KLRG1 expression on natural killer cells is associated with HIV persistence, and its targeting promotes the reduction of the viral reservoir.

Human immunodeficiency virus (HIV) infection induces immunological dysfunction, which limits the elimination of HIV-infected cells during treated infection. Identifying and targeting dysfunctional immune cells might help accelerate the purging of the persistent viral reservoir. Here, we show that chronic HIV infection increases natural killer (NK) cell populations expressing the negative immune regulator KLRG1, both in peripheral blood and lymph nodes. Antiretroviral treatment (ART) does not reestablish these functionally impaired NK populations, and the expression of KLRG1 correlates with active HIV transcription. Targeting KLRG1 with specific antibodies significantly restores the capacity of NK cells to kill HIV-infected cells, reactivates latent HIV present in CD4+ T cells co-expressing KLRG1, and reduces the intact HIV genomes in samples from ART-treated individuals. Our data support the potential use of immunotherapy against the KLRG1 receptor to impact the viral reservoir during HIV persistence.

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.

Limited induction of polyfunctional lung-resident memory T cells against SARS-CoV-2 by mRNA vaccination compared to infection.

Resident memory T cells (TRM) present at the respiratory tract may be essential to enhance early SARS-CoV-2 viral clearance, thus limiting viral infection and disease. While long-term antigen-specific TRM are detectable beyond 11 months in the lung of convalescent COVID-19 patients, it is unknown if mRNA vaccination encoding for the SARS-CoV-2 S-protein can induce this frontline protection. Here we show that the frequency of CD4+ T cells secreting IFNγ in response to S-peptides is variable but overall similar in the lung of mRNA-vaccinated patients compared to convalescent-infected patients. However, in vaccinated patients, lung responses present less frequently a TRM phenotype compared to convalescent infected individuals and polyfunctional CD107a+ IFNγ+ TRM are virtually absent in vaccinated patients. These data indicate that mRNA vaccination induces specific T cell responses to SARS-CoV-2 in the lung parenchyma, although to a limited extend. It remains to be determined whether these vaccine-induced responses contribute to overall COVID-19 control.

Zinc pyrithione is a potent inhibitor of PLPro and cathepsin L enzymes with ex vivo inhibition of SARS-CoV-2 entry and replication.

Zinc pyrithione (1a), together with its analogues 1b-h and ruthenium pyrithione complex 2a, were synthesised and evaluated for the stability in biologically relevant media and anti-SARS-CoV-2 activity. Zinc pyrithione revealed potent in vitro inhibition of cathepsin L (IC50=1.88 ± 0.49 µM) and PLPro (IC50=0.50 ± 0.07 µM), enzymes involved in SARS-CoV-2 entry and replication, respectively, as well as antiviral entry and replication properties in an ex vivo system derived from primary human lung tissue. Zinc complexes 1b-h expressed comparable in vitro inhibition. On the contrary, ruthenium complex 2a and the ligand pyrithione a itself expressed poor inhibition in mentioned assays, indicating the importance of the selection of metal core and structure of metal complex for antiviral activity. Safe, effective, and preferably oral at-home therapeutics for COVID-19 are needed and as such zinc pyrithione, which is also commercially available, could be considered as a potential therapeutic agent against SARS-CoV-2.

Antiretroviral therapy duration and immunometabolic state determine efficacy of ex vivo dendritic cell-based treatment restoring functional HIV-specific CD8+ T cells in people living with HIV.

BACKGROUND: Dysfunction of CD8+ T cells in people living with HIV-1 (PLWH) receiving anti-retroviral therapy (ART) has restricted the efficacy of dendritic cell (DC)-based immunotherapies against HIV-1. Heterogeneous immune exhaustion and metabolic states of CD8+ T cells might differentially associate with dysfunction. However, specific parameters associated to functional restoration of CD8+ T cells after DC treatment have not been investigated. METHODS: We studied association of restoration of functional HIV-1-specific CD8+ T cell responses after stimulation with Gag-adjuvant-primed DC with ART duration, exhaustion, metabolic and memory cell subsets profiles. FINDINGS: HIV-1-specific CD8+ T cell responses from a larger proportion of PLWH on long-term ART (more than 10 years; LT-ARTp) improved polyfunctionality and capacity to eliminate autologous p24+ infected CD4+ T cells in vitro. In contrast, functional improvement of CD8+ T cells from PLWH on short-term ART (less than a decade; ST-ARTp) after DC treatment was limited. This was associated with lower frequencies of central memory CD8+ T cells, increased co-expression of PD1 and TIGIT and reduced mitochondrial respiration and glycolysis induction upon TCR activation. In contrast, CD8+ T cells from LT-ARTp showed increased frequencies of TIM3+ PD1- cells and preserved induction of glycolysis. Treatment of dysfunctional CD8+ T cells from ST-ARTp with combined anti-PD1 and anti-TIGIT antibodies plus a glycolysis promoting drug restored their ability to eliminate infected CD4+ T cells. INTERPRETATION: Together, our study identifies specific immunometabolic parameters for different PLWH subgroups potentially useful for future personalized DC-based HIV-1 vaccines. FUNDING: NIH (R21AI140930), MINECO/FEDER RETOS (RTI2018-097485-A-I00) and CIBERINF grants.

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

Human 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.

Evaluation of SARS-CoV-2 entry, inflammation and new therapeutics in human lung tissue cells.

The development of physiological models that reproduce SARS-CoV-2 infection in primary human cells will be instrumental to identify host-pathogen interactions and potential therapeutics. Here, using cell suspensions directly from primary human lung tissues (HLT), we have developed a rapid platform for the identification of viral targets and the expression of viral entry factors, as well as for the screening of viral entry inhibitors and anti-inflammatory compounds. The direct use of HLT cells, without long-term cell culture and in vitro differentiation approaches, preserves main immune and structural cell populations, including the most susceptible cell targets for SARS-CoV-2; alveolar type II (AT-II) cells, while maintaining the expression of proteins involved in viral infection, such as ACE2, TMPRSS2, CD147 and AXL. Further, antiviral testing of 39 drug candidates reveals a highly reproducible method, suitable for different SARS-CoV-2 variants, and provides the identification of new compounds missed by conventional systems, such as VeroE6. Using this method, we also show that interferons do not modulate ACE2 expression, and that stimulation of local inflammatory responses can be modulated by different compounds with antiviral activity. Overall, we present a relevant and rapid method for the study of SARS-CoV-2.

Poly I:C and STING agonist-primed DC increase lymphoid tissue polyfunctional HIV-1-specific CD8+ T cells and limit CD4+ T-cell loss in BLT mice.

Effective function of CD8+ T cells and enhanced innate activation of DCs in response to HIV-1 is linked to protective antiviral immunity in controllers. Manipulation of DC targeting the master regulator TANK-binding Kinase 1 (TBK1) might be useful to acquire controller-like properties. Here, we evaluated the impact of the combination of 2´3´-c´diAM(PS)2 and Poly I:C as potential adjuvants capable of potentiating DC´s abilities to induce polyfunctional HIV-1 specific CD8+ T-cell responses in vitro and in vivo using a humanized BLT mouse model. Adjuvant combination enhanced TBK-1 phosphorylation and IL-12 and IFN-ß expression on DC and increased their ability to activate polyfunctional HIV-1-specific CD8+ T cells in vitro. Moreover, higher proportions of hBLT mice vaccinated with ADJ-DC exhibited less severe CD4+ T-cell depletion following HIV-1 infection compared to control groups. This was associated with infiltration of CD8+ T cells in the white pulp from the spleen, reduced spread of infected p24+ cells to LN, and with preserved abilities of CD8+ T cells from the spleen and blood of vaccinated animals to induce specific polyfunctional responses upon antigen stimulation. Therefore, priming of DC with PolyI:C and STING agonists might be useful for future HIV-1 vaccine studies.

Entrectinib-A SARS-CoV-2 Inhibitor in Human Lung Tissue (HLT) Cells.

Since the start of the COVID-19 outbreak, pharmaceutical companies and research groups have focused on the development of vaccines and antiviral drugs against SARS-CoV-2. Here, we apply a drug repurposing strategy to identify drug candidates that are able to block the entrance of the virus into human cells. By combining virtual screening with in vitro pseudovirus assays and antiviral assays in Human Lung Tissue (HLT) cells, we identify entrectinib as a potential antiviral drug.

Antibody cooperative adsorption onto AuNPs and its exploitation to force natural killer cells to kill HIV-infected T cells.

HIV represents a persistent infection which negatively alters the immune system. New tools to reinvigorate different immune cell populations to impact HIV are needed. Herein, a novel nanotool for the specific enhancement of the natural killer (NK) immune response towards HIV-infected T-cells has been developed. Bispecific Au nanoparticles (BiAb-AuNPs), dually conjugated with IgG anti-HIVgp120 and IgG anti-human CD16 antibodies, were generated by a new controlled, linker-free and cooperative conjugation method promoting the ordered distribution and segregation of antibodies in domains. The cooperatively-adsorbed antibodies fully retained the capabilities to recognize their cognate antigen and were able to significantly enhance cell-to-cell contact between HIV-expressing cells and NK cells. As a consequence, the BiAb-AuNPs triggered a potent cytotoxic response against HIV-infected cells in blood and human tonsil explants. Remarkably, the BiAb-AuNPs were able to significantly reduce latent HIV infection after viral reactivation in a primary cell model of HIV latency. This novel molecularly-targeted strategy using a bispecific nanotool to enhance the immune system represents a new approximation with potential applications beyond HIV.

Peripheral and lung resident memory T cell responses against SARS-CoV-2.

Resident memory T cells (TRM) positioned within the respiratory tract are probably required to limit SARS-CoV-2 spread and COVID-19. Importantly, TRM are mostly non-recirculating, which reduces the window of opportunity to examine these cells in the blood as they move to the lung parenchyma. Here, we identify circulating virus-specific T cell responses during acute infection with functional, migratory and apoptotic patterns modulated by viral proteins and associated with clinical outcome. Disease severity is associated predominantly with IFNγ and IL-4 responses, increased responses against S peptides and apoptosis, whereas non-hospitalized patients have increased IL-12p70 levels, degranulation in response to N peptides and SARS-CoV-2-specific CCR7+ T cells secreting IL-10. In convalescent patients, lung-TRM are frequently detected even 10 months after initial infection, in which contemporaneous blood does not reflect tissue-resident profiles. Our study highlights a balanced anti-inflammatory antiviral response associated with a better outcome and persisting TRM cells as important for future protection against SARS-CoV-2 infection.

The active human immunodeficiency virus reservoir during antiretroviral therapy: emerging players in viral persistence.

PURPOSE OF REVIEW: To discuss the role of CD4+ T cells with active Human immunodeficiency virus (HIV), meaning infected cells with transcriptional and/or translational viral activity during antiretroviral therapy (ART), focusing on new technologies for its detection, potential cell markers for its characterization, and evidences on the contribution of the active HIV reservoir to long-term viral persistence. RECENT FINDINGS: HIV-infected cells expressing viral ribonucleic acid are systematically detected in subjects on long-term ART. In recent years, powerful new tools have provided significant insights into the nature, quantification, and identification of cells with active HIV, including the identification of new cell markers, and the presence of viral activity in specific cell populations located in different cellular and anatomical compartments. Moreover, studies on viral sequence integrity have identified cell clones with intact viral genomes and active viral transcription that could potentially persist for years. Together, new investigations support the notion that the active reservoir could represent a relevant fraction of long-term infected cells, and therefore, the study of its cell sources and mechanisms of maintenance could represent a significant advance in our understanding of viral persistence and the development of new curative strategies. SUMMARY: The presence of HIV-infected cells with viral expression during ART has been traditionally overlooked for years. Based on recent investigations, this active viral reservoir could play an important role in HIV persistence.

Recommendations for measuring HIV reservoir size in cure-directed clinical trials.

Therapeutic strategies are being clinically tested either to eradicate latent HIV reservoirs or to achieve virologic control in the absence of antiretroviral therapy. Attaining this goal will require a consensus on how best to measure the numbers of persistently infected cells with the potential to cause viral rebound after antiretroviral-therapy cessation in assessing the results of cure-directed strategies in vivo. Current measurements assess various aspects of the HIV provirus and its functionality and produce divergent results. Here, we provide recommendations from the BEAT-HIV Martin Delaney Collaboratory on which viral measurements should be prioritized in HIV-cure-directed clinical trials.

Resident memory T cells are a cellular reservoir for HIV in the cervical mucosa.

HIV viral reservoirs are established very early during infection. Resident memory T cells (TRM) are present in tissues such as the lower female genital tract, but the contribution of this subset of cells to the pathogenesis and persistence of HIV remains unclear. Here, we show that cervical CD4+TRM display a unique repertoire of clusters of differentiation, with enrichment of several molecules associated with HIV infection susceptibility, longevity and self-renewing capacities. These protein profiles are enriched in a fraction of CD4+TRM expressing CD32. Cervical explant models show that CD4+TRM preferentially support HIV infection and harbor more viral DNA and protein than non-TRM. Importantly, cervical tissue from ART-suppressed HIV+ women contain high levels of viral DNA and RNA, being the TRM fraction the principal contributor. These results recognize the lower female genital tract as an HIV sanctuary and identify CD4+TRM as primary targets of HIV infection and viral persistence. Thus, strategies towards an HIV cure will need to consider TRM phenotypes, which are widely distributed in tissues.

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.

Expression of CD20 after viral reactivation renders HIV-reservoir cells susceptible to Rituximab.

The identification of exclusive markers to target HIV-reservoir cells will represent a significant advance in the search for therapies to cure HIV. Here, we identify the B lymphocyte antigen CD20 as a marker for HIV-infected cells in vitro and in vivo. The CD20 molecule is dimly expressed in a subpopulation of CD4-positive (CD4+) T lymphocytes from blood, with high levels of cell activation and heterogeneous memory phenotypes. In lymph node samples from infected patients, CD20 is present in productively HIV-infected cells, and ex vivo viral infection selectively upregulates the expression of CD20 during early infection. In samples from patients on antiretroviral therapy (ART) this subpopulation is significantly enriched in HIV transcripts, and the anti-CD20 monoclonal antibody Rituximab induces cell killing, which reduces the pool of HIV-expressing cells when combined with latency reversal agents. We provide a tool for targeting this active HIV-reservoir after viral reactivation in patients while on ART.

Dendritic Cells From the Cervical Mucosa Capture and Transfer HIV-1 via Siglec-1.

Antigen presenting cells from the cervical mucosa are thought to amplify incoming HIV-1 and spread infection systemically without being productively infected. Yet, the molecular mechanism at the cervical mucosa underlying this viral transmission pathway remains unknown. Here we identified a subset of HLA-DR+ CD14+ CD11c+ cervical DCs at the lamina propria of the ectocervix and the endocervix that expressed the type-I interferon inducible lectin Siglec-1 (CD169), which promoted viral uptake. In the cervical biopsy of a viremic HIV-1+ patient, Siglec-1+ cells harbored HIV-1-containing compartments, demonstrating that in vivo, these cells trap viruses. Ex vivo, a type-I interferon antiviral environment enhanced viral capture and trans-infection via Siglec-1. Nonetheless, HIV-1 transfer via cervical DCs was effectively prevented with antibodies against Siglec-1. Our findings contribute to decipher how cervical DCs may boost HIV-1 replication and promote systemic viral spread from the cervical mucosa, and highlight the importance of including inhibitors against Siglec-1 in microbicidal strategies.

Peering into the HIV reservoir.

The main obstacle to HIV eradication is the establishment of a long-term persistent HIV reservoir. Although several therapeutic approaches have been developed to reduce and eventually eliminate the HIV reservoir, only a few have achieved promising results. A better knowledge of the mechanisms involved in the establishment and maintenance of HIV reservoir is of utmost relevance for the design of new therapeutic strategies aimed at purging it with the ultimate goal of achieving HIV eradication or alternatively a functional cure. In this regard, it is also important to take a close look into the cellular HIV reservoirs other than resting memory CD4 T-cells with key roles in reservoir maintenance that have been recently described. Unraveling the special characteristics of these HIV cellular compartments could aid us in designing new therapeutic strategies to deplete the latent HIV reservoir.

CD32 is expressed on cells with transcriptionally active HIV but does not enrich for HIV DNA in resting T cells.

The persistence of HIV reservoirs, including latently infected, resting CD4+ T cells, is the major obstacle to cure HIV infection. CD32a expression was recently reported to mark CD4+ T cells harboring a replication-competent HIV reservoir during antiretroviral therapy (ART) suppression. We aimed to determine whether CD32 expression marks HIV latently or transcriptionally active infected CD4+ T cells. Using peripheral blood and lymphoid tissue of ART-treated HIV+ or SIV+ subjects, we found that most of the circulating memory CD32+ CD4+ T cells expressed markers of activation, including CD69, HLA-DR, CD25, CD38, and Ki67, and bore a TH2 phenotype as defined by CXCR3, CCR4, and CCR6. CD32 expression did not selectively enrich for HIV- or SIV-infected CD4+ T cells in peripheral blood or lymphoid tissue; isolated CD32+ resting CD4+ T cells accounted for less than 3% of the total HIV DNA in CD4+ T cells. Cell-associated HIV DNA and RNA loads in CD4+ T cells positively correlated with the frequency of CD32+ CD69+ CD4+ T cells but not with CD32 expression on resting CD4+ T cells. Using RNA fluorescence in situ hybridization, CD32 coexpression with HIV RNA or p24 was detected after in vitro HIV infection (peripheral blood mononuclear cell and tissue) and in vivo within lymph node tissue from HIV-infected individuals. Together, these results indicate that CD32 is not a marker of resting CD4+ T cells or of enriched HIV DNA-positive cells after ART; rather, CD32 is predominately expressed on a subset of activated CD4+ T cells enriched for transcriptionally active HIV after long-term ART.

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.IMPORTANCE 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.