Doherty Institute - Research Publications

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    Multiply spliced HIV RNA is a predictive measure of virus production ex vivo and in vivo following reversal of HIV latency
    Zerbato, JM ; Khoury, G ; Zhao, W ; Gartner, MJ ; Pascoe, RD ; Rhodes, A ; Dantanarayana, A ; Gooey, M ; Anderson, J ; Bacchetti, P ; Deeks, SG ; McMahon, J ; Roche, M ; Rasmussen, TA ; Purcell, DFJ ; Lewin, SR (ELSEVIER, 2021-03)
    BACKGROUND: One strategy being pursued to clear latently infected cells that persist in people living with HIV (PLWH) on antiretroviral therapy (ART) is to activate latent HIV infection with a latency reversing agent (LRA). Surrogate markers that accurately measure virus production following an LRA are needed. METHODS: We quantified cell-associated unspliced (US), multiply spliced (MS) and supernatant (SN) HIV RNA by qPCR from total and resting CD4+ T cells isolated from seven PLWH on ART before and after treatment ex vivo with different LRAs, including histone deacetylase inhibitors (HDACi). MS and plasma HIV RNA were also quantified from PLWH on ART (n-11) who received the HDACi panobinostat. FINDINGS: In total and resting CD4+ T cells from PLWH on ART, detection of US RNA was common while detection of MS RNA was infrequent. Primers used to detect MS RNA, in contrast to US RNA, bound sites of the viral genome that are commonly mutated or deleted in PLWH on ART. Following ex vivo stimulation with LRAs, we identified a strong correlation between the fold change increase in SN and MS RNA, but not the fold change increase in SN and US RNA. In PLWH on ART who received panobinostat, MS RNA was significantly higher in samples with detectable compared to non0detectable plasma HIV RNA. INTERPRETATION: Following administration of an LRA, quantification of MS RNA is more likely to reflect an increase in virion production and is therefore a better indicator of meaningful latency reversal. FUNDING: NHMRC, NIH DARE collaboratory.
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    A new way of measuring apoptosis by absolute quantitation of inter-nucleosomally fragmented genomic DNA
    Hooker, DJ ; Mobarok, M ; Anderson, JL ; Rajasuriar, R ; Gray, LR ; Ellett, AM ; Lewin, SR ; Gorry, PR ; Cherry, CL (OXFORD UNIV PRESS, 2012-08)
    Several critical events of apoptosis occur in the cell nucleus, including inter-nucleosomal DNA fragmentation (apoptotic DNA) and eventual chromatin condensation. The generation of apoptotic DNA has become a biochemical hallmark of apoptosis because it is a late 'point of no return' step in both the extrinsic (cell-death receptor) and intrinsic (mitochondrial) apoptotic pathways. Despite investigators observing apoptotic DNA and understanding its decisive role as a marker of apoptosis for over 20 years, measuring it has proved elusive. We have integrated ligation-mediated PCR and qPCR to design a new way of measuring apoptosis, termed ApoqPCR, which generates an absolute value for the amount (picogram) of apoptotic DNA per cell population. ApoqPCR's advances over current methods include a 1000-fold linear dynamic range yet sensitivity to distinguish subtle low-level changes, measurement with a 3- to 4-log improvement in sample economy, and capacity for archival or longitudinal studies combined with high-throughput capability. We demonstrate ApoqPCR's utility in both in vitro and in vivo contexts. Considering the fundamental role apoptosis has in vertebrate and invertebrate health, growth and disease, the reliable measurement of apoptotic nucleic acid by ApoqPCR will be of value in cell biology studies in basic and applied science.
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    Combination Immune Checkpoint Blockade to Reverse HIV Latency
    Van der Sluis, RM ; Kumar, NA ; Pascoe, RD ; Zerbato, JM ; Evans, VA ; Dantanarayana, AI ; Anderson, JL ; Sekaly, RP ; Fromentin, R ; Chomont, N ; Cameron, PU ; Lewin, SR (AMER ASSOC IMMUNOLOGISTS, 2020-03-01)
    In people living with HIV on antiretroviral therapy, HIV latency is the major barrier to a cure. HIV persists preferentially in CD4+ T cells expressing multiple immune checkpoint (IC) molecules, including programmed death (PD)-1, T cell Ig and mucin domain-containing protein 3 (TIM-3), lymphocyte associated gene 3 (LAG-3), and T cell immunoreceptor with Ig and ITIM domains (TIGIT). We aimed to determine whether these and other IC molecules have a functional role in maintaining HIV latency and whether blocking IC molecules with Abs reverses HIV latency. Using an in vitro model that establishes latency in both nonproliferating and proliferating human CD4+ T cells, we show that proliferating cells express multiple IC molecules at high levels. Latent infection was enriched in proliferating cells expressing PD-1. In contrast, nonproliferating cells expressed IC molecules at significantly lower levels, but latent infection was enriched in cells expressing PD-1, TIM-3, CTL-associated protein 4 (CTLA-4), or B and T lymphocyte attenuator (BTLA). In the presence of an additional T cell-activating stimulus, staphylococcal enterotoxin B, Abs to CTLA-4 and PD-1 reversed HIV latency in proliferating and nonproliferating CD4+ T cells, respectively. In the absence of staphylococcal enterotoxin B, only the combination of Abs to PD-1, CTLA-4, TIM-3, and TIGIT reversed latency. The potency of latency reversal was significantly higher following combination IC blockade compared with other latency-reversing agents, including vorinostat and bryostatin. Combination IC blockade should be further explored as a strategy to reverse HIV latency.
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    Limitations of dual-fluorescent HIV reporter viruses in a model of pre-activation latency
    Kim, Y ; Cameron, PU ; Lewin, SR ; Anderson, JL (JOHN WILEY & SONS LTD, 2019-12)
    INTRODUCTION: HIV latency can be established in vitro following direct infection of a resting CD4+ T cell (pre-activation latency) or infection of an activated CD4+ T cell which then returns to a resting state (post-activation latency). We modified a previously published dual-fluorescent reporter virus seeking to track the establishment and reactivation of pre-activation latency in primary CD4+ T cells. METHODS: A previously published dual-fluorescent reporter virus was modified so that expression of enhanced green fluorescent protein (GFP) was under control of the elongation factor 1 alpha (EF1α) promoter to detect latent infection, and E2 crimson (E2CRM) was under control of the nef promoter to detect productive infection. NL4.3 that expressed GFP in place of nef was used as a positive control. We infected the Jurkat T-cell line and primary CD4+ T cells that were either unstimulated or stimulated with either the chemokine CCL19 or phytohaemagglutinin (PHA)/IL-2 and quantified the expression of both fluorescent proteins by flow cytometry. The study was carried out over a period of two years from September 2016 to October 2018. RESULTS AND DISCUSSION: Expression of both fluorophores was detected following infection of the Jurkat T-cell line while only low levels of the latent reporter were observed following infection of primary CD4+ T cells. In unstimulated and CCL19-treated CD4+ T cells, expression of the GFP latent reporter, increased after further activation of the cells with PHA/phorbol 12-myristate 13-acetate (PMA). CONCLUSIONS: Our findings demonstrate that the EF1α promoter has poor constitutive expression in resting CD4+ T cells. Therefore, dual-fluorescent reporter viruses with the EF1α promoter may underestimate the frequency of latent infection in resting CD4+ T cells.
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    Human Immunodeficiency Virus (HIV)-Infected CCR6+ Rectal CD4+ T Cells and HIV Persistence On Antiretroviral Therapy
    Anderson, JL ; Khoury, G ; Fromentin, R ; Solomon, A ; Chomont, N ; Sinclair, E ; Milush, JM ; Hartogensis, W ; Bacchetti, P ; Roche, M ; Tumpach, C ; Gartner, M ; Pitman, MC ; Epling, CL ; Hoh, R ; Hecht, FM ; Somsouk, M ; Cameron, PU ; Deeks, SG ; Lewin, SR (OXFORD UNIV PRESS INC, 2020-03-01)
    BACKGROUND: Identifying where human immunodeficiency virus (HIV) persists in people living with HIV and receiving antiretroviral therapy is critical to develop cure strategies. We assessed the relationship of HIV persistence to expression of chemokine receptors and their chemokines in blood (n = 48) and in rectal (n = 20) and lymph node (LN; n = 8) tissue collected from people living with HIV who were receiving suppressive antiretroviral therapy. METHODS: Cell-associated integrated HIV DNA, unspliced HIV RNA, and chemokine messenger RNA were quantified by quantitative polymerase chain reaction. Chemokine receptor expression on CD4+ T cells was determined using flow cytometry. RESULTS: Integrated HIV DNA levels in CD4+ T cells, CCR6+CXCR3+ memory CD4+ T-cell frequency, and CCL20 expression (ligand for CCR6) were highest in rectal tissue, where HIV-infected CCR6+ T cells accounted for nearly all infected cells (median, 89.7%). Conversely in LN tissue, CCR6+ T cells were infrequent, and there was a statistically significant association of cell-associated HIV DNA and RNA with CCL19, CCL21, and CXCL13 chemokines. CONCLUSIONS: HIV-infected CCR6+ CD4+ T cells accounted for the majority of infected cells in rectal tissue. The different relationships between HIV persistence and T-cell subsets and chemokines in rectal and LN tissue suggest that different tissue-specific strategies may be required to eliminate HIV persistence and that assessment of biomarkers for HIV persistence may not be generalizable between blood and other tissues.
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    HIV latency can be established in proliferating and nonproliferating resting CD4+ T cells in vitro: implications for latency reversal
    Moso, MA ; Anderson, JL ; Adikari, S ; Gray, LR ; Khoury, G ; Chang, JJ ; Jacobson, JC ; Ellet, AM ; Chen, W-J ; Saleh, S ; Zaunders, JJ ; Purcell, DFJ ; Camerona, PU ; Churchill, MJ ; Lewin, SR ; Lu, HK (LIPPINCOTT WILLIAMS & WILKINS, 2019-02-01)
    OBJECTIVE: To determine whether latency can be established and reversed in both proliferating and nonproliferating CD4+ T cells in the same model in vitro. METHODS: Activated CD4+ T cells were infected with either a nonreplication competent, luciferase reporter virus or wild-type full-length enhanced green fluorescent protein (EGFP) reporter virus and cultured for 12 days. The cells were then sorted by flow cytometry to obtain two distinct T-cell populations that did not express the T-cell activation markers, CD69, CD25 and human leukocyte antigen (HLA)-DR: CD69CD25HLA-DR small cells (nonblasts) that had not proliferated in vitro following mitogen stimulation and CD69CD25HLA-DR large cells (which we here call transitional blasts) that had proliferated. The cells were then reactivated with latency-reversing agents and either luciferase or EGFP quantified. RESULTS: Inducible luciferase expression, consistent with latent infection, was observed in nonblasts and transitional blasts following stimulation with either phorbol-myristate-acetate/phytohemagglutinin (3.8 ± 1 and 2.9 ± 0.5 fold above dimethyl sulfoxide, respectively) or romidepsin (2.1 ± 0.6 and 1.8 ± 0.2 fold above dimethyl sulfoxide, respectively). Constitutive expression of luciferase was higher in transitional blasts compared with nonblasts. Using wild-type full-length EGFP reporter virus, inducible virus was observed in nonblasts but not in transitional blasts. No significant difference was observed in the response to latency-reversing agents in either nonblasts or transitional blasts. CONCLUSION: HIV latency can be established in vitro in resting T cells that have not proliferated (nonblasts) and blasts that have proliferated (transitional blasts). This model could potentially be used to assess new strategies to eliminate latency.
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    HIV latency reversing agents act through Tat post translational modifications
    Khoury, G ; Mota, TM ; Li, S ; Tumpach, C ; Lee, MY ; Jacobson, J ; Harty, L ; Anderson, JL ; Lewin, SR ; Purcell, DFJ (BMC, 2018-05-11)
    BACKGROUND: Different classes of latency reversing agents (LRAs) are being evaluated to measure their effects in reactivating HIV replication from latently infected cells. A limited number of studies have demonstrated additive effects of LRAs with the viral protein Tat in initiating transcription, but less is known about how LRAs interact with Tat, particularly through basic residues that may be post-translationally modified to alter the behaviour of Tat for processive transcription and co-transcriptional RNA processing. RESULTS: Here we show that various lysine and arginine mutations reduce the capacity of Tat to induce both transcription and mRNA splicing. The lysine 28 and lysine 50 residues of Tat, or the acetylation and methylation modifications of these basic amino acids, were essential for Tat transcriptional control, and also for the proviral expression effects elicited by histone deacetylase inhibitors (HDACi) or the bromodomain inhibitor JQ1. We also found that JQ1 was the only LRA tested that could induce HIV mRNA splicing in the absence of Tat, or rescue splicing for Tat lysine mutants in a BRD4-dependent manner. CONCLUSIONS: Our data provide evidence that Tat activities in both co-transcriptional RNA processing together with transcriptional initiation and processivity are crucial during reactivation of latent HIV infection. The HDACi and JQ1 LRAs act with Tat to increase transcription, but JQ1 also enables post-transcriptional mRNA splicing. Tat residues K28 and K50, or their modifications through acetylation or methylation, are critical for LRAs that function in conjunction with Tat.
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    Getting the "Kill" into "Shock and Kill": Strategies to Eliminate Latent HIV
    Kim, Y ; Anderson, JL ; Lewin, SR (CELL PRESS, 2018-01-10)
    Despite the success of antiretroviral therapy (ART), there is currently no HIV cure and treatment is life long. HIV persists during ART due to long-lived and proliferating latently infected CD4+ T cells. One strategy to eliminate latency is to activate virus production using latency reversing agents (LRAs) with the goal of triggering cell death through virus-induced cytolysis or immune-mediated clearance. However, multiple studies have demonstrated that activation of viral transcription alone is insufficient to induce cell death and some LRAs may counteract cell death by promoting cell survival. Here, we review new approaches to induce death of latently infected cells through apoptosis and inhibition of pathways critical for cell survival, which are often hijacked by HIV proteins. Given advances in the commercial development of compounds that induce apoptosis in cancer chemotherapy, these agents could move rapidly into clinical trials, either alone or in combination with LRAs, to eliminate latent HIV infection.
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    Cancer therapies in HIV cure research
    Rasmussen, TA ; Anderson, JL ; Wightman, F ; Lewin, SR (LIPPINCOTT WILLIAMS & WILKINS, 2017-01)
    PURPOSE OF REVIEW: This article provides an overview of anticancer therapies in various stages of clinical development as potential interventions to target HIV persistence. RECENT FINDINGS: Epigenetic drugs developed for cancer have been investigated in vitro, ex vivo and in clinical trials as interventions aimed at reversing HIV latency and depleting the amount of virus that persists on antiretroviral therapy. Treatment with histone deacetylase inhibitors induced HIV expression in patients on antiretroviral therapy but did not reduce the frequency of infected cells. Other interventions that may accelerate the decay of latently infected cells, in the presence or absence of latency-reversing therapy, are now being explored. These include apoptosis-promoting agents, nonhistone deacetylase inhibitor compounds to reverse HIV latency and immunotherapy interventions to enhance antiviral immunity such as immune checkpoint inhibitors and Toll-like receptor agonists. SUMMARY: A curative strategy in HIV will likely need to both reduce the amount of virus that persists on antiretroviral therapy and improve anti-HIV immune surveillance. Although we continue to explore advances in the field of oncology including cancer immunotherapy, there are major differences in the risk-benefit assessment between HIV-infected individuals and patients with malignancies. Drug development specifically targeting HIV persistence will be the key to developing effective interventions with an appropriate safety profile.
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    Understanding Factors That Modulate the Establishment of HIV Latency in Resting CD4+T-Cells In Vitro
    Anderson, JL ; Mota, TM ; Evans, VA ; Kumar, N ; Rezaei, SD ; Cheong, K ; Solomon, A ; Wightman, F ; Cameron, PU ; Lewin, SR ; Unutmaz, D (PUBLIC LIBRARY SCIENCE, 2016-07-06)
    Developing robust in vitro models of HIV latency is needed to better understand how latency is established, maintained and reversed. In this study, we examined the effects of donor variability, HIV titre and co-receptor usage on establishing HIV latency in vitro using two models of HIV latency. Using the CCL19 model of HIV latency, we found that in up to 50% of donors, CCL19 enhanced latent infection of resting CD4+ T-cells by CXCR4-tropic HIV in the presence of low dose IL-2. Increasing the infectious titre of CXCR4-tropic HIV increased both productive and latent infection of resting CD4+ T-cells. In a different model where myeloid dendritic cells (mDC) were co-cultured with resting CD4+ T-cells, we observed a higher frequency of latently infected cells in vitro than CCL19-treated or unstimulated CD4+ T-cells in the presence of low dose IL-2. In the DC-T-cell model, latency was established with both CCR5- and CXCR4-tropic virus but higher titres of CCR5-tropic virus was required in most donors. The establishment of latency in vitro through direct infection of resting CD4+ T-cells is significantly enhanced by CCL19 and mDC, but the efficiency is dependent on virus titre, co-receptor usage and there is significant donor variability.