Biology subjects

Sevilla, J.

Publications and source records attributed to Sevilla, J..

7 recordsLinked to original sources

bioRxiv2025

Differential CD8+ T/NK cell-mediated reduction of HIV-1 replication after combination of ART with TIGIT or KLRG1 blockade in humanized mice

Expression of TIGIT and KLRG1 has been associated to an exhausted, dysfunctional state in natural killer (NK) and CD8+ T cells from people with HIV-1 (PWH), limiting the efficacy of immunotherapies aiming at achieving a functional cure of the infection. Antiretroviral therapy (ART) does not completely reverse this immune exhaustion, and its combination with blockade of immune checkpoint receptors such as TIGIT and KLRG1 could be a promising strategy to promote control of viral replication in PWH. However, the impact of targeting these two immune checkpoint receptors has not been evaluated in vivo. In this study, we used a humanized Bone Marrow, Liver and Thymus (hBLT) mouse model of HIV-1 infection to study the impact of ART in combination with aTIGIT or aKLRG1 or a bispecific aTIGIT/aKLRG1 mAbs. Our results indicated that combination of ART with either aTIGIT or aKLRG1 mAbs led to faster reduction of HIV-1 pVL. Furthermore, viral rebound after ART interruption (ATI) was delayed in mice treated with aTIGIT and aKLRG1 mAbs. Histological detection of HIV-1 p24 in the spleen was restricted to the white pulp in the aKLRG1 mAb-treated group, which correlated with higher infiltration of IFN{gamma}+ CD8+ T cells in these histological regions and with increased cytotoxic CD107a+ Granzyme B+ CD8+ T cells in the spleen. In contrast, control of HIV-1 replication induced by the aTIGIT mAb was associated with an increased splenic CD107a+ IFN{gamma}+ NKG2C+ CD57-adaptive NK cells. In contrast, combination of ART with a bispecific aTIGIT/aKLRG1 mAb was unable to efficiently suppress viral replication or delay viral rebound after ATI, potentially by inducing apoptosis of adaptive NKG2C+ NK. Together, these results suggest that combination of ART with individual TIGIT or KLRG1 blockade may be a promising immunotherapy strategy against HIV-1 by eliciting differential immune control mechanisms.

immunology↗

bioRxiv2025

Inhibition of NLRP1 Inflammasome Activation by Tyrosine Kinase Inhibitors Restores Erythropoiesis in Diamond-Blackfan Anemia Syndrome

Diamond-Blackfan Anemia Syndrome (DBAS) is characterized by impaired erythropoiesis due to dysfunctional ribosome biogenesis and aberrant cellular signaling. Here, we investigate how ribosomal stress-induced activation of the NLRP1 inflammasome modulates erythroid differentiation in DBAS. We demonstrate that FDA/EMA-approved tyrosine kinase inhibitors (TKIs) effectively mitigate defective erythropoiesis in Diamond-Blackfan anemia syndrome (DBAS) by inhibiting NLRP1 inflammasome activation. Specifically, nilotinib enhances erythroid differentiation in K562 cells through suppression of the ZAK/P38/NLRP1/CASP1 axis, leading to increased GATA1 protein levels and upregulation of key erythroid genes involved in iron acquisition, hemoglobin synthesis, and erythrocyte structure. These effects were validated in human CD34+ hematopoietic stem and progenitor cells (HSPCs) and zebrafish models, where nilotinib, along with other TKIs (imatinib, dasatinib, and bosutinib), promoted erythropoiesis at the expense of myelopoiesis and reduced caspase-1 activity. Importantly, in RPS19-deficient zebrafish and human models and HSPCs from patients with DBAS, nilotinib, imatinib and dasatinib rescued defective erythroid differentiation and restored hemoglobin levels. These findings highlight the potential of TKIs to address the erythroid defects observed in ribosomopathies like DBAS. Given the limited treatment options available for DBAS and other congenital anemias, our study provides compelling evidence for repurposing TKIs as a novel therapeutic strategy to alleviate pathological NLRP1 activation and improve erythropoiesis. This work opens new avenues for managing ribosome-related disorders and advancing personalized medicine approaches for hematopoietic diseases.

immunology↗

bioRxiv2024

Improved prediction of antimicrobial resistance in Klebsiella pneumoniae using machine learning

Klebsiella pneumoniae is an important cause of healthcare-associated infections, with high levels of antimicrobial resistance (AMR) to critical antibiotics such as carbapenems and third-generation cephalosporins (3GCs). Accurate antimicrobial susceptibility detection is essential for guiding appropriate treatment. In this study, we evaluated the efficacy of machine learning (ML) models for predicting AMR phenotypes in K. pneumoniae particularly for antibiotics for which rule-based approaches fail. We analyzed a dataset of 5,907 K. pneumoniae genomes from public databases and a genomic surveillance project in Spanish hospitals. ML models were trained to predict AMR phenotypes using genomic features, and their performance was compared to ResFinder, which implements a conventional rule-based approach. Models were evaluated based on predictive accuracy across antibiotics. Additionally, we conducted a detailed analysis of the genomic features associated with AMR identified by ML to identify new putative AMR determinants. ResFinder exhibited low prediction accuracy for amikacin, fosfomycin, and piperacillin/tazobactam, whereas ML models significantly improved the prediction accuracy for these antibiotics. Moreover, we provide insights into why rule-based methods failed in these cases, specifically related to the genes acc(6)-Ib-cr, fosA, and blaOXA-1, respectively. Finally, we found possible genetic factors related to resistance for each antibiotic. Our findings underscore the value of ML models in AMR prediction based on genome information for K. pneumoniae, especially in challenging cases where traditional methods have low success rates. Continued evaluation and refinement of ML approaches are essential for applying these methods to enhance AMR detection in clinical and public health contexts. ImportanceTo combat antimicrobial resistance (AMR), the rapid and accurate identification of resistance phenotypes is essential for guiding appropriate therapy. In this study, we demonstrate the significant potential of machine learning (ML) to improve AMR prediction in Klebsiella pneumoniae using genomic data. Our findings reveal that gold standard rule-based methods for predicting AMR from genomic data underperform for antibiotics such as amikacin, fosfomycin, and piperacillin/tazobactam. In this study, we identified the genomic determinants that mislead resistance predictions in rule-based methods providing insights that can refine existing rule-based approaches. Moreover we used ML models that improved the prediction accuracy for these antibiotics and used these models to uncover potential new AMR-associated genes that contribute to a deeper understanding of resistance mechanisms. While these findings are specific to K. pneumoniae, the ML approach is broadly applicable to other pathogens facing similar challenges, enabling improved AMR prediction without reliance on prior knowledge.

genomics↗

bioRxiv2023

VIPERA: Viral Intra-Patient Evolution Reporting and Analysis

Viral mutations within patients nurture the adaptive potential of SARS-CoV-2 during chronic infections, which are a potential source of variants of concern. However, there is no integrated framework for the evolutionary analysis of intra-patient SARS-CoV-2 serial samples. Herein we describe VIPERA (Viral Intra-Patient Evolution Reporting and Analysis), a new software that integrates the evaluation of the intra-patient ancestry of SARS-CoV-2 sequences with the analysis of evolutionary trajectories of serial sequences from the same viral infection. We have validated it using positive and negative control datasets and have successfully applied it to a new case, thus enabling an easy and automatic analysis of intra-patient SARS-CoV-2 sequences.

bioinformatics↗

bioRxiv2022

Tracking the emergence and dissemination of a blaNDM-23 Gene in a Multi-Drug Resistance Plasmid of Klebsiella pneumoniae

ObjectivesSince the discovery of blaNDM-1, NDM beta-lactamases have become one of the most widespread carbapenemases worldwide. To date, 28 different NDM variants have been reported but some, such as blaNDM-23, have not been characterized in detail yet. Here, we describe the emergence of a novel blaNDM-23 allele from a blaNDM-1 ancestor and the multidrug resistant plasmid that has disseminated it through a K. pneumoniae ST407 clone in several Spanish hospitals. MethodsBetween 2016 and 2019, 1,972 isolates were collected in an epidemiological survey for ESBL-producing Klebsiella pneumoniae in the Comunitat Valenciana (Spain). Three carbapenem resistant strains failed to be detected by CPE screening tests. These isolates carried a blaNDM-23 gene. To characterize this gene, its emergence, and dissemination, we performed antimicrobial susceptibility tests, hybrid sequencing with Illumina and Nanopore technologies, and phylogenetic analyses. ResultsThe MICs of the blaNDM-23 variant were identical to those of the blaNDM-1. The blaNDM-23 variant was found in 14 isolates in a 97 Kb non-mobilizable, multidrug-resistant plasmid carrying 19 resistance genes for 9 different antimicrobial families. In this plasmid, the blaNDM-23 gene is located in the variable region of a complex class-1 integron with a singular genetic environment. The short genetic distance between blaNDM-23-producing isolates reflects a 5-year-long clonal dispersion involving several hospitals and interregional spread. ConclusionsWe have characterized the genomic and epidemiological contexts in the emergence and community spread of a new blaNDM-23 allele in an MDR-plasmid of Klebsiella pneumoniae. TweetGenomic, epidemiologic and phylogenetic analyses of the emergence of a new NDM allele provide information on the rapid changes underlying the spread of antimicrobial resistance genes and strains in Klebsiella pneumoniae. ImportanceAt a time when antimicrobial resistance has become one of the biggest concerns worldwide, the emergence of novel alleles and extremely drug-resistant plasmids are a threat to public health worldwide. More so when they produce carbapenem resistance in one of the most problematic pathogens in clinical settings, such as Klebsiella pneumoniae. Here, we have used genomic epidemiology to describe the emergence of a novel NDM-23 allele and identify it in a MDR plasmid that has been disseminated through a K. pneumoniae ST407 clone in several hospitals in a Spanish region. By means of bioinformatic and phylogenetic analyses, we have been able to trace the evolutionary and epidemiological route of the new allele, the hosting plasmid, and the strain that carried both of them from Pakistan to Spain. A better understanding of the NDM-producing K. pneumoniae populations and its plasmids has made evident the spread of this clone through the region, enhancing the importance of genomic surveillance in the control of antimicrobial resistance.

microbiology↗

bioRxiv2020

CRISPR/Cas9 technology abolishes the BCR/ABL1 oncogene effect in chronic myeloid leukemia and restores normal hematopoiesis

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease produced by a unique oncogenic event involving the constitutively active tyrosine kinase (TK) BCR/ABL1. TK activity explains most features of CML, such as tumor development and maintenance. TK-inhibitory (TKI) drugs have changed its prognosis and natural history. Unfortunately, the ABL1 gene persists unaffected by TKIs, leukemic stem cells (LSCs) remains, resistant mutations arise and adverse effects may occur during treatment. To address this problem, we have designed a potential therapeutic alternative with CRISPR/Cas9 genome editing nucleases that target LSCs. The strategy was successfully developed in murine and human cell lines and finally was evaluated in primary LSCs isolated from CML transgenic mice and from CML patients. Mouse CML-LSCs edited were orthotopic transplanted in immunodeficient NSG niches where restored the normal hematopoiesis. Importantly, patient-derived xenografts with CD34+-LSCs edited, repopulated and restored the normal hematopoiesis in immunodeficient NSG niches. We show, for the first time, how CRISPR technology efficiently interrupts the BCR/ABL1 oncogene in murine and human LSCs to provide a significant therapeutic benefit. We propose human CML as a potential candidate for CRISPR therapy, providing proof-of-principle for genome editing in CML patients, and open new avenues for the application of this technique in other fusion genes. Key pointsCRISPR system destroys BCR/ABL oncogene and induces a therapeutic benefit in a CML mouse model and CML patient derived xenografts.

cancer biology↗

bioRxiv2019

Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency

CRISPR/Cas9 enables the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, these mutations do not produce the desired null result in all cases, but instead generate a similar, functionally active protein. This effect could limit the therapeutic efficiency of gene therapy strategies based on abrogating oncogene expression, and therefore needs to be considered carefully. If there is an acceptable degree of efficiency of CRISPR/Cas9 delivery to cells, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene, when only one sgRNA can be used. This study shows that the null effect could be increased with an sgRNA targeting the splice donor site (SDS) of the chosen exon. Following this strategy, the generation of null alleles would be facilitated in two independent ways: the probability of producing a frameshift mutation and the probability of interrupting the canonical mechanism of pre-mRNA splicing. In these contexts, we propose to improve the loss-of-function yield driving the CRISPR system at the SDS of critical exons.

molecular biology↗