Immunomodulatory Activity of the Tyrosine Kinase Inhibitor Dasatinib to Elicit NK Cytotoxicity against Cancer, HIV Infection and Aging

Tyrosine kinase inhibitors (TKIs) have been extensively used as a treatment for chronic myeloid leukemia (CML). Dasatinib is a broad-spectrum TKI with off-target effects that give it an immunomodulatory capacity resulting in increased innate immune responses against cancerous cells and viral infected cells. Several studies reported that dasatinib expanded memory-like natural killer (NK) cells and γδ T cells that have been related with increased control of CML after treatment withdrawal. In the HIV infection setting, these innate cells are associated with virus control and protection, suggesting that dasatinib could have a potential role in improving both the CML and HIV outcomes. Moreover, dasatinib could also directly induce apoptosis of senescence cells, being a new potential senolytic drug. Here, we review in depth the current knowledge of virological and immunogenetic factors associated with the development of powerful cytotoxic responses associated with this drug. Besides, we will discuss the potential therapeutic role against CML, HIV infection and aging.This review was partially supported by grants from the “Programa de becas Gilead a la investigación Biomédica GLD21_00111”; “Fondo Europeo para el Desarrollo Regional” (FEDER); the SPANISH AIDS Research Network RD12/0017/0001, RD16/0025/0002 and RD16/0025/0014-ISCIII-FEDER (RIS); the “Fondo de Investigación Sanitaria” (FIS) PI04/0363, PI20/00676; HIVACAT Programme and the CERCA Programme/“Generalitat de Catalunya” SGR 615 and SGR 653. Our group received funding from “la Caixa” Foundation under agreement, as well as from CIBERINFEC from the “Instituto de Salud Carlos III”, Madrid, Spain.S

Atlas of the HIV-1 Reservoir in Peripheral CD4 T Cells of Individuals on Successful Antiretroviral Therapy

ADN del VIH-1; Cura del VIH-1; Reservorio de VIH-1HIV-1 DNA; HIV-1 cure; HIV-1 reservoirADN del VIH-1; Cura del VIH-1; Reservori del VIH-1Knowing the mechanisms that govern the persistence of infected CD4+ subpopulations could help us to design new therapies to cure HIV-1 infection. We evaluated the simultaneous distribution of the HIV-1 reservoir in 13 CD4+ subpopulations from 14 HIV-1-infected individuals on antiretroviral therapy to analyze its relationship with HIV-1 transcription, immune activation, and cell proliferation. A unique large blood donation was used to isolate CD4, CD4 resting (CD4r), CD4 activated (CD4a), T naive (TN), T stem cell memory (TSCM), T central memory (TCM), T transitional memory (TTM), T effector memory (TEM), circulating T follicular helper (cTFH), TCD20, TCD32, and resting memory TCD2high (rmTCD2high) cells. HIV-1 DNA measured by droplet digital PCR ranged from 3,636 copies/106 in TTM to 244 in peripheral blood mononuclear cells (PBMCs), with no subpopulation standing out for provirus enrichment. Importantly, all the subpopulations harbored intact provirus by intact provirus DNA assay (IPDA). TCD32, cTFH, and TTM had the highest levels of HIV-1 transcription measured by fluorescent in situ hybridization with flow cytometry (FISH/flow), but without reaching statistical differences. The subpopulations more enriched in provirus had a memory phenotype, were less activated (measured by CD38+/HLA-DR+), and expressed more programmed cell death 1 (PD-1). Conversely, subpopulations transcribing more HIV-1 RNA were not necessarily enriched in provirus and were more activated (measured by CD38+/HLA-DR+) and more proliferative (measured by Ki-67). In conclusion, the HIV reservoir is composed of a mosaic of subpopulations contributing to the HIV-1 persistence through different mechanisms such as susceptibility to infection, provirus intactness, or transcriptional status. The narrow range of reservoir differences between the different blood cell subsets tested suggests limited efficacy in targeting only specific cell subpopulations during HIV-1 cure strategies. IMPORTANCE The main barrier for HIV-1 cure is the presence of latently infected CD4+ T cells. Although various cell subpopulations have been identified as major HIV-1 reservoir cells, the relative contribution of infected CD4 subpopulations in the HIV-1 reservoir remains largely unknown. Here, we evaluated the simultaneous distribution of the HIV-1 reservoir in 13 CD4+ T-cell subpopulations in peripheral blood from HIV-1-infected individuals under suppressive antiretroviral therapy. We found that the HIV-1 reservoir is composed of a mosaic of cell subpopulations, with heterogeneous proviral DNA, HIV-1 transcription, and activation status. Hence, each cell subpopulation contributes to the HIV-1 persistence through different mechanisms such as susceptibility to infection, rates of intact provirus, transcriptional status or half-life. This research provides new insights into the composition of the HIV-1 reservoir, suggesting that, to be effective, eradication strategies must simultaneously target multiple cell subpopulations.This work was sponsored in part by Grifols and by Gilead (GLD17/00204). The funding organizations had no input in the design of the study or in the collection, analyses, and interpretation of the data. Similarly, they played no role in the writing of the manuscript or in the decision to submit the study for publication. J.M.-P. is supported by grant PID2019-109870RB-I00 from the Spanish Ministry of Science and Innovation. J.M.-P. and M.J.B. are supported by the RETIC RD16/0025/0041 and RD16/0025/0007 (cofunded by the European Regional Development Fund/European Social Fund). M.J.B. is supported by grant RTI2018-101082-B-I00 (MINECO/FEDER) and by the Miguel Servet program funded by the Spanish Health Institute Carlos III (CP17/00179). C.G. was supported by the Ph.D. fellowship of the Spanish Ministry of Education, Culture, and Sport (FPU15/03698)

Identification of HIV-reservoir cells with reduced susceptibility to antibody-dependent immune response

HIV; Infectious disease; ReservoirVIH; Malalties infeccioses; ReservoriVIH; Enfermedades infecciosas; ReservorioHuman immunodeficiency virus (HIV) establishes a persistent infection in heterogeneous cell reservoirs, which can be maintained by different mechanisms including cellular proliferation, and represent the main obstacle to curing the infection. The expression of the Fcγ receptor CD32 has been identified as a marker of the active cell reservoirs in people on antiretroviral therapy (ART), but if its expression has any role in conferring advantage for viral persistence is unknown. Here, we report that HIV-infected cells expressing CD32 have reduced susceptibility to natural killer (NK) antibody-dependent cell cytotoxicity (ADCC) by a mechanism compatible with the suboptimal binding of HIV-specific antibodies. Infected CD32 cells have increased proliferative capacity in the presence of immune complexes, and are more resistant to strategies directed to potentiate NK function. Remarkably, reactivation of the latent reservoir from antiretroviral-treated people living with HIV increases the pool of infected CD32 cells, which are largely resistant to the ADCC immune mechanism. Thus, we report the existence of reservoir cells that evade part of the NK immune response through the expression of CD32.This study was supported by the Spanish Secretariat of Science and Innovation and FEDER funds (grants SAF2015-67334-R and RTI2018-101082-B-I00 [MINECO/FEDER]), the Spanish “Ministerio de Economia y Competitividad, Instituto de Salud Carlos III” (ISCIII, PI17/01470), GeSIDA and the Spanish AIDS network Red Temática Cooperativa de Investigación en SIDA (RD16/0025/0007), the Fundació La Marató TV3 (grants 201805-10FMTV3 and 201814-10FMTV3) and the Gilead fellowships GLD19/00084 and GLD18/00008. M.B is supported by the Miguel Servet program funded by the Spanish Health Institute Carlos III (CP17/00179). A.A-G is supported by the Spanish Secretariat of Science and Innovation Ph.D. fellowship (BES-2016–076382). The funders had no role in study design, data collection, and analysis, the decision to publish, or preparation of the manuscript

A Novel Single-Cell FISH-Flow Assay Identifies Effector Memory CD4 + T cells as a Major Niche for HIV-1 Transcription in HIV-Infected Patients

Cells that actively transcribe HIV-1 have been defined as the "active viral reservoir" in HIV-infected individuals. However, important technical limitations have precluded the characterization of this specific viral reservoir during both treated and untreated HIV-1 infections. Here, we used a novel single-cell RNA fluorescence in situ hybridization-flow cytometry (FISH-flow) assay that requires only 15 million unfractionated peripheral blood mononuclear cells (PBMCs) to characterize the specific cell subpopulations that transcribe HIV RNA in different subsets of CD4 + T cells. In samples from treated and untreated HIV-infected patients, effector memory CD4 + T cells were the main cell population supporting HIV RNA transcription. The number of cells expressing HIV correlated with the plasma viral load, intracellular HIV RNA, and proviral DNA quantified by conventional methods and inversely correlated with the CD4 + T cell count and the CD4/CD8 ratio. We also found that after ex vivo infection of unstimulated PBMCs, HIV-infected T cells upregulated the expression of CD32. In addition, this new methodology detected increased numbers of primary cells expressing viral transcripts and proteins after ex vivo viral reactivation with latency reversal agents. This RNA FISH-flow technique allows the identification of the specific cell subpopulations that support viral transcription in HIV-1-infected individuals and has the potential to provide important information on the mechanisms of viral pathogenesis, HIV persistence, and viral reactivation. Persons infected with HIV-1 contain several cellular viral reservoirs that preclude the complete eradication of the viral infection. Using a novel methodology, we identified effector memory CD4 + T cells, immune cells preferentially located in inflamed tissues with potent activity against pathogens, as the main cells encompassing the transcriptionally active HIV-1 reservoir in patients on antiretroviral therapy. Importantly, the identification of such cells provides us with an important target for new therapies designed to target the hidden virus and thus to eliminate the virus from the human body. In addition, because of its ability to identify cells forming part of the viral reservoir, the assay used in this study represents an important new tool in the field of HIV pathogenesis and viral persistence

Latency reversal agents affect differently the latent reservoir present in distinct CD4+ t subpopulations

Latency reversal agents (LRAs) have proven to induce HIV-1 transcription in vivo but are ineffective at decreasing the size of the latent reservoir in antiretroviral treated patients. The capacity of the LRAs to perturb the viral reservoir present in distinct subpopulations of cells is currently unknown. Here, using a new RNA FISH/flow ex vivo viral reactivation assay, we performed a comprehensive assessment of the viral reactivation capacity of different families of LRAs, and their combinations, in different CD4 T cell subsets. We observed that a median of 16.28% of the whole HIV-reservoir induced HIV-1 transcripts after viral reactivation, but only 10.10% of these HIV-1 RNA cells produced the viral protein p24. Moreover, none of the LRAs were powerful enough to reactivate HIV-1 transcription in all CD4 T cell subpopulations. For instance, the combination of Romidepsin and Ingenol was identified as the best combination of drugs at increasing the proportion of HIV-1 RNA cells, in most, but not all, CD4 T cell subsets. Importantly, memory stem cells were identified as highly resistant to HIV-1 reactivation, and only the combination of Panobinostat and Bryostatin-1 significantly increased the number of cells transcribing HIV within this subset. Overall, our results validate the use of the RNA FISH/flow technique to assess the potency of LRAs among different CD4 T cell subsets, manifest the intrinsic differences between cells that encompass the latent HIV reservoir, and highlight the difficulty to significantly impact the latent infection with the currently available drugs. Thus, our results have important implications for the rational design of therapies aimed at reversing HIV latency from diverse cellular reservoirs

Antiviral drug candidates for entry inhibition of SARS-CoV-2

Peer reviewe

Schlafen 12 restricts HIV-1 latency reversal by a codon-usage dependent post-transcriptional block in CD4+ T cells

Latency is a major barrier towards virus elimination in HIV-1-infected individuals. Yet, the mechanisms that contribute to the maintenance of HIV-1 latency are incompletely understood. Here we describe the Schlafen 12 protein (SLFN12) as an HIV-1 restriction factor that establishes a post-transcriptional block in HIV-1-infected cells and thereby inhibits HIV-1 replication and virus reactivation from latently infected cells. The inhibitory activity is dependent on the HIV-1 codon usage and on the SLFN12 RNase active sites. Within HIV-1-infected individuals, SLFN12 expression in PBMCs correlated with HIV-1 plasma viral loads and proviral loads suggesting a link with the general activation of the immune system. Using an RNA FISH-Flow HIV-1 reactivation assay, we demonstrate that SLFN12 expression is enriched in infected cells positive for HIV-1 transcripts but negative for HIV-1 proteins. Thus, codon-usage dependent translation inhibition of HIV-1 proteins participates in HIV-1 latency and can restrict the amount of virus release after latency reversal.We thank Drs Yingying Li, Feng Gao and Beatrice H. Hahn for providing codon-optimized HIV-1 Gag expression vector, Drs James Hoxie and Susan Zolla-Pazner for supplying anti-Nef and -p24 antibodies, respectively through the NIH AIDS reagent program. We also thank Dr Song Gao for providing SLFN13-tRNA structure information, and Dr Maria-Eugenia Gas Lopez and Dr Ester Gea-Mallorquí for advise. This work was supported by following grants: M.K.I., JSPS Oversea Research Fellowship and Takeda Science Foundation; A.E.C., PT17/0009/0019 (ISCIII/MINECO and FEDER); M.J.B., RTI2018-101082-B-I00 and PID2021-123321OB-I00 [MINECO/FEDER]), and the Miguel Servet program by ISCIII (CP17/00179 and CPII22/00005); C.B., M.R.R., C.D.C., European Union’s Horizon 2020 research and innovation program under grant agreement 681137-EAVI2020 and NIH grant P01-AI131568; J.D., the Spanish Ministry of Science and Innovation (PID2019106959RB-I00/AEI/10.13039/501100011033); A.M., the Spanish Ministry of Science and Innovation (PID2019-106323RB-I00 AEI//10.13039/501100011033) and the institutional “María de Maeztu” Programme for Units of Excellence in R&D (CEX2018-000792-M).info:eu-repo/semantics/publishedVersio

EC50 and CC50 of 39 antiviral drug candidates

Peer reviewe

Schlafen 12 restricts HIV-1 latency reversal by a codon-usage dependent post-transcriptional block in CD4+ T cells

Latency is a major barrier towards virus elimination in HIV-1-infected individuals. Yet, the mechanisms that contribute to the maintenance of HIV-1 latency are incompletely understood. Here we describe the Schlafen 12 protein (SLFN12) as an HIV-1 restriction factor that establishes a post-transcriptional block in HIV-1-infected cells and thereby inhibits HIV-1 replication and virus reactivation from latently infected cells. The inhibitory activity is dependent on the HIV-1 codon usage and on the SLFN12 RNase active sites. Within HIV-1-infected individuals, SLFN12 expression in PBMCs correlated with HIV-1 plasma viral loads and proviral loads suggesting a link with the general activation of the immune system. Using an RNA FISH-Flow HIV-1 reactivation assay, we demonstrate that SLFN12 expression is enriched in infected cells positive for HIV-1 transcripts but negative for HIV-1 proteins. Thus, codon-usage dependent translation inhibition of HIV-1 proteins participates in HIV-1 latency and can restrict the amount of virus release after latency reversal. In cell lines and HIV-1 patient PBMCs, the Schlafen 12 protein (SLFN12) is shown to be an HIV-1 restriction factor that inhibits HIV-1 replication and virus reactivatio

Posada a punt i aplicació d'una nova tècnica per a la caracterització del reservori del VIH i avaluació d'un nou compost natural com a agent reversor de la latència

Tot i que la teràpia antiretroviral ha suposat un enorme avenç en quan a la cronificació de la infecció pel VIH-1, encara avui dia la malaltia és incurable. L'eficàcia de la TAR és indiscutible, però l'aturada del tractament es tradueix sistemàticament en un rebot de la càrrega viral plasmàtica que serà degut, principalment, a l'existència de reservoris latents que s'estableixen molt ràpidament en el moment de la infecció i que poden romandre en l'organisme llargs períodes de temps. S'han dissenyat diverses estratègies o aproximacions que tenen com a objectiu trobar la cura per la infecció causada pel VIH, ja sigui per una eliminació radical dels reservoris latents o bé, pel control de la replicació en absència de tractament acompanyat d'una millora de la funcionalitat del sistema immunitari de l'hoste. Així doncs, per a que aquesta aproximació tingui èxit és necessari que es reactivin els provirus competents de les cèl·lules infectades, actuant sobre un ampli ventall de subpoblacions; es produeixin efectes mínims sobre altres tipus cel·lulars evitant així possibles toxicitats off-target; s'estimulin mecanismes de mort en les cèl·lules infectades i s'eviti una activació global de les cèl·lules T. Per tots aquests motius és imprescindible una bona caracterització d'aquests reservoris virals per tal de poder dissenyar estratègies dirigides a eliminar-los. A més, és imperatiu trobar nous agents reversors de latència que permetin una reactivació dels virus a nivell de cèl·lules T CD4+ sense tenir un efecte negatiu sobre la resta de cèl·lules del sistema immunitari, com serien les cèl·lules T CD8+ citotòxiques. En la present tesi doctoral s'ha posat a punt una nova tècnica, anomenada RNA/FISH flow, en el context de la infecció pel VIH. Aquesta tècnica permet la detecció de molècules d'ARN mitjançant la hibridació in situ fluorescent (FISH) combinada amb un mètode d'amplificació de la senyal i, per l'altra banda, l'immunofenotipatge mitjançant anticossos conjugats amb fluorocrom contra proteïnes intracel·lulars i de superfície cel·lular. Totes aquestes dades seran posteriorment recollides i analitzades a través d'un citòmetre de flux. L'ús d'aquesta tècnica ha permès identificar les cèl·lules efectores de memòria (TEM) com la subpoblació cel·lular que suporta majoritàriament la transcripció viral activa tant en pacients virèmics com en aquells amb la càrrega viral suprimida. A més, l'estudi i caracterització de les subpoblacions cel·lulars que actuen com a reservori del virus i que poden respondre a l'estimulació exògena per part de diferents agents reversors de la latència (LRAs), fent ús d'aquesta nova tècnica, ha determinat que els agents reversors de la latència indueixen una resposta heterogènia en les diferents subpoblacions de cèl·lules T CD4+ de pacients infectats pel VIH; i s'ha identificat la combinació de Romidepsina i Ingenol com una de les més potents. Finalment, s'ha proposat i l'avaluat l'àcid làuric, un àcid gras de cadena mitjana, com a nou inductor de la reactivació viral. Els resultats obtinguts in vitro demostren la capacitat d'aquest compost natural per induir la transcripció i producció de proteïna viral al mateix temps que promou una reprogramació del metabolisme cel·lular sense afectar a la capacitat citotòxica de les cèl·lules T CD8+. En conclusió, el RNA/FISH flow es determina com una tècnica vàlida per estudiar el reservori transcripcionalment actiu i avaluar compostos que actuïn com a agents reversors de latència, entre d'altres; i es postula l'àcid làuric com un nou LRA prometedor per ser testat en futurs assajos clínics.Aunque la terapia antirretroviral ha supuesto un enorme avance en cuanto a la cronificación de la infección por el VIH-1, hoy en día la enfermedad sigue siendo incurable. La eficacia de la TAR es indiscutible, pero la interrupción del tratamiento se traduce sistemáticamente en un rebote de la carga viral plasmática debido, principalmente, a la existencia de reservorios latentes que se establecen muy rápidamente en el momento de la infección y que pueden permanecer en el organismo largos períodos de tiempo. Se han diseñado varias estrategias o aproximaciones que tienen como objetivo encontrar la cura para la infección causada por el VIH, ya sea por una eliminación radical de los reservorios latentes o bien, por el control de la replicación en ausencia de tratamiento acompañado de una mejora de la funcionalidad del sistema inmunitario del huésped. Así pues, para que esta aproximación tenga éxito es necesario que se reactiven los provirus competentes de las células infectadas, actuando sobre un amplio abanico de subpoblaciones; provoque efectos mínimos sobre otros tipos celulares evitando así posibles toxicidades off-target; se estimulen mecanismos de muerte en las células infectadas y se evite una activación global de las células T. Por todos estos motivos es imprescindible una buena caracterización de los reservorios virales para poder diseñar estrategias dirigidas a eliminarlos. Además, es imperativo encontrar nuevos agentes reversores de la latencia que permitan una reactivación de los virus a nivel de células T CD4+ sin tener un efecto negativo sobre el resto de células del sistema inmunitario, como serían las células T CD8+ citotóxicas. En la presente tesis doctoral se ha puesto a punto una nueva técnica, denominada RNA/FISH flow, en el contexto de la infección por el VIH. Esta técnica permite la detección de moléculas de ARN mediante la hibridación in situ fluorescente (FISH) combinada con un método de amplificación de la señal y, por otro lado, el inmunofenotipaje mediante anticuerpos conjugados con fluorocromos contra proteínas intracelulares y de superficie celular. Todos estos datos serán posteriormente recogidos y analizados por un citómetro de flujo. El uso de esta técnica ha permitido identificar las células efectoras de memoria (TEM) como la subpoblación celular que soporta mayoritariamente la transcripción viral activa tanto en pacientes virémicos como en aquellos con la carga viral suprimida. Además, el estudio y caracterización de las subpoblaciones celulares que actúan como reservorio del virus y que pueden responder a la estimulación exógena por parte de diferentes agentes reversores de la latencia (LRAs), usando esta nueva técnica, ha determinado que los LRA inducen una respuesta heterogénea en las diferentes subpoblaciones de células T CD4+ de pacientes infectados por el VIH y se ha identificado la combinación de Romidepsina y Ingenol como una de las más potentes para reactivar el virus. Finalmente, se ha propuesto y evaluado el ácido láurico, un ácido graso de cadena mediana, como nuevo inductor de la reactivación viral. Los resultados obtenidos in vitro demuestran la capacidad de este compuesto natural para inducir la transcripción y producción de proteína viral a la vez que promueve una reprogramación del metabolismo celular sin afectar a la capacidad citotóxica de las células T CD8+. En conclusión, el RNA/FISH flow se determina como una técnica válida para estudiar el reservorio transcripcionalmente activo y evaluar compuestos que actúen como agentes reversores de latencia, entre otros; y se postula el ácido láurico como un nuevo LRA prometedor para ser testado en futuros ensayos clínicos.Current antiretroviral drugs maintain the viral load under the limit of detection; however, the interruption of the treatment results in an immediate rebound of the virus. The presence of latent cell reservoirs that persist in antiretroviral treated HIV-infected patients is considered the main obstacle to cure HIV. Several strategies are investigated as an additional treatment despite ART, aimed to reduce or eliminate the HIV reservoirs. The "shock and kill" strategy is based on the reactivation of the latent virus using drugs called latency reversal agents (LRAs), and the killing of those reactivated cells by the cytopathic effects or the action of the immune system. To achieve a successful cure, it is needed to reactivate all the competent provirus of infected cells, to induce a broader reactivation in all subpopulations where HIV is hidden without affecting other cell types avoiding off-target toxicities; and to promote cell death of reactivated cells and without inducing a global activation of T cells. Therefore, it is essential to perform an in-deep characterization of the viral reservoir and to find new latency reversal agents able to induce HIV reactivation without impairing the cytotoxic capacity of immune cells. In this PhD thesis, a new technique, so-called RNA/FISH flow, has been developed in the context of the HIV infection. This technique allowed the detection of RNA molecules by fluorescent in situ hybridization (FISH) combined with a signal amplification method and the immunophenotyping of transcriptionally active cells using fluorochrome-conjugated antibodies against intracellular and cell surface proteins. All data were collected and analysed using a flow cytometer. Using this novel technique, we identified memory effector CD4+T cells (TEM) as the main cell population supporting the HIV-RNA transcription both in viremic patients and in those with suppressed viral load. Furthermore, the study and characterization by the RNA/FISH flow method of the cell subpopulations that are susceptible to HIV reactivation by different latency-reversing agents (LRAs) determined that LRAs induced a heterogeneous response in the different subpopulations of CD4+T cells of HIV-infected patients, and identified Romidepsin and Ingenol as one of the most potent combinations at reactivating HIV. Finally, lauric acid, a medium-chain fatty acid, has been proposed and evaluated as a new LRA. The results obtained in vitro demonstrated the ability of this natural compound to induce the HIV transcription and production of viral proteins. Moreover, lauric acid significantly reprogramed the metabolic signatures of host cells preserving the cytotoxic capacity of CD8+ T cells at killing viral-reactivated cells from primary cell reservoirs. In conclusion, the RNA/FISH flow method is determined as a valid technique to study the transcriptionally active HIV reservoir and to evaluate compounds with HIV latency reversal capacity (LRA). Furthermore, lauric acid is postulated, for the first time, as a promising new LRA to be tested in future clinical trials