Evaluating a New Class of AKT/mTOR Activators for HIV Latency Reversing Activity Ex Vivo and In Vivo.

An ability to activate latent HIV-1 expression could benefit many HIV cure strategies, but the first generation of latency reversing agents (LRAs) has proven disappointing. We evaluated AKT/mTOR activators as a potential new class of LRAs. Two glycogen synthase kinase-3 inhibitors (GSK-3i's), SB-216763 and tideglusib (the latter already in phase II clinical trials) that activate AKT/mTOR signaling were tested. These GSK-3i's reactivated latent HIV-1 present in blood samples from aviremic individuals on antiretroviral therapy (ART) in the absence of T cell activation, release of inflammatory cytokines, cell toxicity, or impaired effector function of cytotoxic T lymphocytes or NK cells. However, when administered in vivo to SIV-infected rhesus macaques on suppressive ART, tideglusib exhibited poor pharmacodynamic properties and resulted in no clear evidence of significant SIV latency reversal. Whether alternative pharmacological formulations or combinations of this drug with other classes of LRAs will lead to an effective in vivo latency-reversing strategy remains to be determined.IMPORTANCE If combined with immune therapeutics, latency reversing agents (LRAs) have the potential to reduce the size of the reservoir sufficiently that an engineered immune response can control the virus in the absence of antiretroviral therapy. We have identified a new class of LRAs that do not induce T-cell activation and that are able to potentiate, rather than inhibit, CD8+ T and NK cell cytotoxic effector functions. This new class of LRAs corresponds to inhibitors of glycogen synthase kinase-3. In this work, we have also studied the effects of one member of this drug class, tideglusib, in SIV-infected rhesus monkeys. When tested in vivo, however, tideglusib showed unfavorable pharmacokinetic properties, which resulted in lack of SIV latency reversal. The disconnect between our ex vivo and in vivo results highlights the importance of developing next generation LRAs with pharmacological properties that allow systemic drug delivery in relevant anatomical compartments harboring latent reservoirs.

Everolimus, an mTORC1/2 inhibitor, in ART-suppressed individuals who received solid organ transplantation: A prospective study.

Pharmacologic inhibition of the mammalian target of rapamycin (mTOR) in the setting of renal transplantation has previously been associated with lower human immunodeficiency virus 1 (HIV-1) DNA burden, and in vitro studies suggest that mTOR inhibition may lead to HIV transcriptional silencing. Because prospective clinical trials are lacking, we conducted an open-label, single-arm study to determine the impact of the broad mTOR inhibitor, everolimus, on residual HIV burden, transcriptional gene expression profiles, and immune responses in HIV-infected adult solid organ transplant (SOT) recipients on antiretroviral therapy. Whereas everolimus therapy did not have an overall effect on cell-associated HIV-1 DNA and RNA levels in the entire cohort, participants who maintained everolimus time-averaged trough levels >5 ng/mL during the first 2 months of therapy had significantly lower RNA levels up to 6 months after the cessation of study drug. Time-averaged everolimus trough levels significantly correlated with greater inhibition of mTOR gene pathway transcriptional activity. Everolimus treatment also led to decreased PD-1 expression on certain T cell subsets. These data support the rationale for further study of the effects of mTOR inhibition on HIV transcriptional silencing in non-SOT populations, either alone or in combination with other strategies. Trial Registration: ClinicalTrials.gov NCT02429869.

Cerebrospinal fluid soluble CD30 elevation despite suppressive antiretroviral therapy in individuals living with HIV-1.

OBJECTIVES: The aim of this study was to assess soluble CD30 (sCD30), a protein that colocalises with HIV-1 RNA and DNA in lymphoid cells and tissues, in cerebrospinal fluid (CSF) as a marker of HIV-1 infection in the central nervous system (CNS). METHODS: This was a cross-sectional study using archived samples from two clinical cohorts. Soluble CD30 concentrations were measured in paired CSF and plasma from untreated viraemic individuals (n=52), individuals on suppressive antiretroviral therapy (ART) (n=33), HIV-1 controllers (n=10), participants with CSF HIV-1 'escape' (n=11) and controls without HIV-1 infection (n=16). Nonparametric tests were used to compare levels across groups and evaluate correlations with HIV-1 RNA, CSF neurofilament light chain protein (NFL) and neopterin. RESULTS: Compared with controls (median 30 ng/mL, interquartile range [IRQ] 23-50), plasma sCD30 levels were elevated in viraemic participants (75 ng/mL, 52-116; P<0.001), but not in those on suppressive ART (38 ng/mL, 32-62). In contrast, CSF sCD30 levels were elevated in ART-suppressed individuals (34 ng/mL, 19-46; P=0.001) and in those with CSF 'escape' (33 ng/mL, 27-40; P=0.004) compared with controls (18 ng/mL, 11-23), but not in untreated viraemic individuals. No association was observed between CSF sCD30 and plasma HIV-1 RNA, concurrent or nadir CD4+ T cell count, duration of infection or plasma sCD30. CSF sCD30 correlated with CSF NFL (r=0.34, P=0.001). CONCLUSIONS: In contrast to plasma, sCD30 levels are elevated in the CSF of individuals with HIV-1 infection who are on suppressive ART. Elevated levels of sCD30 in the CSF may be an indicator of persistent CNS HIV-1 infection, although the mechanism underlying this elevation warrants further investigation.

Correction to: Rapid development of HIV elite control in a patient with acute infection.

After publication of the original article [1], we were notified in Table 1 a column should be removed.

Rapid development of HIV elite control in a patient with acute infection.

BACKGROUND: Elite controllers (EC), a small subset of the HIV-positive population (< 1%), suppress HIV viremia below the limit of quantification of clinical viral load assays in the absence of antiretroviral therapy (ART). However, there is a paucity of longitudinal data detailing the viral and immune dynamics or HIV reservoir seeding during acute infection in individuals that go on to become Elite Controllers. CASE PRESENTATION: In this report, we describe a case of a 42 year old woman diagnosed during acute infection who rapidly and permanently suppressed her viremia in the absence of antiretroviral therapy (ART). Rapid antibody/antigen testing was either negative or equivocal during acute infection, despite subsequent viral load testing at that time point with 71,550 plasma HIV RNA copies/mL, making initial diagnosis challenging. The patient subsequently developed detectable anti-HIV antibodies and an increase in HIV-specific CD8+ T cell responses to overlapping subtype C HIV gag peptide; very low-level plasma viremia (0.84 RNA copies/mL) was detected by an ultrasensitive assay 2 years following infection. Subsequently, she was started on ART for multifocal furunculosis despite continued suppression of virus and stable CD4+ T cell counts. Following ART initiation, HIV specific antibody levels and CD8+ T cell responses increased, but no HIV DNA or RNA was able to be isolated from large numbers of peripheral blood CD4+ T cells. CONCLUSION: This case provides important information regarding the establishment of elite HIV control during acute infection and also demonstrates an increase in HIV-specific immune responses following ART despite undetectable peripheral blood cellular measures of HIV persistence. This case also highlights the challenges in diagnosing acute HIV infection without the use of viral load testing in this rare elite controller phenotype.

CD32-RNA Co-localizes with HIV-RNA in CD3+ Cells Found within Gut Tissues from Viremic and ART-Suppressed Individuals.

BACKGROUND: Identifying biomarkers for cells harboring replication-competent HIV is a major research priority. Recently, there have been mixed reports addressing the possibility that CD32-expressing T cells are enriched for HIV. There is growing evidence that CD32 expression increases with cellular activation that may be related to, but not necessarily specific for, infection with HIV. However, the relationship of CD32 expression to HIV-infection in subtypes of tissue-resident leukocytes is unclear. METHODS: First, we used duplex chromogenic in situ hybridization to identify cells actively transcribing RNA for both CD32 and HIV on human gut tissues. Then we performed multiplexed immunofluorescence and in situ hybridization (mIFISH) on sections from the same tissues to determine the phenotype of individual cells co-expressing HIV-RNA and CD32-RNA. RESULTS: HIV-RNA+ cells were more abundant in tissues from viremic individuals than in those receiving suppressive anti-retroviral therapy (ART). However, staining by both methods indicated that a higher proportion of HIV-RNA+ cells co-expressed CD32-RNA in ART-suppressed individuals than in those with viremia. The majority of HIV-RNA+ cells were CD3+. CONCLUSIONS: Our data suggest that the transcription of CD32-RNA is correlated with HIV transcriptional activity in CD3+ cells found within human gut tissue. Whether or not up-regulation of CD32-RNA is a direct result of HIV transcription or more global T-cell activation remains unclear.

Human Herpes Virus 8 in HIV-1 infected individuals receiving cancer chemotherapy and stem cell transplantation.

BACKGROUND: Human Herpes Virus 8 (HHV8) can cause Kaposi's Sarcoma (KS) in immunosuppressed individuals. However, little is known about the association between chemotherapy or hematopoietic stem cell transplantation (HSCT), circulating HHV8 DNA levels, and clinical KS in HIV-1-infected individuals with various malignancies. Therefore, we examined the associations between various malignancies, systemic cancer chemotherapy, T cell phenotypes, and circulating HHV8 DNA in 29 HIV-1-infected participants with concomitant KS or other cancer diagnoses. METHODS: We quantified HHV8 plasma viral loads and cell-associated HHV8 DNA and determined the relationship between circulating HHV8 DNA and lymphocyte counts, and markers of early and late lymphocyte activation, proliferation and exhaustion. RESULTS: There were no significant differences in plasma HHV8 DNA levels between baseline and post-chemotherapy time points or with the presence or absence of clinical KS. However, in two participants circulating HHV8 DNA increased following treatment for KS or HSCT for lymphoma,. We observed an approximately 2-log10 reduction in plasma HHV8 DNA in an individual with KS and multicentric Castleman disease following rituximab monotherapy. Although individuals with clinical KS had lower mean CD4+ T cell counts and percentages as expected, there were no significant associations with these factors and plasma HHV8 levels. We identified increased proportions of CD8+ and CD4+ T cells expressing CD69 (P = 0.01 & P = 0.04 respectively), and increased CD57 expression on CD4+ T cells (P = 0.003) in participants with detectable HHV8. CONCLUSION: These results suggest there is a complex relationship between circulating HHV8 DNA and tissue-based disease in HIV-1 and HHV8 co-infected individuals with various malignancies.

Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+ T cells expressing CD30.

HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection.

Elucidating the Burden of HIV in Tissues Using Multiplexed Immunofluorescence and In Situ Hybridization: Methods for the Single-Cell Phenotypic Characterization of Cells Harboring HIV In Situ.

Persistent tissue reservoirs of HIV present a major barrier to cure. Defining subsets of infected cells in tissues is a major focus of HIV cure research. Herein, we describe a novel multiplexed in situ hybridization (ISH) (RNAscope) protocol to detect HIV-DNA (vDNA) and HIV-RNA (vRNA) in formalin-fixed paraffin-embedded (FFPE) human tissues in combination with immunofluorescence (IF) phenotyping of the infected cells. We show that multiplexed IF and ISH (mIFISH) is suitable for quantitative assessment of HIV vRNA and vDNA and that multiparameter IF phenotyping allows precise identification of the cellular source of the ISH signal. We also provide semi-quantitative data on the impact of various tissue fixatives on the detectability of vDNA and vRNA with RNAscope technology. Finally, we describe methods to quantitate the ISH signal on whole-slide digital images and validation of the quantitative ISH data with quantitative real-time PCR for vRNA. It is our hope that this approach will provide insight into the biology of HIV tissue reservoirs and to inform strategies aimed at curing HIV.

HIV-1 persistence following extremely early initiation of antiretroviral therapy (ART) during acute HIV-1 infection: An observational study.

BACKGROUND: It is unknown if extremely early initiation of antiretroviral therapy (ART) may lead to long-term ART-free HIV remission or cure. As a result, we studied 2 individuals recruited from a pre-exposure prophylaxis (PrEP) program who started prophylactic ART an estimated 10 days (Participant A; 54-year-old male) and 12 days (Participant B; 31-year-old male) after infection with peak plasma HIV RNA of 220 copies/mL and 3,343 copies/mL, respectively. Extensive testing of blood and tissue for HIV persistence was performed, and PrEP Participant A underwent analytical treatment interruption (ATI) following 32 weeks of continuous ART. METHODS AND FINDINGS: Colorectal and lymph node tissues, bone marrow, cerebral spinal fluid (CSF), plasma, and very large numbers of peripheral blood mononuclear cells (PBMCs) were obtained longitudinally from both participants and were studied for HIV persistence in several laboratories using molecular and culture-based detection methods, including a murine viral outgrowth assay (mVOA). Both participants initiated PrEP with tenofovir/emtricitabine during very early Fiebig stage I (detectable plasma HIV-1 RNA, antibody negative) followed by 4-drug ART intensification. Following peak viral loads, both participants experienced full suppression of HIV-1 plasma viremia. Over the following 2 years, no further HIV could be detected in blood or tissue from PrEP Participant A despite extensive sampling from ileum, rectum, lymph nodes, bone marrow, CSF, circulating CD4+ T cell subsets, and plasma. No HIV was detected from tissues obtained from PrEP Participant B, but low-level HIV RNA or DNA was intermittently detected from various CD4+ T cell subsets. Over 500 million CD4+ T cells were assayed from both participants in a humanized mouse outgrowth assay. Three of 8 mice infused with CD4+ T cells from PrEP Participant B developed viremia (50 million input cells/surviving mouse), but only 1 of 10 mice infused with CD4+ T cells from PrEP Participant A (53 million input cells/mouse) experienced very low level viremia (201 copies/mL); sequence confirmation was unsuccessful. PrEP Participant A stopped ART and remained aviremic for 7.4 months, rebounding with HIV RNA of 36 copies/mL that rose to 59,805 copies/mL 6 days later. ART was restarted promptly. Rebound plasma HIV sequences were identical to those obtained during acute infection by single-genome sequencing. Mathematical modeling predicted that the latent reservoir size was approximately 200 cells prior to ATI and that only around 1% of individuals with a similar HIV burden may achieve lifelong ART-free remission. Furthermore, we observed that lymphocytes expressing the tumor marker CD30 increased in frequency weeks to months prior to detectable HIV-1 RNA in plasma. This study was limited by the small sample size, which was a result of the rarity of individuals presenting during hyperacute infection. CONCLUSIONS: We report HIV relapse despite initiation of ART at one of the earliest stages of acute HIV infection possible. Near complete or complete loss of detectable HIV in blood and tissues did not lead to indefinite ART-free HIV remission. However, the small numbers of latently infected cells in individuals treated during hyperacute infection may be associated with prolonged ART-free remission.

Human Immunodeficiency Virus Type 1 Persistence Following Systemic Chemotherapy for Malignancy.

Background: Systemic chemotherapies for various malignancies have been shown to significantly, yet transiently, decrease numbers of CD4+ T lymphocytes, a major reservoir for human immunodeficiency virus type 1 (HIV-1) infection. However, little is known about the impact of cytoreductive chemotherapy on HIV-1 reservoir dynamics, persistence, and immune responses. Methods: We investigated the changes in peripheral CD4+ T-cell-associated HIV-1 DNA and RNA levels, lymphocyte activation, viral population structure, and virus-specific immune responses in a longitudinal cohort of 15 HIV-1-infected individuals receiving systemic chemotherapy or subsequent autologous stem cell transplantation for treatment of hematological malignancies and solid tumors. Results: Despite a transient reduction in CD4+ T cells capable of harboring HIV-1, a 1.7- and 3.3-fold increase in mean CD4+ T-cell-associated HIV-1 RNA and DNA, respectively, were observed months following completion of chemotherapy in individuals on antiretroviral therapy. We also observed changes in CD4+ T-cell population diversity and clonal viral sequence expansion during CD4+ T-cell reconstitution following chemotherapy cessation. Finally, HIV-1 DNA was preferentially, and in some cases exclusively, detected in cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-responsive CD4+ T cells following chemotherapy. Conclusions: Expansion of HIV-infected CMV/EBV-specific CD4 + T cells may contribute to maintenance of the HIV DNA reservoir following chemotherapy.

High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay.

Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs) increase the level of viral transcription from cells producing HIV RNA or harboring transcriptionally-inactive (latent) infection. We therefore developed a microfluidic single-cell-in-droplet (scd)PCR assay to directly measure the number of CD4+ T cells that produce unspliced (us)RNA and multiply spliced (ms)RNA following ex vivo latency reversal with either an histone deacetylase inhibitor (romidepsin) or T cell receptor (TCR) stimulation. Detection of HIV-1 transcriptional activity can also be performed on hundreds of thousands of CD4+ T-cells in a single experiment. The scdPCR method was then applied to CD4+ T cells obtained from HIV-1-infected individuals on antiretroviral therapy. Overall, our results suggest that effects of LRAs on HIV-1 reactivation may be heterogeneous-increasing transcription from active cells in some cases and increasing the number of transcriptionally active cells in others. Genomic DNA and human mRNA isolated from HIV-1 reactivated cells could also be detected and quantified from individual cells. As a result, our assay has the potential to provide needed insight into various reservoir eradication strategies.

A humanized mouse-based HIV-1 viral outgrowth assay with higher sensitivity than in vitro qVOA in detecting latently infected cells from individuals on ART with undetectable viral loads.

Assays that can verify full viral eradication are essential in the context of achieving a cure for HIV/AIDS. In vitro quantitative viral out growth assays (qVOA) are currently the gold standard for measuring latent HIV-1 but these assays often fail to detect very low levels of replication-competent virus. Here we investigated an alternative in vivo approach for sensitive viral detection using humanized mice (hmVOA). Peripheral blood CD4+ T cell samples from HIV subjects on stable ART with undetectable viral loads by RT-PCR were first assayed by in vitro qVOA. Corresponding patient samples in which no virus was detected by qVOA were injected into humanized mice to allow viral outgrowth. Of the five qVOA virus negative samples, four gave positive viral outgrowth in the hmVOA assay suggesting that it is more sensitive in detecting latent HIV-1.

Expression of the innate immune receptor LILRB5 on monocytes is associated with mycobacteria exposure.

Antigen presenting cells (APC) are critical components of innate immunity and consequently shape the adaptive response. Leukocyte Ig Like Receptors (LILR) are innate immune receptors predominantly expressed on myeloid cells. LILR can influence the antigen presenting phenotype of monocytic cells to determine the nature of T cell responses in infections including Mycobaterium leprae. We therefore investigated the relevance of LILR in the context of Mycobacterium tuberculosis. Real-time PCR studies indicated that the transcriptional profile of the orphan receptor LILRB5 was significantly up-regulated following exposure to mycobacteria. Furthermore, LILRA1 and LILRB5 were able to trigger signalling through direct engagement of mycobacteria using tranfectant cells incorporating a reporter system. We describe for the first time the expression of this receptor on T cells, and highlight the potential relevance to mycobacterial recognition. Furthermore, we demonstrate that crosslinking of this receptor on T cells increases proliferation of cytotoxic, but not helper, T cells.