Nuevo inhibidor de serina proteasa y su uso

La invención se refiere a una nueva proteína, identificada mediante rastreo de una genoteca de cDNA de Anisakis simplex, así como a su secuencia codificante y a sus vectores de expresión. Dicha proteína, miembro de la familia de las serpinas, es capaz de inhibir de manera dependiente de dosis a la trombina humana, sin afectar al factor Xa de la coagulación, y sin que la heparina altere su actividad inhibitoria. Así, la invención se refiere también al uso de esta proteína para la preparación de un medicamento, especialmente si se trata de un anticoagulante. Su administración tendría especial interés en los casos de hipercoagulopatías asociadas a la administración de heparina. Además, representa una alternativa a los anticoagulantes derivados de la cumarina, a los que podría sustituir en cualquiera de las situaciones en las que se administran.REIVINDICACIONES: 1. Un polipéptido cuya secuencia de aminoácidos comprende: a) la secuencia representada por SEQ ID NO:3, o b) una secuencia idéntica al menos en un 40% a SEQ ID NO:3 que presenta estructura de serpina con capacidad de inhibir a la trombina humana, o c) la secuencia de aminoácidos representada por SEQ ID NO:2, o d) una secuencia idéntica al menos en un 40% a SEQ ID NO:2, que presenta estructura de serpina con capacidad de inhibir a la trombina humana. 2. Polipéptido según la reivindicación 1, que comprende la secuencia de aminoácidos representada por SEQ ID NO:3. 3. Polipéptido según la reivindicación 1, que comprende una secuencia de aminoácidos que presenta estructura de serpina con capacidad de inhibir a la trombina humana, en el que dicha secuencia es idéntica a SEQ ID NO:3 en un porcentaje que se selecciona del grupo del 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99, 5% y 99, 9%. 4. Polipéptido según la reivindicación 1, que comprende la secuencia de aminoácidos representada por SEQ ID NO:2. 5. Polipéptido según la reivindicación 1, que comprende una secuencia de aminoácidos que presenta estructura de serpina con capacidad de inhibir a la trombina humana, en el que dicha secuencia es idéntica a SEQ ID NO:2 en un porcentaje que se selecciona del grupo del 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99, 5% y 99, 9%. 6. Polipéptido según una cualquiera de las reivindicaciones anteriores, que es una proteína de fusión que comprende la secuencia de una segunda proteína. 7. Polipéptido según la reivindicación 6, en el que la segunda proteína es la glutatión-S-transferasa. 8. Polipéptido según una cualquiera de las reivindicaciones anteriores, cuya capacidad de inhibición de la trombina humana no se ve afectada por la presencia de heparina. 9. Polipéptido según una cualquiera de las reivindicaciones anteriores, que presenta también capacidad de inhibir las catepsinas G y L. 10. Una molécula aislada de ácido nucleico que comprende una secuencia que codifica una serpina con capacidad de inhibir la trombina humana seleccionada del grupo que consiste en: a) una secuencia de ácido nucleico que codifica una secuencia polipeptídica idéntica al menos en un 40% a la secuencia representada por SEQ ID NO:3 b) una secuencia de ácido nucleico que codifica una secuencia polipeptídica idéntica al menos en un 40% a la secuencia representada por SEQ ID NO:2; c) una molécula de ácido nucleico complementaria a una de las anteriores. 11. Molécula de ácido nucleico según la reivindicación 10, que comprende una secuencia que codifica una secuencia polipeptídica idéntica a la representada por SEQ ID NO:3. 12. Molécula de ácido nucleico según la reivindicación 10, que comprende una secuencia que codifica una secuencia polipeptídica idéntica a la representada por SEQ ID NO:2. 13. Molécula de ácido nucleico según la reivindicación 12, que comprende la secuencia representada por SEQ ID NO: 1. 14. Molécula de ácido nucleico según la reivindicación 10, que comprende un fragmento de secuencia que codifica una secuencia polipeptídica que es idéntica a la representada por SEQ ID NO:2 al menos en un porcentaje que se selecciona del grupo de 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99, 5% y 99, 9%. 15. Un vector de expresión que comprende la secuencia de ADN correspondiente a la molécula de ácido nucleico de una cualquiera de las reivindicaciones 10 a 14 unida operativamente a una secuencia de control de la expresión. 16. Una célula hospedadora transformada con un vector de expresión de la reivindicación 15. 17. Célula hospedadora según la reivindicación 16, que es una bacteria, una levadura, o una célula eucariótica. 18. Un método para producir una proteína que comprende las etapas de: a) cultivar la célula hospedadora transformada con un vector de expresión de la reivindicación 15 que permite la expresión del polipéptido con capacidad de inhibir la trombina humana codificado en dicho vector de expresión; b) purificar el polipéptido sintetizado en la etapa a) de la célula o del medio celular. 19. Uso de un polipéptido de una cualquiera de las reivindicaciones 1 a 9 para la preparación de un medicamento. 20. Uso según la reivindicación 19, en el que el medicamento está destinado a ser utilizado como anticoagulante. 21. Uso según la reivindicación 20, en el que el medicamento está destinado a pacientes en los que se sospeche que puede haber alguna alteración en proteínas de la cascada de coagulación distintas de la trombina, a pacientes inmunodeprimidos, a pacientes con trastornos hepáticos, a pacientes con coagulopatías de consumo con coagulación intravascular diseminada, a pacientes que presenten estados de hipercoagulabilidad asociados al uso de heparina o a pacientes con déficit de antitrombina III. 22. Uso según la reivindicación 20, en el que el medicamento está destinado a ser administrado como anticoagulante durante períodos perioperatorios. 23. Uso según la reivindicación 22, en el que el medicamento está destinado a ser administrado durante el período perioperatorio de la cirugía cardiopulmonar, y de la cirugía traumatológica. 24. Uso según la reivindicación 23, en el que el medicamento está destinado a ser administrado durante el período perioperatorio de la cirugía traumatológica en tratamientos de sustitución completa de cadera y rodilla. 25. Uso según la reivindicación 19, en el que el medicamento está destinado a pacientes con síndrome coronario agudo, fibrilación auricular, enfermedad coronaria crónica, prótesis mecánicas o articulares y válvulas cardíacas o vasculares. 26. Uso según una cualquiera de las reivindicaciones 20 a 25, en el que el medicamento está destinado a ser administrado sin la administración simultánea de heparina.Cuando una patente se hace internacional, se puede encontrar en el idioma de cada país en que se ha solicitado. En Espacenet se tiene acceso a los documentos en cada idioma.Instituto de Salud Carlos III; Universidad Central de VenezuelaSolicitud de patent

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

Multiple-bead assay for the differential serodiagnosis of neglected human cestodiases: Neurocysticercosis and cystic echinococcosis

Background: Neurocysticercosis (NCC), and cystic echinococcosis (CE) are two neglected diseases caused by cestodes, co-endemic in many areas of the world. Imaging studies and serological tests are used in the diagnosis of both parasitic diseases, but cross-reactions may confound the results of the latter. The novel multiplex bead-based assay with recombinant antigens has been reported to increases the diagnostic accuracy of serological techniques. Methodology: We set-up an immunoassay based on the multiplex bead-based platform (MBA), using the rT24H (against Cysticercus cellulosae, causing cysticercosis) and r2B2t (against Echinococcus granulosus sensu lato, causing CE) recombinant antigens, for simultaneous and differential diagnosis of these infections. The antigens were tested on 356 sera from 151 patients with CE, 126 patients with NCC, and 79 individuals negative for both diseases. Specificity was calculated including sera from healthy donors, other neurological diseases and the respective NCC or CE sera counterpart. The diagnostic accuracy of this assay was compared with two commercial ELISA tests, Novalisa and Ridascreen, widely used in the routine diagnosis of cysticercosis and CE, respectively. Main findings: For the diagnosis of NCC, sensitivity ranged from 57.94-63.49% for the rT24H-MBA, and 40.48-46.03% for Novalisa ELISA depending on exclusion or inclusion of sera having equivocal results on ELISA from the analysis; specificities ranged from 90.87-91.30% and 70.43-76.96%, respectively. AUC values of the ROC curve were 0.783 (rT24H) and 0.619 (Novalisa) (p-value < 0.001). For the diagnosis of CE, the sensitivity of the r2B2t-MBA ranged from 68.87-69.77% and of Ridascreen ELISA from 50.00-57.62%; specificities from 92.47-92.68% and from 74.15-80.98%, respectively. AUC values were 0.717 and 0.760, respectively. Conclusions/Significance: Overall, the recombinant antigens tested with the bead-based technology showed better diagnostic accuracy than the commercial assays, particularly for the diagnosis of NCC. The possibility of testing the same serum sample simultaneously for the presence of antibodies against both antigens is an added value particularly in seroprevalence studies for cysticercosis linked to control programs in endemic areas where these two parasites coexist.MJP received grants to support this study: “PI17CIII/00019” from ISCIII-AESI and "CIBER Enfermedades infecciosas CB21/13/00120" from ISCIII. https://www.isciii.es. BGB received a Predoctoral Fellowship award for career development by Fundación Rafael Folch (2021/E02). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Evaluation of onchocerciasis seroprevalence in Bioko Island (Equatorial Guinea) after years of disease control programmes

BACKGROUND: Onchocerciasis or "river blindness" is a chronic parasitic disease caused by the filarial worm Onchocerca volvulus, transmitted through infected blackflies (Simulium spp.). Bioko Island (Equatorial Guinea) used to show a high endemicity for onchocerciasis. During the last years, the disease control programmes using different larvicides and ivermectin administration have considerably reduced the prevalence and intensity of infection. Based on this new epidemiological scenario, in the present work we aimed to assess the impact of the strategies applied against onchocerciasis in Bioko Island by an evaluation of IgG4 antibodies specific for recombinant Ov-16 in ELISA. METHODS: A cross-sectional study was conducted in Bioko Island from mid-January to mid-February, 2014. Twenty communities were randomly selected from rural and urban settings. A total of 140 households were chosen. In every selected household, all individuals aged 5 years and above were recruited; 544 study participants agreed to be part of this work. No previous data on onchocerciasis seroprevalence in the selected communities were available. Blood samples were collected and used in an "ELISA in-house" prepared with recombinant Ov-16, expressed and further purified. IgG4 antibodies specific for recombinant Ov-16 were evaluated by ELISA in all of the participants. RESULTS: Based on the Ov-16 ELISA, the onchocerciasis seroprevalence was 7.9 %, mainly concentrated in rural settings; samples from community Catedral Ela Nguema (# 16) were missed during the field work. Among the rural setups, communities Inasa Maule (# 7), Ruiché (# 20) and Barrios Adyacentes Riaba (# 14), had the highest seropositivity percentages (29.2, 26.9 and 23.8 %, respectively). With respect to the urban settings, we did not find any positive case in communities Manzana Casa Bola (# 3), Colas Sesgas (# 6), Getesa (# 8), Moka Bioko (# 9), Impecsa (# 10), Baney Zona Baja (# 12) and Santo Tomás de Aquino (# 1). No onchocerciasis seropositive samples were found in 10-year-old individuals or younger. The IgG4 positive titles increased in older participants. CONCLUSIONS: A significant decline in onchocerciasis prevalence was observed in Bioko Island after years of disease-vector control and CDTI strategy. The seroprevalence increased with age, mainly in rural settings that could be due to previous exposure of population to the filarial parasite, eliminated by the control programmes introduced against onchocerciasis. A new Ov-16 serological evaluation with a larger sample size of children below 10 years of age is required to demonstrate the interruption of transmission of O. volvulus in the human population of Bioko Island (Equatorial Guinea) according to the WHO criteria.We would like to thank the National Program for Control of Onchocerciasis and other Filariasis in Equatorial Guinea for supporting us to obtain the information on which this study is based. We are grateful to the study participants for volunteering to participate in the study and the data collectors for performing field work. Our gratitude to the Unit of Serological Diagnosis, Department of Parasitology, Centro Nacional de Microbiología, and the Spanish Red Cross for providing some control samples. Thanks are also due to Diana Gomez-Barroso, from the National Centre of Epidemiology (ISCIII) for her help with the mapping.S

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

Intraventricular neurocysticercosis in a migrant from Honduras

We report in Madrid (Spain) a case of intraventricular neurocysticercosis in a migrant from Choluteca (Honduras) confirmed by epidemiological, radiological and microbiological criteria.S

Epidemiological Scenario of Anisakidosis in Spain Based on Associated Hospitalizations: The Tip of the Iceberg.

The risk of infection with Anisakis has been recognized for some time, but it is now emerging due to major awareness, better diagnostic techniques, and increasing preference for raw or lightly cooked food. Spain has the second-highest reported incidence after Japan, though the real anisakidosis burden is unknown because of the scarcity of epidemiological data. This study provides a 19-year review of anisakidosis-related hospitalizations describing epidemiological trends and patient characteristics. We performed a retrospective descriptive study using the Spanish Hospitalization Minimum Data Set from 1997 to 2015. Hospitalization rates were calculated and spatial distribution of cases and their temporal behavior were assessed. Clinical characteristics were described, including related codiagnoses and procedures. A total of 2471 hospital discharges were identified. A continuous increasing trend was observed, with several peaks. Most affected communities were located in the northwest inland part of the country. Almost 54% of hospitalized patients were male, with a mean age of 51.3 years. Median length of stay was 5 days, and the hospitalization median cost around €2900. Fatal outcome occurred in 0.5%. Most frequent codiagnoses were digestive diseases, mainly intestinal obstruction. Urticaria, anaphylactic reaction, and angioneurotic edema were only recorded in 2.2%, 2.4%, and 1.2%, respectively. Knowing that hospitalization is unusual in anisakidosis, we offer calculations of the real disease burden. Improving disease surveillance in parallel to disease control will be useful both in gaining extended disease knowledge and reducing morbidity and related costs.This work was supported by the European Regional Development Fund from the European Commission (contract number RD12/0018/0001).S

Delineating distinct heme-scavenging and -binding functions of domains in MF6p/helminth defense molecule (HDM) proteins from parasitic flatworms

MF6p/FhHDM-1 is a small protein secreted by the parasitic flatworm (trematode) Fasciola hepatica that belongs to a broad family of heme-binding proteins (MF6p/helminth defense molecules (HDMs)). MF6p/HDMs are of interest for understanding heme homeostasis in trematodes and as potential targets for the development of new flukicides. Moreover, interest in these molecules has also increased because of their immunomodulatory properties. Here we have extended our previous findings on the mechanism of MF6p/HDM-heme interactions and mapped the protein regions required for heme binding and for other biological functions. Our data revealed that MF6p/FhHDM-1 forms high-molecular-weight complexes when associated with heme and that these complexes are reorganized by a stacking procedure to form fibril-like and granular nanostructures. Furthermore, we showed that MF6p/FhHDM-1 is a transitory heme-binding protein as protein·heme complexes can be disrupted by contact with an apoprotein (e.g. apomyoglobin) with higher affinity for heme. We also demonstrated that (i) the heme-binding region is located in the MF6p/FhHDM-1 C-terminal moiety, which also inhibits the peroxidase-like activity of heme, and (ii) MF6p/HDMs from other trematodes, such as Opisthorchis viverrini and Paragonimus westermani, also bind heme. Finally, we observed that the N-terminal, but not the C-terminal, moiety of MF6p/HDMs has a predicted structural analogy with cell-penetrating peptides and that both the entire protein and the peptide corresponding to the N-terminal moiety of MF6p/FhHDM-1 interact in vitro with cell membranes in hemin-preconditioned erythrocytes. Our findings suggest that MF6p/HDMs can transport heme in trematodes and thereby shield the parasite from the harmful effects of heme.This work was supported in part by Ministerio de Ciencia e Innovación (Spain) Grant AGL2011-30563-C03, Xunta de Galicia (Spain) Grant GPC2014/058, Instituto de Salud Carlos III-Acción Estratégica de Salud Intramural Grant PI14CIII/00076, and the European Fund for Regional Development (FEDER). The authors declare that they have no conflicts of interest with the contents of this article.S

Collaborative Studies for the Detection of spp. Infections in Humans within CYSTINET, the European Network on Taeniosis/Cysticercosis.

Laboratory tools for diagnosing taeniosis/cysticercosis in non-endemic countries are available; however, there is little data on their performance. To provide information on the sensitivity, specificity, and reproducibility of these tools, inter-laboratory studies were organized within the EU COST-Action CYSTINET (TD1302). Two serological and one coprological Ring Trials (RTs) were organized to test a panel of human-derived sera and stool samples using assays routinely conducted by the participating laboratories to detect Taenia spp. infections. Four Western blots (WBs) and five ELISAs were used by nine laboratories for cysticercosis diagnosis. In the first serological RT, the overall sensitivity was 67.6% (95% CI, 59.1-75.4), whereas specificity was 97% (95% CI, 89.8-99.6). WBs recorded the best accuracy. A second serological RT was organized, to assess the three tests most frequently used during the first RT. Two out of six laboratories performed all the three tests. The overall sensitivity and specificity were 52.8% (95% CI, 42.8-62.7) and 98.1% (95% CI, 93.2-99.7), respectively. Laboratory performance strongly affected test results. Twelve laboratories participated in the coprological RT using conventional microscopy and six laboratories used molecular assays. Traditional diagnosis by microscopy yielded better results than molecular diagnosis. This may have been influenced by the lack of standardization of molecular tests across participating laboratories