Clark, C. E. et al. Maternal helminth infection is associated with higher infant immunoglobulin A titers to antigen in orally administered vaccines. The Journal of Infectious Diseases 213 , 1996-2004 (2016). Natukunda, A. et al. The effect of helminth infection on vaccine responses in humans and animal models: A systematic review and meta-analysis. Parasite Immunol 44 , e12939 (2022). https://doi.org/10.1111/pim.12939 Nkurunungi, G. et al. Effect of intensive treatment for schistosomiasis on immune responses to vaccines among rural Ugandan island adolescents: randomised controlled trial protocol A for the ‘POPulation differences in VACcine responses’(POPVAC) programme. BMJ open 11 , e040426 (2021). Zirimenya, L. et al. Impact of BCG revaccination on the response to unrelated vaccines in a Ugandan adolescent birth cohort: randomised controlled trial protocol C for the ‘POPulation differences in VACcine responses’(POPVAC) programme. BMJ open 11 , e040430 (2020). Natukunda, A. et al. Pre-vaccination Schistosoma mansoni and hookworm infections are associated with altered vaccine immune responses: a longitudinal analysis among adolescents living in helminth-endemic islands of Lake Victoria, Uganda. Front Immunol 15 , 1460183 (2024). https://doi.org/10.3389/fimmu.2024.1460183 Dahora, L. C. et al. IgA and IgG1 Specific to Vi Polysaccharide of Salmonella Typhi Correlate With Protection Status in a Typhoid Fever Controlled Human Infection Model. Front Immunol 10 , 2582 (2019). https://doi.org/10.3389/fimmu.2019.02582 Nkurunungi, G. et al. The effect of intensive praziquantel administration on vaccine-specific responses among schoolchildren in Ugandan schistosomiasis-endemic islands (POPVAC A): an open-label, randomised controlled trial. The Lancet Global Health 12 , e1826-e1837 (2024). https://doi.org/10.1016/S2214-109X(24)00280-8 Sanya, R. E. et al. The Impact of Intensive Versus Standard Anthelminthic Treatment on Allergy-related Outcomes, Helminth Infection Intensity, and Helminth-related Morbidity in Lake Victoria Fishing Communities, Uganda: Results From the LaVIISWA Cluster-randomized Trial. Clin Infect Dis 68 , 1665-1674 (2019). https://doi.org/10.1093/cid/ciy761 Nassuuna, J. et al. The effect of BCG revaccination on the response to unrelated vaccines in urban Ugandan adolescents (POPVAC C): an open-label, randomised controlled trial. The Lancet Global Health 12 , e1849-e1859 (2024). https://doi.org/10.1016/S2214-109X(24)00282-1 Nkurunungi, G. et al. Do helminth infections underpin urban-rural differences in risk factors for allergy-related outcomes? Clin Exp Allergy 49 , 663-676 (2019). https://doi.org/10.1111/cea.13335 Lycke, N. Y. & Bemark, M. The regulation of gut mucosal IgA B-cell responses: recent developments. Mucosal Immunol 10 , 1361-1374 (2017). https://doi.org/10.1038/mi.2017.62 Weis, A. M. & Round, J. L. Microbiota-antibody interactions that regulate gut homeostasis. Cell Host Microbe 29 , 334-346 (2021). https://doi.org/10.1016/j.chom.2021.02.009 Sabin, E. A., Araujo, M. I., Carvalho, E. M. & Pearce, E. J. Impairment of tetanus toxoid-specific Thl-like immune responses in humans infected with Schistosoma mansoni. The Journal of infectious diseases 173 , 269-272 (1996). Manurung, M. D. et al. Systems analysis unravels a common rural-urban gradient in immunological profile, function, and metabolic dependencies. Science advances 11 , eadu0419 (2025). Naylor, C. et al. Environmental enteropathy, oral vaccine failure and growth faltering in infants in Bangladesh. EBioMedicine 2 , 1759-1766 (2015). Lauer, J. M. et al. Unsafe drinking water is associated with environmental enteric dysfunction and poor growth outcomes in young children in rural southwestern Uganda. The American journal of tropical medicine and hygiene 99 , 1606 (2018). Mbuyi-Kalonji, L. et al. Non-typhoidal Salmonella intestinal carriage in a Schistosoma mansoni endemic community in a rural area of the Democratic Republic of Congo. PLoS neglected tropical diseases 14 , e0007875 (2020). Melhem, R. & LoVERDE, P. T. Mechanism of interaction of Salmonella and Schistosoma species. Infection and immunity 44 , 274-281 (1984). Nassuuna, J. et al. Helminth driven gut inflammation and microbial translocation associate with altered vaccine responses in rural Uganda. npj Vaccines 10 , 56 (2025). Floudas, A. et al. Schistosoma mansoni Worm Infection Regulates the Intestinal Microbiota and Susceptibility to Colitis. Infect Immun 87 (2019). https://doi.org/10.1128/IAI.00275-19 Jenkins, T. P. et al. Schistosoma mansoni infection is associated with quantitative and qualitative modifications of the mammalian intestinal microbiota. Sci Rep 8 , 12072 (2018). https://doi.org/10.1038/s41598-018-30412-x Walusimbi, B. et al. The gut microbiome and metabolome associate with Schistosoma mansoni infection and cardiovascular disease risk in Uganda. Nature Communications (2026). https://doi.org/10.1038/s41467-026-68983-3 Nkurunungi, G. et al. Population differences in vaccine responses (POPVAC): scientific rationale and cross-cutting analyses for three linked, randomised controlled trials assessing the role, reversibility and mediators of immunomodulation by chronic infections in the tropics. BMJ Open 11 , e040425 (2021). https://doi.org/10.1136/bmjopen-2020-040425 Nkurunungi, G. et al. Effect of intensive treatment for schistosomiasis on immune responses to vaccines among rural Ugandan island adolescents: randomised controlled trial protocol A for the 'POPulation differences in VACcine responses' (POPVAC) programme. BMJ Open 11 , e040426 (2021). https://doi.org/10.1136/bmjopen-2020-040426 Zirimenya, L. et al. The effect of intermittent preventive treatment for malaria with dihydroartemisinin–piperaquine on vaccine-specific responses among schoolchildren in rural Uganda (POPVAC B): a double-blind, randomised controlled trial. The Lancet Global Health 12 , e1838-e1848 (2024). https://doi.org/https://doi.org/10.1016/S2214-109X(24)00281-X Natukunda, A. et al. Effect of intermittent preventive treatment for malaria with dihydroartemisinin-piperaquine on immune responses to vaccines among rural Ugandan adolescents: randomised controlled trial protocol B for the 'POPulation differences in VACcine responses' (POPVAC) programme. BMJ Open 11 , e040427 (2021). https://doi.org/10.1136/bmjopen-2020-040427 Zirimenya, L. et al. Impact of BCG revaccination on the response to unrelated vaccines in a Ugandan adolescent birth cohort: randomised controlled trial protocol C for the 'POPulation differences in VACcine responses' (POPVAC) programme. BMJ Open 11 , e040430 (2021). https://doi.org/10.1136/bmjopen-2020-040430 Webb, E. L. et al. Effect of single-dose anthelmintic treatment during pregnancy on an infant's response to immunisation and on susceptibility to infectious diseases in infancy: a randomised, double-blind, placebo-controlled trial. Lancet 377 , 52-62 (2011). https://doi.org/10.1016/S0140-6736(10)61457-2 Corstjens, P. L. et al. Tools for diagnosis, monitoring and screening of Schistosoma infections utilizing lateral-flow based assays and upconverting phosphor labels. Parasitology 141 , 1841-1855 (2014). https://doi.org/10.1017/s0031182014000626 Verweij, J. J. et al. Simultaneous detection and quantification of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in fecal samples using multiplex real-time PCR. Am J Trop Med Hyg 77 , 685-690 (2007). Emmanuel, I. O. & Ekkehard, D. Epidemiology, of bilharzias (schistosomiasis) in Uganda from 1902 until 2005. Afr Health Sci 8 , 239-243 (2008). Gibani, M. M. et al. Homologous and heterologous re-challenge with Salmonella typhi and Salmonella paratyphi A in a randomised controlled human infection model. PLoS Negl Trop Dis 14 , e0008783 (2020). https://doi.org/10.1371/journal.pntd.0008783 Additional Declarations Competing interest reported. G.N. and A.M.E. report grants from Wellcome Trust. G.N. reports funding from the EDCTP2 programme supported by the European Union. A.M.E. reports funding from Medical Research Council (MRC) of the United Kingdom for conduct of the parent study; A.M.E. reports funding from NIH, Science for Africa Foundation and DELTAS Africa, outside the submitted work. B.W. and A.M.E. report funding from the Royal Society outside the submitted work. A.M.E., B.W. and A.N. report support from UK National Institute of Health and care Research (NIHR). A.M.E. further reports support from the Serum Institute of India, Uganda National Expanded Programme on Immunisation, and Emergent BioSolutions for conduct of the parent study. All other authors declare no competing interests. Supplementary Files 5.SupplementaryinformationcoproCLEANCOPY.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 05 May, 2026 Reviews received at journal 24 Apr, 2026 Reviewers agreed at journal 17 Apr, 2026 Reviewers agreed at journal 17 Apr, 2026 Reviewers invited by journal 16 Apr, 2026 Submission checks completed at journal 15 Apr, 2026 First submitted to journal 08 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8832138","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":619831531,"identity":"aefc843f-a8ae-45ae-b335-c1b54bbbb42a","order_by":0,"name":"Bridgious Walusimbi","email":"data:image/png;base64,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","orcid":"","institution":"MRC/UVRI and LSHTM Uganda Research Unit","correspondingAuthor":true,"prefix":"","firstName":"Bridgious","middleName":"","lastName":"Walusimbi","suffix":""},{"id":619831532,"identity":"ccb89add-8f87-4909-8f7c-6263d7997d15","order_by":1,"name":"Agnes Natukunda","email":"","orcid":"","institution":"MRC/UVRI and LSHTM Uganda Research Unit","correspondingAuthor":false,"prefix":"","firstName":"Agnes","middleName":"","lastName":"Natukunda","suffix":""},{"id":619831533,"identity":"b6129b2e-908b-40d6-8fbf-a3cb314b0244","order_by":2,"name":"Rebecca Amongin","email":"","orcid":"","institution":"MRC/UVRI and LSHTM Uganda Research Unit","correspondingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"","lastName":"Amongin","suffix":""},{"id":619831534,"identity":"91321a46-7ee1-4ed9-970d-bcee71b82aad","order_by":3,"name":"Victoria Heyraud","email":"","orcid":"","institution":"London School of Hygiene \u0026 Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"Victoria","middleName":"","lastName":"Heyraud","suffix":""},{"id":619831535,"identity":"774218d0-5472-4438-a9f7-0c7b8c1bf139","order_by":4,"name":"Claire Baine","email":"","orcid":"","institution":"MRC/UVRI and LSHTM Uganda Research Unit","correspondingAuthor":false,"prefix":"","firstName":"Claire","middleName":"","lastName":"Baine","suffix":""},{"id":619831536,"identity":"f5f9f540-f1e2-4a24-82f3-ec2cca5ecaae","order_by":5,"name":"Jacent Nassuuna","email":"","orcid":"","institution":"MRC/UVRI and LSHTM Uganda Research Unit","correspondingAuthor":false,"prefix":"","firstName":"Jacent","middleName":"","lastName":"Nassuuna","suffix":""},{"id":619831539,"identity":"54c2133b-8a15-4825-8f55-e0bf9413aed0","order_by":6,"name":"Emily L Webb","email":"","orcid":"","institution":"London School of Hygiene \u0026 Tropical Medicine","correspondingAuthor":false,"prefix":"","firstName":"Emily","middleName":"L","lastName":"Webb","suffix":""},{"id":619831541,"identity":"12050a33-720b-489c-9047-e25520577cdc","order_by":7,"name":"Alison M Elliott","email":"","orcid":"","institution":"MRC/UVRI and LSHTM Uganda Research Unit","correspondingAuthor":false,"prefix":"","firstName":"Alison","middleName":"M","lastName":"Elliott","suffix":""},{"id":619831543,"identity":"b9710c7f-24b5-4f6d-b7f3-373dfdcb5d08","order_by":8,"name":"Gyaviira Nkurunungi","email":"","orcid":"","institution":"MRC/UVRI and LSHTM Uganda Research Unit","correspondingAuthor":false,"prefix":"","firstName":"Gyaviira","middleName":"","lastName":"Nkurunungi","suffix":""}],"badges":[],"createdAt":"2026-02-09 15:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8832138/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8832138/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106728795,"identity":"64e661fb-9ef4-4e95-92cb-5b5d2b86692c","added_by":"auto","created_at":"2026-04-12 18:45:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":149008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e\u003cstrong\u003e‡\u003c/strong\u003e\u003c/sup\u003eOne capsule per day taken on 3 alternate days (day 1, 3 and 5)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy schedule.\u003c/strong\u003e Created in BioRender: \u003ca href=\"https://BioRender.com/6pie25g\"\u003ehttps://BioRender.com/6pie25g\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u003cstrong\u003e§\u003c/strong\u003e\u003c/sup\u003eIn the rural trial, baseline \u003cem\u003eS\u003c/em\u003e. Typhi O:LPS-specific copro-antibodies and total copro-IgA were measured at week -6 (screening timepoint) and baseline \u003cem\u003eS\u003c/em\u003e. Typhi O:LPS-specific plasma antibodies were measured at week 0. Baseline (screening) for the urban trial was at week 0, as there was no week -6 timepoint. All baseline timepoints were pre-Ty21a vaccination.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8832138/v1/c225127b220e796fb3a37036.jpg"},{"id":106702455,"identity":"1e9566eb-d0f9-40b9-9885-a53bea69923a","added_by":"auto","created_at":"2026-04-12 07:33:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":127426,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRural-urban comparison of post-Ty21a vaccination levels of plasma and copro-antibody responses.\u003c/strong\u003e Linear regression was used to compare rural and urban individuals. P values are adjusted for age, sex, number of Ty21a doses received and corresponding baseline antibody responses. Horizontal lines in the plots represent medians and boxes denote interquartile ranges (IQR). Whiskers were drawn using the Tukey method (1.5 times IQR). Individual points represent outliers (\u0026gt;1.5 times IQR away from median).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8832138/v1/9e03e364bcd27fca8c1e32a7.jpg"},{"id":106702454,"identity":"6d296ff5-84d1-4b00-92f6-244b413781d6","added_by":"auto","created_at":"2026-04-12 07:33:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssociation of baseline \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSchistosoma mansoni\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection with post-vaccination levels of plasma and copro-antibody responses. \u003c/strong\u003eForest plots show geometric mean ratios (GMRs) and 95% confidence intervals (95%CIs) for associations between post-vaccination antibody concentrations and current \u003cem\u003eS. mansoni\u003c/em\u003e infection (plasma circulating anodic antigen ≥ 30 pg/ml and/or PCR-detectable\u003cem\u003e S. mansoni \u003c/em\u003eDNA). Raw antibody responses were skewed, so log\u003csub\u003e10\u003c/sub\u003e-transformed antibody data were used in our linear regression models; we back-transformed the results to obtain GMRs and 95%CIs. All GMRs and 95%CIs were adjusted for age, sex, setting, number of Ty21a doses received and corresponding baseline responses. Blue colour denotes associations where GMR and 95%CI \u0026lt;1, and black colour denotes lack of a significant association. No significant positive associations were observed.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8832138/v1/e174f36a899c1b8758a37a65.jpg"},{"id":106994209,"identity":"4b5978cd-db5d-4c03-bd22-01c273ef7300","added_by":"auto","created_at":"2026-04-15 15:06:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1793272,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8832138/v1/780f6b90-a3e3-42bd-a54d-4218098b9431.pdf"},{"id":106806331,"identity":"f65f3e55-dad1-46a8-85cc-b3d354d39bbe","added_by":"auto","created_at":"2026-04-13 15:31:57","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1560661,"visible":true,"origin":"","legend":"","description":"","filename":"5.SupplementaryinformationcoproCLEANCOPY.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8832138/v1/8e1dc3d713e61d9df20af740.pdf"}],"financialInterests":"Competing interest reported. G.N. and A.M.E. report grants from Wellcome Trust. G.N. reports funding from the EDCTP2 programme supported by the European Union. A.M.E. reports funding from Medical Research Council (MRC) of the United Kingdom for conduct of the parent study; A.M.E. reports funding from NIH, Science for Africa Foundation and DELTAS Africa, outside the submitted work. B.W. and A.M.E. report funding from the Royal Society outside the submitted work. A.M.E., B.W. and A.N. report support from UK National Institute of Health and care Research (NIHR). A.M.E. further reports support from the Serum Institute of India, Uganda National Expanded Programme on Immunisation, and Emergent BioSolutions for conduct of the parent study. All other authors declare no competing interests.","formattedTitle":"Rural-urban differences in oral typhoid vaccine responses in Uganda: contribution of Schistosoma mansoni","fulltext":[{"header":"Main Text","content":"\u003cp\u003eOral vaccines exhibit heterogeneity in immunogenicity and efficacy across populations, with impaired responses often reported in tropical low-income countries (LICs) compared with high-income countries (HICs),\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e2\u003c/sup\u003e and in rural compared to urban LIC settings.\u003csup\u003e3\u003c/sup\u003e Environmental exposures, including immunomodulators such as helminths, may contribute to this variability yet their effects remain incompletely understood.\u003csup\u003e4\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e5\u003c/sup\u003e Using harmonised protocols from the POPulation differences in VACcine responses (POPVAC) programme in Uganda\u003csup\u003e6\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e7\u003c/sup\u003e, we previously reported rural-urban differences in \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Typhi O-lipopolysaccharide (O:LPS)-specific \u003cstrong\u003eplasma IgG\u003c/strong\u003eresponses following oral typhoid (Ty21a) vaccination,\u003csup\u003e3\u003c/sup\u003e and inverse associations with \u003cem\u003eSchistosoma mansoni\u003c/em\u003e (\u003cem\u003eSm\u003c/em\u003e) infection.\u003csup\u003e8\u003c/sup\u003e Given the importance of \u003cstrong\u003emucosal immunity\u003c/strong\u003ein protection against \u003cem\u003eS. \u003c/em\u003e\u003cem\u003eTyphi\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e\u003csup\u003e9\u003c/sup\u003e we extended these findings by evaluating \u003cstrong\u003esystemic and mucosal antibody responses\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e focusing on \u003cem\u003eS. \u003c/em\u003e\u003cem\u003eTyphi\u003c/em\u003e O:LPS-specific \u003cstrong\u003eplasma IgA\u003c/strong\u003e and \u003cstrong\u003ecopro\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003eIgA and copro-IgG\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e \u003cstrong\u003eacross contrasting POPVAC settings: \u003c/strong\u003e\u003cem\u003eSm\u003c/em\u003e\u003cstrong\u003e-endemic rural islands of Lake \u003c/strong\u003e\u003cstrong\u003eVictoria\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e10\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e,\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e11\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e and urban mainland communities with lower helminth exposure\u003c/strong\u003e.\u003csup\u003e12\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e13\u003c/sup\u003e We used causal mediation analysis to assess whether \u003cem\u003eSm \u003c/em\u003einfection contributed to observed rural-urban differences in these responses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. \u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e and \u003cstrong\u003eSupplementary Fig. S1a\u003c/strong\u003e show the study schedule, with additional details provided in the flowchart (\u003cstrong\u003eSupplementary Fig. S1b\u003c/strong\u003e) and methods section. Of the 778 participants enrolled across the two POPVAC settings, 718 (92%) were seen at the Ty21a vaccination timepoint. Of these, 699 (97%) received at least one Ty21a dose. Baseline (pre-Ty21a vaccination) data were available for \u003cstrong\u003e697 participants (\u003c/strong\u003e403 rural \u0026ndash; POPVAC A; 294 urban \u0026ndash; POPVAC C) for at least one of the following measures: \u003cem\u003eS. Typhi\u003c/em\u003e O:LPS-specific plasma IgA, copro-IgA, copro-IgG, or total copro-IgA (\u003cstrong\u003eSupplementary Fig. S1b\u003c/strong\u003e and \u003cstrong\u003eTable 1\u003c/strong\u003e). Sex distribution was comparable between settings (57% vs 60% male; p=0.560), but rural participants were younger than urban participants (median age 11 vs 15 years; \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001). \u003cem\u003eSm\u003c/em\u003e exposure was markedly higher in rural participants, with 71% testing positive by combined plasma circulating anodic antigen (CAA) and/or stool PCR compared with 43% among urban participants (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001). Ty21a uptake was high (\u003cu\u003e\u0026gt;\u003c/u\u003e92% for each of the three doses), but lower among rural participants for doses 2 and 3 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001; \u003cem\u003ep\u003c/em\u003e=0.009, respectively). \u003c/p\u003e\n\u003ctable style=\"width: 4.7e+2pt\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eRural (POPVAC A)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eUrban (POPVAC C)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003en/N (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003en/N (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ep value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSocio-demographic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \n \u003c/td\u003e\n \u003ctd\u003e\n \n \u003c/td\u003e\n \u003ctd\u003e\n \n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAge in years, median (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11 (9-17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15 (13-17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMale sex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e231/403 (57)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e175/294 (60)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.560\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBaseline \u003cem\u003eS. mansoni\u003c/em\u003e infection\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \n \u003c/td\u003e\n \u003ctd\u003e\n \n \u003c/td\u003e\n \u003ctd\u003e\n \n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePlasma CAA\u0026ge;30pg/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e218/402 (54)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e58/294 (20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePCR positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e237/400 (59)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e106/291 (36)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePCR positive and/or CAA\u0026ge;30pg/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e286/403 (71)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e126/294 (43)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBaseline STH infections\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \n \u003c/td\u003e\n \u003ctd\u003e\n \n \u003c/td\u003e\n \u003ctd\u003e\n \n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003e\u003cu\u003eNecator americanus\u003c/u\u003e\u003c/em\u003e, PCR positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e87/400 (22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3/291 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eStrongyloides stercoralis\u003c/em\u003e, PCR positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e31/400 (8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2/291 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eOral typhoid (Ty21a) vaccination\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eReceived Ty21a dose 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e403/403 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e292/294 (99)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.097\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eReceived Ty21a dose 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e367/398 (92)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e291/294 (99)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eReceived Ty21a dose 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e316/331 (95)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e289/292 (99)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1. Baseline characteristics of study participants. \u003c/strong\u003eData are from POPVAC A (rural, N = 403) and POPVAC C (urban, N = 294) participants who received oral typhoid vaccination and who had baseline data available for at least one of the following measures: total copro-IgA, \u003cem\u003eSalmonella\u003c/em\u003e Typhi O:LPS-specific copro-IgG, \u003cem\u003eS\u003c/em\u003e. Typhi O:LPS-specific plasma IgA, and \u003cem\u003eS\u003c/em\u003e. Typhi O:LPS-specific copro-IgA. Denominators vary due to missing data and are therefore lower than 403 (rural) or 294 (urban) for some variables. P values for comparisons between rural and urban participants were obtained using chi-squared (or Fisher\u0026rsquo;s exact) tests for categorical variables and Wilcoxon rank-sum tests for continuous variables.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAbbreviations: PCR\u003c/strong\u003e, Polymerase Chain Reaction; \u003cstrong\u003eCAA\u003c/strong\u003e, Circulating Anodic Antigen, \u003cstrong\u003eSTH\u003c/strong\u003e, Soil-transmitted Helminths\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThere was no effect of POPVAC trial interventions (rural: intensive versus standard praziquantel treatment;\u003csup\u003e10\u003c/sup\u003e urban: BCG versus no BCG revaccination\u003csup\u003e12\u003c/sup\u003e) on antibody concentrations (\u003cstrong\u003eSupplementary Table S1\u003c/strong\u003e); therefore, data from both trial arms within each setting were combined for subsequent analyses. Analyses\u003cstrong\u003e of post-vaccination antibody responses were restricted to participants who received at least one Ty21a dose.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour weeks after Ty21a vaccination, systemic and mucosal \u003cem\u003eS. \u003c/em\u003e\u003cem\u003eTyphi O:LPS\u003c/em\u003e-specific antibody concentrations were substantially higher in urban than rural participants (\u003cstrong\u003eFig. 2\u003c/strong\u003e, \u003cstrong\u003eSupplementary Table S2\u003c/strong\u003e): after adjustment for age, sex, number of Ty21a doses received and corresponding baseline antibody levels, urban residence was associated with higher \u003cem\u003eS\u003c/em\u003e. Typhi O:LPS-specific plasma IgA (geometric mean ratio [95% confidence interval]: 1.44 [1.12-1.84], \u003cem\u003ep\u003c/em\u003e=0.004), copro-IgA (1.71 [1.09-2.69], \u003cem\u003ep\u003c/em\u003e=0.020)and copro-IgG responses (1.26 [1.05-1.51], \u003cem\u003ep\u003c/em\u003e=0.011).By contrast, total copro-IgA levels did not differ significantly by setting, indicating that rural-urban differences reflected antigen-specific vaccine responses rather than global alterations in mucosal IgA production. This likely reflects the composite nature of total copro-IgA, dominated by steady-state mucosal antibody production and continuous environmental antigen exposure. In contrast, antigen-specific vaccine responses require coordinated antigen processing and T cell-dependent class switching and this might account for greater sensitivity to environmental immunomodulation.\u003csup\u003e14\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e15\u003c/sup\u003e Consistent with mucosal immunobiology, copro-IgA concentrations were generally higher than copro-IgG, although this observation is interpreted qualitatively owing to assay differences.\u003c/p\u003e\n\u003cp\u003eGiven the marked disparity in \u003cem\u003eSm\u003c/em\u003e exposure between the rural and urban settings, we next assessed associations between baseline \u003cem\u003eSm\u003c/em\u003e infection and Ty21a-induced responses using pooled analyses across all participants. In models adjusted for age, sex, geographical setting, number of Ty21a doses, and pre-vaccination plasma IgA responses, no significant associations were observed between baseline \u003cem\u003eSm\u003c/em\u003e infection and Ty21a-induced responses (\u003cstrong\u003eFig. 3a\u003c/strong\u003e; \u003cstrong\u003eSupplementary Table S3a\u003c/strong\u003e). However, interaction tests indicated effect modification by setting for post-vaccination \u003cem\u003eS.\u003c/em\u003e\u003cem\u003e \u003c/em\u003eTyphi O:LPS-specific plasma IgA (interaction \u003cem\u003ep\u003c/em\u003e=0.015) and weaker evidence for copro-IgA (interaction \u003cem\u003ep\u003c/em\u003e=0.082), suggesting that the immunological impact of \u003cem\u003eSm\u003c/em\u003e infection on Ty21a responses differed between rural and urban environments (\u003cstrong\u003eSupplementary Table S3b\u003c/strong\u003e). \u003c/p\u003e\n\u003cp\u003eStratified analyses supported this interpretation. Among rural participants, \u003cem\u003eSm\u003c/em\u003e infection was associated with reduced post-vaccination \u003cem\u003eS.\u003c/em\u003e Typhi O:LPS-specific plasma IgA (aGMR [95% CI]: 0.79 [0.65-0.97], \u003cem\u003ep\u003c/em\u003e=0.021;\u003cstrong\u003e Fig. 3b\u003c/strong\u003e, \u003cstrong\u003eSupplementary Table S3b\u003c/strong\u003e). This finding is consistent with our previous observations of inverse associations between baseline \u003cem\u003eSm \u003c/em\u003einfection and Ty21a-induced plasma IgG in the same rural participants,\u003csup\u003e8\u003c/sup\u003e and with other reports of helminth-associated attenuation of vaccine-specific systemic antibody responses following parenteral immunisation.\u003csup\u003e5\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e16\u003c/sup\u003e However, \u003cem\u003eSm\u003c/em\u003e infection was not associated with post-vaccination copro-IgA responses. In contrast, among urban participants, baseline \u003cem\u003eSm\u003c/em\u003e infection was associated with reduced post-vaccination \u003cem\u003eS.\u003c/em\u003e Typhi O:LPS-specific copro-IgA (0.64 [0.44-0.93], \u003cem\u003ep\u003c/em\u003e=0.018), but showed no effect on plasma IgA responses (\u003cstrong\u003eFig. 3c\u003c/strong\u003e, \u003cstrong\u003eSupplementary Table S3b\u003c/strong\u003e). \u003c/p\u003e\n\u003cp\u003eThese findings indicate that the immunomodulatory effects of \u003cem\u003eSm\u003c/em\u003e infection on oral vaccine-induced immunity are not uniform but are likely contingent on the broader immuno-epidemiological context. In rural settings, sustained exposure to multiple enteric pathogens and chronic immune stimulation may result in \u003cstrong\u003ea more highly conditioned mucosal immune environment\u003c/strong\u003e, reducing the marginal impact of helminth infection on vaccine-induced responses.\u003csup\u003e17-19\u003c/sup\u003e High cumulative schistosome exposure in our rural setting may also obscure true inverse associations with Ty21a-induced copro-antibody responses, particularly given evidence that \u003cem\u003eSm \u003c/em\u003einfection is associated with increased \u003cem\u003eSalmonella\u003c/em\u003e intestinal carriage\u003cem\u003e \u003c/em\u003eand altered gut ecology.\u003csup\u003e20\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e21\u003c/sup\u003e Notably, despite this background, we still observed inverse associations between \u003cem\u003eSm \u003c/em\u003einfection and plasma Ty21a-induced antibody in rural participants, suggesting that helminth-associated immunomodulation is better detected in the systemic compartment. This may reflect the intravascular lifecycle of \u003cem\u003eSm\u003c/em\u003e, with circulating egg antigens driving systemic regulatory immune responses, whereas mucosal effects may be less readily resolved against a background of high local immune activation. In contrast, among urban participants, who experience lower cumulative exposure to helminths and enteric infections, \u003cem\u003eSm\u003c/em\u003e infection may exert a more pronounced immunological effect at the intestinal mucosa, resulting in a detectable suppression of antigen-specific mucosal IgA responses following oral vaccination. This interpretation is supported by evidence from our recent work in rural Uganda demonstrating that \u003cem\u003eSm\u003c/em\u003e infection is associated with gut inflammation, microbial translocation, and impaired vaccine responses.\u003csup\u003e22\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that \u003cem\u003eSm\u003c/em\u003e infection can alter the intestinal microbial community, and mucosal immune function,\u003csup\u003e23-25\u003c/sup\u003e suggesting potential pathways for helminth-mediated modulation of oral vaccine responses. Building on these findings, future studies could evaluate interventions that modulate the gut immune environment such as dietary strategies, microbiome-directed approaches, or mucosal adjuvants to enhance oral vaccine immunogenicity in helminth-endemic populations.\u003c/p\u003e\n\u003cp\u003eTo assess whether \u003cem\u003eSm\u003c/em\u003e infection may have a causal role in rural-urban differences in Ty21a vaccine responses, we performed causal mediation analyses (\u003cstrong\u003eTable 2\u003c/strong\u003e). We observed that rural-urban differences in Ty21a-induced \u003cem\u003eS. Typhi\u003c/em\u003e O:LPS-specific plasma IgA and copro-IgG responses were largely driven by direct effects of setting, with little evidence of a meaningful contribution from \u003cem\u003eSm\u003c/em\u003e infection. However, for \u003cem\u003eS. Typhi\u003c/em\u003e O:LPS-specific copro-IgA, mediation analysis showed that \u003cstrong\u003elower post-vaccination responses in the rural setting were partially mediated by\u003c/strong\u003e \u003cem\u003eSm\u003c/em\u003e \u003cstrong\u003einfection: w\u003c/strong\u003ehile the natural direct effect did not reach statistical significance (GMR [95%CI]: 1.51 [0.91-2.51], \u003cem\u003ep\u003c/em\u003e=0.109), the natural indirect effect via \u003cem\u003eSm\u003c/em\u003e infection was significant (1.15 [1.02-1.30], \u003cem\u003ep\u003c/em\u003e=0.024), corresponding to an estimated 25.52% (95%CI: -6.23\u0026ndash;57.26) of the total effect mediated. These findings suggest that while rural-urban differences in systemic Ty21a responses are predominantly driven by factors other than \u003cem\u003eSm\u003c/em\u003e infection, \u003cstrong\u003ehelminth-associated effects contribute specifically to variation in antigen-specific mucosal IgA responses. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough \u003cem\u003eSm\u003c/em\u003e infection was significantly associated with reduced plasma IgA in rural participants (\u003cstrong\u003eSupplementary Table S3\u003c/strong\u003e), a role for \u003cem\u003eSm \u003c/em\u003ein explaining the observed rural-urban difference was not supported by mediation analyses, which indicated no detectable indirect effect. The finding that a significant mediation effect was observed only for Ty21a-induced copro-IgA is biologically plausible. Copro-IgA represents a proximal measure of mucosal immune function at the gut-luminal interface, where helminth-associated inflammation, epithelial disruption, and microbial translocation are most pronounced. Given that Ty21a is a live attenuated oral vaccine whose immunogenicity relies on effective mucosal antigen processing and IgA class switching, helminth-driven changes of the intestinal immune environment are most likely to manifest as alterations in antigen-specific mucosal IgA responses. Additional environmental exposures, including variation in nutrition, sanitation, enteric infections (including soil-transmitted helminths), microbial diversity, and differences in drinking water quality, are likely to shape mucosal immune conditioning beyond the effects of \u003cem\u003eSm\u003c/em\u003e infection alone. \u003c/p\u003e\n\u003cp\u003eFinally, the absence of differences in post-vaccination responses to Ty21a between intensive and standard praziquantel treatment arms suggests that while baseline \u003cem\u003eSm\u003c/em\u003e infection contributes to heterogeneity in oral vaccine responses as shown in our mediation analysis, praziquantel treatment alone may be insufficient to reverse established helminth-associated immunomodulation in this high-exposure rural setting, and the treatment\u0026ndash;vaccination interval may have been inadequate for such reversal. This likely shows the influence of cumulative environmental exposures, including enteric infections, microbial burden and nutritional factors, that are not addressed by anti-helminthic treatment in isolation.\u003c/p\u003e\n\u003cp\u003eA potential study limitation is pooling of trial arms; although no overall intervention effects were observed, residual or subgroup-specific effects, including sex-specific responses to intensive praziquantel treatment,\u003csup\u003e10\u003c/sup\u003e cannot be excluded. We also did not formally assess the contribution of soil-transmitted helminths, because they were very rare in the urban setting (Table 1). Furthermore, we did not assess functional antibody activity; therefore, it is unclear whether rural-urban differences in concentrations recapitulate differences in protection. Future studies should incorporate functional and cellular and mechanistic immune profiling.\u003c/p\u003e\n\u003cp\u003eTogether, these data demonstrate pronounced rural-urban differences in immune responses to oral typhoid vaccination, characterised by stronger systemic and mucosal \u003cem\u003eS. Typhi\u003c/em\u003e-specific antibody responses in urban participants. Our causal mediation analyses suggest that while rural-urban differences in systemic Ty21a responses are predominantly driven by factors other than \u003cem\u003eSm\u003c/em\u003e infection, the helminth may contribute to differences in antigen-specific mucosal IgA responses. These findings, based on antibody concentrations and mediation analyses, do not establish causality but highlight environmental exposures as key determinants of heterogeneity in oral vaccine responses.\u003c/p\u003e\n\u003ctable style=\"width: 7.7e+2pt\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eResponses, 4 weeks post-vaccination\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTotal Effect\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGMR (95%CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTotal Effect p-\u003c/strong\u003e\u003cstrong\u003evalue \u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNatural Direct Effect\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGMR (95%CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNatural direct Effect p-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNatural Indirect Effect\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGMR (95%CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNatural Indirect Effect p-value \u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eProportion mediated\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e% (95%CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eS. Typhi \u003c/em\u003eO:LPS-specific plasma IgA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.43 (1.15, 1.76)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.48 (1.17, 1.87)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.96 (0.90, 1.04)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.317\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-10.35% (-30.80, 10.09)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eS. \u003c/em\u003eTyphi O:LPS-specific copro-IgA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.74 (1.07, 2.85)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.026\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.51 (0.91, 2.51)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.15 (1.02, 1.30)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25.52% (-6.23, 57.26)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e Typhi O:LPS-specific copro-IgG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.26 (1.03, 1.55)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.027\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.26 (1.02, 1.55)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00 (0.95, 1.06)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.955\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.66% (-22.21, 23.53)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 2: Estimates comparing schistosomiasis-endemic rural to urban mediated by \u003cem\u003eS. mansoni\u003c/em\u003e infection\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe table shows effects of the urban-rural environment on Ty21a responses. Natural indirect effect is the effect of the urban-rural environment mediated though \u003cem\u003eS. mansoni\u003c/em\u003e infection whereas the natural direct effect is the effect through other pathways. The total effect is the sum of the natural direct and indirect effects. All estimates are adjusted for age, sex, number of Ty21a doses received, and corresponding baseline Ty21a responses.\u003c/p\u003e\n \u003cp\u003eGMR: geometric mean ratio, 95%CI: 95% confidence interval\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed assent was obtained from all participants and written informed consent from their respective parents or guardians. This included consent for future use of samples for additional related research. Ethics approval was provided by the Uganda Virus Research Institute Research Ethics Committee (references: GC/127/18/09/680, GC/127/19/05/664, GC/127/18/09/682), the London School of Hygiene and Tropical Medicine Observational/Interventions Research Ethics Committee (reference: 16032, 16034), the Uganda National Council for Science and Technology (references: HS2486, HS2491), and the Uganda National Drug Authority (reference: CTA0093, CTA0094).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParent study design and population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis analysis uses data and samples from the POPulation differences in VACcine responses (POPVAC) programme, a set of randomised controlled trials\u003csup\u003e3\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e26\u003c/sup\u003e among schoolchildren aged 9\u0026ndash;17 years in rural\u003csup\u003e10\u003c/sup\u003e and urban\u003csup\u003e12\u003c/sup\u003e Uganda. \u003c/p\u003e\n\u003cp\u003eThe rural trial considered here (POPVAC A; July 2019\u0026ndash;September 2020; ISRCTN60517191) was conducted in \u003cem\u003eSchistosoma mansoni-\u003c/em\u003eendemic fishing communities on the Koome islands of Lake Victoria. The full trial protocol\u003csup\u003e27\u003c/sup\u003e and primary results\u003csup\u003e10\u003c/sup\u003e have been published. In brief, POPVAC A compared intensive versus standard praziquantel treatment for \u003cem\u003eSm\u003c/em\u003e and its impact on vaccine-specific immune responses. Children in the intensive arm received three praziquantel doses (40 mg/kg) before the first vaccination (BCG) followed by quarterly treatment for one year; the standard arm received a single praziquantel dose at the primary endpoint (8 weeks post-BCG), consistent with national school-based deworming guidelines. The POPVAC programme also included a second rural trial (POPVAC B) evaluating intermittent dihydroartemisinin\u0026ndash;piperaquine (DP) versus placebo on the same set of vaccine responses.\u003csup\u003e28\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e29\u003c/sup\u003e As the present investigation did not use POPVAC B samples, all references to the \u0026ldquo;rural setting\u0026rdquo; relate exclusively to POPVAC A.\u003c/p\u003e\n\u003cp\u003eThe urban trial (POPVAC C; August 2020\u0026ndash;August 2021; ISRCTN10482904)\u003csup\u003e12\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e30\u003c/sup\u003e was undertaken in Entebbe municipality, an area with lower helminth and malaria prevalence. POPVAC C evaluated the effect of BCG revaccination compared with no revaccination on immune responses to subsequently administered unrelated vaccines among participants of the Entebbe Mother and Baby Study (EMaBS) cohort.\u003csup\u003e31\u003c/sup\u003e POPVAC C served as the urban comparator for both the wider POPVAC programme\u003csup\u003e3\u003c/sup\u003e and for this investigation. \u003c/p\u003e\n\u003cp\u003eExcept for the respective trial interventions (praziquantel in POPVAC A and BCG revaccination in POPVAC C), study procedures were harmonised across sites. In both settings, participants received BCG vaccine (Serum Institute of India) at week 0; yellow fever (YF-17D, Sanofi Pasteur, France), oral typhoid (Ty21a, Vivotif, PaxVax, UK; one capsule per day taken on 3 alternate days) and HPV (Gardasil, Merck \u0026amp; Co, USA) at week 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCurrent study design and population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present investigation used samples from randomly selected POPVAC A (rural) and POPVAC C (urban) participants and focuses on the Ty21a vaccine. Vaccine immunogenicity (\u003cem\u003eS\u003c/em\u003e. Typhi\u003cem\u003e \u003c/em\u003eO:LPS-specific plasma IgA; \u003cem\u003eS\u003c/em\u003e. Typhi O:LPS-specific copro-IgG and IgA; procedures detailed below) was assessed at four weeks post-Ty21a vaccination (week 8 of the trials). Total copro-IgA was also measured. Baseline responses were measured at week 0 in the urban trial. In the rural trial, baseline \u003cem\u003eS\u003c/em\u003e. Typhi O:LPS-specific plasma antibodies were measured at week 0 and baseline total copro-IgA and \u003cem\u003eS\u003c/em\u003e. Typhi O:LPS-specific copro-antibodies at week -6 (\u003cstrong\u003eSupplementary Fig. S1\u003c/strong\u003e), reflecting pre-vaccination sample availability within the parent trial design.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSchistosoma mansoni \u003c/em\u003einfection status was assessed retrospectively at baseline, prior to any intervention (week -6 in the rural setting and week 0 in the urban setting), using previously published methods.\u003csup\u003e8\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e10\u003c/sup\u003e Infection was defined by the presence of detectable plasma circulating anodic antigen (CAA), measured using the up-converting phosphor lateral flow SCAA20 assay with a positivity threshold of 30 pg/ml,\u003csup\u003e32\u003c/sup\u003e and/or detection of \u003cem\u003eS. mansoni\u003c/em\u003e DNA in stool by PCR, adapted from established protocols.\u003csup\u003e33\u003c/sup\u003e Infection with other \u003cem\u003eSchistosoma \u003c/em\u003especies has not been documented in our study areas.\u003csup\u003e34\u003c/sup\u003e The stool PCR assay also included primers and probes for \u003cem\u003eNecator americanus\u003c/em\u003e and \u003cem\u003eStrongyloides stercoralis\u003c/em\u003e DNA detection, as previously reported.\u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA measurement of \u003cem\u003eSalmonella\u003c/em\u003e Typhi O-lipopolysaccharide (O:LPS)-specific copro-IgG and IgA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e Typhi O:LPS-specific copro-IgG and IgA were quantified using in-house ELISAs. Firstly, stool samples (0.125g) were suspended in 1 ml of 1X phosphate buffered saline (PBS), vortexed for 1 minute and centrifuged at 3000g for 10 minutes. Supernatants were stored at -80\u0026deg;C until analysis. On the first day of the ELISA assay, Immulon 4HX 96-well plates (Thermo Fisher) were coated overnight at 4 \u0026deg;C with 50 \u0026micro;L/well of S. Typhi O:LPS (Sigma L2387) diluted in bicarbonate buffer (0.1 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e/NaHCO\u003csub\u003e3\u003c/sub\u003e, pH 9.6), excluding the first two columns. Plates were coated at 15 \u0026micro;g/ml for IgA assays and 5 \u0026micro;g/ml for IgG assays. Two-fold serial dilutions of IgA or IgG standards, prepared in coating buffer, were added to the first two columns to generate standard curves. Human colostrum IgA (Sigma I2636; top concentration 500 ng/ml) and human serum IgG (Sigma I2511; top concentration 2000 ng/ml) were used as standards. After coating, plates were washed with 0.05% Tween-20 in PBS (PBST) and blocked for 1 hour at room temperature with 5% skimmed milk powder in PBST. Plates were then washed, and 50 \u0026micro;L of stool supernatants were added and incubated for 2 hours at room temperature. For the standard curve wells (first two columns), 50 \u0026micro;L of 1X PBS was added in place of stool supernatant. Following washing, plates were incubated for 1 hour with 50 \u0026micro;L/well of either goat anti-human IgA-HRP (Bio-Rad, STAR141P; 1:6000 dilution) or polyclonal rabbit anti-human IgG-HRP (Agilent Dako; 1:3000 dilution), diluted in assay buffer (1% skimmed milk powder in PBST). After a final wash, plates were developed in the dark with 100 \u0026micro;L/well of 3,3\u0026prime;,5,5\u0026prime;-tetramethylbenzidine (TMB; Sigma-Aldrich, T0440) for IgA assays or o-phenylenediamine (OPD; Sigma-Aldrich, P9187) for IgG assays. Colour development was stopped with 30 \u0026micro;L/well of 2 M sulphuric acid after 12 minutes (IgA) or 17 minutes (IgG). Optical density was read at 450 nm for IgA assays and 490 nm for IgG assays (reference wavelength 630 nm) using a BioTek ELx808 plate reader. Copro-antibody concentrations (ng/ml) were interpolated from standard curves using Gen5 software (BioTek Instruments Inc., USA) with a five-parameter logistic curve fit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA measurement of total copro-IgA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStool samples were processed as described above. Immulon 4HX 96-well plates (Thermo Fisher) were coated overnight at 4\u0026deg;C with 50 \u0026micro;l/well of purified goat anti-human immunoglobulins (polyvalent; 1 \u0026micro;g/mL; Invitrogen) diluted in carbonate/bicarbonate coating buffer (0.1 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e/NaHCO\u003csub\u003e3\u003c/sub\u003e, pH 9.6). Plates were washed with 1X phosphate-buffered saline containing 0.05% Tween-20 (PBST) and blocked for 2 hours at room temperature with 5% skimmed milk in PBST. After washing, 50 \u0026micro;l/well of standards or samples were added. Standards (human colostrum IgA; Sigma I2636) were prepared in assay buffer (1% skimmed milk in PBST), starting at 500 ng/mL with two-fold serial dilutions, and added to columns 1\u0026ndash;2. Stool supernatants were added to columns 3\u0026ndash;12, and plates were incubated for 2 hours at room temperature. Plates were then washed and incubated for 1 hour at room temperature with 50 \u0026micro;l/well of goat anti-human IgA conjugated to horseradish peroxidase (HRP; Bio-Rad, STAR141P) diluted 1:6000 in assay buffer. After additional washes, plates were developed in the dark with 100 \u0026micro;l/well of o-phenylenediamine substrate (Sigma-Aldrich), and colour development was stopped by addition of 30 \u0026micro;l/well of 2 M sulphuric acid. Optical density was read at 490 nm with a reference wavelength of 630 nm using a BioTek ELx808 microplate reader. Total copro-IgA concentrations (ng/mL) were interpolated from standard curves using Gen5 data collection and analysis software (BioTek Instruments Inc., USA) with a five-parameter logistic curve fit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA measurement of plasma \u003cem\u003eSalmonella\u003c/em\u003e Typhi O-lipopolysaccharide (O:LPS)-specific IgA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasma IgA specific to \u003cem\u003eSalmonella\u003c/em\u003e Typhi O-lipopolysaccharide (O:LPS) was also quantified using an in-house ELISA. Nunc Maxisorp 96-well plates (Thermo Fisher) were coated overnight at 4\u0026deg;C with 50 \u0026micro;l/well of \u003cem\u003eS.\u003c/em\u003e Typhi O:LPS (Sigma L2387) diluted in bicarbonate buffer (0.1 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e/NaHCO\u003csub\u003e3\u003c/sub\u003e, pH 9.6). Plates were coated at 5 \u0026micro;g/ml. Plates were washed with 0.05% Tween-20 in 1X phosphate-buffered saline (PBST) and blocked for 1 hour at room temperature with 5% skimmed milk powder in PBST. After washing, plates were incubated for 2 hours at room temperature with 50 \u0026micro;l/well of diluted plasma samples and two-fold serial dilutions of standard sera. Plasma samples were diluted 1:83.3 in assay buffer (1% skimmed milk in PBST). Standards were derived from pooled sera with known O:LPS-specific antibody titres (top concentration 20 ELISA units [EU]/ml), provided by the Oxford Vaccine Centre Biobank. These sera were obtained from individuals with high O-antigen responses following \u003cem\u003eS.\u003c/em\u003e Typhi challenge in a controlled human infection study.\u003csup\u003e35\u003c/sup\u003e Following sample incubation, plates were washed and antibody binding detected by incubation for 1 hour at room temperature with goat anti-human IgG-HRP (Insight Biotechnology, UK) diluted 1:6000 in assay buffer. Plates were washed and developed with 100 \u0026micro;l/well of o-phenylenediamine (OPD; Sigma-Aldrich). Colour development was stopped with 30 \u0026micro;l/well of 2M sulphuric acid after 10 minutes. Optical density was measured at 490 nm with a reference wavelength of 630 nm using a BioTek ELx808 plate reader. O:LPS-specific plasma IgA titres were expressed as nominal ELISA units (EU/mL) and interpolated from standard curves using Gen5 data collection and analysis software (BioTek Instruments Inc., USA) with a five-parameter logistic curve fit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were conducted in Stata v19.5 (College Station, Texas, USA), with data visualisation done in GraphPad Prism v10.0.0 (GraphPad Software, San Diego, CA, USA). All analyses were conducted only in study participants that were vaccinated with the oral typhoid (Ty21a) vaccine, excluding those who received none of the three Ty21a vaccine doses. Baseline characteristics of study participants were summarised by geographical setting. \u003cem\u003eSchistosoma mansoni\u003c/em\u003e prevalence and intensity at baseline (week -6, pre-Ty21a vaccination), Ty21a uptake and sociodemographic characteristics were compared between rural (POPVAC A) and urban (POPVAC C) settings using chi-squared (or Fisher\u0026rsquo;s exact) tests (categorical variables) and Wilcoxon rank sum tests (continuous variables). Analysis of total copro-IgA and \u003cem\u003eSalmonella \u003c/em\u003eTyphi O:LPS-specific plasma and copro-antibody concentrations revealed skewed distributions; therefore, log\u003csub\u003e10\u003c/sub\u003e transformations (concentration + 1) were applied to the data. The primary analysis outcomes were \u003cem\u003eSalmonella\u003c/em\u003e Typhi O:LPS-specific plasma and copro-antibody concentrations, and total copro-IgA, measured 4 weeks post-Ty21a vaccination, at the week 8 timepoint of the POPVAC trials. \u003c/p\u003e\n\u003cp\u003eFor rural-urban comparisons of log\u003csub\u003e10\u003c/sub\u003e transformed plasma and copro-antibody concentrations, linear regression analyses were conducted. Regression coefficients were back transformed to obtain geometric mean ratios (GMRs) and 95% confidence intervals (95%CIs). Both crude (unadjusted) and adjusted GMRs and 95%CIs are presented. Adjusted analyses controlled for age, sex, number of Ty21a doses received, and baseline (pre-vaccination) plasma and copro-antibody concentrations. \u003c/p\u003e\n\u003cp\u003eAssociations between baseline \u003cem\u003eSchistosoma mansoni\u003c/em\u003e infection and post-vaccination plasma and copro-antibody concentrations were also assessed using linear regression. In the primary model, we adjusted for age, sex, number of Ty21a doses received, geographical setting, and baseline (pre-vaccination) plasma and copro-antibody concentrations. In the second model, we included an interaction term between \u003cem\u003eS. mansoni\u003c/em\u003e infection and geographical setting, and used this model to obtain stratum (setting)-specific estimates.\u003c/p\u003e\n\u003cp\u003eTo evaluate whether \u003cem\u003eSchistosoma mansoni\u003c/em\u003e infection mediated rural-urban differences in Ty21a-induced immune responses, we performed causal mediation analyses within the counterfactual framework using the Stata \u0026ldquo;mediate\u0026rdquo; command. Geographical setting (rural vs urban) was the exposure, baseline \u003cem\u003eS. mansoni\u003c/em\u003e infection (CAA and/or stool PCR positivity) the mediator, and post-vaccination antigen-specific plasma and copro-antibody concentrations the continuous outcomes. Analyses were adjusted for age, sex, number of Ty21a doses received and baseline antibody concentrations, and natural direct, indirect, and total effects were estimated, with the proportion mediated reported when the effects were in the same direction. This approach assumes no uncontrolled confounding of the exposure-outcome, mediator-outcome, or exposure-mediator relationships. Participants with missing vaccine or mediator data were excluded.\u003c/p\u003e\n\u003cp\u003eThe effects on post-vaccination plasma and copro-antibody concentrations of intensive versus standard praziquantel treatment (rural trial) and of BCG versus no BCG revaccination (urban trial) were also assessed using linear regression. These analyses did not adjust for covariates, as POPVAC trials were randomised. However, we controlled for corresponding baseline vaccine responses to increase precision of analyses.\u003c/p\u003e\n\u003cp\u003eA 5% significance level was used for all analyses. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe de-identified individual participant data that underlie the results reported in this article are stored in a non-publicly available repository (LSHTM Data Compass), together with a data dictionary. Data are available on request via \u0026nbsp;https://doi.org/10.17037/DATA.00005144. Researchers who would like to access the data may submit a request through LSHTM Data Compass, detailing the data requested, the intended use for the data, and evidence of relevant experience and other information to support the request. The request will be reviewed by the corresponding authors in consultation with the MRC/UVRI and LSHTM data management committee, with oversight from the UVRI and LSHTM ethics committees. In line with the MRC policy on Data Sharing, there will have to be a good reason for turning down a request. Patient Information Sheets and consent forms specifically referenced making anonymised data available and this has been approved by the relevant ethics committees. Researchers given access to the data will sign data sharing agreements which will restrict the use to answering pre-specified research questions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank study participants, their parents and communities for participation, and the Koome sub-county local council, Mukono District authorities, Entebbe Hospital, and Entebbe Municipal Council authorities for supporting the POPVAC trial work. We thank Elizabeth Jones, Andrew Pollard, the Oxford Vaccine Centre Biobank and their study participants for kindly providing control sera for the \u003cem\u003eS\u003c/em\u003e. Typhi O:LPS-specific antibody assays; Claudia de Dood, Govert van Dam and Paul Corstjens (Leiden University Medical Center) for production of reagents and support in undertaking the CAA assays.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe parent POPVAC programme of work was supported primarily by the Medical Research Council (MRC) of the United Kingdom (grant # MR/R02118X/1 and MC_PC_21034). This award was jointly funded by the UK Medical Research Council (MRC) and the UK Foreign Commonwealth and Development Office (FCDO) under the MRC/FCDO Concordat agreement and is also part of the EDCTP2 programme supported by the European Union. GN received support from the EDCTP2 programme supported by the European Union (grant # TMA2019PF-2707) and from the Wellcome Trust (grant # 224263/Z/21/Z). AN, ELW and AME received support from the UK National Institute of Health and Care Research (NIHR) under its \"NIHR Global Health Research Group on Vaccines for vulnerable people in Africa (VAnguard)” [grant # NIHR134531] using UK aid from the UK Government to support global health research. JN was also supported by the Makerere University – Uganda Virus Research Institute Centre of Excellence for Infection and Immunity Research and Training (MUII-plus), funded under the DELTAS Africa Initiative (grant # DEL-15-004). The DELTAS Africa Initiative was an independent funding scheme of the African Academy of Sciences, Alliance for Accelerating Excellence in Science in Africa and supported by the New Partnership for Africa’s Development Planning and Coordinating Agency with funding from the Wellcome Trust and the UK Government. ELW also received funding through the International Statistics and Epidemiology Group (ISEG) from the UK MRC grant # MR/R010161/1. BW was supported by GCRF collaborative Grant (R120442) from the Royal Society awarded to AME. The work was conducted at the MRC/UVRI and LSHTM Uganda Research Unit; both ISEG and the MRC/UVRI and LSHTM Uganda Research Unit are jointly funded by the UK MRC part of UK Research and Innovation and the UK Foreign, Commonwealth and Development Office (FCDO) under the MRC/FCDO Concordat agreement and are also part of the EDCTP2 programme supported by the European Union. The views expressed in this publication are those of the authors and not necessarily those of the funders or the UK government.\u003c/p\u003e\n\u003cp\u003eThe funders had no role in study design, data collection, data analysis, data interpretation, writing of the report, or in the decision to submit this article for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.N. and A.M.E. conceptualised the study; R.A., V.H., C.B. and J.N. conducted the laboratory work under supervision of G.N.; B.W., A.N., R.A., V.H., J.N., G.N. and E.W. curated the data; B.W., A.N. and G.N. undertook the formal analyses; B.W. and G.N. wrote the original draft, and all authors reviewed and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.N. and A.M.E. report grants from Wellcome Trust. G.N. reports funding from the EDCTP2 programme supported by the European Union. A.M.E. reports funding from Medical Research Council (MRC) of the United Kingdom for conduct of the parent study; A.M.E. reports funding from NIH, Science for Africa Foundation and DELTAS Africa, outside the submitted work. B.W. and A.M.E. report funding from the Royal Society outside the submitted work. A.M.E., B.W. and A.N. report support from UK National Institute of Health and care Research (NIHR). A.M.E. further reports support from the Serum Institute of India, Uganda National Expanded Programme on Immunisation, and Emergent BioSolutions for conduct of the parent study. All other authors declare no competing interests.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJiang, V., Jiang, B., Tate, J., Parashar, U. D. \u0026amp; Patel, M. M. 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M.\u003cem\u003e et al.\u003c/em\u003e Homologous and heterologous re-challenge with Salmonella typhi and Salmonella paratyphi A in a randomised controlled human infection model. \u003cem\u003ePLoS Negl Trop Dis\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, e0008783 (2020). https://doi.org/10.1371/journal.pntd.0008783\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8832138/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8832138/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Vaccine immunogenicity varies across populations, yet drivers remain unclear. We compared systemic (plasma) and mucosal (coprological) antibody responses to the oral typhoid vaccine Ty21a, quantified by ELISA, among Ugandan adolescents from urban and Schistosoma mansoni-endemic rural settings. Urban participants elicited higher responses. Causal mediation analysis indicated that S. mansoni partly accounted for reduced mucosal, but not systemic, responses in rural participants, highlighting implications for oral vaccine effectiveness in endemic settings.","manuscriptTitle":"Rural-urban differences in oral typhoid vaccine responses in Uganda: contribution of Schistosoma mansoni","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-12 07:32:56","doi":"10.21203/rs.3.rs-8832138/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-05T21:14:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-24T08:52:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42992503812671448406272831734153826598","date":"2026-04-17T19:37:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321929285114053749527722079512554302437","date":"2026-04-17T07:57:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-16T14:38:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-15T09:39:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Vaccines","date":"2026-04-08T17:26:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d2fa643-375c-48bd-9622-22416f4d9cce","owner":[],"postedDate":"April 12th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Accepted","date":"2026-05-05T22:49:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-05T21:14:09+00:00","index":37,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":65962851,"name":"Health sciences/Diseases"},{"id":65962852,"name":"Biological sciences/Immunology"},{"id":65962853,"name":"Health sciences/Medical research"},{"id":65962854,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-05-05T22:53:52+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-12 07:32:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8832138","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8832138","identity":"rs-8832138","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}