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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.

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.

Subcutaneous dirofilariasis, caused by the parasitic nematode Dirofilaria repens , is a growing concern in Europe, affecting both dogs and humans. This study focused on D. repens Dr20/22, a protein encoded by an alt (abundant larval transcript) gene family. While well-documented in L3 larvae of other filariae species, this gene family had not been explored in dirofilariasis. The research involved cloning Dr20/22 cDNA, molecular characterization, and evaluating its potential application in the diagnosis of dirofilariasis. Although Real-Time analysis revealed mRNA expression in both adult worms and microfilariae, the native protein remained undetected in lysates from both developmental stages. This suggests the protein’s specificity for L3 larvae and may be related to a process called SLTS (spliced leader trans-splicing), contributing to stage-specific gene expression. The specificity of the antigen for invasive larvae positions it as a promising early marker for dirofilariasis. However, ELISA tests using sera from infected and uninfected dogs indicated limited diagnostic utility. While further research is required, our findings contribute to a deeper understanding of the molecular and immunological aspects of host-parasite interactions and could offer insights into the parasite's strategies for evading the immune system.

Many different Schistosoma antigens have been evaluated as vaccine candidates, including the recombinant form of the Schistosoma mansoni 14-kDa fatty acid-binding protein (rSm14). However, recombinant proteins often lack intrinsic immunostimulatory activity, a limitation that can be addressed by using vaccine formulations that contain adjuvants. In this work, we describe the immune response triggered by rSm14, a vaccine candidate against schistosomiasis currently under clinical trial, formulated with either (i) Monophosphoryl Lipid A (MPLA), (ii) MPLA/Alum, or (iii) Freund's adjuvant. rSm14/MPLA and rSm14/MPLA/Alum formulations induced increased frequency of effector and memory CD4+ T and central memory CD8+ T cells, respectively. Both formulations induced significant production of rSm14-specific IgG and IgG1 antibodies, which could recognize the protein's native form. The rSm14/Freund's formulation elicited a robust immune response characterized by increased levels of IFN-γ, TNF, IgG, IgG1, and IgG2c antibodies, and expansion of memory B cell. These soluble factors have been implicated in the efficacy of Sm14-based vaccines. Despite inducing both humoral and cellular immune responses, the different formulations did not impact worm burden and the number of eggs trapped in the liver and intestine. Altogether, our findings indicate a limitation in the use of the molecules assessed in this study, such as IFN-γ, TNF, and specific antibodies, as correlates of protection and vaccine efficacy.

The antigen Na-GST-1, expressed by the hookworm Necator americanus, plays crucial biochemical roles in parasite survival. This study explores the development of mRNA vaccine candidates based on Na-GST-1, building on the success of recombinant Na-GST-1 (rNa-GST-1) protein, currently assessed as a subunit vaccine candidate, which has shown promise in preclinical and clinical studies. By leveraging the flexible design of RNA vaccines and protein intracellular trafficking signal sequences, we developed three variants of Na-GST-1 as native (cytosolic), secretory, and plasma membrane-anchored (PM) antigens. After one immunization in mice, mRNA vaccines induced an earlier onset of antigen-specific antibodies compared to rNa-GST-1. Following two immunizations, mRNA vaccines induced similar or superior levels of antigen-specific antibodies compared to rNa-GST-1. Secretory Na-GST-1 was comparable to rNa-GST1 in producing neutralizing antibodies against Na-GST-1's thiol transferase activity, while native Na-GST-1 induced a more robust CD8+ T cell response due to its intracellular accumulation. Although PM Na-GST-1 elicited one of highest titers of antigen-specific antibody and a diverse set of memory T-cell populations, it resulted in a lower ratio of neutralizing antibodies after IgG purification compared to the other vaccine candidates. These findings emphasize the importance of antigen localization in tailoring immune responses and suggest that extracellular antigens are more effective for inducing humoral responses, whereas cytosolic antigen accumulation enhances MHC-1 peptide presentation. Future studies will determine if these in vitro and immunogenicity findings translate to in vivo efficacy. Altogether, mRNA vaccines offer numerous possibilities in the development of multivalent vaccines with single or multiple antigens.

Fasciola spp. infection is a significant zoonotic disease. Fasciola gigantica cathepsin L1H (FgCathL1H) is expressed across the life stages of Fasciola gigantica : newly excysted juvenile (NEJ), juvenile, and adult. An emerging tool for diagnosing fasciolosis in humans and cattle involves single-chain variable fragments (scFv) antibodies. These antibodies, consisting of linked variable regions of heavy chains (VHs) and light chains (VLs), retain binding specificity and affinity. This study aims to construct, express, and characterize an scFv antibody for use in a diagnostic kit for fasciolosis. The study successfully constructed and expressed recombinant scFv antibody genes derived from mouse spleen cells in Escherichia coli HB2151. Specific VH and VL fragments targeting recombinant FgCathL1H were amplified, inserted into a phagemid vector (pCANTAB5E), and transformed into E. coli TG1. Infection with the M13KO7 helper phage produced recombinant phages, and scFv clones with a high binding capacity were selected through three rounds of bio-panning. The expression of scFv proteins was induced with 1 mM IPTG, yielding antibodies detectable in the culture supernatant and periplasmic space. The indirect ELISA revealed strong binding in 10 scFv phage clones, which were sequenced and analyzed via computer-guided homology modeling and showed a similar classification to CDR1–3, consisting of VHs and VLs. The scFv DNA construct was approximately 747 bp in length. The SDS-PAGE, ELISA, and western blot confirmed the specificity of the scFv clone 1B, particularly at ~ 29 kDa. Docking studies showed epitopes on the scFv interacting with FgCathL1H. This scFv reacted specifically with F. gigantica antigens at 36 kDa (whole body (WB) of metacercaria and NEJ) and ~ 28 kDa (WB of 4-week-old juveniles and adults, and adult excretory–secretory protein (ES)). Immunolocalization showed positive staining in the cecal epithelium. Thus, scFv anti-rFgCathL1H shows promise for diagnosing fasciolosis.

To investigate the effects of Echinococcus granulosus sensu stricto ( E. granulosus s.s. ) reproduction-associated protein EGr_09888 on the development of E. granulosus s.s. protoscoleces (PSCs) in vitro. SWISS-MODEL, Netphos3.1, and PyMOL bioinformatics analysis software, along with subcellular localization prediction, were used to analyze the structure, phosphorylation sites, epitopes, and distribution of the EGr_09888 protein. The recombinant plasmid pET30a-EGr_09888 was constructed for protein expression and purification, and New Zealand white rabbits were immunized with the recombinant protein. An indirect ELISA method was used to determine serum IgG levels, through Western blotting to verify immune specificity and different stages worm protein extracts EGr_09888 protein expression. The distribution of EGr_09888 in PSCs, adult worms and gravid segments was analyzed by immunofluorescence staining. PSCs cultured in vitro for 3, 6, and 10 weeks were treated with rabbit anti-EGr_09888 high-titer immune serum for 6 days and then their survival rate and the gene expression of EGr_09888 in the PSCs was analyzed. The result revealed that the EGr_09888 protein has diverse secondary-structure amino acid proportions, and its SWISS-MODEL-predicted tertiary structure is reliable, containing 54 potential phosphorylation sites. It has B- and T-cell epitopes at amino acids (aa) 25–44 and 166–185, shows immunogenic potential, and is localized mainly in the cytoplasm. Induced expression of the recombinant EGr_09888 protein (27 kDa) resulted in rabbit anti-EGr_09888 serum with a potency greater than 1:160000. Immunoblotting revealed that the rabbit anti-EGr_09888 serum could specifically bind to the recombinant EGr_09888 protein. And the expression level of EGr_09888 protein was higher in adult worm protein extracts ( P  ≤ 0.001), Immunofluorescence staining analysis revealed that EGr_09888 was localized mainly within the uterus of the gravid segment of E. granulosus s.s adult worms. After 6- and 10-week-old PSCs were treated with rabbit anti-EGr_09888 serum, the survival rate and relative PSC EGr_09888 expression level were significantly reduced ( P  < 0.0001). This study revealed that EGr_09888, a reproduction-related protein in E. granulosus s.s. , has potential as a candidate vaccine for definitive hosts.

Opisthorchis viverrini (Ov) infection caused opisthorchiasis, which posed an important risk for the development of cholangiocarcinoma (CCA). Therefore, it is crucial to focus on the primary prevention and control of opisthorchiasis in order to control CCA effectively in Thailand and other endemic regions. A recent diagnostic method of antigen detection using monoclonal antibody-based enzyme-linked immunosorbent assay (mAb-ELISA) has the potential for rapid mass screening of opisthorchiasis. Nevertheless, the specific antigen(s) in Ov adult worms recognized by mAb have not been determined. In this study, we aimed to identify and characterize the target molecule of our in-house Ov-specific monoclonal antibody (mAb KKU505). The specific antigenic band formed by the reaction of Ov adult worm extract and mAb KKU505 was detected using western blot analysis. The protein band was identified as the myosin heavy chain of Ov using LC-MS/MS analysis. The reactivity of the recombinant full-length myosin heavy chain (rMHC) was comparable to that of the crude Ov antigen when evaluated using mAb-ELISA at similar protein concentrations. Moreover, the binding ability between Ov myosin head domain and mAb KKU505 was confirmed using in silico analysis. The results reported here indicate that rMHC could potentially substitute for Ov crude antigen in antigen detection by mAb-ELISA and as a positive control for Ov-strip in lateral flow assays, thereby avoiding the use of laboratory animals for the production of Ov adult worms.

Barbervax is the first and only available vaccine to protect animals against Haemonchus contortus - one of the most pathogenic parasites of small ruminants. This vaccine contains a kind of native antigen called H11, a glycoprotein complex derived from integral gut of this parasite. Native H11 has been shown to induce high levels (72-95%) of protection, but single or two recombinant molecules of H11 are consistently unsuccessful. An increasing number of aminopeptidases related to H11 have been characterized in the past three decades, but little is known about which ones are the key contributors to protective immunity. Our recent work has revealed that the immunoprotective effect of H11 is primarily associated with its N-glycan moieties. To identify key immunoprotective glycoproteins derived from H11 antigen, we employed glycan-related protective IgG antibodies combined with LC-MS/MS analysis and identified five glycosylated H11 proteins: H11, H11-1, H11-2, H11-4, and H11-5. Subsequently, we utilized the baculovirus-insect cell expression system and successfully expressed four H11 recombinant proteins including rH11, rH11-1, rH11-2 and rH11-4, which demonstrated similar aminopeptidase activity and comparable high-mannose and di-fucosylated N-glycan structures to those found on native H11. Immunization of goats with a cocktail of four rH11s induced a 66.29% reduction ( p > 0.05) in total worm burden and cumulative fecal egg counts. High level of anti-rH11s IgG which could inhibit H. contortus intestinal aminopeptidase activity and larval development. Collectively, our study identified glycoprotein antigens from H11 and assessed their protective efficacy of a recombinant cocktail expressed in insect cells. This work will provide valuable insights into further development of recombinant vaccines against parasitic nematodes.

Current treatments and prevention strategies for echinococcosis are inadequate. Recent advancements in molecular vaccine development show promise against ; however, remains a challenge. A Multi-epitope Vaccine could potentially induce specific B and T lymphocyte responses, thereby offering protection against infection. This study aimed to develop a MEV against alveolar echinococcosis. Key epitopes from the proteins EmTSP3 and EmTIP were identified using immunoinformatics analyses. These analyses were conducted to assess the MEV feasibility, structural characteristics, molecular docking, molecular dynamics simulations, and immune simulations. The immunogenicity and antigenicity of the vaccine were evaluated through and experiments, employing ELISA, Western blotting, FCM, challenge infection experiments, and ELISPOT. The effective antigenicity and immunogenicity of MEV were demonstrated through immunoinformatics, as well as and experiments. experiments revealed that MEV increased the secretion of IFN-γ and IL-4 in PBMC and successfully bound to specific antibodies in patient serum. Furthermore, mice immunized with MEV developed a robust immune response, characterized by elevated levels of CD4+ and CD8+ T-cells, increased secretion of IFN-γ and IL-4 by specific Th1 and Th2 cells, and heightened serum antibody levels. Importantly, MEV reduced the weight of cysts by conferring resistance against echinococcosis. These findings suggest that MEV is a promising candidate for the prevention of infection. A total of 7 CTL, 7 HTL, 5 linear B-cell, and 2 conformational B-cell epitopes were identified. The vaccine has demonstrated effective antigenicity and immunogenicity against AE through molecular docking, immune simulation, molecular dynamics studies, and both and experiments. It provides effective protection against infection, thereby laying a foundation for further development.