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The high morbimortality due to SARS-CoV-2 infection in oncohematological diseases (OHD) and hematopoietic stem cell transplant (HSCT) recipients in the pre-vaccine era has made vaccination a priority in this group. After HSCT, the immune responses against common vaccines such as tetanus, varicella, rubella, and polio may be lost. However, the loss of immunity developed by COVID-19 vaccination after HSCT has not been completely defined. In this study, both humoral and cellular immunity against SARS-CoV-2 were analyzed in 29 individuals with OHD who were vaccinated before receiving allogeneic (n = 11) or autologous (n = 18) HSCT. All participants had low but protective levels of neutralizing IgGs against SARS-CoV-2 after HSCT despite B-cell lymphopenia and immaturity. Although antibody-dependent cellular cytotoxicity was impaired, direct cellular cytotoxicity was similar to healthy donors in participants with autologous-HSCT, in contrast to individuals with allogeneic–HSCT, which severely deteriorated. No significant changes were observed in the immune response before and after HSCT. During follow-up, all reported post-HSCT SARS-CoV-2 infections were mild. This data emphasizes that COVID-19 vaccination is effective, necessary, and safe for individuals with OHD and also supports the persistence of some degree of immune protection after HSCT, at least in the short term, when patients cannot yet be revaccinated. This work was supported by projects PI21/00877 and PI22CIII/00059, funded by the Strategic Action in Health of the Instituto de Salud Carlos III (SICIII) and co-funded by European Regional Development Fund (ERDF), “A way to make Europe”; the Coordinated Research Activities at the National Center of Microbiology (CNM, Instituto de Salud Carlos III) (COV20_00679) to promote an integrated response against SARS-CoV-2 in Spain (Spanish Ministry of Science and Innovation) that is coordinated by Dr Inmaculada Casas (WHO National Influenza Center of the CNM); a generous donation provided by Chiesi España, S.A.U. (Barcelona, Spain); and the Spanish Ministry of Science and Innovation (PID2019-110275RB-I00). The work of Sara Rodríguez-Mora is financed by NIH grant R01AI143567. The work of Montserrat Torres and Guiomar Casado are financed by CIBERINFEC, co-financed by ERDF, “A way to make Europe”. The work of Clara Sánchez-Menéndez is financed by Programa Investigo, FIBio HRC-IRYCIS, co-financed by ERDF, “A way to make Europe”. Sí

The main objective of this study was to determine the influence of the cytotoxic activity of peripheral blood mononuclear cells (PBMCs) on the outcome of unvaccinated individuals with critical COVID-19 admitted to the ICU. Blood samples from 23 individuals were collected upon admission and then every 2 weeks for 13 weeks until death (Exitus group) (n = 13) or discharge (Survival group) (n = 10). We did not find significant differences between groups in sociodemographic, clinical, or biochemical data that may influence the fatal outcome. However, direct cellular cytotoxicity of PBMCs from individuals of the Exitus group against pseudotyped SARS-CoV-2-infected Vero E6 cells was significantly reduced upon admission (−2.69-fold; p = 0.0234) and after 4 weeks at the ICU (−5.58-fold; p = 0.0290), in comparison with individuals who survived, and it did not improve during hospitalization. In vitro treatment with IL-15 of these cells did not restore an effective cytotoxicity at any time point until the fatal outcome, and an increased expression of immune exhaustion markers was observed in NKT, CD4+, and CD8+ T cells. However, IL-15 treatment of PBMCs from individuals of the Survival group significantly increased cytotoxicity at Week 4 (6.18-fold; p = 0.0303). Consequently, immunomodulatory treatments that may overcome immune exhaustion and induce sustained, efficient cytotoxic activity could be essential for survival during hospitalization due to critical COVID-19. This work was supported by the Coordinated Research Activities at the National Center of Microbiology (CNM, Instituto de Salud Carlos III) (COV20_00679) to promote an integrated response against SARS-CoV-2 in Spain (Spanish Ministry of Science and Innovation) that is coordinated by Dr Inmaculada Casas (WHO National Influenza Center of the CNM); a generous donation provided by Chiesi España, S.A.U. (Barcelona, Spain); the Spanish Ministry of Science and Innovation (PID2019-110275RB-I00). The work of Guiomar Casado is financed by CIBERINFEC, co-financed by the European Regional Development Fund (FEDER) “A way to make Europe”. The work of Montserrat Torres is supported by Instituto de Salud Carlos III (COV20_00679). The work of Fernando Ramos Martín is financed by the Spanish Ministry of Science and Innovation (PID2019-110275RB-I00). The work of Mario Manzanares is supported by a pre-doctoral grant from Instituto de Salud Carlos III (ISCIII-PFIS FI20CIII/00021). The work of Lorena Vigón is supported by a pre-doctoral grant from Instituto de Salud Carlos III (FIS PI16CIII/00034-ISCIII-FEDER). The work of Sara Rodríguez-Mora is financed by NIH grant R01AI143567. No

Macrophages are one of the main cellular targets of human immunodeficiency virus type 1 (HIV-1). Macrophage infection by HIV-1 is inefficient due to the presence of the viral restriction factor sterile alpha motif and histidine aspartic acid domain containing protein 1 (SAMHD1). Ex vivo human monocyte-derived macrophages (MDMs) express SAMHD1 in an equilibrium between active (unphosphorylated) and inactive (phosphorylated) states. We and others have shown that treatment of MDMs with the FDA-approved tyrosine kinase inhibitor, dasatinib, ablates SAMHD1 phosphorylation, thus skewing the balance towards a cellular state that is refractory to HIV-1 infection. We hypothesized that dasatinib inhibits a putative tyrosine kinase that is upstream of SAMHD1. In search for this tyrosine kinase, we probed several candidates and were unable to identify a single target that, when inhibited, was sufficient to explain the dephosphorylation of SAMHD1 we observe upon treatment with dasatinib. On the other hand, we probed the ability of dasatinib to directly inhibit the serine/threonine cyclin dependent kinases 1, 2, 4 and 6 and confirmed that dasatinib directly inhibits these kinases. Therefore, our results show that inhibition of the proximal CDKs 1, 2, 4 and 6 by dasatinib is clearly detectable, leads to blockade of infection by HIV-1, and may be sufficient to explain the activity of dasatinib against SAMHD1 phosphorylation.

Up to half of individuals who contract SARS-CoV-2 develop symptoms of long-COVID approximately three months after initial infection. These symptoms are highly variable, and the mechanisms inducing them are yet to be understood. We compared plasma cytokine levels from individuals with long-COVID to healthy individuals and found that those with long-COVID had 100% reductions in circulating levels of interferon gamma (IFNγ) and interleukin-8 (IL-8). Additionally, we found significant reductions in levels of IL-6, IL-2, IL-17, IL-13, and IL-4 in individuals with long-COVID. We propose immune exhaustion as the driver of long-COVID, with the complete absence of IFNγ and IL-8 preventing the lungs and other organs from healing after acute infection, and reducing the ability to fight off subsequent infections, both contributing to the myriad of symptoms suffered by those with long-COVID.

Previously, we identified a stabilization of the HIV reservoir size in HIV + monoinfected and HIV+/HCV + coinfected patients who eliminated HCV with DAAs as well as a decrease in HIV reservoir size in HIV+/HCV- spontaneously clearers. However, variations in the distribution of memory CD4 + T cell subpopulations in coinfected patients after HCV clearance remains unclear. To analyze the expression pattern of T cell maturation (CD45RA and CD27) and senescence (CD28 and CD57) markers by flow cytometry in those patients. Longitudinal study (52 weeks follow-up) of 49 HIV+/HCV+, 38 HIV+/HCV- and 59 HIV + subjects, all of them aviremic. A generalized increase in expression of CD28 was observed for CD8 + and in CD4 + CD27 + cells. The proportion of both CD4 + TEM TH1 and TH2 cells decreased significantly in HIV+/HCV- while remained stable in HIV + and HIV+/HCV + individuals. The general decline in cell senescence of cytotoxic lymphocytes may be due to reduced antigenic stimulation of HIV-specific and HCV-specific lymphocytes. The dynamic of HIV viral reservoir in HIV+/HCV- patients matches the dynamic of CD4 + cells with a TH1 phenotype, which could mean a decrease in the number of circulating TFH1 lymphocytes after an improvement in generalized inflammation state.

Main cause of severe illness and death in COVID-19 patients appears to be an excessive but ineffectual inflammatory immune response that may cause severe acute respiratory distress syndrome (ARDS). Vitamin D may favour an anti-inflammatory environment and improve cytotoxic response against some infectious diseases. A multicenter, single-blind, prospective, randomized clinical trial was approved in patients with COVID-19 pneumonia and levels of 25-hydroxyvitamin D (25(OH)D) of 14.8 ng/ml (SD: 6.18) to test antiviral efficacy, tolerance and safety of 10,000 IU/day of cholecalciferol (vitamin D3) for 14 days, in comparison with 2000 IU/day. After supplementation, mean serum 25(OH)D levels increased to 19 ng/ml on average in 2000 IU/day versus 29 ng/ml in 10,000 IU/day group (p

Factor de impacto: 5,858 Q1 The latent viral reservoir formed by HIV-1, mainly in CD4+ T cells, is responsible for the failure of antiretroviral therapy (ART) to achieve a complete elimination of the virus in infected individuals. We previously determined that CD4+ T cells from individuals with chronic myeloid leukemia (CML) on treatment with dasatinib are resistant to HIV-1 infection ex vivo. The main mechanism for this antiviral effect is the preservation of SAMHD1 activity. In this study, we aimed to evaluate the impact of dasatinib on the viral reservoir of HIV-infected individuals with CML who were on simultaneous treatment with ART and dasatinib. Due to the low estimated incidence of HIV-1 infection and CML (1:65,000), three male individuals were recruited in Spain and Germany. These individuals had been on treatment with standard ART and dasatinib for median 1.3 years (IQR 1.3-5.3 years). Reservoir size and composition in PBMCs from these individuals was analyzed in comparison with HIV-infected individuals on triple ART regimen and undetectable viremia. The frequency of latently infected cells was reduced more than 5-fold in these individuals. The reactivation of proviruses from these cells was reduced more than 4-fold and, upon activation, SAMHD1 phosphorylation was reduced 40-fold. Plasma levels of the homeostatic cytokine IL-7 and CD4 effector subpopulations TEM and TEMRA in peripheral blood were also reduced. Therefore, treatment of HIV-infected individuals with dasatinib as adjuvant of ART could disturb the reservoir reactivation and reseeding, which might have a beneficial impact to reduce its size. This work was supported by NIH grant R01AI143567; the Spanish Ministry of Science and Innovation (PID2019-110275RB- 23I00); the Spanish AIDS Research Network RD16CIII/0002/0001 that is included in Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2016-2020, Instituto de Salud Carlos III, European Region Development Fund (ERDF). The work of María Rosa López-Huertas and Sara Rodríguez-Mora is financed by NIH grant R01AI143567. The work of Lorena Vigón is supported by a pre-doctoral grant from Instituto de Salud Carlos III (FIS PI16CIII/00034-ISCIII-FEDER). Jose M. Miró received a personal 80:20 research grant from Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) (Barcelona, Spain) during 2017–2021. Sí

Background Virus neutralization by antibodies is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal murine leukemia virus genome encoding firefly luciferase. This assay design is intended for use in laboratories with biocontainment level 2 and therefore circumvents the need for the biocontainment level 3 that would be required for replication-competent SARS-CoV-2 virus. To validate the pseudovirion assay, we set up comparisons with other available antibody tests including those from Abbott, Euroimmun and Siemens, using archived, known samples. Results 11 out of 12 SARS-CoV-2-infected patient serum samples showed neutralizing activity against SARS-CoV-2-spike pseudotyped MLV viruses, with neutralizing titers-50 (NT50) that ranged from 1:25 to 1:1,417. Five historical samples from patients hospitalized for severe influenza infection in 2016 tested negative in the neutralization assay (NT50 50 2 = 0.9344). Conclusions SARS-CoV-2 spike/MLV pseudovirions provide a practical means of assessing neutralizing activity of antibodies in serum or plasma from infected patients under laboratory conditions consistent with biocontainment level 2. This assay offers promise also in evaluating immunogenicity of spike glycoprotein-based candidate vaccines in the near future.

BCR-ABL is an aberrant tyrosine kinase responsible for chronic myeloid leukemia (CML). Tyrosine kinase inhibitors (TKIs) induce a potent antileukemic response mostly based on the inhibition of BCR-ABL, but they also increase the activity of Natural Killer (NK) and CD8+ T cells. After several years, patients may interrupt treatment due to sustained, deep molecular response. By unknown reasons, half of the patients relapse during treatment interruption, whereas others maintain a potent control of the residual leukemic cells for several years. In this study, several immunological parameters related to sustained antileukemic control were analyzed. According to our results, the features more related to poor antileukemic control were as follows: low levels of cytotoxic cells such as NK, (Natural Killer T) NKT and CD8&plusmn, TCR&gamma, &beta, + T cells, low expression of activating receptors on the surface of NK and NKT cells, impaired synthesis of proinflammatory cytokines or proteases from NK cells, and HLA-E*0103 homozygosis and KIR haplotype BX. A Random Forest algorithm predicted 90% of the accuracy for the classification of CML patients in groups of relapse or non-relapse according to these parameters. Consequently, these features may be useful as biomarkers predictive of CML relapse in patients that are candidates to initiate treatment discontinuation.

The presence of latent human immunodeficiency virus 1 (HIV-1) in quiescent memory CD4+ T cells represents a major barrier to viral eradication. Proliferation of memory CD4+ T cells is the primary mechanism that leads to persistence of the latent reservoir, despite effective antiretroviral therapy (ART). Memory CD4+ T cells are long-lived and can proliferate through two mechanisms: homeostatic proliferation via γc-cytokine stimulation or antigen-driven proliferation. Therefore, therapeutic modalities that perturb homeostatic and antigen-driven proliferation, combined with ART, represent promising strategies to reduce the latent reservoir. In this study, we investigated a library of FDA-approved oncology drugs to determine their ability to inhibit homeostatic and/or antigen-driven proliferation. We confirmed potential hits by evaluating their effects on proliferation in memory CD4+ T cells from people living with HIV-1 on ART (PLWH) and interrogated downstream signaling of γc-cytokine stimulation. We found that dasatinib and ponatinib, tyrosine kinase inhibitors, and trametinib, MEK inhibitor, reduced both homeostatic and antigen-driven proliferation by 65%, with a reduction in viability less than 45%, ex vivo. In memory CD4+ T cells from PLWH, only dasatinib restricted both homeostatic and antigen-driven proliferation and prevented spontaneous rebound, consistent with promoting a smaller reservoir size. We show that dasatinib restricts IL-7 induced proliferation through STAT5 phosphorylation inhibition. Our results establish that the anti-cancer agent, dasatinib, is an exciting candidate to be used as an anti-proliferative drug in a clinical trial since it efficiently blocks proliferation and is well tolerated in patients with chronic myeloid leukemia (CML).

A potential strategy to cure HIV-1 infection is to use latency reversing agents (LRAs) to eliminate latent reservoirs established in resting CD4+ T (rCD4+) cells. As no drug has been shown to be completely effective, finding new drugs and combinations are of increasing importance. We studied the effect of Maraviroc (MVC), a CCR5 antagonist that activates NF-κB, on HIV-1 replication from latency. HIV-1-latency models based on CCL19 or IL7 treatment, before HIV-1 infection were used. Latently infected primary rCD4+ or central memory T cells were stimulated with MVC alone or in combination with Bryostatin-1, a PKC agonist known to reverse HIV-1 latency. MVC 5 μM and 0.31 μM were chosen for further studies although other concentrations of MVC also increased HIV-1 replication. MVC was as efficient as Bryostatin-1 in reactivating X4 and R5-tropic HIV-1. However, the combination of MVC and Bryostatin-1 was antagonistic, probably because Bryostatin-1 reduced CCR5 expression levels. Although HIV-1 reactivation had the same tendency in both latency models, statistical significance was only achieved in IL7-treated cells. These data suggest that MVC should be regarded as a new LRA with potency similar as Bryostatin-1. Further studies are required to describe the synergistic effect of MVC with other LRAs. The authors would like to acknowledge the staff at the Institute of Health Carlos III (National Centre for Microbiology, Madrid, Spain) for their support. No

Factor de impacto: 5,858 Q1 Tyrosine kinase inhibitors (TKIs) are successfully used in clinic to treat chronic myeloid leukemia (CML). Our group previously described that CD4+ T cells from patients with CML on treatment with TKIs such as dasatinib were resistant to HIV-1 infection ex vivo. The main mechanism for this antiviral activity was primarily based on the inhibition of SAMHD1 phosphorylation, which preserves the activity against HIV-1 of this innate immune factor. Approximately 50% CML patients who achieved a deep molecular response (DMR) may safely withdraw TKI treatment without molecular recurrence. Therefore, it has been speculated that TKIs may induce a potent antileukemic response that is maintained in most patients even one year after treatment interruption (TI). Subsequent to in vitro T-cell activation, we observed that SAMHD1 was phosphorylated in CD4+ T cells from CML patients who withdrew TKI treatment more than one year earlier, which indicated that these cells were now susceptible to HIV-1 infection. Importantly, these patients were seronegative for HIV-1 and seropositive for cytomegalovirus (CMV), but without CMV viremia. Although activated CD4+ T cells from CML patients on TI were apparently permissive to HIV-1 infection ex vivo, the frequency of proviral integration was reduced more than 12-fold on average when these cells were infected ex vivo in comparison with cells isolated from untreated, healthy donors. This reduced susceptibility to infection could be related to an enhanced NK-dependent cytotoxic activity, which was increased 8-fold on average when CD4+ T cells were infected ex vivo with HIV-1 in the presence of autologous NK cells. Enhanced cytotoxic activity was also observed in CD8 + T cells from these patients, which showed 8-fold increased expression of TCRγδ and more than 18-fold increased production of IFNγ upon activation with CMV peptides. In conclusion, treatment with TKIs induced a potent antileukemic response that may also have antiviral effects against HIV-1 and CMV, suggesting that transient use of TKIs in HIV-infected patients could develop a sustained antiviral response that would potentially interfere with HIV-1 reservoir dynamics. This work was supported by NIH grant R01AI143567; the Spanish Ministry of Economy and Competitiveness (SAF2016-78480-R); the Spanish AIDS Research Network RD16CIII/0002/0001 that is included in Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2016-2020, Instituto de Salud Carlos III, European Region Development Fund (ERDF). The work of María Rosa López-Huertas and Sara Rodríguez-Mora is financed by NIH grant R01AI143567. The work of Lorena Vigón is supported by a pre-doctoral grant from Instituto de Salud Carlos III (FIS PI16CIII/00034-ISCIII-FEDER). The work of Elena Mateos is supported by the Spanish Ministry of Economy and Competitiveness SAF2016-78480-R. Sí

Coinfection with hepatitis C virus (HCV) influences HIV reservoir size. However, it is unknown whether this coinfection also induces a higher provirus transcription. Viral transcription is promoted by synergy between cellular factors such as NF-&kappa, B and the viral regulator Tat. The impact of HCV coinfection on HIV provirus transcription was analyzed in resting (r)CD4 T+ cells (CD3+CD4+CD25-CD69-HLADR-) and rCD4 T cells-depleted PBMCs (rCD4 T- PBMCs) from a multicenter cross-sectional study of 115 cART-treated HIV patients: 42 HIV+/HCV+ coinfected individuals (HIV+/HCV+), 34 HIV+ patients with HCV spontaneous clearance (HIV+/HCV&minus, ) and 39 HIV patients (HIV+). Viral transcription was assessed in total RNA through the quantification of unspliced, single spliced, and multiple spliced viral mRNAs by qPCR. Linear correlations between viral reservoir size and viral splicing were determined. A 3-fold increase of multiple spliced transcripts in rCD4 T+ cells of HIV+/HCV+ patients was found compared to HIV+ individuals (p &lt, 0.05). As Tat is synthesized by multiple splicing, the levels of Tat were also quantified in these patients. Significant differences in single and multiple spliced transcripts were also observed in rCD4 T- PBMCs. Levels of multiple spliced mRNAs were increased in rCD4 T+ cells isolated from HIV+/HCV+ subjects, which could indicate a higher Tat activity in these cells despite their resting state.

The causative mutation responsible for limb girdle muscular dystrophy 1F (LGMD1F) is one heterozygous single nucleotide deletion in the stop codon of the nuclear import factor Transportin 3 gene (TNPO3). This mutation causes a carboxy-terminal extension of 15 amino acids, producing a protein of unknown function (TNPO3_mut) that is co-expressed with wild-type TNPO3 (TNPO3_wt). TNPO3 has been involved in the nuclear transport of serine/arginine-rich proteins such as splicing factors and also in HIV-1 infection through interaction with the viral integrase and capsid. We analyzed the effect of TNPO3_mut on HIV-1 infection using PBMCs from patients with LGMD1F infected ex vivo. HIV-1 infection was drastically impaired in these cells and viral integration was reduced 16-fold. No significant effects on viral reverse transcription and episomal 2-LTR circles were observed suggesting that the integration of HIV-1 genome was restricted. This is the second genetic defect described after CCR5Δ32 that shows strong resistance against HIV-1 infection., Author summary TNPO3 has been described as a key factor in the infection by the human immunodeficiency virus (HIV-1), the causative agent of AIDS. In 2013, a relationship between a genetic defect in TNPO3 gene and a rare muscle disease named Limb Girdle Muscular Dystrophy 1F (LGMD1F), with an autosomal dominant transmission, was discovered. LGMD1F patients show a heterozygous single nucleotide deletion in the TNPO3 gene that generates a TNPO3_mut protein. Our results demonstrate that cells from patients with this mutation in TNPO3 are resistant to HIV-1 infection in vitro. We are faced with an in vivo situation in which the genetic defect that causes this rare disease confers resistance to HIV infection. Therefore, TNPO3 mutation represents a natural model to understand the pathogenesis of both diseases. Cells from LGMD1F patients can be used to understand the mechanisms of action of TNPO3 in HIV infection and to design new therapeutic strategies for the treatment of both diseases. The use of HIV-1 as a methodological tool will permit a better understanding of the physiopathological mechanisms derived from the mutation in TNPO3 that causes the muscle disease.

HIV-1 infection remains incurable despite the efficient combined antiretroviral therapy (cART) due to the formation of long-lived viral reservoirs that are mostly settled in CD4+T cells and maintained by homeostatic proliferation. The use of cytostatic drugs such as tyrosine kinase inhibitors (TKIs) as adjuvants to cART could be helpful to avoid the reservoir establishment and replenishment. We determined previously that TKI dasatinib, which is successfully used for treating chronic myeloid leukemia (CML), shows antiviral effect against HIV-1 infection of CD4+ T cells in vitro. HIV-infected subjects that developed CML may safely combine long-term treatment with TKIs and cART but there is no information about the effect of dasatinib on HIV-1 reservoir in vivo. Therefore, we analyzed the ability of dasatinib to protect NSG mice engrafted with human CD34+ hematopoietic stem cells from HIV-1 infection. Mice were randomly assigned to two groups that received dasatinib or placebo daily by oral gavage. After five days, all mice were infected intraperitoneally with HIV-1 and followed up for 21 days in the absence of cART. Daily administration of dasatinib decreased viral and proviral load in all treated mice, showing in 40% of these mice undetectable viral RNA or DNA in blood. Proviral HIV-1 DNA in gut-associated lymphoid tissue (GALT) was also reduced in all dasatinib-treated mice and under the limit of detection in one of these mice. Finally, treatment with dasatinib modified the distribution of CD4+ and CD8+ T-cell subpopulations, delaying their differentiation into memory T-cell subsets that are a major component of the viral reservoir. In conclusion, dasatinib afforded protection of NSG mice from HIV-1 intraperitoneal infection in the absence of cART. Funding for this research was provided by: Ministerio de Economía y Competitividad (SAF2013-44677-R, SAF2016-78480-R) Ministerio de Ciencia, Innovación y Universidades (FIS PI16CIII/00034-ISCIII-FEDER) European Regional Development Fund Bristol-Myers Squibb (BMS AI471-041) Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ANRS 2014-2) Spanish Ministry of Education, Culture and Sport (FPU15/03698). Sí

This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2013-44677-R, SAF2016-78480-R); the Instituto de Salud Carlos III (ISCIII-FIS PI16CIII/00034); and the Spanish AIDS Research Network RD16CIII/0002/0001-ISCIII-FEDER. JAM developed this work in the frame of a ‘Juan de la Cierva 2012’ post-doctoral program, Ministerio de Competitividad, Spain. JMM received a personal intensification research grant #INT15/00168 during 2016 from Instituto de Salud Carlos III, Madrid, Spain.

Monográfico con los resúmenes del Ninth International Workshop on HIV Persistence during Therapy 10-13 December 2019, Miami, Florida, USA Sí

HIV-1 infection cannot be cured due to reservoirs formed early after infection. Decreasing the massive CD4+ T cell activation that occurs at the beginning of the disease would delay reservoir seeding, providing a better prognosis for patients. CD4+ T cell activation is mediated by protein kinase C (PKC) theta (θ), which is involved in T-cell proliferation, as well as NF-κB, NF-AT, and AP-1 activation. We found that PKCθ activity increased viral replication, but also that HIV-1 induced higher activation of PKCθ in infected CD4+ T cells, creating a feedback loop. Therefore, specific inhibition of PKCθ activity could contribute to control HIV-1 replication. We tested the efficacy of seven PKCθ specific inhibitors to control HIV-1 replication in CD4+ T cells and selected two of the more potent and safer: CGX1079 and CGX0471. They reduced PKCθ phosphorylation at T538 and its translocation to the plasma membrane, which correlated with decreased HIV-1 retrotranscription through partial inhibition of SAMHD1 antiviral activity, rendering lower proviral integration. CGX1079 and CGX0471 also interfered with viral transcription, which would reduce the production of new virions, as well as the subsequent spread and infection of new targets that would increase the reservoir size. CGX1079 and CGX0471 did not completely abrogate T-cell functions such as proliferation and CD8-mediated release of IFN-γ in PBMCs from HIV-infected patients, thereby avoiding general immunosuppresion. Consequently, using PKCθ inhibitors as adjuvant of antiretroviral therapy in recently infected patients would decrease the pool of activated CD4+ T cells, thwarting proviral integration and reducing the reservoir size. We greatly appreciate the secretarial assistance of Mrs. Olga Palao. We thank Dr. Monsef Benkirane and Dr. Benjamin Descours who kindly provided the specific antibody against SAMHD1 phosphorylated at T592. We also thank Dr. Javier Martı´nez-Picado and Dr. Maria Carmen Puertas (IrsiCaixa Institute for AIDS Research, Badalona, Spain) for their help with the standardization of qPCRs in our laboratory. This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2010-18388, SAF2013-44677-R, FIS PI12/00506, and FIS PI12/00969); FIPSE (360924/10); the SPANISH AIDS Research Network RD12/0017/ 0015 that is included in the Spanish I + D + I Plan and is co-financed by ISCIII-Subdirección General de Evaluacion and European Funding for Regional Development (FEDER); EUROPRISE Network of Excellence of the EU, grant number LSHP CT-2006-037611, and Agence nationale de recherches sur le sida et les he´ patites virales (ANRS 2014-2). The work of Elena Mateos is supported by a contract of the Instituto de Salud Carlos III (Spain) (MPY 1371/12). The work of Sara Rodrı´guez-Mora is supported by a fellowship of Sara Borrell from Spanish Ministry of Economy and Competitiveness. The work of Marı´a Rosa Lo´ pez-Huertas is supported by a fellowship of the European Union Program Health 2009 (CHAARM). Dr. Montserrat Plana is a researcher at the Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS) and is supported by the Spanish Health Institute Carlos III (ISCIII) and the Health Department of the Catalan Government (Generalitat de Catalunya). Sí

HIV-1 replication is efficiently controlled by the regulator protein Tat (101 amino acids) and codified by two exons, although the first exon (1–72 amino acids) is sufficient for this process. Tat can be released to the extracellular medium, acting as a soluble pro-apoptotic factor in neighboring cells. However, HIV-1-infected CD4+ T lymphocytes show a higher resistance to apoptosis. We observed that the intracellular expression of Tat delayed FasL-mediated apoptosis in both peripheral blood lymphocytes and Jurkat cells, as it is an essential pathway to control T cell homeostasis during immune activation. Jurkat-Tat cells showed impairment in the activation of caspase-8, deficient release of mitochondrial cytochrome c, and delayed activation of both caspase-9 and -3. This protection was due to a profound deregulation of proteins that stabilized the mitochondrial membrane integrity, such as heat shock proteins, prohibitin, or nucleophosmin, as well as to the up-regulation of NF-κB-dependent anti-apoptotic proteins, such as BCL2, c-FLIPS, XIAP, and C-IAP2. These effects were observed in Jurkat expressing full-length Tat (Jurkat-Tat101) but not in Jurkat expressing the first exon of Tat (Jurkat-Tat72), proving that the second exon, and particularly the NF-κB-related motif ESKKKVE, was necessary for Tat-mediated protection against FasL apoptosis. Accordingly, the protection exerted by Tat was independent of its function as a regulator of both viral transcription and elongation. Moreover, these data proved that HIV-1 could have developed strategies to delay FasL-mediated apoptosis in infected CD4+ T lymphocytes through the expression of Tat, thus favoring the persistent replication of HIV-1 in infected T cells.

Dendritic cells (DCs) are professional antigen-presenting cells whose functions are dependent on their degree of differentiation. In their immature state, DCs capture pathogens and migrate to the lymph nodes. During this process, DCs become resident mature cells specialized in antigen presentation. DCs are characterized by a highly limiting environment for human immunodeficiency virus type 1 (HIV-1) replication due to the expression of restriction factors such as SAMHD1 and APOBEC3G. However, uninfected DCs capture and transfer viral particles to CD4 lymphocytes through a trans-enhancement mechanism in which chemokines are involved. We analyzed changes in gene expression with whole-genome microarrays when immature DCs (IDCs) or mature DCs (MDCs) were productively infected using Vpx-loaded HIV-1 particles. Whereas productive HIV infection of IDCs induced expression of interferon-stimulated genes (ISGs), such induction was not produced in MDCs, in which a sharp decrease in ISG- and CXCR3-binding chemokines was observed, lessening trans-infection of CD4 lymphocytes. Similar patterns of gene expression were found when DCs were infected with HIV-2 that naturally expresses Vpx. Differences were also observed under conditions of restrictive HIV-1 infection, in the absence of Vpx. ISG expression was not modified in IDCs, whereas an increase of ISG- and CXCR3-binding chemokines was observed in MDCs. Overall these results suggest that sensing and restriction of HIV-1 infection are different in IDCs and MDCs. We propose that restrictive infection results in increased virulence through different mechanisms. In IDCs avoidance of sensing and induction of ISGs, whereas in MDCs increased production of CXCR3-binding chemokines, would result in lymphocyte attraction and enhanced infection at the immune synapse.IMPORTANCE In this work we describe for the first time the activation of a different genetic program during HIV-1 infection depending on the state of maturation of DCs. This represents a breakthrough in the understanding of the restriction to HIV-1 infection of DCs. The results show that infection of DCs by HIV-1 reprograms their gene expression pattern. In immature cells, productive HIV-1 infection activates interferon-related genes involved in the control of viral replication, thus inducing an antiviral state in surrounding cells. Paradoxically, restriction of HIV-1 by SAMHD1 would result in lack of sensing and IFN activation, thus favoring initial HIV-1 escape from the innate immune response. In mature DCs, restrictive infection results in HIV-1 sensing and induction of ISGs, in particular CXCR3-binding chemokines, which could favor the transmission of HIV to lymphocytes. Our data support the hypothesis that genetic DC reprograming by HIV-1 infection favors viral escape and dissemination, thus increasing HIV-1 virulence. We thank Olga Palao (AIDS Immunopathogenesis Unit) and A. Zaballos (Genomics Unit, Instituto de Salud Carlos III) for their secretarial and technical assistance, respectively. We also greatly appreciate our patients for their willingness to participate.Funding for this research was provided by: Ministerio de Economía y Competitividad (SAF2013-44677-R, PI16CIII/00034, ISCIII-FIS Nº) Instituto de Salud Carlos III (Spanish AIDS Research Network RD16CIII/0002/0001) Sí

La historia de la epidemia del VIH es una historia de dolor y sufrimiento con millones de enfermos y muertos en todo el mundo. Es también una historia de miedos y aislamiento por el estigma y discriminación que sufrieron o sufren muchas personas. Pero a su vez es una historia de lucha, de avances científicos, de organización de la sociedad civil, de puesta en marcha de grandes planes y objetivos mundiales. Desde la aparición a principios de los años ochenta de los primeros casos de neumonía grave en individuos jóvenes sanos, que presentaban una pérdida de las defensas naturales del organismo, para la cual no había nombre, y que suscitó desconcierto en la comunidad científica, los avances en este campo se han producido a gran velocidad (figura 1). En menos de diez años se aisló el virus, se desarrollaron métodos de diagnóstico comerciales y ya se habían descubierto los primeros tratamientos. En pocas enfermedades se produjo tal inversión de recursos destinados a la identificación del agente causal y a la obtención de un tratamiento efectivo. Introducción: La gran epidemia del siglo XX. Capítulo 1: ¿qué es el VIH? Origen y características del virus. Capítulo 2: El sistema inmunitario y el VIH. Capítulo 3: Historia natural de la enfermedad y mecanismos de transmisión. Capítulo 4; La infección por el VIH en cifras. Capítulo 5: El envejecimiento de las personas con infección por el VIH. Capítulo 6: El tratamiento antirretroviral: una historia de fracasos y grandes logros. Capítulo 7: La importancia de la prevención. Bibliografía.

INTRODUCTION: Infection caused by HIV-1 is nowadays a chronic disease due to a highly efficient antiretroviral treatment that is nevertheless, unable to eliminate the virus from the organism. New strategies are necessary in order to impede the formation of the viral reservoirs, responsible for the failure of the antiretroviral treatment to cure the infection. Areas covered: The purpose of this review is to discuss the possibility of using tyrosine kinase inhibitors (TKIs) for the treatment of HIV-1 infection. These inhibitors are successfully used in patients with distinct cancers such as chronic myeloid leukemia. The most relevant papers have been selected and commented. Expert opinion: The family of TKIs are directed against the activation of tyrosine kinases from the Src family. Some of these kinases are essential for the activation of CD4 + T cells, the major target of HIV-1. During acute or primary infection the CD4 + T cells are massively activated, which is mostly responsible for the generation of the reservoirs, the spread of the infection and the destruction of activated CD4 + T cells, infected or not. Consequently, we discuss the possibility of using TKIs as adjuvant of the antiretroviral treatment against HIV-1 infection mostly, but not exclusively, during the acute/recent phase. This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2013-44677-R, SAF2016-78480-R); the Instituto de Salud Carlos III (INT15/00168, ISCIII-FIS PI16CIII/00034); the Spanish AIDS Research Network RD12/0036/0004, RD16CIII/0002/0001-ISCIII-FEDER and the grant RD12/0036/0004; Bristol-Myers Squibb partially financed our study (grant BMS AI471-041). J Ambrosioni developed this work in the frame of a ‘Juan de la Cierva 2012ʹ postdoctoral program, Spanish Ministry of Economy and Competitiveness, Spain. JMM received a personal intensification research grant INT15/00168 in 2016 from Instituto de Salud Carlos III, Madrid, Spain. The European Regional Development Fund (ERDF) ‘A way to build Europe’ also provided funding. Sí

New antiretroviral agents with alternative mechanisms are needed to complement the combination therapies used to treat HIV-1 infections. Here we report the identification of bioavailable molecules that interfere with the gene expression processes of HIV-1. The compounds were detected by screening a small library of FDA-approved drugs with an assay based on measuring the displacement of Rev, and essential virus-encoded protein, from its high-affinity RNA binding site. The antiretroviral activity of two hits was based on interference with post-integration steps of the HIV-1 cycle. Both hits inhibited RRE-Rev complex formation in vitro, and blocked LTR-dependent gene expression and viral transcription in cellular assays. The best compound altered the splicing pattern of HIV-1 transcripts in a manner consistent with Rev inhibition. This mechanism of action is different from those used by current antiretroviral agents. The screening hits recognized the Rev binding site in the viral RNA, and the best compound did so with substantial selectivity, allowing the identification of a new RNA-binding scaffold. These results may be used for developing novel antiretroviral drugs. This project was supported by MINECO of Spain (grant BFU2012-30770 to J.G.), ISCIII of Spain (Intrasalud PI12/0056 to J.A.), the Spanish AIDS Research Network (RD12/0017/0015, co-financed by ISCIII, MINECO and FEDER), the regional government of Valencia Spain (ACOMP/2014/056 to J. G.), and Universidad Católica de Valencia. M. B. was supported by a grant of the Spanish MINECO. The high-throughput screening was performed by the screening service of the CIPF research centre (Valencia Spain). The authors thank LuisGonzález for his involvement in the early stages of the project, as well as Ángel Cantero Camacho and Jesús Beltrán for help with full-length RRE cloning. Sí