Use of an interactomics pipeline to assess the potential of new antivirals against SARS-CoV-2

(Póster 80) Background: In late 2019 SARS-CoV-2 infection appeared in China, becoming a pandemic in 2020. The scientific community reacted rapidly, characterizing the viral genome and its encoded proteins, aiming at interfering with viral spreading with vaccines and antivirals. The receptor binding domain (RBD) of the viral spike (S) protein plays a key role in cell entry of the virus. It interacts with the cellular receptor for SARS-CoV-2, the membrane-bound human Angiotensin Converting Ectoenzyme 2 (ACE2). With the goal of monitoring interference with this interaction by potential antiviral drugs, we have set up at the Institute for Biomedicine of Valencia (IBV-CSIC) an interactomics pipeline targeting the initial step of viral entry. Methods: For the production part of the pipeline (pure RBD/Spike variants and soluble ACE2), see parallel poster. These proteins allowed monitoring of the RBD/Spike-ACE2 interaction in presence or absence of potential inhibitors. Thermal shift assays (thermofluor) were used for initial detection of compound binding at different ligand/protein ratios and media conditions (pH, buffers, chaotropic agents). Next, binding affinity and on/off kinetics were characterized using Biolayer interferometry (BLI), Surface plasmon resonance (SPR), Microscale Thermophoresis (MST) and/or Isothermal titration calorimetry (ITC). For protein-protein interactions, we mostly used BLI or SPR, whereas for proteinsmall compound analysis MST was generally best. Protein aggregation-dissociation was monitored by size exclusion chromatography with multiangle light scattering (SEC-MALS). Results: Candidates proven by thermal shift assays to bind to RBD/spike protein without affecting the integrity of these proteins were subjected to quantitative affinity measurements. We successfully demonstrated that BLI, SPR and MST can be used to follow the interactions between SARS-CoV- 2 proteins and the putative drug candidates, as well as to monitor the interference with Spike-Ace2 binding of potential drug candidates. While BLI and SPR displayed reproducible results in the measurement of protein-protein interaction (applied to soluble ACE2 used as a decoy), they were less suitable for measuring the binding of small molecules. The fact that most small compounds were only soluble in organic solvents made difficult to obtain a low signal/noise while using BLI, necessary for the assessment of the binding. We overcame that problem by using MST. After dilution of the compounds to the final experimental concentrations, the technique could detect a significant binding signal enough to calculate binding parameters. MST also allowed to measure the degree of interference that each compound was having on RBD/Spike-ACE2 interaction. The pipeline has been customized and validated with compounds of very different nature provided by different groups belonging to the PTI and other external laboratories, as well as with different Ace2 decoys designed at the IBV. Conclusions: The interactomics platform at the IBV has been used to successfully develop two different antiviral approaches in order to fight COVID-19. It has allowed technical specialization of the staff as well as the development, in a very short period of time, of two ambitious projects. We have demonstrated that we can perform interactomic characterization for challenging projects as well as provide information about binding of antivirals to potential new SARS-CoV-2 variants of concern

New findings with the IBV decoy for cell entry inhibition of SARS-CoV-2, and unique structural data for soluble dimeric ACE2 bound to the viral S trimer

Resumen del trabajo presentado a las III Jornadas Científicas PTI+ Salud Global, celebradas en el Centro de Ciencias Humanas y Sociales (CCHS), CSIC (Madrid) del 20 al 22 de noviembre de 2023.[Background] The SARS-CoV-2 spike protein (S) mediates the interaction of the virus with cellular membrane receptor (angiotensin-converting enzyme 2, ACE2). In previous PTI meetings, we reported heterologous production in vitro of the ACE2 extracellular domains modified by site-directed mutagenesis to increase its affinity for the S protein, to enable it to be used as viral entry inhibitor (decoy) by competing with the membrane-bound cellular receptor. We now test the value of these decoys for: 1) binding to S variants that emerged during the evolution of the pandemic in viral lineages of concern; and 2) inhibiting experimental cellular infection by pseudotyped virus expressing these S variants. Cellular syncytia formation has been described in several organs as a manifestation of severe COVID-19, and likely has pathogenic impact. To test further our decoys’ effectiveness, we studied their impact on cellular syncytia formation within an experimental in vitro cell culture model. Searching for effective decoys, we produced monomeric and dimeric ACE2 proteins, depending on the respective absence/presence of the extracellular collectrin domain. Interestingly, there are no reported structures of dimeric soluble ACE2 bound to the S protein. After extensive knowledge-guided trial-and-error, we succeeded in visualizing by cryo-electron microscopy (cryoEM) this interaction (~7-Å-resolution), and in understanding the challenges inherent in determining such a complex structural organization.[Methods] 1) Recombinant production and purification of the monomeric or dimeric ACE2, their decoys the receptor binding domain (RBD) and the S protein variants of interest. We used baculovirus/insect cells to produce ACE2s and RBDs, and human Expi293F cells for the S proteins. 2) Biolayer interferometry for assessing protein-protein interactions; 3) Use of a model system for monitoring viral cellular infection and its inhibition by decoys. We used a pseudotyped engineered vesicular stomatitis virus expressing and exposing at its surface the desired S protein variant, to infect appropriate SARS-CoV-2-susceptible mammalian cells; 4) Single-particle cryoEM; 5) Syncytia formation testing using an engineered cultured cell system in which heterologous surface expression of the S protein in one cell type induces syncytium formation in other cells expressing membrane-bound ACE2.[Results] Our decoys proved highly effective in preventing cellular infection by pseudotyped virus expressing the S proteins of different SARS-CoV-2 variants of concern. Biophysical results have validated the maintained interaction between the decoy and the various S protein variants. When introduced into the cellular model system for syncytia formation, the decoys proved capable of decreasing such formation. Puzzlingly, the monomeric decoy was more effective than the dimeric one. The cryoEM images unveiled an ACE2 dimer configuration, where the subunits, resembling the previously reported monomer, were oriented at an angle of >60º, in which the vortex was the interlinked collectrin domains. Both catalytic domains engage with a single RBD of one subunit from different S trimers. The formation of a network at high stoichiometries of both components poses a challenge for structure determination by cryoEM.[Conclusions] Unlike therapeutic antibodies, which proved ineffective on variants not initially used for their production, our decoys should be effective in preventing infection by all widely widespread SARS-CoV-2 variants.Peer reviewe

Glioblastoma Therapy with Cytotoxic Mesenchymal Stromal Cells Optimized by Bioluminescence Imaging of Tumor and Therapeutic Cell Response

Genetically modified adipose tissue derived mesenchymal stromal cells (hAMSCs) with tumor homing capacity have been proposed for localized therapy of chemo- and radiotherapy resistant glioblastomas. We demonstrate an effective procedure to optimize glioblastoma therapy based on the use of genetically modified hAMSCs and in vivo non invasive monitoring of tumor and therapeutic cells. Glioblastoma U87 cells expressing Photinus pyralis luciferase (Pluc) were implanted in combination with hAMSCs expressing a trifunctional Renilla reniformis luciferase-red fluorescent protein-thymidine kinase reporter in the brains of SCID mice that were subsequently treated with ganciclovir (GCV). The resulting optimized therapy was effective and monitoring of tumor cells by bioluminescence imaging (BLI) showed that after 49 days GCV treatment reduced significantly the hAMSC treated tumors; by a factor of 104 relative to controls. Using a Pluc reporter regulated by an endothelial specific promoter and in vivo BLI to image hAMSC differentiation we gained insight on the therapeutic mechanism. Implanted hAMSCs homed to tumor vessels, where they differentiated to endothelial cells. We propose that the tumor killing efficiency of genetically modified hAMSCs results from their association with the tumor vascular system and should be useful vehicles to deliver localized therapy to glioblastoma surgical borders following tumor resection

The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: the case of the spike A222V mutation

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has now reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness.This research work was supported by the European Commission–NextGenerationEU through the CSIC Global Health Platform. Additionally, authors would like to acknowledge economic support from the Spanish Ministry of Science and Innovation through Grants: PID2019-104757RB-I00 funded by MCIN/AEI/ 10.13039/501100011033, RTI2018-094399-A-I00, and “ERDF A way of making Europe”, by the “European Union”, Grant SEV 2017-0712 funded by MCIN/AEI /10.13039/501100011033, the “Comunidad Autónoma de Madrid" through Grant: S2017/BMD3817, and the European Union (EU) and Horizon 2020 through grants: Marie-Curie Fellowship EnLaCES (MSCA IF 2020, Proposal: 101024130) (to JK), HighResCells (ERC - 2018 - SyG, Proposal: 810057), and iNEXT-Discovery (Proposal: 871037). AM, VR, JB and JLL are funded by CIBERER-ISCIII (proposal: COV20/00437), Fondo Supera COVID-19 (proposal: CSICCOVID19-082), Banco Santander (Proposal: BlockAce), and CSIC PTI Salud Global (Proposal: 202080E110). VR is funded by the Spanish Ministry of Science and Innovation through Grant PID2020-120322RB-C21. IC is funded by project PID2019-104477RB-100, Fondo COVID COV20/00140 and ERC CoG 101001038. MC is funded by the RyC program from the Spanish Ministry of Science and Innovation, the Generalitat Valenciana (SEJI/2019/011).N

The role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: the case of the spike A222V mutation

Resumen del trabajo presentado a las II Jornadas Científicas PTI + Salud Global, celebradas los días 5 y 6 de octubre de 2022 en el Auditorio Santiago Grisolía de Valencia (España).Peer reviewe

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Dosis elevada en el dosímetro personal de una auxiliar de enfermería en Electrofisiología A high dose in the personal dosage meter of nursing assistant in Electrophysiology field

En una sala de Electrofisiología del Servicio de Cardiología en donde se efectúan procedimientos guiados con rayos X de ablación con radiofrecuencia y de implantes de desfibrilador biventricular, se registraron dosis mensuales anormalmente elevadas en el dosímetro personal de solapa de una auxiliar de enfermería que trabaja en la misma, con valores de dosis equivalente personal, Hp(10), de 125 mSv y de dosis equivalente personal superficial, Hp(0.07) de 128.1 mSv. Ante la ansiedad producida en la trabajadora al conocer las lecturas de su dosímetro, y dado que no se dispone de precedentes investigados en la instalación, se ha realizado un estudio para dilucidar si la dosis registrada ha podido ser recibida por la trabajadora o si solo ha sido recibida por el dosímetro. Se hacen medidas con un monitor ambiental en condiciones simuladas de los procedimientos, para estimar las dosis reales que pueda recibir el personal de la sala. Se comparan estos valores con los del rendimiento del equipo de RX en haz directo medidos durante la revisión anual de control de calidad del mismo, concluyéndose que ha sido el dosímetro, y no la trabajadora, el que se ha expuesto a radiación directa, siendo la situación más probable la de que dicho dosímetro cayó sobre el tubo de RX, normalmente orientado hacia el techo, un tiempo del orden de 1 minuto. Paralelamente a estas estimaciones dosimétricas, el Servicio de Prevención de Riesgos Laborales realizó la vigilancia de la salud, concluyendo que la trabajadora no tenía ningún indicio de haber recibido dosis de la magnitud registrada por su dosímetro.In a room of electrophysiology from the cardiology service where procedures are made guided with X-Rays of ablation with radiofrequency and from implants ventricular defibrillation; there were registered monthly doses usually high in the same personal dosimeter flap of a nursing assistant who works there with values of equivalent personal doses, Hp (10), of superficial equivalent doses, Hp (0.07) of 128.1mSv. In view of the anxiety produced in the worker upon knowing the readings of its dosimeter, and since there is no disposition of precedents investigated in the facility, and it has been carried out a study for explaining if the doses registered could has been received by the worker if it is only received by the dosimeter

Beneficios socioeconómicos de un programa de monitorización domiciliaria o televigilancia en pacientes con terapia de resincronización cardiaca coordinado por enfermería

Introduction: The advantages that telesurveillance programs offer with respect to face-to-face visits are, on the one hand, avoiding travels, which results in a greater comfort for patients and considerable financial savings, and on the other, relieving face-to-face consultations. Objective: To calculate the travels, their cost and the hours of face-to-face consultation avoided in those patients included in the program. Methodology: The number of kilometers that patients should have traveled from their provinces to our hospital in case they had been revised in a scheduled face-to-face consultation have been calculated, and the economic savings that this has entailed have been estimated, taking the Captio report of mileage 2015 as a reference. The hours of face-to-face consultation avoided have also been evaluated, considering 20 minutes per consultation and the money saved. Results: 1,326 face-to-face consultations have been avoided in 376 patients with cardiac resynchronization therapy. A mileage of 140,184 km and 1,692 hours of travel have been avoided in those 376 patients. The three provinces most benefited in the number of consultations avoided had a reduction of 160, 118 and 173 consultations, and the savings generated were 16,480 euros and 784 hours, 5,900 euros and 283 hours and 4,411 euros and 236 hours, respectively. An optimization of resources has been verified, since 442 hours of face-to-face consultations and 182,974 euros have been saved. Conclusions: The telesurveillance program in the patients of the study has avoided many travels, with important associated economic savings, and has avoided the saturation of face-to-face consultations.Introducción: Las ventajas que ofrecen los programas de televigilancia respecto a las visitas presenciales son, por un lado evita los desplazamientos, lo cual redunda en una mayor comodidad para los pacientes y un ahorro económico considerable, por otro, aligerar las consultas presenciales. Objetivo: Calcular los desplazamientos, su coste y las horas de consulta presencial evitadas en los pacientes incluidos en el programa. Metodología: Se ha calculado los kilómetros de desplazamiento que tendrían que haber realizado los pacientes desde sus provincias hasta nuestro hospital en caso de haber sido revisados en consulta presencial programada y se ha realizado un cálculo del ahorro económico que esto ha supuesto, tomando como referencia el Captio del kilometraje 2015. También se ha evaluado las horas de consulta presencial evitadas, considerando 20 minutos por consulta y el dinero ahorrado. Resultados: Se han evitado 1.326 consultas presenciales en 376 pacientes con terapia de resincronización cardiaca. El kilometraje evitado en esos 376 pacientes ha sido 140.184 km y 1.692 horas de desplazamiento. Las tres provincias más beneficiadas en el número de consultas evitadas fueron 160, 118 y 173, el ahorro generado fue 16.480 euros y 784 horas, 5.900 euros y 283 horas y 4.411 euros y 236 horas, respectivamente. Se ha constatado una optimización de recursos, ya que se han ahorrado 442 horas de consultas presenciales y 182.974 euros. Conclusiones: El programa de televigilancia en los pacientes del estudio ha evitado muchos desplazamientos, con un importante ahorro económico asociado y ha evitado la saturación de las consultas presenciales