Selective miRNA inhibition in CD8+ cytotoxic T lymphocytes enhances HIV-1 specific cytotoxic responses.

miRNAs dictate relevant virus-host interactions, offering new avenues for interventions to achieve an HIV remission. We aimed to enhance HIV-specific cytotoxic responses-a hallmark of natural HIV control- by miRNA modulation in T cells. We recruited 12 participants six elite controllers and six patients with chronic HIV infection on long-term antiretroviral therapy ("progressors"). Elite controllers exhibited stronger HIV-specific cytotoxic responses than the progressors, and their CD8+T cells showed a miRNA (hsa-miR-10a-5p) significantly downregulated. When we transfected ex vivo CD8+ T cells from progressors with a synthetic miR-10a-5p inhibitor, miR-10a-5p levels decreased in 4 out of 6 progressors, correlating with an increase in HIV-specific cytotoxic responses. The effects of miR-10a-5p inhibition on HIV-specific CTL responses were modest, short-lived, and occurred before day seven after modulation. IL-4 and TNF-α levels strongly correlated with HIV-specific cytotoxic capacity. Thus, inhibition of miR-10a-5p enhanced HIV-specific CD8+ T cell capacity in progressors. Our pilot study proves the concept that miRNA modulation is a feasible strategy to combat HIV persistence by enhancing specific cytotoxic immune responses, which will inform new approaches for achieving an antiretroviral therapy-free HIV remission.

Effect of the use of Galectin-9 and blockade of TIM-3 receptor in the latent cellular reservoir of HIV-1.

Reactivation of latent HIV-1 is a necessary step for the purging of the viral reservoir, although it does not seem to be enough. The stimulation of HIV-1 specific cytotoxic T lymphocytes (CTL) may be just as essential for this purpose. In this study, we aimed to show the effect of galectin-9 (Gal-9), known to revert HIV-1 latency, in combination with the blockade of TIM-3, a natural receptor for Gal-9 and an exhaustion marker. We confirmed the ability of Gal-9 to reactivate latent HIV-1 in Jurkat-LAT-GFP cells, as well as in an IL-7-based cellular model. This reactivation was not mediated via the TIM-3 receptor, but rather by the recognition of the Gal-9 of a specific oligosaccharide pattern of resting memory CD4+ T cells' surfaces. The potency of Gal-9 in inducing transcription of latent HIV-1 was equal to or greater than that of other latency-reversing agents (LRA). Furthermore, the combination of Gal-9 with other LRA did not show synergistic effects in the reactivation of the latent virus. To evaluate the impact of TIM-3 inhibition on the CTL-response, different co-culture experiments with CD4+T, CD8+ T, and NK cells were performed. Our data showed that blocking TIM-3 was associated with control of viral replication in both in vitro and ex vivo models in cells from PLWH on antiretroviral therapy. A joint strategy of the use of Gal-9 to reactivate latent HIV-1 and the inhibition of TIM-3 to enhance the HIV-1 CTL specific-response was associated with control of the replication of the virus that was being reactivated, thus potentially contributing to the elimination of the viral reservoir. Our results place this strategy as a promising approach to be tested in future studies. Reactivation of latent-HIV-1 by Gal-9 and reinvigoration of CD8+ T cells by TIM-3 blockade could be used separately or in combination.ImportanceHIV-1 infection is a health problem of enormous importance that still causes significant mortality. Antiretroviral treatment (ART) has demonstrated efficacy in the control of HIV-1 replication, decreasing the morbidity and mortality of the infection, but it cannot eradicate the virus. In our work, we tested a protein, galectin-9 (Gal-9), an HIV-1 latency-reversing agent, using an in vitro cellular model of latency and in cells from people living with HIV-1 (PLWH) on antiretroviral therapy. Our results confirmed the potential role of Gal-9 as a molecule with a potent HIV-1 reactivation capacity. More importantly, using a monoclonal antibody against T cell immunoglobulin and the mucin domain-containing molecule 3 (TIM-3) receptor we were able to enhance the HIV-1 cytotoxic T lymphocytes (CTL) specific response to eliminate the CD4+ T cells in which the virus had been reactivated. When used together, i.e., Gal-9 and TIM-3 blockade, control of the replication of HIV-1 was observed, suggesting a decrease in the cellular reservoir.

Prolonged administration of maraviroc reactivates latent HIV in vivo but it does not prevent antiretroviral-free viral rebound.

Human immunodeficiency virus (HIV) remains incurable due to latent viral reservoirs established in non-activated CD4 T cells that cannot be eliminated via antiretroviral therapy. Current efforts to cure HIV are focused on identifying drugs that will induce viral gene expression in latently infected cells, commonly known as latency reversing agents (LRAs). Some drugs have been shown to reactivate latent HIV but do not cause a reduction in reservoir size. Therefore, finding new LRAs or new combinations or increasing the round of stimulations is needed to cure HIV. However, the effects of these drugs on viral rebound after prolonged treatment have not been evaluated. In a previous clinical trial, antiretroviral therapy intensification with maraviroc for 48 weeks caused an increase in residual viremia and episomal two LTR-DNA circles suggesting that maraviroc could reactivate latent HIV. We amended the initial clinical trial to explore additional virologic parameters in stored samples and to evaluate the time to viral rebound during analytical treatment interruption in three patients. Maraviroc induced an increase in cell-associated HIV RNA during the administration of the drug. However, there was a rapid rebound of viremia after antiretroviral therapy discontinuation. HIV-specific T cell response was slightly enhanced. These results show that maraviroc can reactivate latent HIV in vivo but further studies are required to efficiently reduce the reservoir size.

Maraviroc reactivates HIV with potency similar to that of other latency reversing drugs without inducing toxicity in CD8 T cells.

Human immunodeficiency virus (HIV) remains incurable due to latent reservoirs established in non-activated CD4 T cells. Current efforts to achieve a functional cure rely on immunomodulatory strategies focused on enhancing the functions of cytotoxic cells. Implementation of these actions requires a coordinated activation of the viral transcription in latently infected cells so that the reservoir became visible and accessible to cytotoxic cells. As no latency reversing agent (LRA) has been shown to be completely effective, new combinations are of increasing importance. Recent data have shown that maraviroc is a new LRA. In this work, we have explored how the combination of maraviroc with other LRAs influences on HIV reactivation using in vitro latency models as well as on the cell viability of CD8 T cells from ART-treated patients. Maraviroc reactivated HIV with a potency similar to other LRAs. Triple combinations resulted toxic and were rejected. No dual combination was synergistic. The combination with panobinostat or disulfiram maintained the effect of both drugs without inducing cell proliferation or toxicity. Maraviroc does not alter the viability of CD8 T cells isolated from patients under antiretroviral treatment. This finding enhances the properties of maraviroc as a LRA.

Selective miRNA Modulation Fails to Activate HIV Replication in In Vitro Latency Models.

HIV remains incurable because of viral persistence in latent reservoirs that are inaccessible to antiretroviral therapy. A potential curative strategy is to reactivate viral gene expression in latently infected cells. However, no drug so far has proven to be successful in vivo in reducing the reservoir, and therefore new anti-latency compounds are needed. We explored the role of microRNAs (miRNAs) in latency maintenance and their modulation as a potential anti-latency strategy. Latency models based on treating resting CD4 T cells with chemokine (C-C motif) ligand 19 (CCL19) or interleukin-7 (IL7) before HIV infection and next-generation sequencing were used to identify the miRNAs involved in HIV latency. We detected four upregulated miRNAs (miRNA-98, miRNA-4516, miRNA-4488, and miRNA-7974). Individual or combined inhibition of these miRNAs was performed by transfection into cells latently infected with HIV. Viral replication, assessed 72 h after transfection, did not increase after miRNA modulation, despite miRNA inhibition and lack of toxicity. Furthermore, the combined modulation of five miRNAs previously associated with HIV latency was not effective in these models. Our results do not support the modulation of miRNAs as a useful strategy for the reversal of HIV latency. As shown with other drugs, the potential of miRNA modulation as an HIV reactivation strategy could be dependent on the latency model used.

Maraviroc Is Associated with Latent HIV-1 Reactivation through NF-κB Activation in Resting CD4+ T Cells from HIV-Infected Individuals on Suppressive Antiretroviral Therapy.

Maraviroc is a CCR5 antagonist used in the treatment of HIV-1 infection. We and others have suggested that maraviroc could reactivate latent HIV-1. To test the latency-reversing potential of maraviroc and the mechanisms involved, we performed a phase II, single-center, open-label study in which maraviroc was administered for 10 days to 20 HIV-1-infected individuals on suppressive antiretroviral therapy (EudraCT registration no. 2012-003215-66). All patients completed full maraviroc dosing and follow-up. The primary endpoint was to study whether maraviroc may reactivate HIV-1 latency, eliciting signaling pathways involved in the viral reactivation. An increase in HIV-1 transcription in resting CD4+ T cells, estimated by levels of HIV-1 unspliced RNA, was observed. Moreover, activation of the NF-κB transcription factor was observed in these cells. To elucidate the mechanism of NF-κB activation by maraviroc, we have evaluated in HeLa P4 C5 cells, which stably express CCR5, whether maraviroc could be acting as a partial CCR5 agonist, with no other mechanisms or pathways involved. Our results show that maraviroc can induce NF-κB activity and that NF-κB targets gene expression by CCR5 binding, since the use of TAK779, a CCR5 inhibitor, blocked NF-κB activation and functionality. Taking the results together, we show that maraviroc may have a role in the activation of latent virus transcription through the activation of NF-κB as a result of binding CCR5. Our results strongly support a novel use of maraviroc as a potential latency reversal agent in HIV-1-infected patients.IMPORTANCE HIV-1 persistence in a small pool of long-lived latently infected resting CD4+ T cells is a major barrier to viral eradication in HIV-1-infected patients on antiretroviral therapy. A potential strategy to cure HIV-1-infection is the use of latency-reversing agents to eliminate the reservoirs established in resting CD4+ T cells. As no drug has been shown to be completely effective so far, the search for new drugs and combinations remains a priority for HIV cure. We examined the ability of maraviroc, a CCR5 antagonist used as an antiretroviral drug, to activate latent HIV-1 in infected individuals on antiretroviral therapy. The study showed that maraviroc can activate NF-κB and, subsequently, induce latent HIV-1-transcription in resting CD4+ T cells from HIV-1-infected individuals on suppressive antiretroviral therapy. Additional interventions will be needed to eliminate latent HIV-1 infection. Our results suggest that maraviroc may be a new latency-reversing agent to interfere with HIV-1 persistence during antiretroviral therapy.

The CCR5-antagonist Maraviroc reverses HIV-1 latency in vitro alone or in combination with the PKC-agonist Bryostatin-1.

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.

Bryostatin-1 for latent virus reactivation in HIV-infected patients on antiretroviral therapy.

OBJECTIVE: The protein kinase C (PKC) agonist bryostatin-1 has shown significant ex-vivo potency to revert HIV-1 latency, compared with other latency reversing agents (LRA). The safety of this candidate LRA remains to be proven in treated HIV-1-infected patients. METHODS: In this pilot, double-blind phase I clinical-trial (NCT 02269605), we included aviraemic HIV-1-infected patients on triple antiretroviral therapy to evaluate the effects of two different single doses of bryostatin-1 (10 or 20 µg/m) compared with placebo. RESULTS: Twelve patients were included, four in each arm. Bryostatin-1 was well tolerated in all participants. Two patients in the 20 µg/m arm developed grade 1 headache and myalgia. No detectable increases of cell-associated unspliced (CA-US) HIV-1-RNA were observed in any study arm, nor differences in HIV-1 mRNA dynamics between arms (P = 0.44). The frequency of samples with low-level viraemia did not differ between arms and low-level viraemia did not correlate with CA-US HIV-1-RNA levels (P = 0.676). No changes were detected on protein kinase C (PKC) activity and in biomarkers of inflammation (sCD14 and interleukin-6) in any study arm. After the single dose of bryostatin-1, plasma concentrations were under detection limits in all the patients in the 10 µg/m arm, and below 50 pg/ml (0.05 nmol/l) in those in the 20 µg/m arm. CONCLUSION: Bryostatin-1 was safe at the single doses administered. However, the drug did not show any effect on PKC activity or on the transcription of latent HIV, probably due to low plasma concentrations. This study will inform next trials aimed at assessing higher doses, multiple dosing schedules or combination studies with synergistic drugs.

Virological response to short-course maraviroc monotherapy does not predict viral tropism in HIV-1-infected treatment-naive patients.

OBJECTIVES: We aimed to evaluate whether virological response to a short course of maraviroc monotherapy could predict HIV-1 tropism. METHODS: A clinical trial was performed in HIV-1 treatment-naive patients infected with R5- or non-R5-tropic virus determined using the Trofile(®) assay, with >1000 HIV-1 RNA copies/mL. Maraviroc was administered for 10 days. Viral load was measured at baseline and days 4, 7, 10 and 28. The main outcome measurement was the decline in HIV-1 RNA at day 10. The trial was registered in the ClinicalTrials.gov database (NCT01060618; TROPISMVC). RESULTS: Forty patients [30 R5 and 10 dual/mixed (D/M)] were recruited. There was a significant decrease in HIV-1 RNA after 10 days of maraviroc treatment in patients with R5-tropic virus (median 1.52 log10 RNA copies/mL; 95% CI 1.23-1.63; P < 0.0001), but also in patients with D/M-tropic virus (median 1.62 log(10) RNA copies/mL; 95% CI 0.33-1.88; P = 0.00024). The difference in the HIV-1 RNA decrease (-0.16 log(10) RNA copies/mL; 95% CI -0.53 to 0.22) was not significant (P = 0.410). A decrease >0.5 log(10) RNA copies/mL was found in 96.3% of patients with R5-tropic virus and in 70% of patients with D/M-tropic virus (P = 0.052). The differences were not significant when a decline of 1 log(10) RNA copies/mL was considered (92.6% versus 70%; P = 0.11). CONCLUSIONS: Treatment-naive patients infected with R5- or D/M-tropic virus have similar virological responses to a short course of maraviroc monotherapy. This clinical test thus cannot be used as a surrogate marker of viral tropism in this population.

Raltegravir pharmacokinetics in HIV/HCV-coinfected patients with advanced liver cirrhosis (Child-Pugh C).

OBJECTIVES: To describe raltegravir pharmacokinetics at steady-state in HIV/hepatitis C virus (HCV)-coinfected patients under antiretroviral (ARV) treatment with (n = 5) and without (n = 5) advanced liver cirrhosis (Child-Pugh C). METHODS: This was a non-randomized, Phase I, parallel-assignment, open-label pharmacokinetic study in HIV/HCV-coinfected patients with Child-Pugh grade C hepatic cirrhosis. We recruited clinically stable HIV/HCV-coinfected adult patients with controlled HIV viraemia (<50 copies/mL) for at least 6 months. Raltegravir (400 mg twice daily) was added under fasting conditions for 5 days to the successful ritonavir-boosted protease inhibitor-based ARV regimen. The trial was registered in the ClinicalTrials.gov database (NCT01289951) (LIVERAL). RESULTS: Raltegravir AUC0-12 and C12 were increased 1.72-fold (90% CI, 1.02 to 2.92) and 6.58-fold (90% CI, 2.92 to14.85), respectively, in patients with advanced liver cirrhosis. No safety issues were identified and raltegravir was well tolerated by all patients. CONCLUSIONS: Raltegravir plasma levels are increased in HIV/HCV-coinfected patients with advanced liver cirrhosis (Child-Pugh C). Despite the higher exposure, raltegravir was safe and well tolerated.

Mechanisms of resistance and susceptibility to experimental visceral leishmaniosis: BALB/c mouse versus Syrian hamster model.

Several animal models have been established to study visceral leishmaniosis (VL), a worldwide vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem. BALB/c mice and Syrian hamsters are the most widely used experimental models. In this paper, we summarize the advantages and disadvantages of these two experimental models and discuss the results obtained using these models in different studies of VL. Studies using the BALB/c mouse model have underscored differences between the liver and spleen in the course of VL, indicating that pathological evaluation of the visceral organs is essential for understanding the immune mechanisms induced by Leishmania infantum infection. The main goal of this review is to collate the relevant literature on Leishmania pathogenesis into a sequence of events, providing a schematic view of the main components of adaptive and innate immunity in the liver and spleen after experimental infection with L. infantum or L. donovani. This review also presents several viewpoints and reflections about some controversial aspects of Leishmania research, including the choice of experimental model, route of administration, inoculum size and the relevance of pathology (intimately linked to parasite persistence): a thorough understanding of which is essential for future VL research and the successful development of efficient control strategies for Leishmania spp.