Doherty Institute - Research Publications
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ItemNo Preview AvailableCombination Immune Checkpoint Blockade Enhances IL-2 and CD107a Production from HIV-Specific T Cells Ex Vivo in People Living with HIV on Antiretroviral Therapy(AMER ASSOC IMMUNOLOGISTS, 2022-01-01)In people with HIV (PWH) on antiretroviral therapy (ART), immune dysfunction persists, including elevated expression of immune checkpoint (IC) proteins on total and HIV-specific T cells. Reversing immune exhaustion is one strategy to enhance the elimination of HIV-infected cells that persist in PWH on ART. We aimed to evaluate whether blocking CTL-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), T cell Ig domain and mucin domain 3 (TIM-3), T cell Ig and ITIM domain (TIGIT) and lymphocyte activation gene-3 (LAG-3) alone or in combination would enhance HIV-specific CD4+ and CD8+ T cell function ex vivo. Intracellular cytokine staining was performed using human PBMCs from PWH on ART (n = 11) and expression of CD107a, IFN-γ, TNF-α, and IL-2 was quantified with HIV peptides and Abs to IC. We found the following: 1) IC blockade enhanced the induction of CD107a and IL-2 but not IFN-γ and TNF-α in response to Gag and Nef peptides; 2) the induction of CD107a and IL-2 was greatest with multiple combinations of two Abs; and 3) Abs to LAG-3, CTLA-4, and TIGIT in combinations showed synergistic induction of IL-2 in HIV-specific CD8+ and CD107a and IL-2 production in HIV-specific CD4+ and CD8+ T cells. These results demonstrate that the combination of Abs to LAG-3, CTLA-4, or TIGIT can increase the frequency of cells expressing CD107a and IL-2 that associated with cytotoxicity and survival of HIV-specific CD4+ and CD8+ T cells in PWH on ART. These combinations should be further explored for an HIV cure.
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ItemMemory CD4+ T cells that co-express PD1 and CTLA4 have reduced response to activating stimuli facilitating HIV latency(CELL PRESS, 2022-10-18)Programmed cell death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) suppress CD4+ T cell activation and may promote latent HIV infection. By performing leukapheresis (n = 21) and lymph node biopsies (n = 8) in people with HIV on antiretroviral therapy (ART) and sorting memory CD4+ T cells into subsets based on PD1/CTLA4 expression, we investigate the role of PD1 and CTLA 4 in HIV persistence. We show that double-positive (PD1+CTLA4+) cells in blood contain more HIV DNA compared with double-negative (PD1-CTLA4-) cells but still have a lower proportion of cells producing multiply spliced HIV RNA after stimulation as well as reduced upregulation of T cell activation and proliferation markers. Transcriptomics analyses identify differential expression of key genes regulating T cell activation and proliferation with MAF, KLRB1, and TIGIT being upregulated in double-positive compared with double-negative cells, whereas FOS is downregulated. We conclude that, in addition to being enriched for HIV DNA, double-positive cells are characterized by negative signaling and a reduced capacity to respond to stimulation, favoring HIV latency.
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ItemA clinical trial of non-invasive imaging with an anti-HIV antibody labelled with copper-64 in people living with HIV and uninfected controls(ELSEVIER, 2021-03)BACKGROUND: A research priority in finding a cure for HIV is to establish methods to accurately locate and quantify where and how HIV persists in people living with HIV (PLWH) receiving suppressive antiretroviral therapy (ART). Infusing copper-64 (64Cu) radiolabelled broadly neutralising antibodies targeting HIV envelope (Env) with CT scan and positron emission tomography (PET) identified HIV Env in tissues in SIV infected non-human primates . We aimed to determine if a similar approach was effective in people living with HIV (PLWH). METHODS: Unmodified 3BNC117 was compared with 3BNC117 bound to the chelator MeCOSar and 64Cu (64Cu-3BNC117) in vitro to assess binding and neutralization. In a clinical trial 64Cu-3BNC117 was infused into HIV uninfected (Group 1), HIV infected and viremic (viral load, VL >1000 c/mL; Group 2) and HIV infected aviremic (VL <20 c/mL; Group 3) participants using two dosing strategies: high protein (3mg/kg unlabeled 3BNC117 combined with <5mg 64Cu-3BNC117) and trace (<5mg 64Cu-3BNC117 only). All participants were screened for 3BNC117 sensitivity from virus obtained from viral outgrowth. Magnetic resonance imaging (MRI)/PET and pharmacokinetic assessments (ELISA for serum 3BNC117 concentrations and gamma counting for 64Cu) were performed 1, 24- and 48-hours post dosing. The trial (clincialtrials.gov NCT03063788) primary endpoint was comparison of PET standard uptake values (SUVs) in regions of interest (e.g lymph node groups and gastrointestinal tract). FINDINGS: Comparison of unmodified and modified 3BNC117 in vitro demonstrated no difference in HIV binding or neutralisation. 17 individuals were enrolled of which 12 were dosed including Group 1 (n=4, 2 high protein, 2 trace dose), Group 2 (n=6, 2 high protein, 4 trace) and Group 3 (n=2, trace only). HIV+ participants had a mean CD4 of 574 cells/microL and mean age 43 years. There were no drug related adverse effects and no differences in tissue uptake in regions of interest (e.g lymph node gut, pharynx) between the 3 groups. In the high protein dosing group, serum concentrations of 3BNC117 and gamma counts were highly correlated demonstrating that 64Cu-3BNC117 remained intact in vivo. INTERPRETATION: In PLWH on or off ART, the intervention of infusing 64Cu-3BNC117 and MRI/PET imaging over 48 hours, was unable to detect HIV-1 env expression in vivo. Future studies should investigate alternative radiolabels such as zirconium which have a longer half-life in vivo. FUNDING: Funded by the Alfred Foundation, The Australian Centre for HIV and Hepatitis Virology Research with additional support from the Division of AIDS, National Institute of Allergy and Infectious Disease, US National Institutes of Health (USAI126611). JHM and SRL are supported by the Australian National Health and Medical Research Council.
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ItemA randomized trial of vorinostat with treatment interruption after initiating antiretroviral therapy during acute HIV-1 infection(MEDISCRIPT LTD, 2020-09)OBJECTIVE AND DESIGN: A randomized, open-label pilot study in individuals treated with antiretroviral therapy (ART) since acute HIV infection (AHI) with a regimen including a histone deacetylase inhibitor to induce HIV from latency and control HIV replication during subsequent treatment interruption (TI). METHODS: Fifteen participants who initiated ART at AHI were randomized to vorinostat/hydroxychloroquine/maraviroc (VHM) plus ART (n = 10) or ART alone (n = 5). The VHM arm received three 14-day vorinostat cycles within 10 weeks before TI. ART was resumed for plasma viral load (VL) > 1,000 HIV RNA copies/mL. Primary outcome was proportion of participants on VHM + ART versus ART only with VL < 50 copies/mL for 24 weeks after TI. RESULTS: Fifteen participants on ART (median: 178 weeks: range 79-295) enrolled. Two on VHM + ART experienced serious adverse events. Fourteen participants underwent TI; all experienced VL rebound with no difference in time between arms: VHM + ART (n = 9) median: 4 weeks and ART only (n = 5) median: 5 weeks. VHM induced a 2.2-fold increase in VL (p = 0.008) by single-copy HIV RNA assay after the first cycle. Neopterin levels increased significantly following the first two cycles. After VHM treatment, the frequencies of peripheral blood mononuclear cells harboring total HIV DNA and cell-associated RNA were unchanged. All participants achieved VL suppression following ART re-initiation. CONCLUSIONS: Administration of VHM increased HIV VL in plasma, but this was not sustained. VHM did not impact time to viral rebound following TI and had no impact on the size of the HIV reservoir, suggesting that HIV reservoir elimination will require alternative treatment strategies.
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ItemDiverse effects of interferon alpha on the establishment and reversal of HIV latency(PUBLIC LIBRARY SCIENCE, 2020-02)HIV latency is the major barrier to a cure for people living with HIV (PLWH) on antiretroviral therapy (ART) because the virus persists in long-lived non-proliferating and proliferating latently infected CD4+ T cells. Latently infected CD4+ T cells do not express viral proteins and are therefore not visible to immune mediated clearance. Therefore, identifying interventions that can reverse latency and also enhance immune mediated clearance is of high interest. Interferons (IFNs) have multiple immune enhancing effects and can inhibit HIV replication in activated CD4+ T cells. However, the effects of IFNs on the establishment and reversal of HIV latency is not understood. Using an in vitro model of latency, we demonstrated that plasmacytoid dendritic cells (pDC) inhibit the establishment of HIV latency through secretion of type I IFNα, IFNβ and IFNω but not IFNε or type III IFNλ1 and IFNλ3. However, once latency was established, IFNα but no other IFNs were able to efficiently reverse latency in both an in vitro model of latency and CD4+ T cells collected from PLWH on suppressive ART. Binding of IFNα to its receptor expressed on primary CD4+ T cells did not induce activation of the canonical or non-canonical NFκB pathway but did induce phosphorylation of STAT1, 3 and 5 proteins. STAT5 has been previously demonstrated to bind to the HIV long terminal repeat and activate HIV transcription. We demonstrate diverse effects of interferons on HIV latency with type I IFNα; inhibiting the establishment of latency but also reversing HIV latency once latency is established.
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ItemLongitudinal analysis of subtype C envelope tropism for memory CD4+T cell subsets over the first 3 years of untreated HIV-1 infection(BMC, 2020-08-06)BACKGROUND: HIV-1 infects a wide range of CD4+ T cells with different phenotypic properties and differing expression levels of entry coreceptors. We sought to determine the viral tropism of subtype C (C-HIV) Envelope (Env) clones for different CD4+ T cell subsets and whether tropism changes during acute to chronic disease progression. HIV-1 envs were amplified from the plasma of five C-HIV infected women from three untreated time points; less than 2 months, 1-year and 3-years post-infection. Pseudoviruses were generated from Env clones, phenotyped for coreceptor usage and CD4+ T cell subset tropism was measured by flow cytometry. RESULTS: A total of 50 C-HIV envs were cloned and screened for functionality in pseudovirus infection assays. Phylogenetic and variable region characteristic analysis demonstrated evolution in envs between time points. We found 45 pseudoviruses were functional and all used CCR5 to mediate entry into NP2/CD4/CCR5 cells. In vitro infection assays showed transitional memory (TM) and effector memory (EM) CD4+ T cells were more frequently infected (median: 46% and 25% of total infected CD4+ T cells respectively) than naïve, stem cell memory, central memory and terminally differentiated cells. This was not due to these subsets contributing a higher proportion of the CD4+ T cell pool, rather these subsets were more susceptible to infection (median: 5.38% EM and 2.15% TM cells infected), consistent with heightened CCR5 expression on EM and TM cells. No inter- or intra-participant changes in CD4+ T cell subset tropism were observed across the three-time points. CONCLUSIONS: CD4+ T cell subsets that express more CCR5 were more susceptible to infection with C-HIV Envs, suggesting that these may be the major cellular targets during the first 3 years of infection. Moreover, we found that viral tropism for different CD4+ T cell subsets in vitro did not change between Envs cloned from acute to chronic disease stages. Finally, central memory, naïve and stem cell memory CD4+ T cell subsets were susceptible to infection, albeit inefficiently by Envs from all time-points, suggesting that direct infection of these cells may help establish the latent reservoir early in infection.