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

Impact of Transcriptome and Gut Microbiome on the Response of HIV-1 Infected Individuals to a Dendritic Cell-Based HIV Therapeutic Vaccine

Therapeutic vaccines based on dendritic cells offer a good approach to HIV-specific T-cell responses and partial control of the viral load after antiretroviral therapy interruption. The aim of the present study was to identify mRNA expression profiles and to assess the impact of the gut microbiome composition for predicting the viral load control after antiretroviral therapy interruption. We enrolled 29 patients to receive either placebo or a monocyte-derived dendritic cell vaccine. Patients with a decrease in their viral load of >0.5 log10 copies/mL by 12 weeks after antiretroviral therapy interruption were considered responders. In total, 66 genes were considered differentially expressed between responders and non-responders. Enrichment analysis revealed several upregulated pathways involved in the host defense response to a virus via the type I interferon signaling pathway. Regarding the gut microbiota, responders showed enriched levels of Bacteroidetes (p < 0.005) and Verrucomicrobia (p = 0.017), while non-responders were enriched with Tenericutes (p = 0.049) and Actinobacteria (p < 0.005). We also found important differences at the genus level. However, we did not discover any effect of the dendritic cell vaccine on the transcriptome or the gut microbiota. An alternative analysis did characterize that the microbiota from responders were associated with the metabolic production of short-chain fatty acids, which are key metabolites in the regulation of intestinal homeostasis. The evidence now consistently shows that short-chain fatty acid depletion occurs in HIV-infected individuals receiving antiretroviral treatment.This study was partially supported by the SPANISH AIDS Research Network (RIS) projects RD16/0025/0002 and RD16/0025/0013 integrated in the State Plan for Scientific and Technical Research and Innovation from the General Sub-Directorate for research assessment and promotion; Spanish Carlos III Institute of Health (ISCIII) co-funded by the European Regional Development Fund (FEDER); the Fondo de Investigación Sanitaria (FIS), AC16/00051 and PI18/00699; Gilead grants in Biomedical research in HIV, hepatic, and hemo-oncology from the Spanish Ministry of Economy (MINECO) (grants: SAF2015-66193-R and RTI2018-096309-B-I00); and the income found from Europe in the Regional Development (FEDER) and the CERCA Programme/Generalitat de Catalunya SGR 615 and SGR 653. Felipe García received the support of José María Segovia de Arana contracts.S

Highly Efficient Autologous HIV-1 Isolation by Coculturing Macrophage With Enriched CD4+ T Cells From HIV-1 Patients

We described a novel HIV autologous isolation method based in coculturing macrophages and CD4+T-cell-enriched fractions from peripheral blood collected from antiretroviral-treated (ART) HIV patients. This method allows the isolation of high viral titers of autologous viruses, over 1010HIV RNA copies/ml, and reduces the time required to produce necessary amounts for virus for use as antigens presented by monocyte-derived myeloid cells in HIV therapeutic vaccine approaches. By applying these high titer and autologous virus produced in the patient-derived cells, we intended to elicit a boost of the immunological system response in HIV therapeutic vaccines in clinical trials.This study was partially supported by grants from the Spanish Ministry of Economy (MINECO) (grants: SAF2015-66193-R, SAF-2017-88089-R, RTI2018-096309-B-I00); the Fondo Europeo para el Desarrollo Regional (FEDER); the SPANISH AIDS Research Network RD16/0025/0002 and RD16/0025/0014-ISCIII-FEDER (RIS); the Fondo de Investigación Sanitaria (FIS) PI12/01247 and PI20/00676; and HIVACAT program and the CERCA Programme/Generalitat de Catalunya SGR 615 and SGR 653. The project leading to these results has received funding from “la Caixa” Foundation under agreement. This study was also supported in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract Nos. HHSN261200800001E and 75N91019D00024.S

Generation and characterization of a defective HIV-1 Virus as an immunogen for a therapeutic vaccine

Background The generation of new immunogens able to elicit strong specific immune responses remains a major challenge in the attempts to obtain a prophylactic or therapeutic vaccine against HIV/AIDS. We designed and constructed a defective recombinant virus based on the HIV-1 genome generating infective but non-replicative virions able to elicit broad and strong cellular immune responses in HIV-1 seropositive individuals. Results Viral particles were generated through transient transfection in producer cells (293-T) of a full length HIV-1 DNA carrying a deletion of 892 base pairs (bp) in the pol gene encompassing the sequence that codes for the reverse transcriptase (NL4-3/ΔRT clone). The viral particles generated were able to enter target cells, but due to the absence of reverse transcriptase no replication was detected. The immunogenic capacity of these particles was assessed by ELISPOT to determine γ-interferon production in a cohort of 69 chronic asymptomatic HIV-1 seropositive individuals. Surprisingly, defective particles produced from NL4-3/ΔRT triggered stronger cellular responses than wild-type HIV-1 viruses inactivated with Aldrithiol-2 (AT-2) and in a larger proportion of individuals (55% versus 23% seropositive individuals tested). Electron microscopy showed that NL4-3/ΔRT virions display immature morphology. Interestingly, wild-type viruses treated with Amprenavir (APV) to induce defective core maturation also induced stronger responses than the same viral particles generated in the absence of protease inhibitors. Conclusions We propose that immature HIV-1 virions generated from NL4-3/ΔRT viral clones may represent new prototypes of immunogens with a safer profile and stronger capacity to induce cellular immune responses than wild-type inactivated viral particles

Treatment with integrase inhibitors alters SARS-CoV-2 neutralization levels measured with HIV-based pseudotypes in people living with HIV

The presence of neutralizing antibodies (NAbs) is a major correlate of protection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Thus, different in vitro pseudoviruses-based assays have been described to detect NAbs against SARS-CoV-2. However, the determination of NAbs against SARS-CoV-2 in people living with HIV (PLWH) through HIV-based pseudoparticles could be influenced by cross-neutralization activity or treatment, impeding accurate titration of NAbs. Two assays were compared using replication-defective HIV or VSV-based particles pseudotyped with SARS-CoV-2 spike to measure NAbs in COVID-19-recovered and COVID-19-naïve PLWH. The assay based on HIV-pseudoparticles displayed neutralization activity in all COVID-19-recovered PLWH with a median neutralizing titer 50 (NT50) of 1417.0 (interquartile range [IQR]: 450.3-3284.0), but also in 67% of COVID-19-naïve PLWH (NT50: 631.5, IQR: 16.0-1535.0). Regarding VSV-pseudoparticles system, no neutralization was observed in COVID-19-naïve PLWH as expected, whereas in comparison with HIV-pseudoparticles assay lower neutralization titers were measured in 75% COVID-19-recovered PLWH (NT50: 100.5; IQR: 20.5-1353.0). Treatment with integrase inhibitors was associated with inaccurate increase in neutralization titers when HIV-based pseudoparticles were used. IgG purification and consequent elimination of drugs from samples avoided the interference with retroviral cycle and corrected the lack of specificity observed in HIV-pseudotyped assay. This study shows methodological alternatives based on pseudoviruses systems to determine specific SARS-CoV-2 neutralization titers in PLWH

Prevalence, clinical characteristics and outcome of severe primary HIV-1 infection: a prospective cohort study

Background: Severe cases of primary HIV infection have been described in patients presenting with neurological involvement, AIDS defining events or other life-threatening events. These severe forms have not been fully studied. Objectives: To determine the prevalence and characteristics of severe PHI in a hospital-based cohort of primary HIV infection, and the response to the early initiation of antiretroviral therapy (ART) at 12 months. Methods: Every patient with PHI attending Hospital Clínic of Barcelona (1997-2015) was evaluated. Severe PHI was defined using clinical, analytical and immunological criteria. Chi-squared test was used for categorical variables and Student's t-test for quantitative variables. Results: 33% of 224 PHI patients (95% CI: 26.84%-39.16%) had a severe PHI. These patients had more symptoms, abnormal analytical parameters and hospital admissions. The severe PHI group had a significantly higher viral load although no differences were observed at 12 months in terms of viral suppression or CD4 count recovery. None died during PHI. Conclusions: Up to one third of patients in our cohort presented with a severe PHI, which was associated with higher hospitalization rates and higher plasma HIV RNA viral load. However, severe forms were not associated to a worse clinical, immunological or virological outcome at 12 months

Use of RT-defective HIV virions: new tool to evaluate specific response in chronic asymptomatic HIV-infected individuals

Background Generation of new reagents that can be used to screen or monitor HIV-1-specific responses constituted an interesting field in the development of HIV vaccines to improve their efficacy. Methods We have evaluated the specific T cell response against different types of NL4-3 virions (including NL4-3 aldrithiol-2 treated, NL4-3/ΔRT and R5 envelopes: NL4-3/ΔRT/ΔEnv[AC10] and NL4-3/ΔRT/ΔEnv[Bal]) and against pools of overlapping peptides (15 mer) encompassing the HIV-1 Gag and Nef regions. Cryopreserved PBMC from a subset of 69 chronic asymptomatic HIV positive individuals have been employed using different techniques including IFN-γ ELISPOT assay, surface activation markers and intracellular cytokine staining (ICS) by flow cytometry. Results The differential response obtained against NL4-3 aldrithiol-2 treated and NL4-3/ΔRT virions (25% vs 55%, respectively) allow us to divide the population in three groups: "full-responders" (positive response against both viral particles), "partial-responders" (positive response only against NL4-3/ΔRT virions) and "non-responders" (negative responses). There was no difference between X4 and R5 envelopes. The magnitude of the total responses was higher against NL4-3/ΔRT and was positively correlated with gender and inverse correlated with viral load. On the contrary CD4+ T cell count was not associated with this response. In any case responses to the viruses tended to be lower in magnitude than those detected by the overlapping peptides tested. Finally we have found an increased frequency of HLA-B27 allele (23% vs 9%) and a significant reduction in some activation markers (CD69 and CD38) on T cells surface in responders vs non-responders individuals. Conclusions In summary these virions could be considered as alternative and useful reagents for screening HIV-1-specific T cell responses in HIV exposed uninfected people, HIV infected patients and to assess immunogenicity of new prototypes both in vitro and in vaccine trials, by a feasible, simply, effective and low cost assay

HIV-1 promonocytic and lymphoid cell lines: an in vitro model of in vivo mitochondrial and apoptotic lesion.

To characterize mitochondrial/apoptotic parameters in chronically human immunodeficiency virus (HIV-1)-infected promonocytic and lymphoid cells which could be further used as therapeutic targets to test pro-mitochondrial or anti-apoptotic strategies as in vitro cell platforms to deal with HIV-infection. Mitochondrial/apoptotic parameters of U1 promonocytic and ACH2 lymphoid cell lines were compared to those of their uninfected U937 and CEM counterparts. Mitochondrial DNA (mtDNA) was quantified by rt-PCR while mitochondrial complex IV (CIV) function was measured by spectrophotometry. Mitochondrial-nuclear encoded subunits II-IV of cytochrome-c-oxidase (COXII-COXIV), respectively, as well as mitochondrial apoptotic events [voltage-dependent-anion-channel-1(VDAC-1)-content and caspase-9 levels] were quantified by western blot, with mitochondrial mass being assessed by spectrophotometry (citrate synthase) and flow cytometry (mitotracker green assay). Mitochondrial membrane potential (JC1-assay) and advanced apoptotic/necrotic events (AnexinV/propidium iodide) were measured by flow cytometry. Significant mtDNA depletion spanning 57.67% (P < 0.01) was found in the U1 promonocytic cells further reflected by a significant 77.43% decrease of mitochondrial CIV activity (P < 0.01). These changes were not significant for the ACH2 lymphoid cell line. COXII and COXIV subunits as well as VDAC-1 and caspase-9 content were sharply decreased in both chronic HIV-1-infected promonocytic and lymphoid cell lines (<0.005 in most cases). In addition, U1 and ACH2 cells showed a trend (moderate in case of ACH2), albeit not significant, to lower levels of depolarized mitochondrial membranes. The present in vitro lymphoid and especially promonocytic HIV model show marked mitochondrial lesion but apoptotic resistance phenotype that has been only partially demonstrated in patients. This model may provide a platform for the characterization of HIV-chronicity, to test novel therapeutic options or to study HIV reservoirs

Genetic Study of SARS-CoV-2 Non Structural Protein 12 in COVID-19 Patients Non Responders to Remdesivir

Remdesivir (RDV) was the first antiviral drug approved by the FDA to treat severe coronavirus disease-2019 (COVID-19) patients. RDV inhibits SARS-CoV-2 replication by stalling the non structural protein 12 (nsp12) subunit of the RNA-dependent RNA polymerase (RdRp). No evidence of global widespread RDV-resistance mutations has been reported, however, defining genetic pathways to RDV resistance and determining emergent mutations prior and subsequent antiviral therapy in clinical settings is necessary. This study identified 57/149 (38.3%) patients who did not respond to one course (5-days) (n = 36/111, 32.4%) or prolonged (5 to 20 days) (n = 21/38, 55.3%) RDV therapy by subgenomic RNA detection. Genetic variants in the nsp12 gene were detected in 29/49 (59.2%) non responder patients by Illumina sequencing, including the de novo E83D mutation that emerged in an immunosuppressed patient after receiving 10 + 8 days of RDV, and the L838I detected at baseline and/or after prolonged RDV treatment in 9/49 (18.4%) non responder subjects. Although 3D protein modeling predicted no interference with RDV, the amino acid substitutions detected in the nsp12 involved changes on the electrostatic outer surface and in secondary structures that may alter antiviral response. It is important for health surveillance to study potential mutations associated with drug resistance as well as the benefit of RDV retreatment, especially in immunosuppressed patients and in those with persistent replication. IMPORTANCE This study provides clinical and microbiologic data of an extended population of hospitalized patients for COVID-19 pneumonia who experienced treatment failure, detected by the presence of subgenomic RNA (sgRNA). The genetic variants found in the nsp12 pharmacological target of RDV bring into focus the importance of monitoring emergent mutations, one of the objectives of the World Health Organization (WHO) for health surveillance. These mutations become even more crucial as RDV keeps being prescribed and new molecules are being repurposed for the treatment of COVID-19. The present article offers new perspectives for the clinical management of non responder patients treated and retreated with RDV and emphasizes the need of further research of the benefit of combinatorial therapies and RDV retreatment, especially in immunosuppressed patients with persistent replication after therapy.This work was financed by a Gilead Sciences grant (IN-ES-540-6089) and CIBER Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, España (CB21/13/00081). This work was financed by ad hoc patronage funds for research on COVID-19 from donations from citizens and organizations to the Hospital Clínic de Barcelona-Fundació Clínic per a la Recerca Biomèdica.S

Generation and characterization of a defective HIV-1 Virus as an immunogen for a therapeutic vaccine

BACKGROUND: The generation of new immunogens able to elicit strong specific immune responses remains a major challenge in the attempts to obtain a prophylactic or therapeutic vaccine against HIV/AIDS. We designed and constructed a defective recombinant virus based on the HIV-1 genome generating infective but non-replicative virions able to elicit broad and strong cellular immune responses in HIV-1 seropositive individuals. RESULTS: Viral particles were generated through transient transfection in producer cells (293-T) of a full length HIV-1 DNA carrying a deletion of 892 base pairs (bp) in the pol gene encompassing the sequence that codes for the reverse transcriptase (NL4-3/ΔRT clone). The viral particles generated were able to enter target cells, but due to the absence of reverse transcriptase no replication was detected. The immunogenic capacity of these particles was assessed by ELISPOT to determine γ-interferon production in a cohort of 69 chronic asymptomatic HIV-1 seropositive individuals. Surprisingly, defective particles produced from NL4-3/ΔRT triggered stronger cellular responses than wild-type HIV-1 viruses inactivated with Aldrithiol-2 (AT-2) and in a larger proportion of individuals (55% versus 23% seropositive individuals tested). Electron microscopy showed that NL4-3/ΔRT virions display immature morphology. Interestingly, wild-type viruses treated with Amprenavir (APV) to induce defective core maturation also induced stronger responses than the same viral particles generated in the absence of protease inhibitors. CONCLUSIONS: We propose that immature HIV-1 virions generated from NL4-3/ΔRT viral clones may represent new prototypes of immunogens with a safer profile and stronger capacity to induce cellular immune responses than wild-type inactivated viral particles.This study was supported by grants FIS PI050265, FIS PI040503, FIS PI070291, FIS Intrasalud 080752, FIS PS09/01297, FIS PI10/02984, SAF2006-26667-E, FIT 09-010-205-9, FIPSE 36780/08, Fundación Mútua Madrileña, TRA-094, EC10-153, ISCIII-RETIC RD06/0006, HIVACAT–HIV Development Program in Catalonia, FIPSE 36630/07, UE Program Health 2009 CHAARM. Spanish Health Institute Carlos III (ISCIII) and the Health Department of the Catalan Government (Generalitat de Catalunya). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S