Biology subjects

Criscuolo, E.

Publications and source records attributed to Criscuolo, E..

8 recordsLinked to original sources

bioRxiv2025

Fully Human Stem Cell-Derived BBB Model for Clinical Disease Modeling and Neurotropic AAV selection

The blood-brain barrier (BBB) is a highly functionalized vascular interface which regulates the exchange of substances between the neural parenchyma and its periphery. BBB leakage, leading to its uncontrolled permeability, is increasingly recognized to facilitate the onset of neuropathologies and aggravate their clinical progression. In vitro models of the BBB have rapidly evolved into elaborated structures that mimic its spatial architecture and multicellular nature. However, their cellular components are currently highly heterogeneous in origin and maturation state. Here, we have developed novel procedures to establish reproducible and scalable sources of endothelial, mural and astroglial cells generating a fully human pluripotent stem cell (hPSC)-derived BBB model, termed thBBBA. hPSC-derived BBB cell types are readily assembled into thBBBAs that develop mature functional properties with high barrier impermeability. Mature thBBBAs can also be generated by frozen hPSC-derived cell samples, providing a simple and scalable off-the-shelf system for general use. thBBBAs were instrumental in identifying the critical pathological role of an IL-6 autocrine source in disrupting thBBBA integrity, increasing its permeability to NMDAR antibodies from autoimmune encephalitis patients, and revealing the therapeutic effects of tocilizumab in this setting. Additionally, we have shown that thBBBAs are an invaluable system for ranking the clinical readiness of novel engineered AAV neurotropic capsids, previously selected in animal models or in vitro systems.

neuroscience↗

bioRxiv2022

Discovering host protein interactions specific for SARS-CoV-2 RNA genome

SARS-CoV-2, a positive single-stranded RNA virus, interacts with host cell proteins throughout its life cycle. These interactions are necessary for the host to recognize and hinder the replication of SARS-CoV-2. For the virus, to translate, transcribe and replicate its genetic material. However, many details of these interactions are still missing. We focused on the proteins binding to the highly structured 5 and 3 end regions of SARS-CoV-2 RNA that were predicted by the catRAPID algorithm to attract numerous proteins, exploiting RNA-Protein Interaction Detection coupled with Mass Spectrometry (RaPID-MS) technology. The validated interactors, which agreed with our predictions, include pseudouridine synthase PUS7 that binds to both ends of the viral RNA. Nanopore direct-RNA sequencing confirmed that the RNA virus is heavily modified, and PUS7 consensus regions were found in both SARS-CoV-2 RNA end regions. Notably, a modified site was detected in the viral Transcription Regulatory Sequence - Leader (TRS-L) and can influence the viral RNA structure and interaction propensity. Overall, our data map host protein interactions within SARS-CoV-2 UTR regions, pinpointing to a potential role of pseudouridine synthases and post-transcriptional modifications in the viral life cycle. These findings contribute to understanding virus-host dynamics and may guide the development of targeted therapies.

molecular biology↗

bioRxiv2021

Nanopore ReCappable Sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested sub genomic RNAs used to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

genomics↗

bioRxiv2021

SARS-CoV-2 Spike Affinity and Dynamics Exclude the Strict Requirement of an Intermediate Host

SARS-CoV-2 proximal origin is still unclear, limiting the possibility of foreseeing other spillover events with pandemic potential. Here we propose an evolutionary model based on the thorough dissection of SARS-CoV-2 and RaTG13 - the closest bat relative - spike dynamics, kinetics and binding to ACE2. Our results indicate that both spikes share nearly identical, high affinities for Rhinolophus affinis bat and human ACE2, pointing out to negligible species barriers directly related to receptor binding. Also, SARS-CoV-2 spike shows a higher degree of dynamics and kinetics optimization that favors ACE2 engagement. Therefore, we devise an affinity-independent evolutionary process that likely took place in R. affinis bats and limits the eventual involvement of other animal species in initiating the pandemic to the role of vector.

microbiology↗

bioRxiv2021

COVID-eVax, an electroporated plasmid DNA vaccine candidate encoding the SARS-CoV-2 Receptor Binding Domain, elicits protective immune responses in animal models of COVID-19

The COVID-19 pandemic caused by the {beta}-coronavirus SARS-CoV-2 has made the development of safe and effective vaccines a critical global priority. To date, four vaccines have already been approved by European and American authorities for preventing COVID-19 but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle, a technology previously utilized for cancer vaccines. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 Spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax - a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein RBD - induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function and significantly lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started in Italy.

immunology↗

bioRxiv2021

Differential plasmacytoid dendritic cell phenotype and type I Interferon response in asymptomatic and severe COVID-19 infection

SARS-CoV-2 fine-tunes the interferon (IFN)-induced antiviral responses, which play a key role in preventing coronavirus disease 2019 (COVID-19) progression. Indeed, critically ill patients show an impaired type I IFN response accompanied by elevated inflammatory cytokine and chemokine levels, responsible for cell and tissue damage and associated multi-organ failure. Here, the early interaction between SARS-CoV-2 and immune cells was investigated by interrogating an in vitro human peripheral blood mononuclear cell (PBMC)-based experimental model. We found that, even in absence of a productive viral replication, the virus mediates a vigorous TLR7/8-dependent production of both type I and III IFNs and inflammatory cytokines and chemokines, known to contribute to the cytokine storm observed in COVID-19. Interestingly, we observed how virus-induced type I IFN secreted by PBMC enhances anti-viral response in infected lung epithelial cells, thus, inhibiting viral replication. This type I IFN was released by plasmacytoid dendritic cells (pDC) via an ACE-2-indipendent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PD-L1 marker, a feature of type I IFN producing cells. Coherently to what observed in vitro, asymptomatic SARS-CoV-2 infected subjects displayed a similar pDC phenotype associated to a very high serum type I IFN level and induction of anti-viral IFN-stimulated genes in PBMC. Conversely, hospitalized patients with severe COVID-19 display very low frequency of circulating pDC with an inflammatory phenotype and high levels of chemokines and pro-inflammatory cytokines in serum. This study further shed light on the early events resulting from the interaction between SARS-CoV-2 and immune cells occurring in vitro and confirmed ex vivo. These observations can improve our understanding on the contribution of pDC/type I IFN axis in the regulation of the anti-viral state in asymptomatic and severe COVID-19 patients. Author summarySARS-CoV-2 pandemic has resulted in millions of infections and deaths worldwide, yet the role of host innate immune responses in COVID-19 pathogenesis remains only partially characterized. Innate immunity represents the first line of host defense against viruses. Upon viral recognition, the secretion of type I and III interferons (IFN) establishes the cellular state of viral resistance, and contributes to induce the specific adaptive immune responses. Moving from in vitro evidences on the protective role played by plasmacytoid dendritic cells (pDC)-released type I IFN in the early phase of SARS-CoV-2 infection, here we characterized ex vivo the pDC phenotype and the balance between anti-viral and pro-inflammatory cytokines of COVID-19 patients stratified according to disease severity. Our study confirms in COVID-19 the crucial and protective role of pDC/type I IFN axis, whose deeper understanding may contribute to the development of novel pharmacological strategies and/or host-directed therapies aimed at boosting pDC response since the early phases of SARS-CoV-2 infection.

immunology↗

bioRxiv2020

A human stem cell-derived neurosensory-epithelial circuitry on-a-chip to model herpes simplex virus reactivation

Both emerging viruses and well-known viral pathogens endowed with neurotropism can either impair directly the neuronal functions or induce physio-pathological changes by diffusing from the periphery through neurosensory-epithelial connections. However, the current lack of an in vitro system modeling the connectivity between human neurons and peripheral tissues excludes the analysis of viral latency and reactivation and the assessment of natural/artificial induced anti-viral immunity. In this study, we developed the first stable topographic neurosensory-epithelial connection on-a-chip using human stem cell derived dorsal root ganglia (DRG) sensory neurons. Bulk and single cell transcriptomics showed that different combinations of key receptors for Herpes Simplex Virus 1 (HSV-1) are expressed by each sensory neuronal cell type. This neuronal-epithelial circuitry enabled a detailed analysis of the HSV infectivity faithfully modeling its dynamics and cell type specificity. The reconstitution of an organized connectivity between human sensory neurons and keratinocytes into microfluidic chips provides for the first time a powerful in vitro platform to model viral latency and reactivation of human viral pathogens.

microbiology↗

bioRxiv2020

Combined prophylactic and therapeutic use maximizes hydroxychloroquine anti-SARS-CoV-2 effects in vitro

While the SARS-CoV-2 pandemic is hardly hitting the world, it is of extreme importance that significant in vitro observations guide the quick set up of clinical trials. In this study, we evidence that the anti-SARS-CoV2 activity of a clinically achievable hydroxychloroquine concentration is maximized only when administered before and after the infection of Vero E6 cells. This strongly suggests that only a combined prophylactic and therapeutic use of hydroxychloroquine may be effective in limiting viral replication in patients.

microbiology↗