Proteína F del VRSH en conformación pre-fusión estabilizada y anticuerpos neutralizantes específicos frente a la misma.

La presente invención proporciona una proteína de fusión (proteína F) del virus respiratorio sincitial humano (VRSH) en conformación pre-fusión estabilizada, útil para identificar o diseñar anticuerpos y otras moléculas que se unan a ella para el diagnóstico, prevención y/o tratamiento de infecciones producidas por virus de la familia Paramyxoviridae, preferiblemente del género Pneumovirus, más preferiblemente por el VRSH. La presente invención se refiere también a un método de obtención de esta proteína así como a anticuerpos y a aptámeros frente a la misma, los cuales son útiles para el diagnóstico, tratamiento y/o prevención de las infecciones mencionadas.REIVINDICACIONES: 1. Proteína F del VRSH en conformación pre-fusión que presenta al menos un 80% de homología con la SEQ ID NO: 1, que comprende: a. al menos un puente disulfuro intermonomérico en la región que comprende los aminoácidos 450 a 550 de su secuencia aminoacídica, y b. al menos dos aminoácidos básicos del punto de corte de furina que comprende los aminoácidos 106 a 109 de su secuencia aminoacídica y al menos cuatro aminoácidos básicos del punto de corte de furina que comprende los aminoácidos 131 a 136 de su secuencia aminoacídica, sustituidos por asparagina o por glutamina. 2. Proteína según la reivindicación 1 que comprende dos puentes disulfuro intermonoméricos formados mediante la sustitución de los aminoácidos Leu481, Asp489, Ser509 y Asp510 por cisteínas. 3. Proteína según cualquiera de las reivindicaciones 1 ó 2, que comprende los aminoácidos básicos Arg106, Arg108, Arg109, Lys131, Lys132, Arg133, Lys134, Arg135 y Arg136 sustituidos por asparagina. 4. Proteína según cualquiera de las reivindicaciones 1 a 3 que además comprende una cola de histidinas en el extremo C-terminal. 5. Proteína según la reivindicación 4 donde la cola de histidinas se ha formado mediante la adición de la SEQ ID NO: 2 en el extremo C-terminal de la proteína. 6. Proteína según cualquiera de las reivindicaciones 1 a 5 que consiste en la secuencia aminoacídica SEQ ID NO: 3. 7. Secuencia nucleotídica aislada que codifica para la proteína según cualquiera de las reivindicaciones 1 a 6. 8. Anticuerpo frente a la proteína según cualquiera de las reivindicaciones 1 a 6. 9. Aptámero frente a la proteína según cualquiera de las reivindicaciones 1 a 6. 10. Método de obtención de la proteína según cualquiera de las reivindicaciones 1 a 6 que comprende: a. sustituir, en una secuencia aminoacídica de la proteína F del VRSH que presenta al menos un 80% de homología con la SEQ ID NO: 1, al menos dos aminoácidos básicos del punto de corte de furina que comprende los aminoácidos 106 a 109 y al menos cuatro aminoácidos básicos del punto de corte de furina que comprende los aminoácidos 131 a 136, por asparagina o por glutamina, b. introducir al menos un puente disulfuro intermonomérico en la región que comprende los aminoácidos 450 a 550 de la secuencia aminoacídica de la proteína F del VRSH del paso (a) , c. expresar la proteína F del VRSH modificada en los pasos (a) y (b) en un sistema de expresión, y d. purificar la proteína expresada en el paso (c) . 11. Método según la reivindicación 10, donde los aminoácidos básicos del paso (a) son Arg106, Arg108, Arg109, Lys131, Lys132, Arg133, Lys134, Arg135 y Arg136 y se sustituyen por asparagina. 12. Método según cualquiera de las reivindicaciones 10 u 11, donde en el paso (b) se introducen dos puentes disulfuro intermonoméricos mediante la sustitución de los aminoácidos Leu481, Asp489, Ser509 y Asp510 de la secuencia aminoacídica de la proteína F del VRSH del paso (a) por cisteínas. 13. Método según cualquiera de las reivindicaciones 10 a 12, que además comprende añadir una cola de histidinas en el extremo C-terminal de la secuencia aminoacídica de la proteína F del VRSH del paso (b) . 14. Método según la reivindicación 13, donde la cola de histidinas se añade mediante la adición de la SEQ ID NO: 2 en el extremo C-terminal de la secuencia aminoacídica de la proteína F del VRSH del paso (b) . 15. Método según cualquiera de las reivindicaciones 10 a 14 donde el sistema de expresión del paso (c) es el virus vaccinia. 16. Uso de la proteína según cualquiera de las reivindicaciones 1 a 6 para la identificación o diseño de compuestos o composiciones para el diagnóstico, prevención y/o tratamiento de infecciones producidas por virus pertenecientes al género Pneumovirus. 17. Uso de la proteína según la reivindicación 16 donde el virus es el VRSH. 18. Uso de la proteína según cualquiera de las reivindicaciones 1 a 6 para la elaboración de un medicamento. 19. Uso de la proteína según la reivindicación 18 para la elaboración de un medicamento para el diagnóstico, prevención y/o tratamiento de infecciones producidas por virus pertenecientes al género Pneumovirus. 20. Uso de la proteína según la reivindicación 19 donde el virus es el VRSH. 21. Uso de la proteína según cualquiera de las reivindicaciones 18 a 20 donde el medicamento es una vacuna. 22. Uso del anticuerpo según la reivindicación 8 o del aptámero según la reivindicación 9 para la detección de la proteína F de virus pertenecientes al género Pneumovirus. 23. Uso del anticuerpo o del aptámero según la reivindicación 22 donde el virus es el VRSH. 24. Uso del anticuerpo según la reivindicación 8 o del aptámero según la reivindicación 9 para la elaboración de un medicamento. 25. Uso del anticuerpo o del aptámero según la reivindicación 24 para la elaboración de un medicamento para el tratamiento y/o prevención de infecciones producidas por virus pertenecientes al género Pneumovirus. 26. Uso del anticuerpo o del aptámero según la reivindicación 25 donde el virus es el VRSH.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 idiomaInstituto de Salud Carlos IIISolicitud de Patent

Structure-based design of prefusion-stabilized human metapneumovirus fusion proteins

The human metapneumovirus (hMPV) fusion (F) protein is essential for viral entry and is a key target of neutralizing antibodies and vaccine development. The prefusion conformation is thought to be the optimal vaccine antigen, but previously described prefusion F proteins expressed poorly and were not well stabilized. Here, we use structures of hMPV F to guide the design of 42 variants containing stabilizing substitutions. Through combinatorial addition of disulfide bonds, cavity-filling substitutions, and improved electrostatic interactions, we describe a prefusion-stabilized F protein (DS-CavEs2) that expresses at 15 mg/L and has a melting temperature of 71.9 °C. Crystal structures of two prefusion-stabilized hMPV F variants reveal that antigenic surfaces are largely unperturbed. Importantly, immunization of mice with DS-CavEs2 elicits significantly higher neutralizing antibody titers against hMPV A1 and B1 viruses than postfusion F. The improved properties of DS-CavEs2 will advance the development of hMPV vaccines and the isolation of therapeutic antibodies.This work was funded in part by Welch Foundation grant number F-0003-19620604 (J.S.M). Argonne is operated by UChicago Argonne, LLC, for the US Department of Energy (DOE), Office of Biological and Environmental Research under Contract DE-AC02-06CH11357.S

Immunoproteomic analysis of a Chikungunya poxvirus-based vaccine reveals high HLA class II immunoprevalence

BACKGROUND: Efficient adaptive antiviral cellular and humoral immune responses require previous recognition of viral antigenic peptides bound to human leukocyte antigen (HLA) class I and II molecules, which are exposed on the surface of infected and antigen presenting cells, respectively. The HLA-restricted immune response to Chikungunya virus (CHIKV), a mosquito-borne Alphavirus of the Togaviridae family responsible for severe chronic polyarthralgia and polyarthritis, is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a high-throughput mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of human cells infected with a vaccinia virus (VACV) recombinant expressing CHIKV structural proteins was carried out. Twelve viral ligands from the CHIKV polyprotein naturally presented by different HLA-A, -B, and -C class I, and HLA-DR and -DP class II molecules were identified. CONCLUSIONS/SIGNIFICANCE: The immunoprevalence of the HLA class II but not the HLA class I-restricted cellular immune response against the CHIKV structural polyprotein was greater than that against the VACV vector itself. In addition, most of the CHIKV HLA class I and II ligands detected by mass spectrometry are not conserved compared to its closely related O'nyong-nyong virus. These findings have clear implications for analysis of both cytotoxic and helper immune responses against CHIKV as well as for the future studies focused in the exacerbated T helper response linked to chronic musculoskeletal disorders in CHIKV patients.This work was supported by the Spanish Ministry of Economy grants SAF2014-58052 and “Acción Estratégica en Salud” 2018 to DL, SAF-2013-45232-R and SAF-2017-88089-R to ME, and by Israel Science Foundation, grant No. 1435/16 to AA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Natural Spleen Cell Ligandome in Transporter Antigen Processing-Deficient Mice.

Peptides generated by proteases in the cytosol must be translocated to endoplasmic reticulum lumen by the transporter associated with antigen processing (TAP) prior to their assembly with major histocompatibility complex (MHC) class I molecules. Nonfunctional TAP complexes produce a drastic decrease of the MHC class I/peptide complexes presented on the cell surface. Previously, the cellular MHC class I ligandome from TAP-deficient cell lines was determined, but similar analysis from normal tissues remains incomplete. Using high-throughput mass spectrometry to analyze the MHC-bound peptide pools isolated from ex vivo spleen cells of TAP-deficient mice, we identified 210 TAP-independent ligands naturally presented by murine MHC class I molecules. This ligandome showed increased peptide lengths, presence of multiple nested set peptides, and low theoretical MHC binding affinity. The gene ontology enrichment analysis of parental proteins of this TAP-independent subligandome showed almost exclusively enrichment in tissue-specific biological processes related to the immune system as would be expected. Also, cellular components of the extracellular space (namely proteins outside the cell but still within the organism excluding the extracellular matrix) were specifically associated with TAP-independent antigen processing from these ex vivo mice cells. In addition, functional protein association network analysis revealed low protein-protein interactions between parental proteins from the TAP-independent ligandome. Finally, predominant endoproteolytic peptidase specificity for Leu/Phe residues in the P1 position of the scissile bond at both ligand termini was found for the ex vivo TAP-independent ligands. These data indicate that the TAP-independent ligandome from ex vivo cells derives from a more diverse collection of both endoprotease activities and parental proteins and where the cell origin and contribution of the extracellular environment are more relevant than in its equivalent cell lines.This work was supported by the Spanish Ministry of Economy grants SAF2014-58052 and “Acción Estratégica en Salud” MPY 388/18 to D.L., and by Israel Science Foundation, grant No. 1435/16 to A. A. The funding agencies had no role in the study design, data collection, analysis decision to publish, or preparation of the manuscript.S

Proteomics Analysis Reveals That Structural Proteins of the Virion Core and Involved in Gene Expression Are the Main Source for HLA Class II Ligands in Vaccinia Virus-Infected Cells

Protective cellular and humoral immune responses require previous recognition of viral antigenic peptides complexed with human leukocyte antigen (HLA) class II molecules on the surface of the antigen presenting cells. The HLA class II-restricted immune response is important for the control and the clearance of poxvirus infection including vaccinia virus (VACV), the vaccine used in the worldwide eradication of smallpox. In this study, a mass spectrometry analysis was used to identify VACV ligands bound to HLA-DR and -DP class II molecules present on the surface of VACV-infected cells. Twenty-six naturally processed viral ligands among the tens of thousands of cell peptides bound to HLA class II proteins were identified. These viral ligands arose from 19 parental VACV proteins: A4, A5, A18, A35, A38, B5, B13, D1, D5, D7, D12, D13, E3, E8, H5, I2, I3, J2, and K2. The majority of these VACV proteins yielded one HLA ligand and were generated mainly, but not exclusively, by the classical HLA class II antigen processing pathway. Medium-sized and abundant proteins from the virion core and/or involved in the viral gene expression were the major source of VACV ligands bound to HLA-DR and -DP class II molecules. These findings will help to understand the effectiveness of current poxvirus-based vaccines and will be important in the design of new ones.This work was supported by the Spanish Ministry of Economy Grants SAF2014-58052 and “Acción Estratégica en Salud” to D.L., SAF-2013-45232-R and SAF-2017-88089-R to M.E., and by Israel Science Foundation, Grant No. 1435/16 to A.A. The funding agencies had no role in the study design, data collection, analysis decision to publish, or preparation of the manuscript.S

Vitamin K antagonist control in patients with atrial fibrillation in Asia compared with other regions of the world: Real-world data from the GARFIELD-AF registry

Objective: To compare the distribution of international normalized ratios (INRs) in patients receiving vitamin K antagonist (VKA) for newly diagnosed atrial fibrillation in Eastern and Southeastern Asia and in other regions of the world (ORW) represented in the ongoing, global observational study GARFIELD-AF

Evolving antithrombotic treatment patterns for patients with newly diagnosed atrial fibrillation

Objective We studied evolving antithrombotic therapy patterns in patients with newly diagnosed non-valvular atrial fibrillation (AF) and >= 1 additional stroke risk factor between 2010 and 2015

Two-year outcomes of patients with newly diagnosed atrial fibrillation: results from GARFIELD-AF

The relationship between outcomes and time after diagnosis for patients with non-valvular atrial fibrillation (NVAF) is poorly defined, especially beyond the first year