Cross-sectional and longitudinal assays were performed to investigate differences among aspects of the immune response induced after vaccination, infection and challenge. C57BL/6 mice were distributed into five groups: control (C), non-treated animals; vaccinated once (1V) with 500 attenuated cercariae and challenged with 120 normal cercariae; vaccinated three times (3V) with 500 attenuated cercariae (4-week intervals) and challenged with 120 normal cercariae; only challenged with 120 normal cercariae at 84 days (Chc); infected with 500 normal cercariae (Inf) in parallel with the 1V group. The Inf group was perfused 35 days after infection (91 days after the beginning of the experiment; Figure 1 ). The inclusion of the Inf group was designed to detect quantitative and qualitative differences in the immune response induced by identical numbers of normal and attenuated cercariae. Mice were anaesthetized using 80 mg/kg of ketamine (Dopalen) and 10 mg/kg of xylazine (Anasedan; both from Ceva Santé Animale, Paulínia, SP) and infected or vaccinated by percutaneous exposure of shaved abdominal skin for 30 min (29) with 500 normal or 500 attenuated cercariae at the time points indicated in Figure 1 . Four weeks after the final vaccination, the 1V and 3V mice were challenged with 120 normal cercariae by the tail method (30). Forty-five days after challenge, all animals were euthanized with a lethal dose of anesthetic (ketamine and xylazine), and perfusion fluid (saline solution plus 500 units/L of heparin) was pumped into the dorsal aorta allowing for the collection of perfused worms from the ruptured hepatic portal vein. The intestinal mesenteric vessels were examined for residual trapped worms, after which all collected worms were counted using a stereomicroscope.

For the Inf group sampling beyond 5 weeks was not possible because of the lethality of this parasite burden.

The choice of sampling time points was based mainly on the work of Ratcliffe and Wilson (31), who described the peak of the systemic response (response of parasite-specific T-lymphocytes) as occurring 17 days after vaccination, and the peak of recall reactivity after a challenge as occurring seven days after secondary exposure. Cross-sectional and longitudinal assays were performed as follows ( Figure 1 ):

For the three independent longitudinal assays, the protection level (%) in vaccinated mice was calculated after perfusion by comparing the number of worms recovered from each vaccinated group with the control group, as previously described (32). To evaluate the liver-trapped egg burden, a piece of the liver from each mouse was removed, weighed, digested and homogenized for 1 h at 37 °C in 5 ml of 10% KOH, the number of eggs per gram of liver was compared to the control group. Worm fecundity was calculated by dividing the number of eggs/gram of liver tissue by the number of female worms collected. Worm size was assessed by measuring the area of each worm using ImageJ software (http://rsb.info.nih.gov/ij) following the acquisition of images using a camera (DINO-Eye, AM423X, Anmo Electronics Corporation, Taiwan) coupled to a light microscope (NIKON – Eclipse E200, Japan).

At Days 7, 17 and Day 7 pc, six mice per group per time point derived from cross-sectional assay➀ were euthanized by an i.p. injection of ketamine and xylazine and several immunological parameters were assessed. After euthanasia, the trachea was cannulated and each animal’s lungs were washed twice with 0.5 and 1.0 ml of cold PBS (phosphate-buffered saline, pH 7.4), and the recovered material collected in microtubes. After total cell counting, cytospin preparations of BAL cells were stained with Instant-Prov (Newprov), and differential cell counting was performed on 200 cells according to respective morphological and staining characteristics.

Blood cell phenotyping was performed by flow cytometry using cells derived from six mice per group per time point (cross-sectional assay➀). Cells were stained with monoclonal antibodies against CD3 (APC-Cy7™ Rat anti-mouse CD3 Molecular Complex, clone17 A2, BD Pharmingen™), CD4 (PE-Cy™ 5 Rat anti-Mouse CD4, clone H129.19, BD Pharmingen™), CD8 (PE™ Rat anti-Mouse CD8a, clone 53–6.7, BD Pharmingen™) and the early T cell activation marker CD69 (FITC Hamster anti-Mouse CD69, clone H1.2F3, BD Pharmingen™). All immunophenotyping and gate strategy was performed as previously described (33) and exemplified in Supplementary Figure 3A . Flow cytometry measurements were performed on a FACSCanto II ^® instrument (Becton Dickinson, Moutain View, CA) with 15,000 events acquired for each preparation. FlowJo Software (Flow Cytometry Analysis Software 7.6, Tree Star, Inc., Ashland, OR) was used for data analysis.

Six mice per group per time point derived from cross-sectional assay➀ were bled from the retro orbital plexus and sera were collected for ELISA assays to assess anti-schistosome IgG, IgG1, IgG2c and total IgE antibody levels. For the quantification of IgG, IgG1 and IgG2c, 96-well plates (Maxisorp 96-well microtiter, Nunc) were coated with 1 µg/ml of 7-day-old schistosomule total protein extract (34) in carbonate-bicarbonate buffer (pH 9.6) as described previously (27). Total IgE ELISA was carried out using a Mouse IgE ELISA kit (BD OptEIA TM, BD Biosciences, USA) using sample dilutions (1:40) in accordance with manufacturer recommendations. IgG, IgG1, and IgG2c antibody levels were calculated using a standard curve and horseradish peroxidase (HRP)-conjugated antibody anti-mouse IgG, IgG1, or IgG2c (Southern Biotechnology). IgE antibody levels were expressed as OD (optical density) with readings performed at 492 nm in an ELISA plate reader (Epoch Biotek, Biotek Instruments Inc., USA). The correlation analysis of antibody levels at Day 28 and parasitic load, as well as the ratio analysis of IgG1/IgG2c were performed with data collected from longitudinal assay➁ with 15 mice per group.

Cytokine assays were assessed in whole blood (without stimulus), from six mice per group per time point derived from cross-sectional assay➀, as previously described (35). Spleens were removed aseptically and processed as previously described (36). Cell viability counts were performed after staining with Trypan blue. Cells (1x10⁶ per well) were plated on 24-well plates and stimulated with schistosomula extract (15 µg/ml) or concanavalin A (5 µg/ml, Sigma) as a positive control for cell reactivity, or saline as a negative control. After 72 h, culture supernatants were collected for cytokine assay using a Mouse Th1/Th2/Th17 CBA kit (BD™ Cytometric Bead Array). All procedures were conducted according to the manufacturer’s recommendations. Data was plotted by subtracting the baseline levels of cytokine production (no stimulus) for each group, and then displayed as a heatmap (z-score).

Fifty microliters of peripheral blood were collected from the retro-orbital complex of 10 mice per group derived from longitudinal assay➂ and transferred to tubes containing 10% EDTA (Sigma Chemical Co) as an anticoagulant. An Auto Hematology Analyzer apparatus (Mindray BC-2800Vet, Hamburg, Germany) was used to evaluate parameters related to white blood cells (total leukocytes, lymphocytes, monocytes and polymorphonuclear cells) and platelets. Data was calculated as the fold change relative to the control group and then displayed as a heatmap.

To obtain the immune transcriptional profiles of PBMCs after infection, immunization, and challenge, the NanoString nCounter Gene Expression Platform (NanoString Technologies, Inc.) was employed. The Nanostring system utilizes a digital barcode technology for the multiplexed measurement of analytes and offers high levels of precision and sensitivity (< 1 copy per cell) (37). The nCounter^® Mouse Immunology panel used herein covers ~546 general immunology genes. For this experimental block, nine mice per group derived from the longitudinal assay➀ were evaluated ( Supplementary Table 1 ). Blood collection, PBMC separation and RNA extraction were performed as described in Supplementary Methods . These assays were performed at a NanoString Technologies facility (Seattle, WA, USA) using 200 ng of total RNA in accordance with the manufacturer’s recommendations. Three independent biological replicates were assessed per group per time point, in which each individual biological replicate consisted of a pool of PBMCs (containing equal amounts of total RNA) from three mice ( Supplementary Table 1 ). In summary, gene expression levels were measured by counting the number of times the color-coded barcode for a given gene was detected (digital counting). Normalization, expression values (fold change calculations) and statistical analyses were performed using nSolver™ Analysis Software (version 2.6).

To obtain a comprehensive overview of the pathways operating under different experimental conditions, we analyzed the PBMC transcriptional profiles obtained with Agilent DNA arrays after vaccination, infection and challenge (SurePrint G3 Mouse GE 8x60K Microarray, Agilent, USA). This DNA array comprises 39,430 Entrez Gene coding transcripts and 16,251 non-coding transcripts from the mouse genome (RefSeq Build 37). For this experimental block, 12–15 mice per group (Inf, 1V, 3V and Chc) and 22 mice for control group (C) derived from the longitudinal assay➁ were evaluated. Blood collection, PBMC separation and RNA extraction were performed as described in Supplementary Methods . Data revealed a high correlation between the number of PBMCs counted and the amount of RNA extracted ( Supplementary Figure 1 ). Four to five independent biological replicates were assessed per group (Inf, 1V, 3V, and Chc) per time point, and 16 biological replicates from control group (C) were used to generate the baseline of expression for PBMC microarray analysis. All biological replicate consisted of a pool of PBMCs (containing equal amounts of total RNA) from three mice ( Supplementary Table 2 ).

We also used the Agilent array to evaluate gene expression profiles in the skin draining lymph nodes at Day 5 after vaccination/infection. For this, 10 mice per group were euthanized and the sdLNs (axillary and inguinal) were collected, fixed overnight in RNAlater solution (Ambion) and then stored in liquid nitrogen until RNA extraction, performed using a RNeasy Micro Kit (QIAGEN) as per manufacturer’s recommendations. Three independent biological replicates were assessed per group for sdLN microarray analysis, in which each biological replicate consisted of a pool of sdLNs from three mice (containing equal amounts of total RNA – RIN > 7) ( Supplementary Table 2 ).

To produce Cy3-labeled cRNA, each pool of total RNA was amplified and labeled using an Agilent Low Input Quick Amplification Labeling Kit in strict accordance with manufacturer instructions ( Supplementary Methods ). Data were corrected using a quantile normalization method and transformed into logarithmic (log₂) values, which allowed for direct comparisons between up-regulated and down-regulated genes.

To identify differentially expressed genes (DEG), a standard pipeline was implemented for probe filtering and expression values obtained from normalized data were consolidated, and then corrected for batch effects. First, the control probes from Agilent were eliminated, followed by the probes that did not map to known genes according to http://biodbnet.abcc.ncifcrf.gov/db/db2db.php. Next, the probes with no significant signal (flag gIsPosAndSignif Agilent) in at least half of the analyzed replicates were removed. Finally, median values were considered for probes replicated within a given array, while in the case of different probes that mapped to the same gene, the greatest mean expression value was considered. These procedures were performed separately with respect to each experimental time point, resulting in a final list of between 17,000 and 18,000 genes. The fold change calculations and differentially expressed gene (DEG) identification, relative to the control group for each condition was performed using the limma R package. A moderate t-test for each gene in each condition was performed, and then the generated p-value was adjusted by false discovery rate (FDR). Due to the small range in gene expression observed for the Volcano plots and Venn diagram analysis we have selected DEGs using a cutoff p ≤ 0.001 (not adjusted by FDR).

To evaluate the genome-wide expression profiles and determine whether a priori a defined set of genes showed statistically significant, cumulative changes in gene expression correlated with the different vaccination regimens, Gene Set Enrichment Analysis (GSEA, Broad Institute; https://www.broadinstitute.org/) was performed at each time point, using the normalized values of signal intensity in the microarray of all genes in the samples of one test condition and the control samples. This tool evaluates whether the cumulative changes in gene expression in one tested condition is correlated with a particular previously characterized group of multiple genes (a gene set) defined based on prior biological knowledge. Procedures for GSEA were performed as described in Supplementary Methods . Pathway analysis was performed using Ingenuity Pathway Analysis software (IPA) (Qiagen) as described in Supplementary Methods .

Differences in worm and egg burdens, worm size, antibody levels and cell counting were analyzed using a one-way ANOVA followed by Tukey’s multiple post-hoc comparison test. Differences were considered statistically significant when P ≤ 0.05. All graphs, Pearson’s correlation coefficient and statistical analyses were performed using PRISM version 5.02 software (GraphPad, San Diego, CA).

Initially, the protection levels and consistency of the RA vaccine were validated. The animals immunized with a single dose of attenuated cercariae (1V) showed on average of 46% worm burden reduction (47% female, 45% male worms). Protection was higher in the 3V group, representing a 71% worm burden reduction (85% female, 65% male worms). No gender bias reduction was detected, as differences between male and female reduction were proportional to the differences observed in the Chc group ( Figures 2A, B ). Egg count analysis in the liver revealed oviposition reduction, with a 71% decrease in the 1V and 90% in 3V groups, relative to the Chc group ( Figure 2C ). Furthermore, when these data were normalized to the number of females, a 49% reduction of fecundity was observed in the 3V relative to Chc group ( Figure 2D ). Lastly, the body lengths of worms recovered from 3V were 28% shorter than Chc animals under morphometric analysis and several presented a stunted phenotype ( Figures 2E, F ). This immunization and challenge assay was performed two more times and presented very similar results, reinforcing the consistency of the RA cercarial vaccine ( Supplementary Figure 2 ).

The analysis of bronchoalveolar lavage (BAL) revealed that mice vaccinated with attenuated cercariae (1V and 3V) presented increased immune cell migration in comparison to infected (Inf and Chc) and control animals ( Supplementary Figure 3B ). The 3V recruitment was already elevated by Day 7, and in both immunized groups at Day 17, which was maintained at Day 7 pc ( Supplementary Figure 3B ). There was a predominance of macrophages and lymphocytes, with increased neutrophils at Day 7 and increased eosinophils at Day 17 ( Supplementary Figure 4 ). Notably, the infection and challenge protocols involving two different doses of normal cercariae (Inf or Chc) did not elicit significantly altered BAL profiles.

We anticipated that parasite migration through the skin and bloodstream would promote alterations (e.g. tissue damage, inflammation and the healing process) that could be detected systemically by blood analysis, without the need for in vitro stimulation. Phenotypic blood analysis revealed the higher levels of activated T cells (CD4⁺ and CD8⁺) on Day 17 in the 3V than 1V group, which were in turn higher than Inf and Control groups ( Supplementary Figure 3C ). In general, the impact of vaccination was to generate higher total leucocyte and platelet levels in the blood (3V > 1V > Inf) as revealed by the hematological evaluation ( Supplementary Figure 3D ).

In the interpretation of antibody responses, it is important to note that time point 0 is in relation to 1V/Inf, so 3V animals were sensitized by two previous exposures to 500 RA cercariae ( Figure 1 ). Thus, high levels of total anti-Schistosoma IgG were observed in the 3V group at all time points analyzed, whereas levels increased gradually in parallel in the 1V and Inf groups to Day 28, and again after challenge of the 1V animals ( Figure 3A ). The pattern for the IgE isotype followed a similar trajectory with a maximum reached at Day 28, but the differences between groups were not statistically significant ( Figure 3B ). Also, at Day 28, a significant negative correlation between antibody levels and final worm burden was observed for IgG, but not for IgE ( Figure 3C ). Our analysis of the IgG1/IgG2c subclass ratio at Day 28 indicated an immune response biased towards a Th1 profile in the 1V group, whereas the Inf group was more polarized towards a Th2 profile, and the 3V group was intermediate between the two ( Figure 3D ).

The cytokine production by cultured splenocytes after in vitro stimulation revealed an early and intense immune response at Day 7, which was maintained until Day 17, with little distinction between the 1V, 3V and Inf groups; this was characterized by the production of both proinflammatory, Th1 and regulatory cytokines ( Supplementary Figure 5A ). The analysis of plasma cytokines revealed that the previously primed 3V group already had the highest circulating levels of inflammatory (IL-6, TNF-α), Th1 (IFN-γ), Th2 (IL-4) and regulatory (IL-10) cytokines at Day 7; this pattern was maintained at Day 17, and Day 7 post-challenge ( Supplementary Figure 5B ). The 1V group also showed increases in IL-4, IFN-γ and IL-6 at Day 17 ( Supplementary Figure 5B ).

We analyzed gene expression profiles in PBMCs of immunized and infected animals in the circumscribed Mouse Immunology panel of the Nanostring nCounter^® platform; we reasoned that key genes should be revealed between groups at different time points ( Figure 1 ). The Venn diagrams revealed a higher number of differentially expressed genes (DEGs) in the 3V group at Day 7 compared to the other groups, whereas DEGs were higher in the 1V group at Day 17 and Day 7 pc ( Figure 4A ). The Inf and Chc groups presented a lower degree of gene perturbation at all time points, suggesting the poor secondary response elicited by normal parasites.

Several genes for Fc receptors, T-cell cytotoxicity, monocyte/granulocyte maturation, complement, chemokine and chemokine receptors were upregulated in the immunized groups at Day 7 and 17 ( Figure 4B ). Conversely, genes involved in Th2 responses were upregulated and some complement proteins downregulated in the infected group; many of the genes involved in T-cell cytotoxicity were downregulated in the Chc group at Day 7 pc ( Figure 4B ). All this amounts to a considerable degree of differential gene expression elicited by the vaccinating parasites, with the 3V group showing the largest number of up-regulated genes. In contrast, the Inf group shows very few up-regulated genes at Day 7, all of which declined by Day 17, thus indicating only a transitory response to the migrating normal schistosomula; the Chc group showed mostly downregulated genes ( Figure 4B ). Of note, are the upregulation of two genes for granzymes (Gzma and Gzmb), alarmins (S100a8 and S100a9) and six killer cell lectin receptors (Klr) in the 3V group before challenge, with many of these downregulated in the Chc group.

At the Day 7 pc sampling time, only the 1V group showed a significant up-regulation of genes, virtually all involved in the arming of monocytes and granulocytes (three Fc receptors, Complement C3, three chemokines and one chemokine receptor), or in the stimulation of their production in the bone marrow (CSF 1, 2 and 3 receptors). One gene, Selplg (P-selectin glycoprotein ligand 1), which plays a crucial role in leucocyte trafficking into tissues, was down-regulated in the infected Inf and Chc groups at all time points analyzed ( Figure 4B ).

Application of the IPA^® upstream regulators prediction tool revealed a series of regulators in the 1V and 3V groups and very few in the Inf group. The STAT-1 (transcription activator of interferon-stimulated genes) and PLAU (Plasminogen Activator, Urokinase) were prominent in both vaccinated groups. Activation of interferon regulators (IFN-γ, IFN-α/β and IFN-αr) and inhibition of IL10RA (receptor α chain) appeared in the 1V group. While regulators of IL-12 complex, Interferon alpha, TNFSF12 and SPI1 were active in the 3V group. On the other hand, the ITK and IL-15 regulators involved in T-cell proliferation and cytotoxicity of lymphocytes and NK, respectively, were inhibited in the Chc group ( Supplementary Figure 6 ).

Aiming to evaluate early signals of immune response in the lymphoid organs, we assessed the gene expression profile in the sdLN at Day 5 after infection or vaccination in an independent cross-sectional assay ( Figure 1 ). Volcano plots show the highest number of DEGs in Inf (464), followed by groups 3V (369) and 1V (186) ( Supplementary Figure 8 ). The Venn diagram of the DEGs showed that only 33 genes were shared between all three groups, while groups 1V and Inf revealed more similarity (82 shared genes) than the 1V and 3V groups (18 shared genes) ( Figure 6A ).

GSEA was performed on the entire microarray dataset of sdLN in order to reveal early pathways associated with vaccination and infection processes. The pathways were manually curated for the elimination of redundant/overlapping gene sets and those clearly unrelated to immune response or representative of an overly general classification were excluded from further analysis as performed for PBMC (all identified pathways are listed in Supplementary Table 2 ). A heatmap generated revealed distinct signatures in the Inf, 1V and 3V groups ( Figure 6B ). The only gene sets related to immune response shared between all groups was the antigen dependent B-cell activation and the proteasome. Multiple pathways indicative of a Th1 profile were prominent in the 1V group, including gene sets related to signaling of IL-1/IL-1R and signaling related to Leishmania infection (which has a Th1 bias), and signaling and regulation of IFN-γ. In addition, several pathways involved in immune activation were shared between the 1V and Inf groups: antigen presentation; dendritic cells; Th1/Th2 polarization; T-cell co-stimulatory signal 4-1BB (CD137). The differentially modulated gene sets exclusive to the Inf group included: hemostasis, collagen formation, and negative regulation of FGFR. The Inf group also showed negatively modulated gene sets involved in leukocyte transendothelial migration and signaling by GPCR. Reflecting the low number of common DEGs, few pathways were shared between the immunized 1V and 3V groups, namely IFN-γ. Among the gene sets confined to the 3V group were granzyme A and apoptosis execution phase. Notably, the 3V group also displayed down-regulated TCR signaling, and co-stimulation by CD28.

Differences of gene expression in the sdLN may reflect the reasons why attenuated parasites elicit protection, but normal parasites do not. Initially, there is a marked contrast in parasite migration through the nodes, with around 15% of normal parasites peaking at day 5, but quickly passing through (53) and attenuated parasites being slower to arrive, but persisting there at least to day 15. This persistence brings them into direct contact with antigen-presenting dendritic cells (54), which go on to stimulate strong proliferation of T cells, but not B cells (55). The infected groups displayed early up-regulation of pathways associated with Th2 skewing in antigen presenting cells, revealing a very early predisposition towards a Th2-polarized response, previously thought to depend on the deposition of eggs around week 5; the Th2 polarization is the hallmark of chronic schistosomiasis (56, 57, 58). Our data on the IgG1/IgG2c ratio at Day 28 also support this early polarization towards a Th2 profile, before any egg deposition, and it occurs even in mice with a single sex infection (59).

A further distinction is apparent between Inf and vaccinated groups in the regulation of the “pathways of hemostasis”, indicating an important element in the protective immune response. While gene sets for hemostasis (including platelet aggregation) were up-regulated in the Inf and 1V groups at Day 5 in the sdLN, this did not translate to PBMC in the circulation. In the Inf and Chc groups at Day 7 and again at Day 7 post-challenge, respectively, multiple pathways related to hemostasis (activation, signaling and platelet aggregation) were exclusively down-regulated in the PBMCs. We also observed an association between platelet numbers and worm burden, with higher platelet values in vaccinated than infected mice ( Supplementary Figure 9 ). Support for this observation comes from one report that S. mansoni cercariae depress circulating platelet numbers after infection of mice (60). It has also been proposed that schistosome tegument surface enzymes such as ATPDase 1 can inhibit platelet activation (61). Thus, negative regulation of hemostasis may be a hitherto unsuspected mechanism assisting parasite migration through capillary beds en route to the portal vasculature, a process that takes days to weeks. Transit along the tight pulmonary capillaries is a major obstacle (14, 62) and the failure to deploy this evasion mechanism in vaccinated mice could partially explain why their migration is blocked in the lungs (63, 64). It should not be surprising that a migrating schistosome, as a foreign body in intimate contact with the vascular endothelium, would deploy mechanisms to minimize damage that could trigger local hemostasis and impede its progress. Such mechanisms become an obvious target for vaccine-induced protection.

The lymphocytes in sdLN and spleen primed by both normal and attenuated parasites are available for recruitment to all tissues, along with inflammatory cells. The current study confirmed recruitment of leucocytes to the lungs of 1V and 3V groups, but not infected mice, as previously determined (49, 65). The schistosome-reactive T cells with a memory/effector phenotype accumulate and arm the lungs against the subsequent arrival of challenge parasites (66), and orchestrate the formation of effector foci to cause their elimination. This element is missing in mice exposed to normal parasites, either because CD4+ T cells with the right characteristics are not generated or because the conditions for large scale pulmonary recruitment are not created.

PBMC reveal a clear dichotomy in chemokine ligand and receptor expression between vaccinated and infected animals. Peak reactivity of 1V animals was observed at Day 17 and again at Day 7 post challenge (5 and 4 genes), while the 3V group peak was earlier at Day 7 (5 genes). In complete contrast, the Inf and Chc mice expressed only 1 or no genes at the three sampling times, revealing their much lower capacity to mount an inflammatory response against the developing parasites. Lung schistosomula are known to modify the functionality of capillary endothelia inducing an anti-inflammatory phenotype (67) and to down-regulate the expression of the adhesion molecules E-Selectin and VCAM-1, induced by TNF-α on vascular endothelia in vitro (68). Thus, it appears that the absolute requirement for TNF-α production induced by vaccination (45), overrides an anti-inflammatory mechanism deployed by normal schistosomula in the lungs.

Also relevant to leucocyte migration, is the modulation of Selplg, involved in platelet adhesion to endothelia at sites of inflammation (69). In the Inf and Chc groups it is notable that Selplg is down-regulated at Day 7, limiting early cell recruitment. Paradoxically, at Day 17, there was down-regulation of Selplg in all three mouse groups, indicating a strong inhibitory signal for leucocyte diapedesis into the lungs. Plausibly, this initiates the down-regulation of cell recruitment to the lungs.

The detection of a series of Th1 markers by GSEA analysis of the sdLN and PBMC in the 1V group, confirmed the pre-disposition to a Th1 induction pathway as early as five days after exposure to the attenuated parasites, maintained at least until day 17. However, Th2 pathways such as B cell survival and intriguingly, mast cells, were also detected in the PBMC. Finally, IPA upstream regulator analysis of the 1V and 3V Nanostring data supported the induction of the Th1 profile at Day 7 and down-regulation of the IL-10-R alpha chain, removing a potential brake from Th1 expansion. In addition, the regulator effects networks analysis revealed several pathways in the 3V group related to activation and migration of leukocytes ( Figure 7 ), considered a sensitive indicator of vaccine immunogenicity (70). On the contrary, the challenged group exhibits negative regulation of cytotoxic T cells ( Figure 7 ). We conclude that the extended residence of attenuated, as distinct from normal larvae, in sdLN creates the conditions for prolonged expansion of the Th1 arm in the absence of down-regulatory factors such as IL-10, and the subsequent circulation of these schistosome reactive T cells in the bloodstream.

One of the mysteries of the RA vaccine model in mice is why multiple vaccinations do not dramatically increase the level of protection. The sdLN of the 3V group displayed down-regulation of pathways associated with expansion of T cell populations, such as CD28 costimulation and T cell receptor. Day 7 and 17 revealed the pathway for B cell survival, while the Nanostring analysis revealed up-regulation of IgG-Fc receptors. Our data confirm that after three vaccinations, by Day 28, the IgG1:IgG2c ratio has moved towards the Th2 pole, but not as far as in the Inf group. These observations point to a more active involvement of antibodies in the effector response with increasing vaccinations, correlating with the higher levels of IgG observed in 3V versus 1V animals and lower worm burden.

The GSEA analysis at Day 17 revealed many more active pathways in the 3V than in the 1V group. Given the lack of activity in the sdLN of 3V animals, a possible explanation may be that the 3V circulating PBMC have been mobilized from other sites. Several pathways related to growth factors and their corresponding receptors (NGF, EGF, FGFR, EGFR, and VEGF) were especially enriched in the 3V group. It is unclear what this plethora of growth factors and their receptors might have on vaccine efficacy, but analysis of other vaccines has also revealed several pathways related to cell proliferation, such as EGFR (70, 71).

The Nanostring analysis also highlighted up-regulation of two Alarmin genes (S100A8 and A9) in PBMCs from both 1V and 3V groups at Day 7. Their primary source is neutrophils and they are part of the innate immune response. Alarmins were also observed in immunized baboons (26). A role in protection against schistosomes remains to be established, but their detection in the Chc group at Day 7 suggests a bystander effect from neutrophilia.

Analysis of the sdLN of 3V animals highlighted Granzyme A pathways together with Apoptosis. Additionally, Nanostring analysis of PBMC from the 3V group at Day 7 showed up-regulated expression of GzmA and GzmB and six klr (killer cell lectin) genes, while at Day 17 the two granzymes were still up-regulated. These genes and pathways are all indicators of cellular cytotoxicity. GzmA and GzmB genes encode two Granzyme proteases secreted primarily from cytotoxic (CD8⁺) T cells and NK cells. However, selective ablation of CD8+ T cytotoxic cells in mice immunized once with the RA vaccine revealed they were not essential for protection (72). Similar experiments have not been performed with NK cells, but lymph node NK cells stimulated by IL-12 shortly after vaccination are believed critical to the subsequent development of antigen-specific Th1 cells (73). Recently, a PBMC transcriptomic study in schoolchildren revealed that infection with S. hematobium is associated with reduced gene signatures for natural killer (NK) cells, DCs, monocytes and T-cell activation (74). There is also a suggestion from an experiment in pigs immunized with one dose of UV-attenuated Schistosoma japonicum, that Granzyme B increase in skin-draining lymph nodes was associated with protection (25).

These observations on NK cell activity and apoptosis in the sdLN of the 3V animals, coupled with down-regulation of T cell costimulatory pathways point to the development of a hyporesponsive state at this site. This would explain why an increase in the number of vaccinations beyond three does not drive the protective response towards sterile immunity. The nodes have been made refractory by down-regulatory mechanisms at the location where priming of T cells occurs. Indeed, multiple exposures of skin to normal schistosome cercariae have been shown to induce hyporesponsiveness in sdLN, implicating IL-10 as the active agent (75).

The 1V and 3V mouse groups showed moderate and strong protection, respectively, against establishment of normal challenge parasites, but a striking lack of secondary responses was observed at Day 7 after challenge. Indeed, the genes highlighted by Nanostring analysis in the 1V PBMC were largely directed towards mobilization of antibody effector pathways. Furthermore, the more highly protected 3V group showed minimal perturbation of gene expression after challenge. In earlier studies no recruitment of ⁵¹Cr-labelled cells to the lungs was observed after percutaneous challenge (65). Additionally, LN draining the site of a challenge in once-vaccinated mice did not generate a secondary Th1 response; instead, cell proliferation and IFN-γ production were profoundly down-regulated (52). Collectively, these observations indicate that the tendency towards a low anamnestic response, already evident after a single vaccination, is so firmly established after three vaccinations as to preclude any further boosting. The persistence of a Th1 signal after three vaccinations (e.g., STAT-1 and IL-12 pathways revealed by IPA analysis of Nanostring PBMC data) may provide an explanation. Experiments using IFNγ R KO mice showed a moderate level of protection after three to five vaccinations (76), even with antibody titers threefold that of vaccinated wild-type animals (13). This observation has clear implications for the type of immune response that might be elicited by vaccination with antigen preparations.

Our data indicate that the migration of normal schistosomula is associated with the downregulation of hemostasis that likely facilitates their passage through capillary beds. This mechanism is disabled in vaccinated animals, increasing the difficulty of onward migration. This poses a question about the identity of the parasite products that mediate the process.

Recent analysis of gene transcription in ex vivo lung schistosomula confirmed the differential expression of the MEG-3 genes and highlighted five members of the MEG-2 family (77). Coincidentally, these same proteins were secreted by mature eggs embolized in intestinal capillaries (78). It is surely more than coincidence that both the intravascular schistosomulum and egg secrete a similar mixture of MEG-2 and MEG-3 proteins. Indeed, a structural analysis of the MEG-3 proteins suggested they interact with receptors on capillary endothelia to facilitate intravascular migration, and they have been likened to the virulence factors facilitating parasite establishment (79). Collectively, the proteins secreted up to the lung stage at Day 7 provide a novel cohort of antigens that need to be evaluated for protective capacity. Vaccine strategies targeting these proteins may inhibit the down-regulation of hemostasis that facilitates parasite migration through the capillaries, increasing vaccine protection. We have recently used epitope mapping with peptide arrays to investigate the reactivity of 44 S. mansoni exposed proteins including the MEG-3 proteins, using sera from the 3V immunized group (28).

Certain limitations need to be borne in mind when interpreting the data from this study. We have not analyzed the lung’s transcriptome (the site of parasite elimination) and the dynamic range of gene alterations in the PBMC and even sdLN was quite low. However, data provided here can guide future experiments using RNAseq or scRNAseq to analyze PBMC, sdLN and lungs after immunization or infection to investigate differences in host and parasite gene expression at high-resolution rate.

Thus, Systems Vaccinology through transcriptomics allowed us to confirm and extend the involvement of genes associated with Th1-immune responses in one-dose and both Th1 and Th2 responses in three-dose vaccination, none of which were activated promptly by infection. Furthermore, it is clear that higher protection was associated with a broader range of activated immune response genes and pathways. The lymph node data revealed early predisposition towards a Th2-polarized response by infection and expansion of Th1 immune responses by vaccination. On the other hand, the PBMC data revealed the regulation of hemostasis pathways as an important mechanism for parasite survival and the role of growth factor signaling in protection, which can help elucidate molecular events responsible for susceptibility and resistance in schistosomiasis, paving the way to immune intervention.