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.

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.

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.

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.

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.

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.

Episomal HIV-1 DNA and its relationship to other markers of HIV-1 persistence.

Reverse transcription of HIV-1 results in the generation of a linear cDNA that serves as the precursor to the integrated provirus. Other classes of extrachromosomal viral cDNA molecules can be found in acutely infected cells including the 1-LTR and 2-LTR circles of viral DNA, also referred as episomal HIV-1 DNA. Circulating CD4+ T-cells of treatment-naïve individuals contain significant levels of unintegrated forms of HIV-1 DNA. However, the importance of episomal HIV-1 DNA in the study of viral persistence during antiviral therapy (ART) is debatable. 2-LTR circles are preferentially observed in the effector memory CD4+ T cell subset of long-term treated subjects. Treatment intensification of standard regimens has been used to determine if more potent ART can impact viral reservoir activity. Adding a potent antiretroviral drug to a stable triple-drug regimen has no measurable impact on plasma HIV-1 RNA levels, suggesting that ongoing cycles of HIV-1 replication are not a major mechanism driving persistent plasma viremia during triple-drug ART. However, in randomized clinical trials of HIV-1-infected adults on apparently effective ART, the addition of an integrase inhibitor (raltegravir) to stable regimens resulted in a transient increase in 2-LTR circles in some patients, suggesting a pre-intensification steady-state in which the processes of virion generation and de novo infection were occurring. Mathematical modeling of 2-LTR production during integrase inhibitor intensification suggests the coexistence, at different levels, of ongoing de novo infection and de novo replication mechanisms, specifically in inflamed lymphoid drug sanctuaries. Most reports looking into potential changes in 2-LTR circles in interventional clinical studies have simultaneously assessed other potential surrogate markers of viral persistence. Transient increases in 2-LTR circles have been correlated to decreases in CD8+ T-cell activation, transient CD45RA-CD4+ T-cell redistribution, and decreases in the hypercoagulation biomarker D-dimer in ART-intensified individuals. It is difficult, however, to establish a systematic association because the level of correlation with different types of markers differs significantly among studies. In conclusion, despite suppressive ART, a steady-state of de novo infection may persist in some infected individuals and that this may drive immune activation and inflammation changes reflecting residual viral reservoir activity during otherwise apparently suppressive ART.

Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth.

The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4-2.6) between time point 1 and 2; and median of 31 days (IQR: 28-36) between time point 2 and 3. Patients were median of 6 years (IQR: 3-12) on ART, and plasma viral load (<50 copies/ml) was suppressed for median of 4 years (IQR: 2-8). Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA) was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85), us HIV-1 RNA (p = 0.029, R² = 0.40), and VOA (p = 0.041, R2 = 0.44). Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54). The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1). Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the replication-competent virus in ART suppressed patients.

A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4+ T-Cells to Recognition by Cytotoxic T-Lymphocytes.

Resting CD4+ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8+ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8+ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8+ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8+ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam3CSK4. In contrast, we did not observe CD8+ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist 'ALT-803', an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8+ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8+ T-cells in HIV eradication strategies.

Short-Course Toll-Like Receptor 9 Agonist Treatment Impacts Innate Immunity and Plasma Viremia in Individuals With Human Immunodeficiency Virus Infection.

BACKGROUND.: Treatment with latency reversing agents (LRAs) enhances human immunodeficiency virus type 1 (HIV-1) transcription in vivo but leads to only modest reductions in the size of the reservoir, possibly due to insufficient immune-mediated elimination of infected cells. We hypothesized that a single drug molecule-a novel Toll-like receptor 9 (TLR9) agonist, MGN1703-could function as an enhancer of innate immunity and an LRA in vivo. METHODS.: We conducted a single-arm, open-label study in which 15 virologically suppressed HIV-1-infected individuals on antiretroviral therapy received 60 mg MGN1703 subcutaneously twice weekly for 4 weeks. We characterized plasmacytoid dendritic cell, natural killer (NK), and T-cell activation using flow cytometry on baseline and after 4 weeks of treatment. HIV-1 transcription was quantified by measuring plasma HIV-1 RNA during MGN1703 administration. RESULTS.: In accordance with the cell type-specific expression of TLR9, MGN1703 treatment led to pronounced activation of plasmacytoid dendritic cells and substantial increases in plasma interferon-α2 levels (P < .0001). Consistently, transcription of interferon-stimulated genes (eg, OAS1, ISG15, Mx1; each P < .0001) were upregulated in CD4+ T cells as demonstrated by RNA sequencing. Further, proportions of activated cytotoxic NK cells and CD8+ T cells increased significantly during MGN1703 dosing, suggesting an enhancement of cellular immune responses. In 6 of 15 participants, plasma HIV-1 RNA increased from <20 copies/mL to >1500 copies/mL (range, 21-1571 copies/mL) during treatment. CONCLUSIONS.: TLR9 agonist treatment in HIV infection has a dual potential by increasing HIV-1 transcription and enhancing cytotoxic NK cell activation, both of which are key outcomes in HIV-1 eradication therapy. CLINICAL TRIALS REGISTRATION.: NCT02443935.

Potent Cell-Intrinsic Immune Responses in Dendritic Cells Facilitate HIV-1-Specific T Cell Immunity in HIV-1 Elite Controllers.

The majority of HIV-1 elite controllers (EC) restrict HIV-1 replication through highly functional HIV-1-specific T cell responses, but mechanisms supporting the evolution of effective HIV-1-specific T cell immunity in these patients remain undefined. Cytosolic immune recognition of HIV-1 in conventional dendritic cells (cDC) can facilitate priming and expansion of HIV-1-specific T cells; however, HIV-1 seems to be able to avoid intracellular immune recognition in cDCs in most infected individuals. Here, we show that exposure of cDCs from EC to HIV-1 leads to a rapid and sustained production of type I interferons and upregulation of several interferon-stimulated effector genes. Emergence of these cell-intrinsic immune responses was associated with a reduced induction of SAMHD1 and LEDGF/p75, and an accumulation of viral reverse transcripts, but inhibited by pharmacological blockade of viral reverse transcription or siRNA-mediated silencing of the cytosolic DNA sensor cGAS. Importantly, improved cell-intrinsic immune recognition of HIV-1 in cDCs from elite controllers translated into stronger abilities to stimulate and expand HIV-1-specific CD8 T cell responses. These data suggest an important role of cell-intrinsic type I interferon secretion in dendritic cells for the induction of effective HIV-1-specific CD8 T cells, and may be helpful for eliciting functional T cell immunity against HIV-1 for preventative or therapeutic clinical purposes.

Lack of concordance between residual viremia and viral variants driving de novo infection of CD4(+) T cells on ART.

BACKGROUND: In most patients, current antiretroviral therapy (ART) regimens can rapidly reduce plasma viral load. However, even after years of effective treatment, a significant proportion of patients show residual plasma viremia below the clinical detection limit. Although residual viremia might be associated with increased chronic immune activation and morbidity, its origin and its potential role in the replenishment of the viral reservoir during suppressive ART is not completely understood. We performed an in-depth genetic analysis of the total and episomal cell-associated viral DNA (vDNA) repertoire in purified CD4(+) T cell subsets of three HIV-infected individuals, and used phylogenetic analysis to explore its relationship with plasma viruses. RESULTS: The predominant proviral reservoir was established in naïve or memory (central and transitional) CD4(+) T cell subsets in patients harboring X4- or R5-tropic viruses, respectively. Regardless of the viral tropism, most plasma viruses detected under suppressive ART resembled the proviral reservoir identified in effector and transitional memory CD4(+) T-cell subsets in blood, suggesting that residual viremia originates from these cells in either blood or lymphoid tissue. Most importantly, sequences in episomal vDNA in CD4(+) T-cells were not well represented in residual viremia. CONCLUSIONS: Viral tropism determines the differential distribution of viral reservoir among CD4(+) T-cell subsets. In spite of viral tropism, the effector and transitional memory CD4(+) T-cells subsets are the main source of residual viremia during suppressive ART, even though their contribution to the total proviral pool is small. However, the lack of concordance between residual viremia and viral variants driving de novo infection of CD4(+) T cells on ART may reflect the predominance of defective plasma HIV RNA genomes. These findings highlight the need for monitoring the multiple viral RNA/DNA persistence markers, based on their differential contribution to viral persistence.

Innate Immune Activity Correlates with CD4 T Cell-Associated HIV-1 DNA Decline during Latency-Reversing Treatment with Panobinostat.

UNLABELLED: The pharmaceutical reactivation of dormant HIV-1 proviruses by histone deacetylase inhibitors (HDACi) represents a possible strategy to reduce the reservoir of HIV-1-infected cells in individuals treated with suppressive combination antiretroviral therapy (cART). However, the effects of such latency-reversing agents on the viral reservoir size are likely to be influenced by host immune responses. Here, we analyzed the immune factors associated with changes in proviral HIV-1 DNA levels during treatment with the potent HDACi panobinostat in a human clinical trial involving 15 cART-treated HIV-1-infected patients. We observed that the magnitude, breadth, and cytokine secretion profile of HIV-1-specific CD8 T cell responses were unrelated to changes in HIV-1 DNA levels in CD4 T cells during panobinostat treatment. In contrast, the proportions of CD3(-) CD56(+) total NK cells and CD16(+) CD56(dim) NK cells were inversely correlated with HIV-1 DNA levels throughout the study, and changes in HIV-1 DNA levels during panobinostat treatment were negatively associated with the corresponding changes in CD69(+) NK cells. Decreasing levels of HIV-1 DNA during latency-reversing treatment were also related to the proportions of plasmacytoid dendritic cells, to distinct expression patterns of interferon-stimulated genes, and to the expression of the IL28B CC genotype. Together, these data suggest that innate immune activity can critically modulate the effects of latency-reversing agents on the viral reservoir and may represent a target for future immunotherapeutic interventions in HIV-1 eradication studies. IMPORTANCE: Currently available antiretroviral drugs are highly effective in suppressing HIV-1 replication, but the virus persists, despite treatment, in a latent form that does not actively express HIV-1 gene products. One approach to eliminate these cells, colloquially termed the "shock-and-kill" strategy, focuses on the use of latency-reversing agents that induce active viral gene expression in latently infected cells, followed by immune-mediated killing. Panobinostat, a histone deacetylase inhibitor, demonstrated potent activities in reversing HIV-1 latency in a recent pilot clinical trial and reduced HIV-1 DNA levels in a subset of patients. Interestingly, we found that innate immune factors, such as natural killer cells, plasmacytoid dendritic cells, and the expression patterns of interferon-stimulated genes, were most closely linked to a decline in the HIV-1 DNA level during treatment with panobinostat. These data suggest that innate immune activity may play an important role in reducing the residual reservoir of HIV-1-infected cells.

Long-term antiretroviral treatment initiated at primary HIV-1 infection affects the size, composition, and decay kinetics of the reservoir of HIV-1-infected CD4 T cells.

UNLABELLED: Initiation of antiretroviral therapy during the earliest stages of HIV-1 infection may limit the seeding of a long-lasting viral reservoir, but long-term effects of early antiretroviral treatment initiation remain unknown. Here, we analyzed immunological and virological characteristics of nine patients who started antiretroviral therapy at primary HIV-1 infection and remained on suppressive treatment for >10 years; patients with similar treatment duration but initiation of suppressive therapy during chronic HIV-1 infection served as controls. We observed that independently of the timing of treatment initiation, HIV-1 DNA in CD4 T cells decayed primarily during the initial 3 to 4 years of treatment. However, in patients who started antiretroviral therapy in early infection, this decay occurred faster and was more pronounced, leading to substantially lower levels of cell-associated HIV-1 DNA after long-term treatment. Despite this smaller size, the viral CD4 T cell reservoir in persons with early treatment initiation consisted more dominantly of the long-lasting central-memory and T memory stem cells. HIV-1-specific T cell responses remained continuously detectable during antiretroviral therapy, independently of the timing of treatment initiation. Together, these data suggest that early HIV-1 treatment initiation, even when continued for >10 years, is unlikely to lead to viral eradication, but the presence of low viral reservoirs and durable HIV-1 T cell responses may make such patients good candidates for future interventional studies aiming at HIV-1 eradication and cure. IMPORTANCE: Antiretroviral therapy can effectively suppress HIV-1 replication to undetectable levels; however, HIV-1 can persist despite treatment, and viral replication rapidly rebounds when treatment is discontinued. This is mainly due to the presence of latently infected CD4 T cells, which are not susceptible to antiretroviral drugs. Starting treatment in the earliest stages of HIV-1 infection can limit the number of these latently infected cells, raising the possibility that these viral reservoirs are naturally eliminated if suppressive antiretroviral treatment is continued for extremely long periods of time. Here, we analyzed nine patients who started on antiretroviral therapy within the earliest weeks of the disease and continued treatment for more than 10 years. Our data show that early treatment accelerated the decay of infected CD4 T cells and led to very low residual levels of detectable HIV-1 after long-term therapy, levels that were otherwise detectable in patients who are able to maintain a spontaneous, drug-free control of HIV-1 replication. Thus, long-term antiretroviral treatment started during early infection cannot eliminate HIV-1, but the reduced reservoirs of HIV-1 infected cells in such patients may increase their chances to respond to clinical interventions aiming at inducing a drug-free remission of HIV-1 infection.

Immuno-modulatory strategies for reduction of HIV reservoir cells.

Antiretroviral therapy is able to suppress the viral load to below the detection limit, but it is not able to eradicate HIV reservoirs. Thus, there is a critical need for a novel treatment to eradicate (or reduce) the reservoir in order to eliminate the need for a lifelong adherence to antiretroviral therapy, which is expensive and potentially toxic. In this paper, we investigate the possible pharmacological strategies or combinations of strategies that may be beneficial to reduce or possibly eradicate the latent reservoir. We do this via studies with a validated mathematical model, where the parameter values are obtained with newly acquired clinical data for HIV patients. Our findings indicate that the strategy of reactivating the reservoir combined with enhancement of the killing rate of HIV-specific CD8+ T cells is able to eradicate the reservoir. In addition, our analysis shows that a targeted suppression of the immune system is also a possible strategy to eradicate the reservoir.

Hepatitis C therapy with interferon-α and ribavirin reduces CD4 T-cell-associated HIV-1 DNA in HIV-1/hepatitis C virus-coinfected patients.

Combined treatment with interferon alpha (IFN-α) and ribavirin (RBV) can effectively cure HCV infection in a significant proportion of patients, but effects of this regimen on cellular reservoirs for human immunodeficiency virus type 1 (HIV-1) are unknown. Here, we show that treatment with IFN-α/RBV led to a moderate but significant and sustained decline of HIV-1 DNA in CD4 T cells from HIV-1/hepatitis C virus-coinfected patients receiving highly active antiretroviral therapy (n = 12). However, in vitro experiments failed to demonstrate an effect of pharmacological doses of IFN-α on HIV-1 reactivation. Together, these data suggest that treatment with IFN-α/RBV can moderately reduce the reservoir of HIV-1-infected CD4 T cells that persists despite suppressive antiretroviral therapy.

HIV-1 capture and antigen presentation by dendritic cells: enhanced viral capture does not correlate with better T cell activation.

During HIV-1 infection, dendritic cells (DC) facilitate dissemination of HIV-1 while trying to trigger adaptive antiviral immune responses. We examined whether increased HIV-1 capture in DC matured with LPS results in more efficient Ag presentation to HIV-1-specific CD4(+) and CD8(+) T cells. To block the DC-mediated trans-infection of HIV-1 and maximize Ag loading, we also evaluated a noninfectious integrase-deficient HIV-1 isolate, HIV(NL4-3ΔIN). We showed that higher viral capture of DC did not guarantee better Ag presentation or T cell activation. Greater HIV(NL4-3) uptake by fully LPS-matured DC resulted in higher viral transmission to target cells but poorer stimulation of HIV-1-specific CD4(+) and CD8(+) T cells. Conversely, maturation of DC with LPS during, but not before, viral loading enhanced both HLA-I and HLA-II HIV-1-derived Ag presentation. In contrast, DC maturation with the clinical-grade mixture consisting of IL-1ß, TNF-α, IL-6, and PGE(2) during viral uptake only stimulated HIV-1-specific CD8(+) T cells. Hence, DC maturation state, activation stimulus, and time lag between DC maturation and Ag loading impact HIV-1 capture and virus Ag presentation. Our results demonstrate a dissociation between the capacity to capture HIV-1 and to present viral Ags. Integrase-deficient HIV(NL4-3ΔIN) was also efficiently captured and presented by DC through the HLA-I and HLA-II pathways but in the absence of viral dissemination. HIV(NL4-3ΔIN) seems to be an attractive candidate to be explored. These results provide new insights into DC biology and have implications in the optimization of DC-based immunotherapy against HIV-1 infection.

Radiation therapy for the management of painful bone metastases: Results from a randomized trial.

AIM: The aim of this study was to compare the effectiveness of two radiotherapy schedules in patients with bone metastases. BACKGROUND: We analyzed the need for re-irradiation, rates of pain control, pathological fractures, and functionality in patients randomized to single-fraction (8 Gy 1×) or multiple-fraction radiotherapy (3 Gy 10×) with at least 12 months follow-up, during five years. The hypothesis was that the two radiotherapy schedules are equally effective. MATERIALS AND METHODS: Ninety patients with painful skeletal metastases were randomized to receive single fraction (8 Gy) or multiple fraction (3 Gy 10×) radiotherapy. RESULTS: In the single-fraction group, seven pathological fractures occurred (15.5%) versus two (4.4%) in the multiple-fraction group. There was no statistically significant difference between the time it took to suffer a pathological fracture in both groups (p = 0.099). Patients in the single-fraction group received twelve re-irradiations (26.6%), four in the multiple-fraction group (8.8%), with no significant difference between time elapsed before the first re-irradiation (p = 0.438). CONCLUSION: This study shows no difference between the two groups for the majority of patients with painful bone metastases. Patients were followed up during five years, and the trial showed no disadvantage for 8 Gy 1× compared to 3 Gy 10×. Despite the fact that the pathological fracture rate is 3.75 times higher in the single-fraction group, this schedule is considered more convenient for patients and more cost-effective for radiotherapy departments.

Intraepithelial CD15 infiltration identifies high grade anal dysplasia in people with HIV.

Men who have sex with men (MSM) with HIV are at high risk for squamous intraepithelial lesion (SIL) and anal cancer. Identifying local immunological mechanisms involved in the development of anal dysplasia could aid treatment and diagnostics. Here we studied 111 anal biopsies obtained from 101 MSM with HIV, who participated in an anal screening program. We first assessed multiple immune subsets by flow cytometry, in addition to histological examination, in a discovery cohort (n = 54). Selected molecules were further evaluated by immunohistochemistry in a validation cohort (n = 47). Pathological samples were characterized by the presence of Resident Memory T cells with low expression of CD103 and by changes in Natural Killer cell subsets, affecting residency and activation. Furthermore, potentially immune suppressive subsets, including CD15+CD16+ mature neutrophils, gradually increased as the anal lesion progressed. Immunohistochemistry confirmed the association between the presence of CD15 in the epithelium and SIL diagnosis, with a sensitivity of 80% and specificity of 71% (AUC 0.762) for the correlation with high-grade SIL. A complex immunological environment with imbalanced proportions of resident effectors and immune suppressive subsets characterizes pathological samples. Neutrophil infiltration, determined by CD15 staining, may represent a valuable pathological marker associated with the grade of dysplasia.