Mutantes replicativos y atenuados del virus vaccinia como candidatos vacunales frente a la leishmaniasis

Tesis doctoral inédita, leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 19-10-201

SEVA 4.0: an update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes

10 Pág.The SEVA platform (https://seva-plasmids.com) was launched one decade ago, both as a database (DB) and as a physical repository of plasmid vectors for genetic analysis and engineering of Gram-negative bacteria with a structure and nomenclature that follows a strict, fixed architecture of functional DNA segments. While the current update keeps the basic features of earlier versions, the platform has been upgraded not only with many more ready-to-use plasmids but also with features that expand the range of target species, harmonize DNA assembly methods and enable new applications. In particular, SEVA 4.0 includes (i) a sub-collection of plasmids for easing the composition of multiple DNA segments with MoClo/Golden Gate technology, (ii) vectors for Gram-positive bacteria and yeast and [iii] off-the-shelf constructs with built-in functionalities. A growing collection of plasmids that capture part of the standard-but not its entirety-has been compiled also into the DB and repository as a separate corpus (SEVAsib) because of its value as a resource for constructing and deploying phenotypes of interest. Maintenance and curation of the DB were accompanied by dedicated diffusion and communication channels that make the SEVA platform a popular resource for genetic analyses, genome editing and bioengineering of a large number of microorganisms.The SEVA repository has been developed and maintained with funds of the SYCOLIM [ERA-COBIOTECH 2018-PCI2019-111859-2] Project of the Spanish Ministry of Science and Innovation, SYNBIO4FLAV [H2020-NMBP-TR-IND/H2020-NMBP-BIO-2018-814650]; MIX-UP [MIX-UP H2020-BIO-CN-2019-870294] Contracts of the European Union; BIOSINT-CM [Y2020/TCS-6555] Project of the Comunidad de Madrid-European Structural and Investment Funds (FSE, FECER); P.I.N. acknowledges financial support by the Novo Nordisk Foundation [NNF20CC0035580, TARGET (NNF21OC0067996]; European Union's Horizon 2020 Research and Innovation Programme [814418 (SinFonia)]; M.H.H.N. acknowledges funding by the Novo Nordisk Foundation [NNF20CC0035580]; P.D. was funded by Czech Science Foundation Project 22-12505S; A.G.M. was supported by the Grants BioSinT-CM [Y2020/TCS-6555]; CONTEXT (Atracción de Talento Program) [2019-T1/BIO-14053] Projects of the Comunidad de Madrid, MULTI-SYSBIO [PID2020-117205GA-I00]; Severo Ochoa Program for Centres of Excellence in R&D [CEX2020-000999-S] funded by MCIN/AEI/10.13039/501100011033 and the ECCO (ERC-2021-COG-101044360) Contract of the EU. Funding for open access charge: European Commission Grant SYNBIO4FLAV [H2020-NMBP-TR-IND/H2020-NMBP-BIO-2018-814650].With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2020‐000999‐S) .Peer reviewe

High Quality Long-Term CD4+ and CD8+ Effector Memory Populations Stimulated by DNA-LACK/MVA-LACK Regimen in Leishmania major BALB/c Model of Infection

Heterologous vaccination based on priming with a plasmid DNA vector and boosting with an attenuated vaccinia virus MVA recombinant, with both vectors expressing the Leishmania infantum LACK antigen (DNA-LACK and MVA-LACK), has shown efficacy conferring protection in murine and canine models against cutaneus and visceral leishmaniasis, but the immune parameters of protection remain ill defined. Here we performed by flow cytometry an in depth analysis of the T cell populations induced in BALB/c mice during the vaccination protocol DNA-LACK/MVA-LACK, as well as after challenge with L. major parasites. In the adaptive response, there is a polyfunctional CD4+ and CD8+ T cell activation against LACK antigen. At the memory phase the heterologous vaccination induces high quality LACK-specific long-term CD4+ and CD8+ effector memory cells. After parasite challenge, there is a moderate boosting of LACK-specific CD4+ and CD8+ T cells. Anti-vector responses were largely CD8+-mediated. The immune parameters induced against LACK and triggered by the combined vaccination DNA/MVA protocol, like polyfunctionality of CD4+ and CD8+ T cells with an effector phenotype, could be relevant in protection against leishmaniasis

Tratamientos psicológicos empíricamente apoyados para adultos: Una revisión selectiva

Antecedentes: los tratamientos psicológicos han mostrado su eficacia, efectividad y eficiencia para el abordaje de los trastornos mentales; no obstante, considerando el conocimiento científico generado en los últimos años, no se dispone de trabajos de actualización en español sobre cuáles son los tratamientos psicológicos con respaldo empírico. El objetivo fue realizar una revisión selectiva de los principales tratamientos psicológicos empíricamente apoyados para el abordaje de trastornos mentales en personas adultas. Método: se recogen niveles de evidencia y grados de recomendación en función de los criterios propuestos por el Sistema Nacional de Salud de España (en las Guías de Práctica Clínica) para diferentes trastornos psicológicos. Resultados: los resultados sugieren que los tratamientos psicológicos disponen de apoyo empírico para el abordaje de un amplio elenco de trastornos psicológicos. El grado de apoyo empírico oscila de bajo a alto en función del trastorno psicológico analizado. La revisión sugiere que ciertos campos de intervención necesitan una mayor investigación. Conclusiones: a partir de esta revisión selectiva, los profesionales de la psicología podrán disponer de información rigurosa y actualizada que les permita tomar decisiones informadas a la hora de implementar aquellos procedimientos psicoterapéuticos empíricamente fundamentados en función de las características de las personas que demandan ayuda. Background: Psychological treatments have shown their efficacy, effectiveness, and efficiency in dealing with mental disorders. However, considering the scientific knowledge generated in recent years, in the Spanish context, there are no updating studies about empirically supported psychological treatments. The main goal was to carry out a selective review of the main empirically supported psychological treatments for mental disorders in adults. Method: Levels of evidence and degrees of recommendation were collected based on the criteria proposed by the Spanish National Health System (Clinical Practice Guidelines) for different psychological disorders. Results: The results indicate that psychological treatments have empirical support for the approach to a wide range of psychological disorders. These levels of empirical evidence gathered range from low to high depending on the psychological disorder analysed. The review indicates the existence of certain fields of intervention that need further investigation. Conclusions: Based on this selective review, psychology professionals will be able to have rigorous, up-to-date information that allows them to make informed decisions when implementing empirically based psychotherapeutic procedures based on the characteristics of the people who require help

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

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Antigenicity of Leishmania-Activated C-Kinase Antigen (LACK) in Human Peripheral Blood Mononuclear Cells, and Protective Effect of Prime-Boost Vaccination With pCI-neo-LACK Plus Attenuated LACK-Expressing Vaccinia Viruses in Hamsters

Leishmania-activated C-kinase antigen (LACK) is a highly conserved protein among Leishmania species and is considered a viable vaccine candidate for human leishmaniasis. In animal models, prime-boost vaccination with LACK-expressing plasmids plus attenuated vaccinia viruses (modified vaccinia Ankara [MVA] and mutant M65) expressing LACK, has been shown to protect against cutaneous leishmaniasis (CL). Further, LACK demonstrated to induce the production of protective cytokines in patients with active CL or cured visceral leishmaniasis, as well as in asymptomatic individuals from endemic areas. However, whether LACK is capable to trigger cytokine release by peripheral blood mononuclear cells from patients cured of CL due to Leishmania infantum (L. infantum) or induce protection in L. infantum-infected hamsters [visceral leishmaniasis (VL) model], has not yet been analyzed. The present work examines the ex vivo immunogenicity of LACK in cured VL and CL patients, and asymptomatic subjects from an L. infantum area. It also evaluates the vaccine potential of LACK against L. infantum infection in hamsters, in a protocol of priming with plasmid pCI-neo-LACK (DNA-LACK) followed by a booster with the poxvirus vectors MVA-LACK or M65-LACK. LACK-stimulated PBMC from both asymptomatic and cured subjects responded by producing IFN-γ, TNF-α, and granzyme B (Th1-type response). Further, 78% of PBMC samples that responded to soluble Leishmania antigen showed IFN-γ secretion following stimulation with LACK. In hamsters, the protocol of DNA-LACK prime/MVA-LACK or M65-LACK virus boost vaccination significantly reduced the amount of Leishmania DNA in the liver and bone marrow, with no differences recorded between the use of MVA or M65 virus vector options. In summary, the Th1-type and cytotoxic responses elicited by LACK in PBMC from human subjects infected with L. infantum, and the parasite protective effect of prime/boost vaccination in hamsters with DNA-LACK/MVA-LACK and DNA-LACK/M65-LACK, revealed the significance of LACK in activating human and hamster immune responses and support LACK to be a valuable candidate for inclusion in a vaccine against human VL.EC was supported by a contract from RD16CIII/0003/0002 Red de Enfermedades Tropicales, Subprograma RETICS del Plan Estatal de I + D + I 2013-2016, co-funded by FEDER “Una manera de hacer Europa” funds. ME was supported by AEI MINECO/FEDER grant SAF2013-45232-R.S

The Evolution of Poxvirus Vaccines

After Edward Jenner established human vaccination over 200 years ago, attenuated poxviruses became key players to contain the deadliest virus of its own family: Variola virus (VARV), the causative agent of smallpox. Cowpox virus (CPXV) and horsepox virus (HSPV) were extensively used to this end, passaged in cattle and humans until the appearance of vaccinia virus (VACV), which was used in the final campaigns aimed to eradicate the disease, an endeavor that was accomplished by the World Health Organization (WHO) in 1980. Ever since, naturally evolved strains used for vaccination were introduced into research laboratories where VACV and other poxviruses with improved safety profiles were generated. Recombinant DNA technology along with the DNA genome features of this virus family allowed the generation of vaccines against heterologous diseases, and the specific insertion and deletion of poxvirus genes generated an even broader spectrum of modified viruses with new properties that increase their immunogenicity and safety profile as vaccine vectors. In this review, we highlight the evolution of poxvirus vaccines, from first generation to the current status, pointing out how different vaccines have emerged and approaches that are being followed up in the development of more rational vaccines against a wide range of diseases

Analysis of humoral and cellular responses triggered by the MVA vector.

<p>A. IgG total antibodies at different points post-immunization where measured by ELISA using extracts of BSC40 cells infected with VACV. B. Analysis of the E3-specific IFNγ secreting cells by splenocytes measured by ELISPOT 11 days after the booster (prechallenge), 10 days after experimental challenge (postchallenge) or 53 days after the booster (memory). Among the lymphocyte population, T cells were gated and analyzed for IFNγ, TNFα and/or IL-2 production. C. Cytokine production by E3-specific CD8⁺ T cells 11 or 53 days after the booster.</p