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Sensitive diagnostics are needed to improve management and surveillance of opisthorchiasis and opisthorchiasis-associated cholangiocarcinoma (CCA) throughout East Asia. Herein we generate and screen an Opisthorchis viverrini recombinant secreted proteome to identity antibody biomarkers of liver fluke infection and CCA with sera from study participants in endemic populations and evaluate their utility as point-of-care immunochromatographic tests (PoC-ICTs). We incorporate two of the most promising antigens from the proteome array screen, P1 and P9, into PoC-ICTs to further validate their diagnostic performance. The P9-IgG4 PoC-ICT is superior amongst the single recombinant antigen tests for diagnosing fluke infection as well as fluke-induced CCA, and out-performs parasite crude extract-IgG ICTs. Here we identify two biomarkers of O. viverrini infection and infection-associated CCA that could form the basis of novel antibody serodiagnostic tests for human liver fluke infection and associated cancer. Sensitive diagnostics are needed to detect carcinogenic human liver fluke infections. Sadaow et al. here use an immunomics approach to develop field-deployable tests for diagnosing liver fluke infection and its associated liver cancer.

Vaccine approaches for controlling Fasciola hepatica present a promising avenue, particularly considering increasing resistance to anthelmintic treatments and concerns over chemical residues. Targeting vaccine candidates that are expressed and secreted during the early infective stage of F. hepatica could offer an effective alternative. This approach aims to inhibit the invasion and migration of juvenile parasites, which have not yet fully developed their immune evasion mechanisms, thereby preventing parasite establishment and development in the host. In this study, we evaluated the host immune response and the protective efficacy of a vaccine cocktail comprising four antigens -KTSPIDP, VGHC1, CRTA, and CAL- in sheep infected with F. hepatica . These parasitic antigens were selected based on a proteomic analysis coupled with an “in vitro” interaction model between newly excysted juvenile worms and mouse intestinal epithelial cell cultures. Despite inducing a strong IgG1 response, vaccination did not reduce liver fluke burden nor faecal egg counts. However, it reduced liver pathology caused by the parasite. Our findings highlight the need for further research into early-stage interactions between F. hepatica and the host. Understanding these interactions could facilitate the progress of vaccines capable of disrupting parasite development and transmission in livestock, potentially reducing the economic and health impacts associated with fasciolosis.

Fasciola gigantica (F. gigantica) is a vital parasite that causes fasciolosis. Liver fluke infections affect livestock animals, and the Fasciola species (Fasciola spp.) vaccine has been tested for many types of these diseases. Currently, computer-based vaccine design represents an attractive alternative for constructing vaccines. Thus, this study aimed to design the epitopes of linear B-cells (BCL) and helper T lymphocytes (HTL) using an immunoinformatic approach and to investigate in silico and the mice’s immune response. A non-conserved host region, overlapping F. gigantica cathepsin B proteins (FgCatB), and the highest conserved residue percentages were the criteria used to construct epitopes. The GPGPG linker was used to link epitopes in the multi-epitope Fasciola gigantica cathepsin B (MeFgCatB) peptide. The MeFgCatB peptide has high antigenicity, non-allergenicity, non-toxicity, good solubility, and a high-quality structure. The molecular docking between the MeFgCatB peptide and Toll-like receptor 2 (TLR-2) was evaluated. The IgM, IgG1, and IgG2 levels were elevated in silico. In mice, the MeFgCatB peptide was synthesized and administered as an injection. The MeFgCatB-specific IgG1 and IgG2a levels were elevated after week 2, showing a predominance of IgG1. The rFgCatB1, rFgCatB2, and rFgCatB3 were detected using the MeFgCatB peptide-immunized sera. The MeFgCatB peptide-immunized sera were detected at approximately 28–34 kDa in the whole body. In addition, the MeFgCatB immunized sera can positively signal at the caecal epithelium in the NEJ, 4WKJ, and adult stages. In summary, the MeFgCatB peptide is able to induce mixed Th1/Th2 immune responses with Th2 dominating and to detect the native protein of F. gigantica. The MeFgCatB peptide should help against F. gigantica in future experiments.

Cystic Echinococcosis (CE) is a zoonotic disease caused by Echinococcus granulosus sensu lato , forming cysts in ruminants and humans with major health and economic impacts. The immune response to CE cysts is complex, with fertility linked to the host’s inflammatory reaction. This study examines γδ T cell distribution and role within the adventitial layer of non-fertile CE cysts in cattle, including cases co-infected with the trematode Fasciola hepatica (FH), a known immune response modulator. Using immunohistochemistry and double immunofluorescence, we observed γδ T cells dispersed in the adventitial layer, enriched in inflammatory zones. Co-infected cases (CE + FH+) showed a reduced γδ T cell proportion among CD3+ T cells compared to non-coinfected cases, suggesting an immunoregulatory effect of FH. Our findings align with prior studies showing γδ T cell recruitment in granulomatous diseases in ruminants but reveal that co-infection alters this response. This study provides the first detailed characterization of γδ T cells in cattle CE cysts, emphasizing their potential role in granulomatous immune responses. It highlights the need for further research into mechanisms influencing CE cyst fertility and immune modulation in helminth co-infections, advancing our understanding of host-pathogen interactions and informing disease management strategies.

Spatial heterogeneity in susceptibility and exposure to parasites is a common source of confounding variation in disease ecology studies. However, it is not known whether spatial autocorrelation acts on immunity at small scales, within wild animal populations, and whether this predicts spatial patterns in infection. Here we used a well-mixed wild population of individually recognized red deer (Cervus elaphus) inhabiting a heterogeneous landscape to investigate fine-scale spatial patterns of immunity and parasitism. We noninvasively collected 842 fecal samples from 141 females with known ranging behavior over 2 years. We quantified total and helminth-specific mucosal antibodies and counted propagules of three gastrointestinal helminth taxa. These data were analyzed with linear mixed models using the Integrated Nested Laplace Approximation, using a Stochastic Partial Differentiation Equation approach to control for and quantify spatial autocorrelation. We also investigated whether spatial patterns of immunity and parasitism changed seasonally. We discovered substantial spatial heterogeneity in general and helminth-specific antibody levels and parasitism with two helminth taxa, all of which exhibited contrasting seasonal variation in their spatial patterns. Notably, Fasciola hepatica intensity appeared to be strongly influenced by the presence of wet grazing areas, and antibody hotspots did not correlate with distributions of any parasites. Our results suggest that spatial heterogeneity may be an important factor affecting immunity and parasitism in a wide range of study systems. We discuss these findings with regards to the design of sampling regimes and public health interventions, and suggest that disease ecology studies investigate spatial heterogeneity more regularly to enhance their results, even when examining small geographic areas.

Fasciola hepatica is a parasitic trematode responsible for fascioliasis, a significant zoonotic disease affecting livestock worldwide, as well as humans. This study identifies peptides with potential for use in vaccines against Fasciola hepatica and validates multi-epitope constructs from those peptides in vitro. Putative protein sequences derived from the genome of F. hepatica were integrated with phase-specific transcriptomic data to prioritize highly expressed proteins. Among these, extracellular proteins were selected using DeepLoc 2.0 and strong binding affinities across diverse human and murine alleles were predicted with the IEDB MHC II tool. Peptides were further selected based on their toxicity, immunogenicity, and allergenicity. Finally, 55 high-priority candidates were obtained. To express these candidates, mRNA constructs encoding various combinations of these peptides were designed, synthesized using in vitro transcription with T7 or SP6 RNA polymerases, and transfected into cells for expression analysis. SP6 polymerase produced proper capping using CleanCapAG and was far superior in transcribing peptide constructs. Peptides fused in frame with eGFP were expressed efficiently, particularly when peptides were positioned at the 3′ terminus, opening a new field of peptide vaccines created using mRNA technology.

Fasciolosis, a globally re-emerging zoonotic disease, is mostly caused by the parasitic infection with Fasciola hepatica, often known as the liver fluke. This disease has a considerable impact on livestock productivity. This study aimed to evaluate the fluke burdens and faecal egg counts in goats that were administered phage clones of cathepsin L mimotopes and then infected with F. hepatica metacercariae. Additionally, the impact of vaccination on the histology of the reproductive system, specifically related to egg generation in adult parasites, was examined. A total of twenty-four goats, which were raised in sheds, were divided into four groups consisting of six animals each. These groups were randomly assigned. The goats were then subjected to two rounds of vaccination. Each vaccination involved the administration of 1 × 1013 phage particles containing specific mimotopes for cathepsin L2 (group 1: PPIRNGK), cathepsin L1 (group 2: DPWWLKQ), and cathepsin L1 (group 3: SGTFLFS). The immunisations were carried out on weeks 0 and 4, and the Quil A adjuvant was used in combination with the mimotopes. The control group was administered phosphate-buffered saline (PBS) (group 4). At week 6, all groups were orally infected with 200 metacercariae of F. hepatica. At week 22 following the initial immunisation, the subjects were euthanised, and adult F. hepatica specimens were retrieved from the bile ducts and liver tissue, and subsequently quantified. The specimens underwent whole-mount histology for the examination of the reproductive system, including the testis, ovary, vitellaria, Mehlis’ gland, and uterus. The mean fluke burdens following the challenge were seen to decrease by 50.4%, 62.2%, and 75.3% (p < 0.05) in goats that received vaccinations containing cathepsin L2 PPIRNGK, cathepsin L1 DPWWLKQ, and cathepsin L1 SGTFLFS, respectively. Animals that received vaccination exhibited a significant reduction in the production of parasite eggs. The levels of IgG1 and IgG2 isotypes in vaccinated goats were significantly higher than in the control group, indicating that protection is associated with the induction of a mixed Th1/Th2 immune response. The administration of cathepsin L to goats exhibits a modest level of efficacy in inducing histological impairment in the reproductive organs of liver flukes, resulting in a reduction in egg output.

The identification of B-cell epitopes (BCEs) is fundamental to advancing epitope-based vaccine design, therapeutic antibody development, and diagnostics, such as in neglected tropical diseases caused by parasitic pathogens. However, the structural complexity of parasite antigens and the high cost of experimental validation present certain challenges. Advances in Artificial Intelligence (AI)-driven protein engineering, particularly through machine learning and deep learning, offer efficient solutions to enhance prediction accuracy and reduce experimental costs. Here, we present deepBCE-Parasite, a Transformer-based deep learning model designed to predict linear BCEs from peptide sequences. By leveraging a state-of-the-art self-attention mechanism, the model achieved remarkable predictive performance, achieving an accuracy of approximately 81% and an AUC of 0.90 in both 10-fold cross-validation and independent testing. Comparative analyses against 12 handcrafted features and four conventional machine learning algorithms (GNB, SVM, RF, and LGBM) highlighted the superior predictive power of the model. As a case study, deepBCE-Parasite predicted eight BCEs from the leucine aminopeptidase (LAP) protein in Fasciola hepatica proteomic data. Dot-blot immunoassays confirmed the specific binding of seven synthetic peptides to positive sera, validating their IgG reactivity and demonstrating the model's efficacy in BCE prediction. deepBCE-Parasite demonstrates excellent performance in predicting BCEs across diverse parasitic pathogens, offering a valuable tool for advancing the design of epitope-based vaccines, antibodies, and diagnostic applications in parasitology.

Gene expression for Th1/Th2 cytokines (IL-4 and IFN-ɣ), regulatory cytokines (TGF - β and IL - 10) and the transcriptional factor FoxP3 was analyzed in the liver and hepatic lymph nodes (HLN) from sheep immunized with partially protective and non-protective vaccine candidates and challenged with Fasciola hepatica . FoxP3 T cells were also evaluated by immunohistochemistry (IHQ). The most remarkable difference between the partially protected vaccinated (V1) group and the non-protected vaccinated (V2) group was a more severe expansion of FoxP3 T cells recorded by IHQ in both the liver and HLN of the V2 group as compared to the V1 group, whereas no differences were found between the V2 group and the infected control (IC) group. Similar results were recorded for FoxP3 gene expression although significant differences among V1 and V2 groups were only significant in the HLN, while FoxP3 gene expression was very similar in the V2 and IC groups both in the liver and HLN. No significant differences for the remaining cytokines were recorded between the V1 and V2 groups, but in the liver the V2 group shows significant increases of IFN-ɣ and IL-10 as compared to the uninfected control (UC) group whereas the V1 group did not. The lower expansion of FoxP3 T cells and lower increase of IFN-ɣ and IL-10 in the partially protected vaccinated group may be related with lower hepatic lesions and fluke burdens recorded in this group as compared to the other two infected groups. The most relevant change in regulatory cytokine gene expression was the significant increase of TGF-β in the liver of IC, V1 and V2 groups as compared to the UC group, which could be related to hepatic lesions.

In this work we report the protection found in a vaccination trial performed in sheep with two different vaccines composed each one by a cocktail of antigens (rCL1, rPrx, rHDM and rLAP) formulated in two different adjuvants (Montanide ISA 61 VG (G1) and Alhydrogel ® (G2)). The parameters of protection tested were fluke burden, faecal egg count and evaluation of hepatic lesions. In vaccinated group 1 we found a significant decrease in fluke burden in comparison to both unimmunised and infected control group (37.2%; p  = 0.002) and to vaccinated group 2 (Alhydrogel ® ) (27.08%; p  = 0.016). The lower fluke burden found in G1 was accompanied by a decrease in egg output of 28.71% in comparison with the infected control group. Additionally, gross hepatic lesions found in vaccine 1 group showed a significant decrease ( p  = 0.03) in comparison with unimmunised-infected group. The serological study showed the highest level for both IgG1 and IgG2 in animals from group 1. All these data support the hypothesis of protection found in vaccine 1 group.