Influencia de la diversidad inicial y la pauta de cambio ambiental en a evolución del bacteriófago Qβ

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Física de la Materia Condensada. Fecha de Lectura: 29-06-2021La evolución viral está condicionada por multitud de factores entre los que se encuentran la velocidad de cambio en las presiones selectivas y la diversidad genética preexistente en las poblaciones. En general, cuanto más intenso sea el cambio ambiental mayor será la reducción del tamaño de la población, aumentando así la dificultad para generar y seleccionar mutaciones adaptativas, lo que puede conducir a la extinción. Por el contrario, los cambios graduales, que aumentan las presiones selectivas de forma progresiva, suelen ser más compatibles con la adaptación. La diversidad genética preexistente puede facilitar el proceso si en el espectro de mutantes que componen las poblaciones virales se encuentra ya algún genoma con ventajas adaptativas en las nuevas condiciones. En esta tesis doctoral hemos planteado varios experimentos de evolución en los que hemos estudiado los cambios fenotípicos y genotípicos que experimenta un bacteriófago de RNA (Qβ) cuando se propaga a temperaturas diferentes de la óptima, siguiendo diferentes patrones de cambio. A lo largo del proceso se han determinado los valores de la tasa de crecimiento, las secuencias consenso de las poblaciones y también se ha caracterizado el espectro de mutantes mediante tecnologías de secuenciación masiva. Los resultados más relevantes obtenidos muestran que las poblaciones que contienen mayor diversidad genética inicial se adaptan más rápido a un aumento de temperatura (de 37 ᵒC a 43 ᵒC) que las que parten de una población menos diversa. Los análisis de secuenciación masiva indican que en el primer caso se produce un cuello de botella poblacional que podría ser debido a la rápida selección de algún genoma minoritario que estuviera ya presente en la población ancestral. La velocidad de la adaptación también aumenta con la velocidad de cambio de temperatura. A pesar de que hay bastante coincidencia entre las mutaciones que se seleccionan al final del proceso en todos los casos analizados, las vías adaptativas seguidas no son las mismas, como se demuestra por las diferencias en las mutaciones que se fijan en primer lugar y el número de mutaciones de la población ancestral que se mantienen en el nuevo ambiente. La estructura de la población y la diversidad genética contenida en ella también experimentan cambios de distinta intensidad en función de la pauta de cambio y el estado inicial de la población. Por último, la propagación de Qβ en condiciones que alternan la replicación a dos temperaturas subóptimas favorece la adaptación a la que genera la presión selectiva más intensa y lo hace a través de las mismas mutaciones que se seleccionan durante la adaptación constante a esa misma temperatura. Sin embargo, la estructura de la población alternante se asemeja más a la evolucionada de forma constante a la temperatura que ejerce menor presión selectiva, mostrando así que ambas temperaturas influyen en las propiedades de la población evolucionada

Fitness-Dependent, Mild Mutagenic Activity of Sofosbuvir for Hepatitis C Virus

The concept of a mild mutagen was coined to describe a minor mutagenic activity exhibited by some nucleoside analogues that potentiated their efficacy as antiretroviral agents. In the present study, we report the mild mutagen activity of sofosbuvir (SOF) for hepatitis C virus (HCV). Serial passages of HCV in human hepatoma cells, in the presence of SOF at a concentration well below its cytotoxic concentration 50 (CC) led to pre-extinction populations whose mutant spectra exhibited a significant increase of C!U transitions, relative to populations passaged in the absence of SOF. This was reflected in an increase in several diversity indices that were used to characterize viral quasispecies. The mild mutagenic activity of SOF was largely absent when it was tested with isogenic HCV populations that displayed high replicative fitness. Thus, SOF can act as a mild mutagen for HCV, depending on HCV fitness. Possible mechanisms by which the SOF mutagenic activity may contribute to its antiviral efficacy are discussed.The work at CBMSO was supported by grants SAF2014-52400-R from Ministerio de Economía y Competitividad (MINECO); SAF2017-87846-R and BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU); project 525/C/2021 from Fundació La Marató de TV3; PID2020-113888RB-I00 and 202220I116 from Ministerio de Ciencia e Innovación (MICINN); PI18/00210, PI19/00301, PI21/00139, and PI22/00258 from Instituto de Salud Carlos III (ISCIII); S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER); and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). This research work was also funded by the European Commission-NextGenerationEU (regulation EU 2020/ 2094) through the CSIC’s Global Health Platform (PTI Salud Global). C.P. is supported by the Miguel Servet program of the ISCIII (CP14/00121 and CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). B.M.-G. is supported by the predoctoral contract PFIS FI19/00119 from ISCIII, cofinanced by Fondo Social Europeo (FSE), “El FSE invierte en tu futuro”. C.G.-C. is supported by the predoctoral contract PRE2018-083422 from MCIU. P.S. is supported by the postdoctoral contract Margarita Salas, CA1/RSUE/ 2021 from MCIU. A.D.-P. is supported by the contract 13-2022-008566 cofinanced by the Comunidad de Madrid, through the Programa Investigo, en el marco del Plan de Recuperación, Transformación y Resiliencia, financed by the European Union-Next Generation EU. CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas), which is funded by ISCIII. The work at CAB was supported by the MICINN grant no. PID2019-104903RB-I00. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN)

SARS-CoV-2 mutant spectra reveal differences between COVID-19 severity categories

Trabajo presentado en el XVI Congreso Nacional de Virología, celebrado en Málaga (España) del 06 al 09 de septiembre de 2022.RNA virus populations are composed of complex mixtures of genomes that are termed mutant spectra. SARS-CoV-2 replicates as a viral quasispecies, and mutations that are detected at low frequencies in a host can be dominant in subsequent variants. We have studied mutant spectrum complexities of SARS-CoV-2 populations derived from thirty nasopharyngeal swabs of patients infected during the first wave (April 2020) in the Hospital Universitario Fundación Jiménez Díaz. The patients were classified according to the COVID-19 severity in mild (non-hospitalized), moderate (hospitalized) and exitus (hospitalized with ICU admission and who passed away due to COVID-19). Using ultra-deep sequencing technologies (MiSeq, Illumina), we have examined four amplicons of the nsp12 (polymerase)-coding region and two amplicons of the spike-coding region. Ultra-deep sequencing data were analyzed with different cut-off frequency for mutation detection. Average number of different point mutations, mutations per haplotype and several diversity indices were significantly higher in SARS-CoV-2 isolated from patients who developed mild disease. A feature that we noted in the SARS-CoV-2 mutant spectra from diagnostic samples is the remarkable absence of mutations at intermediate frequencies, and an overwhelming abundance of mutations at frequencies lower than 10%. Thus, the decrease of the cut-off frequency for mutation detection from 0.5% to 0.1% revealed an increasement (50- to 100 fold) in the number of different mutations. The significantly higher frequency of mutations in virus from patients displaying mild than moderate or severe disease was maintained with the 0.1% cut- off frequency. To evaluate whether the frequency repertoire of amino acid substitutions differed between SARS-CoV-2 and the well characterized hepatitis C virus (HCV), we performed a comparative study of mutant spectra from infected patients using the same bioinformatics pipelines. HCV did not show the deficit of intermediate frequency substitutions that was observed with SARS-CoV-2. This difference was maintained when two functionally equivalent proteins, the corresponding viral polymerases, were compared. In conclusion, SARS-CoV-2 mutant spectra are rich reservoirs of mutants, whose complexity is not uniform among clinical isolates. Virus from patients who developed mild disease may be a source of new variants that may acquire epidemiological relevance.This work was supported by Instituto de Salud Carlos III, Spanish Ministry of Science and In-novation (COVID-19 Research Call COV20/00181), and co-financed by European Development Regional Fund ‘A way to achieve Europe’. The work was also supported by grants CSIC-COV19-014 from Consejo Superior de Investigaciones Científicas (CSIC), project 525/C/2021 from Fundació La Marató de TV3, PID2020-113888RB-I00 from Ministerio de Ciencia e Innovación, BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 and PI21/00139 from Instituto de Salud Carlos III, and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P., M.C., and P.M. are supported by the Miguel Servet programme of the Instituto de Salud Carlos III (CPII19/00001, CPII17/00006, and CP16/00116, respectively) co-financed by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investi-gación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo) cofinanced by Fondo Social Europeo (FSE). R.L.-V. is supported by predoctoral contract PEJD-2019-PRE/BMD-16414 from Comunidad de Madrid. C.G.-C. is sup-ported by predoctoral contract PRE2018-083422 from MCIU. BS was supported by a predoctoral research fellowship (Doctorados Industriales, DI-17-09134) from Spanish MINECO

SARS-CoV-2 mutant spectra as variant of concern nurseries: endless variation?

SARS-CoV-2 isolates of a given clade may contain low frequency genomes that encode amino acids or deletions which are typical of a different clade.Peer reviewe

Atypical Mutational Spectrum of SARS-CoV-2 Replicating in the Presence of Ribavirin

We report that ribavirin exerts an inhibitory and mutagenic activity on SARS-CoV-2-infecting Vero cells, with a therapeutic index higher than 10. Deep sequencing analysis of the mutant spectrum of SARS-CoV-2 replicating in the absence or presence of ribavirin indicated an increase in the number of mutations, but not in deletions, and modification of diversity indices, expected from a mutagenic activity. Notably, the major mutation types enhanced by replication in the presence of ribavirin were A→G and U→C transitions, a pattern which is opposite to the dominance of G→A and C→U transitions previously described for most RNA viruses. Implications of the inhibitory activity of ribavirin, and the atypical mutational bias produced on SARS-CoV-2, for the search for synergistic anti-COVID-19 lethal mutagen combinations are discussed.This work was supported by Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID-19 Research Call COV20/00181) and cofinanced by the European Development Regional Fund, “A way to achieve Europe.” The work was also supported by grants CSIC-COV19-014 from Consejo Superior de Investigaciones Científicas (CSIC), project 525/C/2021 from Fundació La Marató de TV3, PID2020-113888RB-I00 and 202220I116 from Ministerio de Ciencia e Innovación, BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 and PI21/00139 from Instituto de Salud Carlos III, and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). This research work was also funded by the European Commission-NextGenerationEU (regulation EU 2020/2024) through the CSIC’s Global Health Platform (PTI Salud Global). C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121 and CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). C.G.-C. is supported by predoctoral contract PRE2018-083422 from MCIU. P.S. is supported by postdoctoral contract Margarita Salas, CA1/RSUE/2021 from MCIU. B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from ISCIII, cofinanced by Fondo Social Europeo (FSE).Peer reviewe

Synergism between remdesivir and ribavirin leads to SARS-CoV-2 extinction in cell culture

There is a need for effective anti-COVID-19 treatments, mainly for individuals at risk of severe disease such as the elderly and the immunosuppressed. Drug repositioning has proved effective in identifying drugs that can find a new application for the control of coronavirus disease, in particular COVID-19. The purpose of the present study was to find synergistic antiviral combinations for COVID-19 based on lethal mutagenesis.Peer reviewe

SARS-CoV-2 Mutant Spectra at Different Depth Levels Reveal an Overwhelming Abundance of Low Frequency Mutations

Populations of RNA viruses are composed of complex and dynamic mixtures of variant genomes that are termed mutant spectra or mutant clouds. This applies also to SARS-CoV-2, and mutations that are detected at low frequency in an infected individual can be dominant (represented in the consensus sequence) in subsequent variants of interest or variants of concern. Here we briefly review the main conclusions of our work on mutant spectrum characterization of hepatitis C virus (HCV) and SARS-CoV-2 at the nucleotide and amino acid levels and address the following two new questions derived from previous results: (i) how is the SARS-CoV-2 mutant and deletion spectrum composition in diagnostic samples, when examined at progressively lower cut-off mutant frequency values in ultra-deep sequencing; (ii) how the frequency distribution of minority amino acid substitutions in SARS-CoV-2 compares with that of HCV sampled also from infected patients. The main conclusions are the following: (i) the number of different mutations found at low frequency in SARS-CoV-2 mutant spectra increases dramatically (50- to 100-fold) as the cut-off frequency for mutation detection is lowered from 0.5% to 0.1%, and (ii) that, contrary to HCV, SARS-CoV-2 mutant spectra exhibit a deficit of intermediate frequency amino acid substitutions. The possible origin and implications of mutant spectrum differences among RNA viruses are discussed.This work was supported by Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID-19 Research Call COV20/00181), and co-financed by European Development Regional Fund ‘A way to achieve Europe’. The work was also supported by grants CSIC-COV19-014 from Consejo Superior de Investigaciones Científicas (CSIC), project 525/C/2021 from Fundació La Marató de TV3, PID2020-113888RB-I00 from Ministerio de Ciencia e Innovación, BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 and PI21/00139 from Instituto de Salud Carlos III, and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P., M.C., and P.M. are supported by the Miguel Servet programme of the Instituto de Salud Carlos III (CPII19/00001, CPII17/00006, and CP16/00116, respectively) cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo) cofinanced by Fondo Social Europeo (FSE). R.L.-V. is supported by predoctoral contract PEJD-2019-PRE/BMD-16414 from Comunidad de Madrid. C.G.-C. is supported by predoctoral contract PRE2018-083422 from MCIU. P.S. is supported by postdoctoral contract “Margarita Salas” CA1/RSUE/2021 from MCIU. B.S. was supported by a predoctoral research fellowship (Doctorados Industriales, DI-17-09134) from Spanish MINECO.Peer reviewe

Newer generations of multi-target CAR and STAb-T immunotherapeutics: NEXT CART Consortium as a cooperative effort to overcome current limitations

Adoptive T cellular immunotherapies have emerged as relevant approaches for treating cancer patients who have relapsed or become refractory (R/R) to traditional cancer treatments. Chimeric antigen receptor (CAR) T-cell therapy has improved survival in various hematological malignancies. However, significant limitations still impede the widespread adoption of these therapies in most cancers. To advance in this field, six research groups have created the “NEXT Generation CART MAD Consortium” (NEXT CART) in Madrid’s Community, which aims to develop novel cell-based immunotherapies for R/R and poor prognosis cancers. At NEXT CART, various basic and translational research groups and hospitals in Madrid concur to share and synergize their basic expertise in immunotherapy, gene therapy, and immunological synapse, and clinical expertise in pediatric and adult oncology. NEXT CART goal is to develop new cell engineering approaches and treatments for R/R adult and pediatric neoplasms to evaluate in multicenter clinical trials. Here, we discuss the current limitations of T cell-based therapies and introduce our perspective on future developments. Advancement opportunities include developing allogeneic products, optimizing CAR signaling domains, combining cellular immunotherapies, multi-targeting strategies, and improving tumor-infiltrating lymphocytes (TILs)/T cell receptor (TCR) therapy. Furthermore, basic studies aim to identify novel tumor targets, tumor molecules in the tumor microenvironment that impact CAR efficacy, and strategies to enhance the efficiency of the immunological synapse between immune and tumor cells. Our perspective of current cellular immunotherapy underscores the potential of these treatments while acknowledging the existing hurdles that demand innovative solutions to develop their potential for cancer treatment fully

Evolutionary dynamics in the RNA bacteriophage Qβ depends on the pattern of change in selective pressures

© 2019 by the authors.The rate of change in selective pressures is one of the main factors that determines the likelihood that populations can adapt to stress conditions. Generally, the reduction in the population size that accompanies abrupt environmental changes makes it difficult to generate and select adaptive mutations. However, in systems with high genetic diversity, as happens in RNA viruses, mutations with beneficial effects under new conditions can already be present in the population, facilitating adaptation. In this work, we have propagated an RNA bacteriophage (Qβ) at temperatures higher than the optimum, following different patterns of change. We have determined the fitness values and the consensus sequences of all lineages throughout the evolutionary process in order to establish correspondences between fitness variations and adaptive pathways. Our results show that populations subjected to a sudden temperature change gain fitness and fix mutations faster than those subjected to gradual changes, differing also in the particular selected mutations. The life-history of populations prior to the environmental change has great importance in the dynamics of adaptation. The conclusion is that in the bacteriophage Qβ, the standing genetic diversity together with the rate of temperature change determine both the rapidity of adaptation and the followed evolutionary pathways.This research was funded by the Spanish Ministerio de Ciencia, Innovación y Universidades, grant number FIS2017-89773-P.Peer reviewe

Standing Genetic Diversity and Transmission Bottleneck Size Drive Adaptation in Bacteriophage Qβ

A critical issue to understanding how populations adapt to new selective pressures is the relative contribution of the initial standing genetic diversity versus that generated de novo. RNA viruses are an excellent model to study this question, as they form highly heterogeneous populations whose genetic diversity can be modulated by factors such as the number of generations, the size of population bottlenecks, or exposure to new environment conditions. In this work, we propagated at nonoptimal temperature (43 °C) two bacteriophage Qβ populations differing in their degree of heterogeneity. Deep sequencing analysis showed that, prior to the temperature change, the most heterogeneous population contained some low-frequency mutations that had previously been detected in the consensus sequences of other Qβ populations adapted to 43 °C. Evolved populations with origin in this ancestor reached similar growth rates, but the adaptive pathways depended on the frequency of these standing mutations and the transmission bottleneck size. In contrast, the growth rate achieved by populations with origin in the less heterogeneous ancestor did depend on the transmission bottleneck size. The conclusion is that viral diversification in a particular environment may lead to the emergence of mutants capable of accelerating adaptation when the environment changes