Fasciola spp: Mapping of the MF6 epitope and antigenic analysis of the MF6p/HDM family of heme-binding proteins

MF6p/FhHDM-1 is a small cationic heme-binding protein which is recognized by the monoclonal antibody (mAb) MF6, and abundantly present in parenchymal cells and secreted antigens of Fasciola hepatica. Orthologs of this protein (MF6p/HDMs) also exist in other causal agents of important foodborne trematodiasis, such as Clonorchis sinensis, Opisthorchis viverrini and Paragonimus westermani. Considering that MF6p/FhHDM-1 is relevant for heme homeostasis in Fasciola and was reported to have immunomodulatory properties, this protein is expected to be a useful target for vaccination. Thus, in this study we mapped the epitope recognized by mAb MF6 and evaluated its antigenicity in sheep. The sequence of the MF6p/FhHDM-1 ortholog from F. gigantica (MF6p/FgHDM-1) was also reported. By means of ELISA inhibitions with overlapping synthetic peptides, we determined that the epitope recognized by mAb MF6 is located within the C-terminal moiety of MF6p/FhHDM-1, which is the most conserved region of MF6p/HDMs. By immunoblotting analysis of parasite extracts and ELISA inhibitions with synthetic peptides we also determined that mAb MF6 reacted with the same intensity with F. hepatica and F. gigantica, and in decreasing order of intensity with C. sinensis, O.viverrini and P. westermani orthologs. On the contrary, mAb MF6 showed no reactivity against Dicrocoelium dendriticum and Schistosoma mansoni. The study of the recognition of peptides covering different regions of MF6p/FhHDM-1 by sera from immunized sheep revealed that the C-terminal moiety is the most antigenic, thus being of potential interest for vaccination. We also demonstrated that the production of antibodies to MF6p/FhHDM-1 in sheep infected by F. hepatica occurs relatively early and follows the same pattern as those produced against L-cathepsins.This work was funded by the Ministerio de Ciencia e Innovación, Spain (Grants AGL2011-30563-C03-01, AGL2011-30563-C03-02 and AGL2011-30563-C03-03); Xunta de Galicia, Spain (Grants GPC 2014/058 and ED431B 2017/18); Network of Biomedical Research on Tropical Diseases (RICET), Spain (Grant RD12/0018/0013) and the European Fund for Regional Development (FEDER). VMS holds a predoctoral fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (Programa de Formación del Profesorado Universitario). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Synergism between prior anisakis simplex infections and intake of NSAIDs, on the risk of upper digestive bleeding: A case-control study

Background: The aim of this study was to investigate the relationship between prior Anisakis infections and upper gastrointestinal bleeding (UGIB), and its interaction with non-steroidal anti-inflammatory drug (NSAID) intake. Methods/Principal Findings: We conducted a hospital-based case-control study covering 215 UGIB cases and 650 controls. Odds ratios (ORs) with their confidence intervals (95% CIs) were calculated, as well as the ratio of the combined effects to the sum of the separate effects of Anisakis allergic sensitization and NSAIDs intake. Prior Anisakis infections were revealed by the presence of anti-Anisakis IgE antibodies specific to the recombinant Ani s 1 and Ani s 7 allergens used as the targets in indirect ELISA. Prior Anisakis infections (OR 1.74 [95% CI: 1.10 to 2.75]) and the intake of NSAIDs (OR 6.63 [95% CI: 4.21 to 10.43]) increased the risk of bleeding. Simultaneous NSAIDs intake and Anisakis allergic sensitization increased the risk of UGIB 14-fold (OR = 14.46 [95% CI: 6.08 to 34.40]). This interaction was additive, with a synergistic index of 3.01 (95% CI: 1.18–7.71). Conclusions: Prior Anisakis infection is an independent risk factor for UGIB, and the joint effect with NSAIDs is 3 times higher than the sum of their individual effectsAuthor Summary: Anisakiasis is a worldwide re-emerging disease produced by the consumption of raw, lightly cooked, smoked or marinated fish containing live Anisakis larvae. In acute anisakiasis, mucosal lesions generated by the larvae may provoke upper gastrointestinal bleeding (UGIB). However, the effect of past unnoticed Anisakis infections as a risk factor for UGIB, and a possible synergism with other risk factors such as NSAIDs intake, have never been investigated. In this case-control study we observed that: i) prior Anisakis infections and NSAIDs intake are two independent risk factors for UGIB, and ii) that both risk factors act synergistically to the extent that their joint effect is 3 times higher than the sum of their individual effects. We concluded that, in countries where Anisakis infections are frequent, it would be wise to determine parasite-specific IgE antibodies and to conduct a closer follow-up of patients who consume raw or lightly cooked fish and who are prescribed NSAIDs for long periodsAuthor Summary Anisakiasis is a worldwide re-emerging disease produced by the consumption of raw, lightly cooked, smoked or marinated fish containing live Anisakis larvae. In acute anisakiasis, mucosal lesions generated by the larvae may provoke upper gastrointestinal bleeding (UGIB). However, the effect of past unnoticed Anisakis infections as a risk factor for UGIB, and a possible synergism with other risk factors such as NSAIDs intake, have never been investigated. In this case-control study we observed that: i) prior Anisakis infections and NSAIDs intake are two independent risk factors for UGIB, and ii) that both risk factors act synergistically to the extent that their joint effect is 3 times higher than the sum of their individual effects. We concluded that, in countries where Anisakis infections are frequent, it would be wise to determine parasite-specific IgE antibodies and to conduct a closer follow-up of patients who consume raw or lightly cooked fish and who are prescribed NSAIDs for long periodsThis study was supported by grants PI021512, PI021364, PI020661, PI021572 (Health Research Fund /Fondo de Investigación Sanitaria), SAF2002-04057 (Ministry of Health and Consumer Affairs, Spain), PGIDIT03PXIC20806PN (Galician Regional Authority, Spain) and 02/1572 (Basque Regional Authority, Spain)S

Analysis of Ani s 7 and Ani s 1 allergens as biomarkers of sensitization and allergy severity in human anisakiasis

The high frequency of infection by Anisakis simplex (A. simplex) has led to an increase in IgE sensitization, turning allergy to this parasite a relevant contemporary health problem. Improving the lack of conventional diagnosis test specificity is crucial to better understand these clinical scenarios. Specific IgE (sIgE) to A. simplex extract by ImmunoCAP (Anisakis-sIgE) was determined in sera from 403 blood donors (BD) from Cantabria (North of Spain) of which 51 subjects resulted sensitized. Among these latter, 47 were asymptomatic (sABD). The values of total IgE, prick-test, Anisakis-sIgE, and sIgE to Ani s 1 (anti-rAni s 1) and Ani s 7 (anti-rAni s 7) were compared between 46 sABD and 49 A. simplex allergic patients. The IgE seroprevalence by ImmunoCAP among BD was 12.65%. Allergic patients and sABD showed significant differences in all serum biomarkers evaluated. The area under the curve was assessed for Anisakis-sIgE (0.892), sIgE-rAni s 1 (0.672) and sIgE-rAni s 7 (0.668). After a severe reaction, significantly higher levels of Anisakis-sIgE and sIgE anti-rAni s 1 were detected. Determinations of sIgE by ImmunoCAP, Ani s 1 and Ani s 7 presented different sensitization patterns between allergic and asymptomatic individuals. The Ani s 1 allergen arises as a possible biomarker to detect patients at risk of suffering severe allergic reactions

ANISERP: a new serpin from the parasite Anisakis simplex

Background: Serine proteinase inhibitors (serpins) finely regulate serine proteinase activity via a suicide substrate-like inhibitory mechanism. In parasitic nematodes, some serpins interact with host physiological processes; however, little is known about these essential molecules in Anisakis. This article reports the gene sequencing, cloning, expression and preliminary biochemical and bioinformatically-based structural characterization of a new Anisakis serpin (ANISERP). Methods: The full AniSerp gene was cloned by specific RACE-PCR after screening an Anisakis simplex (L3) cDNA library. For biochemical assays, the AniSerp gene was subcloned into both prokaryotic and eukaryotic vectors, and the recombinant proteins were purified. The inhibitory properties of the proteins were tested in classical biochemical assays using human serine peptidases and AMC substrates. Immunolocalization of ANISERP, theoretical structural analysis and bioinformatically-based structural modelling of the ANISERP protein were also conducted. Results: The AniSerp gene was found to have 1194 nucleotides, coding for a protein of 397 amino acid residues plus a putative N-terminal signal peptide. It showed significant similarity to other nematode, arthropod and mammalian serpins. The recombinant ANISERP expressed in the prokaryotic and eukaryotic systems inhibited the human serine proteases thrombin, trypsin and cathepsin G in a concentration-dependent manner. No inhibitory activity against Factor Xa, Factor XIa, Factor XIIa, elastase, plasmin or chymotrypsin was observed. ANISERP also acted on the cysteine protease cathepsin L. ANISERP was mainly localized in the nematode pseudocoelomic fluid, somatic muscle cell bodies and intestinal cells. The findings of molecular dynamics studies suggest that ANISERP inhibits thrombin via a suicide substrate-like inhibitory mechanism, similar to the mechanism of action of mammalian coagulation inhibitors. In contrast to findings concerning human antithrombin III, heparin had no effect on ANISERP anticoagulant inhibitory activity. Conclusions: Our findings suggest that ANISERP is an internal Anisakis regulatory serpin and that the inhibitory activity against thrombin depends on a suicide substrate-like inhibitory mechanism, similar to that described for human antithrombin (AT)-III. The fact that heparin does not modulate the anticoagulant activity of ANISERP might be explained by the absence in the latter of five of the six positively charged residues usually seen at the AT-III-heparin binding site.We thank Biomol-Informatics SL (http://www.biomol-informatics.com/) for bioinformatic consultation. We also thank Juan Francisco Alcaide and Julia Medrano for their invaluable help in the ANISERP patenting process. We thank Dr Raúl Iglesias (Laboratorio de Parasitología, Facultad de Biología, Universidad de Vigo) for his helpful comments on the interpretation of the IHQ studies

In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA

Recombinant proteins expressed in E. coli are frequently purified by immobilized metal affinity chromatography (IMAC). By means of this technique, tagged proteins containing a polyhistidine sequence can be obtained up to 95% pure in a single step, but some host proteins also bind with great affinity to metal ions and contaminate the sample. A way to overcome this problem is to include a second tag that is recognized by a preexistent monoclonal antibody (mAb) in the gene encoding the target protein, allowing further purification. With this strategy, the recombinant protein can be directly used as target in capture ELISA using plates sensitized with the corresponding mAb. As a proof of concept, in this study we engineered a Trichinella-derived tag (MTFSVPIS, recognized by mAb US9) into a His-tagged recombinant Fasciola antigen (rFhLAP) to make a new chimeric recombinant protein (rUS9-FhLAP), and tested its specificity in capture and indirect ELISAs with sera from sheep and cattle. FhLAP was selected since it was previously reported to be immunogenic in ruminants and is expressed in soluble form in E. coli, which anticipates a higher contamination by host proteins than proteins expressed in inclusion bodies. Our results showed that a large number of sera from non-infected ruminants (mainly cattle) reacted in indirect ELISA with rUS9-FhLAP after single-step purification by IMAC, but that this reactivity disappeared testing the same antigen in capture ELISA with mAb US9. These results demonstrate that the 6XHis and US9 tags can be combined when double purification of recombinant proteins is required.This work was supported by: Ministerio de Economía y Competitividad (Spain) [grant number AGL2011-30563-C03 and AGL2014-57125R], Ministerio de Economía, Industria y Competitividad (INIA, Spain) [grants numbers RTA2017-00010-C02-01 and RTA2017-00010-C02-02] and the Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia, Spain) [grant number ED431B 2017/18]. RAOM holds a predoctoral fellowship from the Spanish Ministerio de Economía y Competitividad (Programa de Formación de Personal Investigador). VMS is supported by a contract under the grant ED431B 2017/18. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Different Fish-Eating Habits and Cytokine Production in Chronic Urticaria with and without Sensitization against the Fish-Parasite Anisakis simplex

Background:Anisakis simplexsensitization has been associated with acute, but also with chronic urticaria.The objective of this study is to characterize chronic urticaria with (CU+) and without sensitization (CU-) againstthe ubiquitous fish parasiteA. simplexin a transversal and longitudinal evaluation.Methods:16 CU+ and 22 CU- patients were included and assessed for Urticaria activity score (UAS), fish-eating habits by standardized questionnaire and cytokine production (assessed by flow cytometric bead-basedarray) of peripheral blood mononuclear cells after stimulation withA. simplexextract or Concanavalin A (ConA). Patients were randomly put on a fish-free diet for three months and UAS, as well as cytokine productionwere again assessed. A difference of"1 in UAS was defined as improvement.Results:There was no difference in UAS in both groups.Anisakisinduced IL-2, IL-4 and IFN-γproduction washigher in CU+. Con A induced IL-6 and IL-10 production was higher in CU+. CU+ was associated with highertotal fish intake, whereas CU- was associated with oily fish intake. The correlation of UAS was positive with oilyfish, but negative with total fish intake.There was a better UAS-based prognosis in CU+ without diet. Improvement was associated with higher ConA induced IL-10!IFN-γas well as IL-10!IL-6 ratios. Further, previous higher oily fish intake was associated withimprovement.Conclusions:Our data confirm the different clinical and immunological phenotype of CU+. Our results show acomplex relationship between fish-eating habits, cytokine production and prognosis, which could have impor-tant consequences in dietary advice in patients with CU. When encounteringA. simplexsensitization, patientsshould not be automatically put on a diet without fish in order to reduce contact withA. simplexproductsThe study was funded by grants from Fundación Sociedad Española de Alergología e Inmunología Clínica (SEAIC) 2009 and Fundación Mutua Madrileña 2009, SpainS

Development and Evaluation of a New Lateral Flow Immunoassay for Serodiagnosis of Human Fasciolosis

Fasciolosis is an important plant-borne trematode zoonosis. This disease is of both clinical and veterinary relevance and, according to the WHO, is considered a re-emerging disease that is spreading around the world. Fasciolosis has a serious impact on health because of the large size of the parasite and the effects of the parasite in down-regulating the host immune response. Human fasciolosis can be distinguished by an acute phase, in which the parasite migrates through different tissues, and a chronic phase in which it invades the bile ducts. Here we describe the development of a rapid, simple and inexpensive immunochromatographic diagnostic method, based on the use of a recombinant cathepsin L1 protein, which performs better than other more complex indirect methods, providing similar specificity and higher sensitivity. The simplicity of the method represents a great advantage for the intervention systems applied in different endemic areas by WHO, such as passive case finding (e.g. Vietnam) and selective treatment (e.g. Egypt). Because of its characteristics, the system can be applied to both phases of the disease, and in holo, meso and hyperendemic areas where point-of-care testing is required

In Vitro Effects of Resveratrol on the Viability and Infectivity of the Microsporidian Encephalitozoon cuniculi

Microsporidians of the genus Encephalitozoon are an important cause of disease in immunocompromised patients, and there are currently no completely effective treatments. The present study investigated the viability and infectivity of spores of Encephalitozoon cuniculi that had been exposed to resveratrol (RESV), a natural phytoalexin found in grapes and red wine. RESV at 50 μM showed significant sporicidal activity, and at 10 to 50 μM it reduced the capacity of the spores to infect dog kidney epithelial cells of the MDCK line. At 10 μM RESV also significantly inhibited intracellular development of the parasite, without affecting host cell viability. These results suggest that RESV may be useful in the treatment of Encephalitozoon infections

QSPR-Perturbation Models for the Prediction of B-Epitopes from Immune Epitope Database: A Potentially Valuable Route for Predicting “In Silico” New Optimal Peptide Sequences and/or Boundary Conditions for Vaccine Development

In the present study, three different physicochemical molecular properties for peptides were calculated using the program MARCH-INSIDE: atomic polarizability, partition coefficient, and polarity. These measures were used as input parameters of a linear discriminant analysis (LDA) in order to develop three different quantitative structure–property relationship (QSPR)-perturbation models for the prediction of B-epitopes reported in the immune epitope database (IEDB) given perturbations in peptide sequence, in vivo process, experimental techniques, and source or host organisms. The accuracy, sensitivity and specificity of the models were >90 % for both training and cross-validation series. The statistical parameters of the models were compared to the results achieved with the electronegativity QSPR-perturbation model previously reported by González-Díaz et al. (J Immunol Res. doi:10.1155/2014/768515, 2014). The results indicate that this type of approach may constitute a potentially valuable route for predicting “in silico” new optimal peptide sequences and/or boundary conditions for vaccine development.Fil: Vázquez Prieto, Severo. Universidad Autónoma de Chile; Chile. Universidad de Santiago de Compostela; España. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Paniagua Crespo, María Esperanza. Universidad de Santiago de Compostela; EspañaFil: Ubeira, Florencio M.. Universidad de Santiago de Compostela; EspañaFil: González Díaz, Humberto. Universidad del País Vasco; España. IKERBASQUE; Españ