Doherty Institute - Research Publications
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ItemLimitations of dual-fluorescent HIV reporter viruses in a model of pre-activation latency(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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ItemNo Preview AvailableHIV latency can be established in proliferating and nonproliferating resting CD4+ T cells in vitro: implications for latency reversal(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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ItemQuantifying Adaptive and Innate Immune Responses in HIV-Infected Participants Using a Novel High Throughput Assay(PUBLIC LIBRARY SCIENCE, 2016-12-09)OBJECTIVES: HIV infection is characterised by persistent immune dysfunction of both the adaptive and innate immune responses. The aim of this study was to evaluate these responses using a novel high throughput assay in healthy controls and HIV-infected individuals prior to and following anti-retroviral treatment (ART). DESIGN: Cross-sectional study. METHODS: Whole blood was assessed using the QuantiFERON Monitor® (QFM) assay containing adaptive and innate immunostimulants. Interferon (IFN)-γ levels (IU/mL) were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: We recruited HIV-infected participants (n = 20 off ART and viremic; n = 59 on suppressive ART) and HIV-uninfected controls (n = 229). Median IFN-γ production was significantly higher in HIV-infected participants compared to controls (IFN-γ 512 vs 223 IU/ml, p<0.0001), but within the HIV-infected participants there was no difference between those on or off ART (median IFN-γ 512 vs 593 IU/ml p = 0.94). Amongst the HIV-infected participants, IFN-γ production was higher in individuals with CD4 count>350 compared to <350 cells/μL (IFN-γ IU/ml 561 vs 259 p = 0.02) and in males compared to females (IFN-γ 542 vs 77 IU/ml p = 0.04). There were no associations between IFN-γ production and age, plasma HIV RNA, nadir CD4 count or duration of HIV infection. Using a multivariable analysis, neither CD4 nor sex were independently predictive of IFN-γ production. CONCLUSION: Using a high throughput assay which assesses both adaptive and innate immune function, we showed elevated IFN-γ production in HIV-infected patients both on and off ART. Further research is warranted to determine if changes in QuantiFERON Monitor® are associated with clinical outcomes.
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ItemHIV integration sites in latently infected cell lines: evidence of ongoing replication(BIOMED CENTRAL LTD, 2017-01-13)BACKGROUND: Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position. RESULTS: We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells. CONCLUSION: Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.
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ItemUnderstanding Factors That Modulate the Establishment of HIV Latency in Resting CD4+T-Cells In Vitro(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.
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ItemHIV integration and the establishment of latency in CCL19-treated resting CD4+ T cells require activation of NF-κB(BMC, 2016-07-26)BACKGROUND: Eradication of HIV cannot be achieved with combination antiretroviral therapy (cART) because of the persistence of long-lived latently infected resting memory CD4(+) T cells. We previously reported that HIV latency could be established in resting CD4(+) T cells in the presence of the chemokine CCL19. To define how CCL19 facilitated the establishment of latent HIV infection, the role of chemokine receptor signalling was explored. RESULTS: In resting CD4(+) T cells, CCL19 induced phosphorylation of RAC-alpha serine/threonine-protein kinase (Akt), nuclear factor kappa B (NF-κB), extracellular-signal-regulated kinase (ERK) and p38. Inhibition of the phosphoinositol-3-kinase (PI3K) and Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/ERK signalling pathways inhibited HIV integration, without significant reduction in HIV nuclear entry (measured by Alu-LTR and 2-LTR circle qPCR respectively). Inhibiting activation of MEK1/ERK1/2, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1) and NF-κB, but not p38, also inhibited HIV integration. We also show that HIV integrases interact with Pin1 in CCL19-treated CD4(+) T cells and inhibition of JNK markedly reduced this interaction, suggesting that CCL19 treatment provided sufficient signals to protect HIV integrase from degradation via the proteasome pathway. Infection of CCL19-treated resting CD4(+) T cells with mutant strains of HIV, lacking NF-κB binding sites in the HIV long terminal repeat (LTR) compared to infection with wild type virus, led to a significant reduction in integration by up to 40-fold (range 1-115.4, p = 0.03). This was in contrast to only a modest reduction of 5-fold (range 1.7-11, p > 0.05) in fully activated CD4(+) T cells infected with the same mutants. Finally, we demonstrated significant differences in integration sites following HIV infection of unactivated, CCL19-treated, and fully activated CD4(+) T cells. CONCLUSIONS: HIV integration in CCL19-treated resting CD4(+) T cells depends on NF-κB signalling and increases the stability of HIV integrase, which allow subsequent integration and establishment of latency. These findings have implications for strategies needed to prevent the establishment, and potentially reverse, latent infection.
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ItemPolymorphisms in the CD14 and TLR4 genes independently predict CD4+ T-cell recovery in HIV-infected individuals on antiretroviral therapy(LIPPINCOTT WILLIAMS & WILKINS, 2016-09-10)BACKGROUND: Chronic HIV infection leads to marked depletion of CD4 T cells in the gastrointestinal tract and increased microbial translocation measured by an increase in circulating lipopolysaccharide (LPS) levels. Here, we hypothesized that single-nucleotide polymorphisms (SNPs) in genes encoding the Toll-like receptor 4 (TLR4) and CD14, the principal receptors for LPS, were associated with CD4 T-cell recovery postantiretroviral therapy (ART). METHODS: Prospective study of predominantly white HIV-infected participants receiving suppressive ART for at least 12 months. We analysed the CD14 SNPs C-260T and the TLR4 SNPs A+896G, C+1196T. We also determined the levels of LPS and soluble CD14 in plasma samples collected pre-ART and post-ART initiation. CD4 T-cell recovery was assessed by linear mixed models. RESULTS: Following ART, individuals with a TT genotype compared with a CT or CC genotype for CD14 C-260T SNP showed higher levels of soluble CD14 (P = 0.008 and 0.003, respectively). The CC genotype for the CD14 C-260T SNP, compared with CT or TT, and the TLR4 SNP (AC/GT), compared with the homozygous genotype (AA/CC), were both independently associated with enhanced long-term CD4 T-cell recovery (>3 months; P < 0.001). CONCLUSION: Polymorphisms in CD14 and TLR4 are independently associated with long-term CD4 T-cell recovery in HIV-infected individuals post-ART.