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Treatment of urinary schistosomiasis by chemotherapy remains challenging due to rapid re-infection and possibly to limited susceptibility to praziquantel treatment. Therefore, therapeutic vaccines represent an attractive alternative control strategy. The objectives of this study were to assess the safety and tolerability profile of the recombinant 28 kDa glutathione S-transferase of Schistosoma haematobium (rSh28GST) in healthy volunteers, and to determine its immunogenicity. Volunteers randomly received 100 µg rSh28GST together with aluminium hydroxide (Alum) as adjuvant (n = 8), or Alum alone as a comparator (n = 8), twice with a 28-day interval between doses. A third dose of rSh28GST or Alum alone was administered to this group at day 150. In view of the results obtained, another group of healthy volunteers (n = 8) received two doses of 300 µg of rSh28GST, again with a 28-day interval. A six-month follow-up was performed with both clinical and biological evaluations. Immunogenicity of the vaccine candidate was evaluated in terms of specific antibody production, the capacity of sera to inhibit enzymatic activity of the antigen, and in vitro cytokine production. Among the 24 healthy male participants no serious adverse events were reported in the days or weeks after administration. Four subjects under rSh28GST reported mild reactions at the injection site while a clinically insignificant increase in bilirubin was observed in 8/24 subjects. No hematological nor biochemical evidence of toxicity was detected. Immunological analysis showed that rSh28GST was immunogenic. The induced Th2-type response was characterized by antibodies capable of inhibiting the enzymatic activity of rSh28GST. rSh28GST in Alum did not induce any significant toxicity in healthy adults and generated a Th2-type immune response. Together with previous preclinical results, the data of safety, tolerability and quality of the specific immune response provide evidence that clinical trials with rSh28GST could be continued in humans as a potential vaccine candidate against urinary schistosomiasis.

Two hookworm vaccine candidates, Na-GST-1 and Na-APR-1, formulated with Glucopyranosyl Lipid A (GLA-AF) adjuvant, have been shown to be safe, well tolerated, and to induce antibody responses in a Phase 1 clinical trial (Clinicaltrials.gov NCT02126462) conducted in Gabon. Here, we characterized T cell responses in 24 Gabonese volunteers randomized to get vaccinated three times with Na-GST-1 and Na-APR-1 at doses of 30μg (n = 8) or 100μg (n = 10) and as control Hepatitis B (n = 6). Blood was collected pre- and post-vaccination on days 0, 28, and 180 as well as 2-weeks after each vaccine dose on days 14, 42, and 194 for PBMCs isolation. PBMCs were stimulated with recombinant Na-GST-1 or Na-APR-1, before (days 0, 28 and 180) and two weeks after (days 14, 42 and 194) each vaccination and used to characterize T cell responses by flow and mass cytometry. A significant increase in Na-GST-1 -specific CD4 + T cells producing IL-2 and TNF, correlated with specific IgG antibody levels, after the third vaccination (day 194) was observed. In contrast, no increase in Na-APR-1 specific T cell responses were induced by the vaccine. Mass cytometry revealed that, Na-GST-1 cytokine producing CD4 + T cells were CD161 + memory cells expressing CTLA-4 and CD40-L. Blocking CTLA-4 enhanced the cytokine response to Na-GST-1. In Gabonese volunteers, hookworm vaccine candidate, Na-GST-1, induces detectable CD4 + T cell responses that correlate with specific antibody levels. As these CD4 + T cells express CTLA-4, and blocking this inhibitory molecules resulted in enhanced cytokine production, the question arises whether this pathway can be targeted to enhance vaccine immunogenicity.

Helminth parasites defy immune exclusion through sophisticated evasion mechanisms, including activation of host immunosuppressive regulatory T (Treg) cells. The mouse parasite Heligmosomoides polygyrus can expand the host Treg population by secreting products that activate TGF-β signalling, but the identity of the active molecule is unknown. Here we identify an H. polygyrus TGF-β mimic ( Hp- TGM) that replicates the biological and functional properties of TGF-β, including binding to mammalian TGF-β receptors and inducing mouse and human Foxp3 + Treg cells. Hp- TGM has no homology with mammalian TGF-β or other members of the TGF-β family, but is a member of the complement control protein superfamily. Thus, our data indicate that through convergent evolution, the parasite has acquired a protein with cytokine-like function that is able to exploit an endogenous pathway of immunoregulation in the host. Heligmosomoides polygyrus can activate mammalian TGF-β signalling pathways, but how it does so is not known. Here the authors identify and isolate a H. polygyrus TFG-β mimic that can bind both mammalian TGF-β receptor subunits, activate Smad signalling and generate inducible regulatory T cells.

BACKGROUNDPartial protective immunity to schistosomiasis develops over time, following repeated praziquantel (PZQ) treatment. Moreover, animals develop protective immunity after repeated immunization with irradiated cercariae. Here, we evaluated the development of natural immunity through consecutive exposure-treatment cycles with Schistosoma mansoni in healthy, Schistosoma-naive participants using single-sex, controlled human S. mansoni infection.METHODSTwenty-four participants were randomized in a double-blinded (1:1) manner to either the reinfection group, which received 3 exposures (weeks 0, 9, and 18) to 20 male cercariae, or to the infection control group, which received 2 mock exposures with water (weeks 0 and 9) prior to cercariae exposure (week 18). Participants were treated with PZQ (or placebo) at weeks 8, 17, and 30. Attack rates (ARs) after the final exposure (weeks 19-30) using serum circulating anodic antigen (CAA) positivity were compared between groups. Adverse events (AEs) were collected for safety.RESULTSTwenty-three participants completed the follow-up. No protective efficacy was observed, given an 82% (9 of 11) AR after the final exposure in the reinfection group and 92% (11 of 12) in the infection control group (protective efficacy 11%; 95% CI -24% to 35%; P = 0.5). Related AEs were higher after the first infection (45%) compared with the second (27%) and third (28%) infections. Severe acute schistosomiasis was observed after the first infections only (2 of 12 in the reinfection group and 2 of 12 in the infection control group).CONCLUSIONRepeated Schistosoma exposure and treatment cycles resulted in apparent clinical tolerance, with fewer symptoms reported following subsequent infections, but did not result in protection against reinfection.TRIAL REGISTRATIONClinicalTrials.gov NCT05085470.FUNDINGEuropean Research Council (ERC) Starting Grant (no. 101075876).

The potential of various quantitative lateral flow (LF) based assays utilizing up-converting phosphor (UCP) reporters for the diagnosis of schistosomiasis is reviewed including recent developments. Active infections are demonstrated by screening for the presence of regurgitated worm antigens (genus specific polysaccharides), whereas anti-Schistosoma antibodies may indicate ongoing as well as past infections. The circulating anodic antigen (CAA) in serum or urine (and potentially also saliva) is identified as the marker that may allow detection of single-worm infections. Quantitation of antigen levels is a reliable method to study effects of drug administration, worm burden and anti-fecundity mechanisms. Moreover, the ratio of CAA and circulating cathodic antigen (CCA) is postulated to facilitate identification of either Schistosoma mansoni or Schistosoma haematobium infections. The UCP-LF assays allow simultaneous detection of multiple targets on a single strip, a valuable feature for antibody detection assays. Although antibody detection in endemic regions is not a useful tool to diagnose active infections, it gains potential when the ratio of different classes of antibody specific for the parasite/disease can be determined. The UCP-LF antibody assay format allows this type of multiplexing, including testing a linear array of up to 20 different targets. Multiple test spots would allow detection of specific antibodies, e.g. against different Schistosoma species or other pathogens as soil-transmitted helminths. Concluding, the different UCP-LF based assays for diagnosis of schistosomiasis provide a collection of tests with relatively low complexity and high sensitivity, covering the full range of diagnostics needed in control programmes for mapping, screening and monitoring.

Most studies assessing praziquantel (PZQ) efficacy have used relatively insensitive diagnostic methods, thereby overestimating cure rate (CR) and intensity reduction rate (IRR). To determine accurately PZQ efficacy, we employed more sensitive DNA and circulating antigen detection methods. A sub-analysis was performed based on a previously published trial conducted in children from Côte d'Ivoire with a confirmed Schistosoma mansoni infection, who were randomly assigned to a standard (single dose of PZQ) or intense treatment group (4 repeated doses of PZQ at 2-week intervals). CR and IRR were estimated based on PCR detecting DNA in a single stool sample and the up-converting particle lateral flow (UCP-LF) test detecting circulating anodic antigen (CAA) in a single urine sample, and compared with traditional Kato-Katz (KK) and point-of-care circulating cathodic antigen (POC-CCA). Individuals positive by all diagnostic methods (i.e., KK, POC-CCA, PCR, and UCP-LF CAA) at baseline were included in the statistical analysis (n = 125). PCR showed a CR of 45% (95% confidence interval (CI) 32-59%) in the standard and 78% (95% CI 66-87%) in the intense treatment group, which is lower compared to the KK results (64%, 95% CI 52-75%) and 88%, 95% CI 78-93%). UCP-LF CAA showed a significantly lower CR in both groups, 16% (95% CI 11-24%) and 18% (95% CI 12-26%), even lower than observed by POC-CCA (31%, 95% CI 17-35% and 36%, 95% CI 26-47%). A substantial reduction in DNA and CAA-levels was observed after the first treatment, with no further decrease after additional treatment and no significant difference in IRR between treatment groups. The efficacy of (repeated) PZQ treatment was overestimated when using egg-based diagnostics (i.e. KK and PCR). Quantitative worm-based diagnostics (i.e. POC-CCA and UCP-LF CAA) revealed that active Schistosoma infections are still present despite multiple treatments. These results stress the need for using accurate diagnostic tools to monitor different PZQ treatment strategies, in particular when moving toward elimination of schistosomiasis. www.clinicaltrials.gov, NCT02868385.

Trichinellosis is a serious zoonositc parasitosis worldwide. Because its clinical manifestations aren't specific, the diagnosis of trichinellosis is not easy to be made. Trichinella spiralis muscle larva (ML) excretory-secretory (ES) antigens are the most widely applied diagnostic antigens for human trichinellosis, but the major drawback of the ES antigens for assaying anti-Trichinella antibodies is the false negative in the early Trichinella infection period. The aim of this study was to characterize the T. spiralis putative serine protease (TsSP) and to investigate its potential use for diagnosis of trichinellosis. The full-length TsSP sequence was cloned and expressed, and recombinant TsSP (rTsSP) was purified by Ni-NTA-Sefinose Column. On Western blotting analysis the rTsSP was recognized by T. spiralis-infected mouse serum, and the natural TsSP was identified in T. spiralis ML crude and ES antigens by using anti-rTsSP serum. Expression of TsSP was detected at various T. spiralis developmental stages (newborn larvae, muscle larvae, intestinal infective larvae and adult worms). Immunolocalization identified the TsSP principally in cuticles and stichosomes of the nematode. The sensitivity of rTsSP-ELISA and ES-ELISA was 98.11% (52/53) and 88.68% (47/53) respectively (P > 0.05) when the sera from trichinellosis patients were examined. However, while twenty-one serum samples of trichinellosis patients' sera at 19 days post-infection (dpi) were tested, the sensitivity (95.24%) of rTsSP-ELISA was distinctly higher than 71.43% of ES-ELISA (P < 0.05). The specificity (99.53%) of rTsSP-ELISA was remarkably higher than 91.98% of ES-ELISA (P < 0.01). Only one out of 20 serum samples of cysticercosis patients cross-reacted with the rTsSP. Specific anti-Trichinella IgG in infected mice was first detected by rTsSP-ELISA as soon as 7 dpi and antibody positive rate reached 100% on 10 dpi, whereas the ES-ELISA did not permit detection of 100% of infected mice before 16 dpi. The rTsSP is a potential early diagnostic antigen for human trichinellosis.

Urinary schistosomiasis, the result of infection by Schistosoma haematobium (Sh), remains a major global health concern. A schistosome vaccine could represent a breakthrough in schistosomiasis control strategies, which are presently based on treatment with praziquantel (PZQ). We report the safety and efficacy of the vaccine candidate recombinant 28-kDa glutathione S-transferase of Sh (rSh28GST) designated as Bilhvax, in a phase 3 trial conducted in Senegal. After clearance of their ongoing schistosomiasis infection with two doses of PZQ, 250 children aged 6-9 years were randomized to receive three subcutaneous injections of either rSh28GST/Alhydrogel (Bilhvax group) or Alhydrogel alone (control group) at week 0 (W0), W4, and W8 and then a booster at W52 (one year after the first injection). PZQ treatment was given at W44, according to previous phase 2 results. The primary endpoint of the analysis was efficacy, evaluated as a delay of recurrence of urinary schistosomiasis, defined by a microhematuria associated with at least one living Sh egg in urine from baseline to W152. During the 152-week follow-up period, there was no difference between study arms in the incidence of serious adverse events. The median follow-up time for subjects without recurrence was 22.9 months for the Bilhvax group and 18.8 months for the control group (log-rank p = 0.27). At W152, 108 children had experienced at least one recurrence in the Bilhvax group versus 112 in the control group. Specific immunoglobulin (Ig)G1, IgG2, and IgG4, but not IgG3 or IgA titers, were increased in the vaccine group. While Bilhvax was immunogenic and well tolerated by infected children, a sufficient efficacy was not reached. The lack of effect may be the result of several factors, including interference by individual PZQ treatments administered each time a child was found infected, or the chosen vaccine-injection regimen favoring blocking IgG4 rather than protective IgG3 antibodies. These observations contrasting with results obtained in experimental models will help in the design of future trials. ClinicalTrials.gov NCT 00870649.

A human hookworm vaccine is being developed to protect children against iron deficiency and anaemia associated with chronic infection with hookworms. Necator americanus aspartic protease-1 (Na-APR-1) and N americanus glutathione S-transferase-1 (Na-GST-1) are components of the blood digestion pathway critical to hookworm survival in the host. Recombinant Na-GST-1 and catalytically inactive Na-APR-1 (Na-APR-1[M74]) adsorbed to Alhydrogel were safe and immunogenic when delivered separately or co-administered to adults in phase 1 trials in non-endemic and endemic areas. We aimed to investigate the safety and immunogenicity of these antigens in healthy children in a hookworm-endemic area. This was a randomised, controlled, observer-blind, phase 1, dose-escalation trial, conducted in a clinical research centre, in 60 children aged six to ten years in Lambaréné, a hookworm-endemic region of Gabon. Healthy children (determined by clinical examination and safety laboratory testing) were randomised 4:1 to receive co-administered Na-GST-1 on Alhydrogel plus Na-APR-1(M74) on Alhydrogel and glucopyranosyl lipid A in aqueous formulation (GLA-AF), or co-administered ENGERIX-B hepatitis B vaccine (HBV) and saline placebo, injected into the deltoid of each arm. Allocation to vaccine groups was observer-masked. In each vaccine group, children were randomised 1:1 to receive intramuscular injections into each deltoid on two vaccine schedules, one at months 0, 2, and 4 or at months 0, 2, and 6. 10 μg, 30 μg, and 100 μg of each antigen were administered in the first, second, and third cohorts, respectively. The intention-to-treat population was used for safety analyses; while for immunogenicity analyses, the per-protocol population was used (children who received all scheduled vaccinations). The primary outcome was to evaluate the vaccines' safety and reactogenicity in healthy children aged between six and ten years. The secondary outcome was to measure antigen-specific serum IgG antibody levels at pre-vaccination and post-vaccination timepoints by qualified ELISAs. The trial is registered with ClinicalTrials.gov, NCT02839161, and is completed. Between Jan 23 and Oct 3, 2017, 137 children were screened, of whom 76 were eligible for this trial. 60 children were recruited, and allocated to either 10 μg of the co-administered antigens (n=8 for each injection schedule), 30 μg (n=8 for each schedule), 100 μg (n=8 for each schedule), or HBV and placebo (n=6 for each schedule) in three sequential cohorts. Co-administration of the vaccines was well tolerated; the most frequent solicited adverse events were mild-to-moderate injection-site pain, observed in up to 12 (75%) of 16 participants per vaccine group, and mild headache (12 [25%] of 48) and fever (11 [23%] of 48). No vaccine-related serious adverse events were observed. Significant anti-Na-APR-1(M74) and anti-Na-GST-1 IgG levels were induced in a dose-dependent manner, with peaks seen 14 days after the third vaccinations, regardless of dose (for Na-APR-1[M74], geometric mean levels [GML]=2295·97 arbitrary units [AU] and 726·89 AU, while for Na-GST-1, GMLs=331·2 AU and 21·4 AU for the month 0, 2, and 6 and month 0, 2, and 4 schedules, respectively). The month 0, 2, and 6 schedule induced significantly higher IgG responses to both antigens (p=0·01 and p=0·04 for Na-APR-1[M74] and Na-GST-1, respectively). Co-administration of recombinant Na-APR-1(M74) and Na-GST-1 to school-aged Gabonese children was well tolerated and induced significant IgG responses. These results justify further evaluation of this antigen combination in proof-of-concept controlled-infection and efficacy studies in hookworm-endemic areas. European Union Seventh Framework Programme.

Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of some helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct the helminth glycoproteins omega-1 and kappa-5, two major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein’s Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens.