TAG HIV Basic Science, Vaccines, and Cure Project Blog

By Richard Jefferys, Project Director at Treatment Action Group (TAG).

recent posts

  • New Data on the Latency-Reversing Activity of Disulfiram

    Several years ago, the laboratory of Robert Siliciano at Johns Hopkins University reported that the FDA-approved drug disulfiram (Antabuse) could reverse HIV latency in laboratory experiments. The discovery led Steve Deeks and colleagues to conduct a small pilot study in HIV-positive people on ART, which found that a standard dose of 500mg/day of disulfiram showed some hints of latency-reversing activity that appeared to be linked to whether drug levels were detectable in blood. In turn, the pilot study spurred the launch of a larger clinical trial investigating the effects of higher disulfiram doses (up to 2000mg/day) in a cohort of 30 HIV-positive individuals on ART. Results from this trial have now been published in The Lancet HIV.

    The researchers report that disulfiram showed evidence of latency-reversing activity at all doses tested. Measurements of unspliced cell-associated HIV RNA—a standard approach for assessing the activity of candidate latency-reversing agents—revealed small but significant increases after dosing compared to baseline. However only the highest dose of 2000mg/day led to a detectable increase in plasma HIV RNA (as measured by an ultrasensitive viral load test with a lower limit of detection of 0.19 copies).

    The magnitude of the effect of disulfiram was considerably smaller than has been reported with the HDAC inhibitor romidepsin, but the authors point out that disulfiram may be more suitable for long-term dosing due to its well-established safety record (it is used to induce sensitivity to alcohol as a means to treat chronic alcoholism). In contrast, the toxicity profile of HDAC inhibitors is less favorable to extended dosing, and there are concerns about the safety implications of the long-term changes in gene expression that have been observed in clinical trials. The ability to administer disulfiram for a prolonged period could be an advantage in studies involving combinations of approaches to depleting the HIV reservoir. 

    The paper also describes an interesting incidental finding from the trial. To establish baseline measures of HIV RNA for each participant, three samples were taken on different days and the results were averaged. The researchers were surprised to discover that the third measurement—taken earlier in the day than the prior two—showed significantly higher levels of unspliced cell-associated HIV RNA. The finding suggests circadian rhythm may influence HIV transcription, and this needs to be considered when attempting to evaluate the effects of latency-reversing agents (these data were also presented at the IAS 2015 conference).

    A final point worth highlighting is that the initial discovery of disulfiram’s latency-reversing potential and a significant portion of the subsequent clinical evaluation was made possible by funding from amfAR’s HIV cure research program. Today on World AIDS Day, a further expansion of this program was announced with the launch of the amfAR Institute for HIV Cure Research at the University of California San Francisco (see amfAR’s press release for more information).

    Lancet HIV 2015

    Published Online November 16, 2015

    http://dx.doi.org/10.1016/ S2352-3018(15)00226-X

    Short-term administration of disulfiram for reversal of latent HIV infection: a phase 2 dose-escalation study

    Julian H Elliott, MBBS, James H McMahon, MBBS, Christina C Chang, MBBS, Sulggi A Lee, MD, Wendy Hartogensis, PhD, Namandje Bumpus, PhD, Rada Savic, PhD, Janine Roney, MPH, Rebecca Hoh, BA, Ajantha Solomon, BSc, Michael Piatak, PhD†, Robert J Gorelick, PhD, Jeff Lifson, PhD, Prof Peter Bacchetti, PhD, Prof Steven G Deeks, MD, Prof Sharon R Lewin, MBBS

    †Died on Sept 19, 2014

    Summary

    Background

    In vitro, disulfiram activated HIV transcription in a primary T-cell model of HIV latency and in a pilot clinical study increased plasma HIV RNA in individuals with adequate drug exposure. We assessed the effect of disulfiram on HIV transcription in a dose-escalation study.

    Methods

    In this prospective dose-escalation study, to optimise disulfiram exposure we included adults with HIV on suppressive antiretroviral therapy, with plasma HIV RNA of less than 50 copies per mL and a CD4 cell count greater than 350 cells per μL. Participants were allocated sequentially to one of three dosing groups (500 mg, 1000 mg, and 2000 mg) and received disulfiram daily for 3 days. Only the staff who did laboratory assays were masked to group assignment. The primary endpoint was change in cell-associated unspliced HIV RNA in CD4 cells. The primary analysis method was a negative binomial regression, with the number of copies as the outcome variable and the input total RNA or plasma volume as an exposure variable, which is equivalent to modelling copies or input. We used these models to estimate changes from before disulfiram to timepoints during and after disulfiram administration. This study is registered with ClinicalTrials.gov, number NCT01944371.

    Findings

    Of 34 participants screened for eligibility at The Alfred Hospital (Melbourne, VIC, Australia), and San Francisco General Hospital (San Francisco, CA, USA), 30 people were enrolled between Sept 24, 2013, and March 31, 2014. The estimated fold increases in cell-associated unspliced HIV RNA from baseline were 1·7 (95% CI 1·3–2·2; p<0·0001) to the timepoint during disulfiram treatment and 2·1 (1·5–2·9; p<0·0001) to the timepoint after disulfiram in the 500 mg group; 1·9 (1·6–2·4; p<0·0001) and 2·5 (1·9–3·3; p<0·0001) in the 1000 mg group; and 1·6 (1·2–2·1; p=0·0026) and 2·1 (1·5–3·1; p=0·0001) in the 2000 mg group. No deaths occurred, and no serious adverse events were noted. Disulfiram was well tolerated at all doses.

    Interpretation

    Short-term administration of disulfiram resulted in increases in cell-associated unspliced HIV RNA at all doses, consistent with activating HIV latency. Disulfiram may be suited for future studies of combination and prolonged therapy to activate latent HIV.

  • Developing Multi-Pronged Antibodies to Target the HIV Reservoir

    In an example of publication kismet, three recent open access articles all converge in describing a new strategy for depleting the HIV reservoir. The research involves engineering antibodies or antibody-like molecules capable of simultaneously binding two targets: the CD3 receptor, a human protein expressed on T cells, and parts of the HIV envelope (Env) protein, which are typically displayed on the outside of infected CD4 T cells when the virus is active. The rationale is that the region of the antibody that targets HIV Env binds to infected cells, while the CD3-targeting region binds to passing T cells and activates them to kill the cell. The idea for this two-pronged—termed bispecific—antibody attack originated in cancer research, and one candidate that targets CD19 (a protein expressed by B cells) and CD3 is already FDA-approved as a second-line treatment for certain forms of acute lymphoblastic leukemia. 

    In the Journal of Clinical Investigation, Julia Sung and colleagues report results obtained in vitro with “Dual-Affinity Re-Targeting” (DART) proteins designed to bind CD3 and HIV Env epitopes targeted by the broadly binding (but non-neutralizing) monoclonal antibodies A32 or 7B2. These DART proteins were able to significantly reduce HIV levels in cultured, HIV-infected CD4 T cells and also showed activity against latently infected CD4 T cells isolated from individuals on ART and exposed to the latency-reversing agent vorinostat.

    In PLoS Pathogens, a research team from Gilead Sciences and MacroGenics, Inc. describe similar studies conducted with a larger array of candidate DART proteins targeting CD3 and one of several different HIV Env epitopes, including those bound by A32, 7B2 and the broadly neutralizing antibodies PGT121, PGT145, VRC01 and 10E8.  The most active DART proteins were those derived from PGT121, PGT145, A32 and 7B2, confirming and extending the results of Sung et al. Of potential importance for clinical development, modified versions of the DART proteins designed to allow for relatively infrequent dosing in humans maintained activity. 

    The third study, by Amarendra Pegu and colleagues from the Vaccine Research Center at the National Institutes of Health, employed a bispecific antibody that attaches to CD3 and the HIV Env epitope recognized by the broadly neutralizing antibody VRC07. In experiments using latently infected CD4 T cells isolated from individuals on ART, the antibody was found to both activate HIV gene expression and reduce HIV DNA levels in most donor samples. 

    An important, overarching concern with all these bispecific antibodies is the extent to which the CD3-binding region might cause generalized T cell activation. Many years ago, anti-CD3 antibodies were tested in people with HIV with the aim of broadly activating CD4 T cells as means to reverse latency and deplete the HIV reservoir, but the results were disastrous: the treatment led to a massive depletion of T cells, inflammatory cytokine release (leading to transient renal failure in one case) and no evident HIV reservoir reduction (see Prins et al,1999).

    All three research groups conducted laboratory assessments of T cell activation, and found little or no evidence that it occurred in the absence of HIV Env expression; in other words, binding of the bispecific antibodies to both targets appeared necessary for inducing activation. However, Pegu et al also performed a safety assessment in SHIV-infected macaques receiving antiretroviral therapy and this revealed a sharp but short-lived drop in peripheral blood CD3+ T cells and a sizable, albeit transient, increase in levels of several inflammation-related cytokines (TNF-α, MIP-1β and IL-10). These immunological perturbations resolved within 24 hours and there were no increases in SHIV viral load or clinically evident adverse events, leading the researchers to conclude that: “overall, these short-term toxicity studies indicate that the treatment was well tolerated.”

    Taken together, the three papers suggest that bispecific antibodies may have promise as an anti-reservoir strategy. A particular appeal of the approach is the capacity to recruit T cells to destroy HIV-infected cells without regard to their antigen specificity—this could be important because HIV-specific T cells are typically functionally compromised in HIV-positive people. Tempering enthusiasm somewhat are the macaque data showing transient T cell depletion and inflammation, a phenomenon that will need to be evaluated in further animal studies. Barring additional safety concerns arising, human trials appear likely given the involvement of Gilead Sciences (a company with a large HIV cure research program). 

    J Clin Invest. 2015 Sep 28. pii: 82314. doi: 10.1172/JCI82314. [Epub ahead of print]

    Dual-Affinity Re-Targeting proteins direct T cell-mediated cytolysis of latently HIV-infected cells.

    Sung JA, Pickeral J, Liu L, Stanfield-Oakley SA, Lam CK, Garrido C, Pollara J, LaBranche C, Bonsignori M, Moody MA, Yang Y, Parks R, Archin N, Allard B, Kirchherr J, Kuruc JD, Gay CL, Cohen MS, Ochsenbauer C, Soderberg K, Liao HX, Montefiori D, Moore P, Johnson S, Koenig S, Haynes BF, Nordstrom JL, Margolis DM, Ferrari G.

    Abstract

    Enhancement of HIV-specific immunity is likely required to eliminate latent HIV infection. Here, we have developed an immunotherapeutic modality aimed to improve T cell-mediated clearance of HIV-1-infected cells. Specifically, we employed Dual-Affinity Re-Targeting (DART) proteins, which are bispecific, antibody-based molecules that can bind 2 distinct cell-surface molecules simultaneously. We designed DARTs with a monovalent HIV-1 envelope-binding (Env-binding) arm that was derived from broadly binding, antibody-dependent cellular cytotoxicity-mediating antibodies known to bind to HIV-infected target cells coupled to a monovalent CD3 binding arm designed to engage cytolytic effector T cells (referred to as HIVxCD3 DARTs). Thus, these DARTs redirected polyclonal T cells to specifically engage with and kill Env-expressing cells, including CD4+ T cells infected with different HIV-1 subtypes, thereby obviating the requirement for HIV-specific immunity. Using lymphocytes from patients on suppressive antiretroviral therapy (ART), we demonstrated that DARTs mediate CD8+ T cell clearance of CD4+ T cells that are superinfected with the HIV-1 strain JR-CSF or infected with autologous reservoir viruses isolated from HIV-infected-patient resting CD4+ T cells. Moreover, DARTs mediated CD8+ T cell clearance of HIV from resting CD4+ T cell cultures following induction of latent virus expression. Combined with HIV latency reversing agents, HIVxCD3 DARTs have the potential to be effective immunotherapeutic agents to clear latent HIV-1 reservoirs in HIV-infected individuals. 

    Nat Commun. 2015 Oct 20;6:8447. doi: 10.1038/ncomms9447.

    Activation and lysis of human CD4 cells latently infected with HIV-1.

    Pegu A, Asokan M, Wu L, Wang K, Hataye J, Casazza JP, Guo X, Shi W, Georgiev I, Zhou T, Chen X, O'Dell S, Todd JP, Kwong PD, Rao SS, Yang ZY, Koup RA, Mascola JR, Nabel GJ.

    Abstract

    The treatment of AIDS with combination antiretroviral therapy (cART) remains lifelong largely because the virus persists in latent reservoirs. Elimination of latently infected cells could therefore reduce treatment duration and facilitate immune reconstitution. Here we report an approach to reduce the viral reservoir by activating dormant viral gene expression and directing T lymphocytes to lyse previously latent, HIV-1-infected cells. An immunomodulatory protein was created that combines the specificity of a HIV-1 broadly neutralizing antibody with that of an antibody to the CD3 component of the T-cell receptor. CD3 engagement by the protein can stimulate T-cell activation that induces proviral gene expression in latently infected T cells. It further stimulates CD8 T-cell effector function and redirects T cells to lyse these previously latent-infected cells through recognition of newly expressed Env. This immunomodulatory protein could potentially help to eliminate latently infected cells and deplete the viral reservoir in HIV-1-infected individuals. 

    PLoS Pathogens

    Published: November 5, 2015DOI: 10.1371/journal.ppat.1005233 

    Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells

    Derek D. Sloan, Chia-Ying Kao Lam, Alivelu Irrinki, Liqin Liu, Angela Tsai, Craig S. Pace, Jasmine Kaur, Jeffrey P. Murry, Mini Balakrishnan, Paul A. Moore, Syd Johnson, Jeffrey L. Nordstrom, Tomas Cihlar, Scott Koenig

    Abstract

    HIV reservoirs and production of viral antigens are not eliminated in chronically infected participants treated with combination antiretroviral therapy (cART). Novel therapeutic strategies aiming at viral reservoir elimination are needed to address chronic immune dysfunction and non-AIDS morbidities that exist despite effective cART. The HIV envelope protein (Env) is emerging as a highly specific viral target for therapeutic elimination of the persistent HIV-infected reservoirs via antibody-mediated cell killing. Dual-Affinity Re-Targeting (DART) molecules exhibit a distinct mechanism of action via binding the cell surface target antigen and simultaneously engaging CD3 on cytotoxic T lymphocytes (CTLs). We designed and evaluated Env-specific DARTs (HIVxCD3 DARTs) derived from known antibodies recognizing diverse Env epitopes with or without broadly neutralizing activity. HIVxCD3 DARTs derived from PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies, mediated potent CTL-dependent killing of quiescent primary CD4 T cells infected with diverse HIV isolates. Similar killing activity was also observed with DARTs structurally modified for in vivo half-life extension. In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART. Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures. Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs. 

    Author Summary

    Current HIV therapies prevent AIDS by dramatically reducing, but not eliminating, HIV infection. A reservoir of HIV-infected cells persists during long-term antiviral therapy, and individuals are at increased risk to develop non-AIDS illnesses, e.g., accelerated heart, bone, or kidney disease. Novel strategies are thus needed to safely kill HIV-infected cells and reduce or eliminate the HIV reservoir. An emerging strategy to kill HIV-infected cells involves antibodies (Abs) that bind the HIV envelope protein (Env). Env can distinguish HIV-infected cells from uninfected cells, and some Env-specific Abs can kill HIV-infected cells by recruiting immune cells, e.g., NK cells and macrophages. Here, we developed a strategy to kill HIV-infected cells that is complementary to Env-specific Abs. We designed and evaluated Dual-Affinity Re-Targeting (DART) molecules that incorporate Env-binding specificities with a CD3-binding specificity to recruit and activate cytotoxic T cells. We report that HIVxCD3 DARTs potently and selectively kill HIV-infected cells. Furthermore, HIV DARTs perturb resting and activated viral reservoirs in cells isolated from individuals on antiviral therapy. This novel strategy may be an important element of future antiviral therapies that target the HIV reservoir.

  • T-Cell Exhaustion Biomarkers Linked to Time to Viral Load Rebound in the SPARTAC Trial

    A study published in Nature Communications has drawn a fair amount of press coverage—and some hyperbolic headlines—by reporting that certain immunological biomarkers may be able to predict the likelihood of maintaining a low viral load after an antiretroviral therapy (ART) interruption.

    The research involves a subset of participants from the SPARTAC trial, which randomized individuals with primary HIV infection to one of three arms: no treatment (the standard of care at the time the trial was conducted) or 48 or 12 weeks of ART followed by an interruption (the primary results were published in NEJM in 2013). Drawing on samples from a subset of participants who received 48 weeks of ART, analyses were conducted to assess if there were associations between a multitude of immunological biomarkers and the time it took for viral load to rebound to detectable levels after ART interruption (these analyses were not planned in the original SPARTAC protocol and are thus considered exploratory). Senior author John Frater from Oxford University originally described the work at CROI earlier this year (the webcast of his talk is available online). 

    The crux of the results is that baseline (pre-ART) levels of three biomarkers linked to T cell exhaustion—PD-1, Tim-3 and Lag-3—were statistically associated with time to viral load rebound to over 400 copies after ART interruption. Participants with baseline levels of these exhaustion biomarkers on CD4 and CD8 T cells that were below the median for the cohort took longer to rebound, on average, compared to those with levels above the median. However, no such association was seen when the biomarkers were measured in samples taken immediately prior to the ART interruption (the only predictor of time to viral load rebound at this timepoint was total HIV DNA, as reported previously).

    The researchers suggest that the study represents a step toward the development of biomarker algorithms capable of identifying ideal candidates for ART interruptions, based on the likelihood of post-treatment control of viral load. However, it is a very early step, as is stressed by the UK website NHS Choices, which published a commentary in response to a wildly overstated Daily Mail headline about the paper (“HIV breakthrough could lead to a CURE: Scientists identify markers on immune cells that 'predict who can stop drug therapy and stay well'”). There are also uncertainties as to whether post-treatment control necessarily equates to a state of health equivalent to that obtained by ART-mediated HIV suppression (see “The Challenge of Defining HIV Remission” in the most recent TAGline). 

    In the discussion section of the paper, the authors note that anti-PD-1 antibodies have shown success as cancer immunotherapies and that there is interest in studying their therapeutic effects in HIV. In laboratory studies, anti-PD-1 antibodies have been shown to stimulate HIV production by latently infected CD4 T cells, and restore functionality to exhausted HIV-specific T cells. There are concerns, however, about the potential for adverse effects, particularly the induction of autoimmune responses (which has been observed in some cancer trials).

    The uncertainty about the risk/benefit of anti-PD-1 antibodies in otherwise healthy HIV-positive people has prompted a research team led by Thomas Uldrick at the National Cancer Institute to design a trial that will enroll HIV-positive individuals with relapsed, refractory, or disseminated cancers. The main aims are to evaluate safety and anticancer effects; assessments of the HIV reservoir and HIV-specific T cell immunity are included as tertiary endpoints. The anti-PD-1 antibody that will be administered is pembrolizumab (trade name Keytruda), which recently gained FDA approval for the treatment of certain forms of melanoma and non-small cell lung cancer. With helpfully coincidental timing, the trial was recently entered in clinicaltrials.gov and is due to launch in January.

    Nat Commun. 2015 Oct 9;6:8495. doi: 10.1038/ncomms9495.

    Immunological biomarkers predict HIV-1 viral rebound after treatment interruption.

    Hurst J, Hoffmann M, Pace M, Williams JP, Thornhill J, Hamlyn E, Meyerowitz J, Willberg C, Koelsch KK, Robinson N, Brown H, Fisher M, Kinloch S, Cooper DA, Schechter M, Tambussi G, Fidler S, Babiker A, Weber J, Kelleher AD, Phillips RE, Frater J.

    Abstract

    Treatment of HIV-1 infection with antiretroviral therapy (ART) in the weeks following transmission may induce a state of 'post-treatment control' (PTC) in some patients, in whom viraemia remains undetectable when ART is stopped. Explaining PTC could help our understanding of the processes that maintain viral persistence. Here we show that immunological biomarkers can predict time to viral rebound after stopping ART by analysing data from a randomized study of primary HIV-1 infection incorporating a treatment interruption (TI) after 48 weeks of ART (the SPARTAC trial). T-cell exhaustion markers PD-1, Tim-3 and Lag-3 measured prior to ART strongly predict time to the return of viraemia. These data indicate that T-cell exhaustion markers may identify those latently infected cells with a higher proclivity to viral transcription. Our results may open new avenues for understanding the mechanisms underlying PTC, and eventually HIV-1 eradication.

  • A Trio of Papers Provide New Insights Into Host Cell Manipulation by HIV

    Viruses are notorious for manipulating host cells in ways that favor viral replication, and HIV is no exception. The HIV protein Nef has a well-described capacity to subvert normal cellular biology, including down-regulating cell surface class I HLA molecules that might otherwise allow CD8 T cells to recognize that the cell is infected. Last week saw the publication of three independent studies that all report the discovery of another important Nef function.

    Two papers in Nature describe the use different techniques to converge on the finding that Nef diverts cellular proteins named serine incorporator (SERINC) 5 and SERINC3 away from the cell membrane, thus preventing their incorporation into budding HIV virions. In the absence of this diversionary Nef activity, the proteins—particularly SERINC5—dramatically impair HIV infectivity. The mechanism by which incorporation of the SERINC proteins compromises HIV is now under investigation. The phenomenon appears relevant to a broad range of enveloped retroviruses, because occlusion of SERINC5 was found to be a conserved function of Nef proteins encoded by multiple primate immunodeficiency viruses and was also accomplished by the structurally distinct Gag protein of murine leukemia virus.

    The third paper, an open access publication in Cell Host & Microbe, also identified SERINC3/5 as targets of Nef using a proteomic analysis of alterations to the T cell surface caused by HIV infection. Additionally, the researchers discovered that HIV’s Vpu protein significantly downregulates SNAT1 (Sodium Coupled Neutral Amino Acid Transporter 1), a protein involved in amino acid metabolism, thereby impairing T cell proliferation. Interestingly, this capacity of Vpu appears to have been acquired relatively recently in evolutionary time as it is restricted to viral variants from the SIVcpz/HIV-1 lineage. Given that this lineage is responsible for the HIV pandemic, the researchers note that Vpu’s acquisition of the ability to downregulate SNAT1 may have contributed to the spread of the virus in the human population.

    The apparent importance of these host cell manipulations in the HIV life cycle suggests that targeted inhibition of Nef and/or Vpu might have therapeutic potential, and further studies are planned.

    Cell Host & Microbe 18, 1–15

    Open Access

    DOI: http://dx.doi.org/10.1016/j.chom.2015.09.003

    Cell Surface Proteomic Map of HIV Infection Reveals Antagonism of Amino Acid Metabolism by Vpu and Nef

    Nicholas J. Mathesonl, Jonathan Sumner, Kim Wals, Radu Rapiteanu, Michael P. Weekes, Raphael Vigan, Julia Weinelt, Michael Schindler, Robin Antrobus, Ana S.H. Costa, Christian Frezza, Clary B. Clish, Stuart J.D. Neil, Paul J. Lehner 

    Summary 

    Critical cell surface immunoreceptors downregulated during HIV infection have previously been identified using non-systematic, candidate approaches. To gain a comprehensive, unbiased overview of how HIV infection remodels the T cell surface, we took a distinct, systems-level, quantitative proteomic approach. >100 plasma membrane proteins, many without characterized immune functions, were downregulated during HIV infection. Host factors targeted by the viral accessory proteins Vpu or Nef included the amino acid transporter SNAT1 and the serine carriers SERINC3/5. We focused on SNAT1, a β-TrCP-dependent Vpu substrate. SNAT1 antagonism was acquired by Vpu variants from the lineage of SIVcpz/HIV-1 viruses responsible for pandemic AIDS. We found marked SNAT1 induction in activated primary human CD4+ T cells, and used Consumption and Release (CoRe) metabolomics to identify alanine as an endogenous SNAT1 substrate required for T cell mitogenesis. Downregulation of SNAT1 therefore defines a unique paradigm of HIV interference with immunometabolism.

    Nature. 2015 Sep 30. doi: 10.1038/nature15400. [Epub ahead of print]

    SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef.

    Usami Y, Wu Y, Göttlinger HG.

    Abstract

    HIV-1 Nef and the unrelated mouse leukaemia virus glycosylated Gag (glycoGag) strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of both SERINC3 and SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivity. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4+ T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS.

    Nature. 2015 Sep 30. doi: 10.1038/nature15399. [Epub ahead of print]

    HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation.

    Rosa A, Chande A, Ziglio S, De Sanctis V, Bertorelli R, Goh SL, McCauley SM, Nowosielska A, Antonarakis SE, Luban J, Santoni FA, Pizzato M.

    Abstract

    HIV-1 Nef, a protein important for the development of AIDS, has well-characterized effects on host membrane trafficking and receptor downregulation. By an unidentified mechanism, Nef increases the intrinsic infectivity of HIV-1 virions in a host-cell-dependent manner. Here we identify the host transmembrane protein SERINC5, and to a lesser extent SERINC3, as a potent inhibitor of HIV-1 particle infectivity that is counteracted by Nef. SERINC5 localizes to the plasma membrane, where it is efficiently incorporated into budding HIV-1 virions and impairs subsequent virion penetration of susceptible target cells. Nef redirects SERINC5 to a Rab7-positive endosomal compartment and thereby excludes it from HIV-1 particles. The ability to counteract SERINC5 was conserved in Nef encoded by diverse primate immunodeficiency viruses, as well as in the structurally unrelated glycosylated Gag from murine leukaemia virus. These examples of functional conservation and convergent evolution emphasize the fundamental importance of SERINC5 as a potent anti-retroviral factor.

  • SMAC Mimetics and HIV Latency

    SMAC (Second Mitochondrial-derived Activator of Caspases) mimetics are a class of compounds that have recently entered clinical trials as potential cancer treatments. The drugs antagonize anti-apoptotic proteins, thereby promoting the apoptotic death of cancer cells. Earlier this year at CROI, a poster presentation offered evidence that SMAC mimetics may also selectively promote the apoptosis of CD4 T cells latently infected by HIV. In a new open access paper published by Cell Host & Microbe, Lars Pache and colleagues add another string to SMAC mimetics bow, reporting that the drugs can work synergistically with HDAC inhibitors to reverse HIV latency. In an experiment using resting CD4 T cells sampled from individuals on suppressive ART, the combination of the SMAC mimetic SBI-0637142 and the HDAC inhibitor panobinostat activated latent HIV as efficiently as T cell activation. Preliminary results from a phase I cancer trial suggest that the SMAC mimetic birinapant has an acceptable toxicity profile for testing in humans, potentially opening the door for studies of these compounds in HIV. 

    Cell Host Microbe. 2015 Sep 9;18(3):345-53. doi: 10.1016/j.chom.2015.08.009.

    BIRC2/cIAP1 Is a Negative Regulator of HIV-1 Transcription and Can Be Targeted by Smac Mimetics to Promote Reversal of Viral Latency.

    Pache L, Dutra MS, Spivak AM, Marlett JM, Murry JP, Hwang Y, Maestre AM, Manganaro L, Vamos M, Teriete P, Martins LJ, König R, Simon V, Bosque A, Fernandez-Sesma A, Cosford ND, Bushman FD, Young JA, Planelles V, Chanda SK.

    Abstract

    Combination antiretroviral therapy (ART) is able to suppress HIV-1 replication to undetectable levels. However, the persistence of latent viral reservoirs allows for a rebound of viral load upon cessation of therapy. Thus, therapeutic strategies to eradicate the viral latent reservoir are critically needed. Employing a targeted RNAi screen, we identified the ubiquitin ligase BIRC2 (cIAP1), a repressor of the noncanonical NF-κB pathway, as a potent negative regulator of LTR-dependent HIV-1 transcription. Depletion of BIRC2 through treatment with small molecule antagonists known as Smac mimetics enhanced HIV-1 transcription, leading to a reversal of latency in a JLat latency model system. Critically, treatment of resting CD4+ T cells isolated from ART-suppressed patients with the histone deacetylase inhibitor (HDACi) panobinostat together with Smac mimetics resulted in synergistic activation of the latent reservoir. These data implicate Smac mimetics as useful agents for shock-and-kill strategies to eliminate the latent HIV reservoir.

  • Attempt to Oversell New Findings on Pyroptosis in HIV Infection Prompts Headline Debacle

    A new open access paper from the laboratory of Warner Greene at the Gladstone Institutes was published online yesterday in the journal Cell Reports, accompanied by a poorly conceived press release that prompted some of the most egregiously inaccurate media headlines about HIV research in recent history. 

    The study, led by Nicole Galloway and Gilad Doitsh, is a continuation of the lab’s work on pyroptosis, an inflammatory form of cell death that can be triggered by abortive infection of CD4 T cells by HIV (the virus gains entry but does not complete further steps in the viral life cycle). The crux of the new findings is that cell-free HIV particles cannot cause pyroptosis; rather, cell-to-cell transmission of virions is required to initiate the cascade of events that ends in pyroptotic CD4 T cell death. Regrettably, whoever at Gladstone Institutes was responsible for the press release decided the best way to try and hook the media into covering the paper was to use the headline “HIV particles do not cause AIDS, our own immune cells do.” Not only is this ripe for misunderstanding, it is not even an accurate representation of what the study shows – the immune cells don’t do anything to cause AIDS unless HIV is spreading between them. Yet worse, the notion that this research entirely explains how HIV causes AIDS—which is treated as fact in both press release and paper—is still unproven.

    The resulting media coverage has thus been misleading in two ways. First there was a jaw-droppingly inaccurate version of the press release headline offered by United Press International: “Study: HIV does not cause AIDS, human immune cells do.” To their credit, UPI have since made corrections (the original headline can still be seen in the URL) but the original circulated far and wide on the internet and several other outlets have offered variants (e.g. “AIDS: HIV Not Culprit”). Inevitably, these headlines will be picked up and used as propaganda by AIDS denialists, who are always on the lookout for material that can be quote-mined and will have no interest in the actual content of the news articles or the paper.

    Secondly, news stories are credulously repeating the claims in the Gladstone Institutes press release that pyroptosis underlies progression to AIDS. But there are uncertainties about this because the data almost entirely derives from studies involving X4-tropic HIV isolates (see prior blog post on the 2013 papers). Specifically, the claim in the press release that “95% of cell death from HIV is caused by immune cells committing suicide in self-defense after an unsuccessful infection” derives from findings in the ex vivo human lymphoid aggregate culture (HLAC) system showing that X4-tropic and dual R5/X4 HIV isolates cause massive depletion of bystander CD4 T cells that are not productively infected. But the original studies that reported this finding also showed that R5-tropic HIV isolates cause very little depletion of bystander CD4 T cells (see figure 2 of this 2003 Journal of Virology paper). Like the press release, the Cell Reports paper also glosses over this distinction, simply stating: “infection of HLACs with HIV-1 produces extensive loss of CD4 T cells––less than 5% of the cells die as a result of productive viral infection, while >95% of them die as a consequence of abortive infection.” The experiments reported in the new paper only involve an X4-tropic laboratory HIV isolate.

    This is not to argue that pyroptosis plays no role in disease progression (there is a strong case to be made that it explains the consistent association between X4 tropism and rapid CD4 T cell loss), but rather that it is not yet clear how it contributes in R5-tropic HIV infection. For example, many individuals progress to AIDS without showing evidence of the emergence of X4-tropic variants, and R5-tropic SHIVs cause simian AIDS in macaques. As far as is known at this juncture, progression in these settings occurs without 95% of the CD4 T cell death resulting from pyroptosis. There has been a preliminary report that pyroptosis is involved in the death of CCR5-expressing CD4 T cells in the gut, but more research is needed. 

    It is understandable that scientists and their institutions might want to raise the profile of their work, particularly in a time of shrinking research funding, but in this case I think the significance of largely in vitro research findings has been hugely oversold and it has resulted in some horribly misleading news stories. For community-based advocates like TAG who try to promote public understanding of—and support for—scientific research, this type of debacle is extremely unwelcome, because no responses, caveats, or explanations we offer will ever reach the vast numbers of people who will read the misleading media reports.

    Cell Reports 12, 1–9, September 8, 2015

    Report

    Cell-to-Cell Transmission of HIV-1 Is Required to Trigger Pyroptotic Death of Lymphoid-Tissue-Derived CD4 T Cells

    Nicole L.K. Galloway5, Gilad Doitsh5, Kathryn M. Monroe, Zhiyuan Yang, Isa Muñoz-Arias, David N. Levy, Warner C. Greene

    5Co-first author

    Publication stage: In Press Corrected Proof

    Open Access

    DOI: http://dx.doi.org/10.1016/j.celrep.2015.08.011

    Summary 

    The progressive depletion of CD4 T cells underlies clinical progression to AIDS in untreated HIV-infected subjects. Most dying CD4 T cells correspond to resting nonpermissive cells residing in lymphoid tissues. Death is due to an innate immune response against the incomplete cytosolic viral DNA intermediates accumulating in these cells. The viral DNA is detected by the IFI16 sensor, leading to inflammasome assembly, caspase-1 activation, and the induction of pyroptosis, a highly inflammatory form of programmed cell death. We now show that cell-to-cell transmission of HIV is obligatorily required for activation of this death pathway. Cell-free HIV-1 virions, even when added in large quantities, fail to activate pyroptosis. These findings underscore the infected CD4 T cells as the major killing units promoting progression to AIDS and highlight a previously unappreciated role for the virological synapse in HIV pathogenesis.

  • Combining PKC Agonists and Bromodomain Inhibitors to Reverse HIV Latency

    Two recent papers in PLoS Pathogens report that combinations of candidate latency-reversing agents can potently activate HIV production by latently infected CD4 T cells in laboratory experiments. Pairings of the PKC agonists bryostatin-1 or ingenol with the bromodomain inhibitor JQ1 were most effective, generating levels of virus production by latently infected cells similar to those achieved by maximal T cell activation. The results appear consistent with those published earlier this year by the research group of Robert Siliciano at Johns Hopkins University, and are encouraging because there had been some skepticism as to whether any latency reversing strategy could match the effects of maximal T cell activation (which is known to be too dangerous to use in people). But there are caveats: the compounds do affect T cell activation pathways and it is not yet known if they will be safe in HIV-positive individuals; currently, they are being tested (and in the case of ingenol, used topically) as cancer treatments.

    The two new publications derive from independent research laboratories led by Satya Dandekar at UC Davis and Carine Van Lint at Université Libre de Bruxelles (ULB), respectively. The experimental findings are broadly consistent but involve two different variants of ingenol: ingenol-B, which has previously been reported to have latency-reversing activity, and ingenol-3-angelate (PEP005), the active component of an FDA-approved topical treatment for precancerous actinic keratosis named PICATO. The opinions of the two groups regarding which version of ingenol might be safer for systemic use in humans differ somewhat. Van Lint’s group writes:

    “Importantly, ingenol-3-angelate appears to be more toxic than ing-B when orally delivered to rats and dogs (Luiz Pianowski, Kyolab, Brazil, personal communication).”

    While Dandekar’s states:

    “Similar to the safety of the topical application of PICATO, the systemic (intravenous) use of PEP005 in small animals (mini pig and rat model) was reported to be relatively safe, with the maximum nonlethal dose >73 μg/kg (See Assessment report of PICATO to European Medicines Agency, Sept 20, 2012). While additional safety data with systemic administration in non-human primates is needed, these existing data support further investigation of PEP005 as a potential candidate in HIV cure studies.”

    Further animal model studies should help resolve this uncertainty. The UK website NHS Choices, which provides commentary on research-related stories in the mainstream media, takes a rather dim view of Dandekar’s suggestion (reported by the BBC) that the FDA-approved status of PICATO is relevant to the prospects of this approach in people with HIV, countering that “although the drug is being used on patients, it is currently just applied to the skin. The effects may be very different if the whole body is exposed to the drug, as would be required to locate hidden reservoirs of HIV.” 

    The other PKC agonist studied, bryostatin-1, is already being tested in HIV-positive people in a small, single-dose clinical trial in Spain supported by a biotech company named Aphios; results are pending. While some researchers have expressed concern about the potential toxicities of bryostatin-1, Van Lint’s group is more sanguine, noting that in a phase I trial in children with cancers (published in 1999) “only few patients have experienced myalgia, photophobia or eye pain.” But the report from this trial also states “toxicities of bryostatin-1 occurred days after the infusion and lasted for prolonged periods” and I think it’s fair to say that opinions about the acceptability of these types of side effects in healthy HIV-positive people are likely to vary. There are ongoing efforts to develop safer and more targeted analogs of bryostatin-1, notably those led by Paul Wender at Stanford University.

    Bromodomain inhibitors are also being studied in cancer. The compound used in these studies, JQ1, has too short a half-life for human use but Van Lint and colleagues note: “Clinical trials with JQ1-derivative called TEN-010 (also called JQ2) and another BETi called GSK525762 have been initiated recently to characterize their safety, tolerability, pharmacokinetics and anti-cancer activity (clinicaltrials.gov).” Safety information from these trials will be important in determining whether studies in HIV are appropriate. 

    PLoS Pathog. 2015 Jul 30;11(7):e1005063. doi: 10.1371/journal.ppat.1005063. eCollection 2015 Jul.

    An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression.

    Darcis G, Kula A, Bouchat S, Fujinaga K, Corazza F, Ait-Ammar A, Delacourt N, Melard A, Kabeya K, Vanhulle C, Van Driessche B, Gatot JS, Cherrier T, Pianowski LF, Gama L, Schwartz C, Vila J, Burny A, Clumeck N, Moutschen M, De Wit S, Peterlin BM, Rouzioux C, Rohr O, Van Lint C.

    Abstract

    The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

    PLoS Pathog. 2015 Jul 30;11(7):e1005066. doi: 10.1371/journal.ppat.1005066. eCollection 2015 Jul.

    Synergistic Reactivation of Latent HIV Expression by Ingenol-3-Angelate, PEP005, Targeted NF-kB Signaling in Combination with JQ1 Induced p-TEFb Activation.

    Jiang G, Mendes EA, Kaiser P, Wong DP, Tang Y, Cai I, Fenton A, Melcher GP, Hildreth JE, Thompson GR, Wong JK, Dandekar S.

    Abstract

    Although anti-retroviral therapy (ART) is highly effective in suppressing HIV replication, it fails to eradicate the virus from HIV-infected individuals. Stable latent HIV reservoirs are rapidly established early after HIV infection. Therefore, effective strategies for eradication of the HIV reservoirs are urgently needed. We report that ingenol-3-angelate (PEP005), the only active component in a previously FDA approved drug (PICATO) for the topical treatment of precancerous actinic keratosis, can effectively reactivate latent HIV in vitro and ex vivo with relatively low cellular toxicity. Biochemical analysis showed that PEP005 reactivated latent HIV through the induction of the pS643/S676-PKCδ/θ-IκBα/ε-NF-κB signaling pathway. Importantly, PEP005 alone was sufficient to induce expression of fully elongated and processed HIV RNAs in primary CD4+ T cells from HIV infected individuals receiving suppressive ART. Furthermore, PEP005 and the P-TEFb agonist, JQ1, exhibited synergism in reactivation of latent HIV with a combined effect that is 7.5-fold higher than the effect of PEP005 alone. Conversely, PEP005 suppressed HIV infection of primary CD4+ T cells through down-modulation of cell surface expression of HIV co-receptors. This anti-cancer compound is a potential candidate for advancing HIV eradication strategies.

  • Estimating How Frequently Latent HIV Reactivates

    The primary barrier to curing HIV infection is the persistence of the virus in a latent form in long-lived resting memory CD4 T cells. The number of latently infected resting memory CD4 T cells in a typical individual on ART is estimated to be in the range of 1-60 million. An important strand of HIV cure research involves attempting to reduce the size of this persistent HIV reservoir, in hopes of delaying or—better yet—preventing viral load rebound when ART is interrupted. In order to gain insight into how feasible this might be, researchers have employed mathematical modeling to estimate how the size of the HIV reservoir relates to time to viral load rebound.

    A widely cited model created Alison Hill and colleagues has suggested that reservoir reductions on the order of 5-6 logs (100,000-1 million fold) would be necessary to delay viral load rebound for 30 years or more in most individuals (an outcome that would approach a lifelong cure), while a yearlong delay would likely require a drop of at least 3 logs (1,000 fold). Last month in PLoS Pathogens, a new model was published that argues that significant delays in viral load rebound might be achieved with far more modest declines in HIV reservoir size.

    The study draws on data from several different clinical trials in which ART was interrupted and time to viral load rebound assessed. A series of calculations are used to generate an estimate of how often—on average—a latently infected resting memory CD4 T cell would have to start producing virus to explain the kinetics of the rise in viral load observed after ART interruption. The math is complex and opaque to a non-mathematician, but produces a result of one successful reactivation of latent HIV every six days, considerably less frequent than a previous estimate of five times per day based on studies involving drug resistance mutations (this prior estimate is used in the Alison Hill model). A separate analysis in the new paper, using different methodology based on the genetic characteristics of rebounding HIV, arrives at a reasonably similar estimate of once every 3.6 days.

    The researchers extrapolate that a yearlong delay in viral load rebound might therefore be achievable with a reduction in the HIV reservoir of 60-70 fold, a more optimistic scenario than proposed previously by Alison Hill et al. But, on the surface at least, the Hill model appears more consistent with the well-publicized cases of the Boston patients, two individuals who were reported to have experienced HIV reservoir reductions of at least 3 logs as a result of receiving stem cell transplants to treat cancers. After a carefully conducted ART interruption, viral load rebound occurred after around three months in one case and eight months in the other. The authors of the new model suggest that this apparent discrepancy might be explained by the presence of a larger, unmeasured HIV reservoir in the tissues of the Boston patients.

    The Alison Hill et al PNAS paper also cites a case report by Tae-Wook Chun and colleagues describing an individual with an HIV reservoir approximately 1,500 fold lower than a typical person on ART in whom viral load rebound occurred 50 days after ART interruption. Although it would be premature to draw conclusions from so few case reports, it has to be noted that a 60-70 fold decline in the HIV reservoir doesn’t appear to have delayed viral load rebound for a year in anybody as yet. And in three examples where greater reservoir reductions appear to have occurred, viral load rebound was not delayed for a year.

    The cautious interpretation would be that additional data are needed to help refine the mathematical modeling and ascertain which models most closely approximate the biological reality. An intervention (beyond stem cell transplantation, which cannot be studied on a large scale) that significantly lowered HIV reservoir levels would also allow for a more direct assessment of the impact on time to viral load rebound.

    Remission Definitions

    A final comment on the use of the term  “remission” in this context: it’s important to note the distinction between the type remission being described in this work and the “virological remission” reported in the recent case of the teenage post-treatment controller and the similar VISCONTI cohort participants. The former involves a complete absence of HIV activity due to remaining latently infected cells staying in a non-activated resting state for the duration of the remission, and any eventual viral load rebound occurring as a result of a latently infected cell becoming activated and producing infectious virus (as best as anyone can tell with available technology, this appears to be the type of remission that occurred in the Boston patients and Mississippi baby). The latter scenario of post-treatment control of viral load is different because it involves ongoing limitation of HIV replication by immune responses; in other words, low-level HIV replication activity that is kept in check by the immune system. Current evidence implies that the former type of remission is more likely to be associated with a state of health comparable to being HIV-negative (and therefore consistent with most people’s understanding of the term remission), whereas post-treatment control might be associated with some degree of inflammation-mediated risk of disease, making it perhaps questionable as to whether the term remission should be applied. 

    PLoS Pathog. 2015 Jul 2;11(7):e1005000. doi: 10.1371/journal.ppat.1005000. eCollection 2015.

    HIV Reactivation from Latency after Treatment Interruption Occurs on Average Every 5-8 Days-Implications for HIV Remission.

    Pinkevych M, Cromer D, Tolstrup M, Grimm AJ, Cooper DA, Lewin SR, Søgaard OS, Rasmussen TA, Kent SJ, Kelleher AD, Davenport MP.

    Abstract

    HIV infection can be effectively controlled by anti-retroviral therapy (ART) in most patients. However therapy must be continued for life, because interruption of ART leads to rapid recrudescence of infection from long-lived latently infected cells. A number of approaches are currently being developed to 'purge' the reservoir of latently infected cells in order to either eliminate infection completely, or significantly delay the time to viral recrudescence after therapy interruption. A fundamental question in HIV research is how frequently the virus reactivates from latency, and thus how much the reservoir might need to be reduced to produce a prolonged antiretroviral-free HIV remission. Here we provide the first direct estimates of the frequency of viral recrudescence after ART interruption, combining data from four independent cohorts of patients undergoing treatment interruption, comprising 100 patients in total. We estimate that viral replication is initiated on average once every ≈6 days (range 5.1- 7.6 days). This rate is around 24 times lower than previous thought, and is very similar across the cohorts. In addition, we analyse data on the ratios of different 'reactivation founder' viruses in a separate cohort of patients undergoing ART-interruption, and estimate the frequency of successful reactivation to be once every 3.6 days. This suggests that a reduction in the reservoir size of around 50-70-fold would be required to increase the average time-to-recrudescence to about one year, and thus achieve at least a short period of anti-retroviral free HIV remission. Our analyses suggests that time-to-recrudescence studies will need to be large in order to detect modest changes in the reservoir, and that macaque models of SIV latency may have much higher frequencies of viral recrudescence after ART interruption than seen in human HIV infection. Understanding the mean frequency of recrudescence from latency is an important first step in approaches to prolong antiretroviral-free viral remission in HIV.

  • Innate Immunity and HIV DNA Declines in Panobinostat Recipients

    Results from a phase I trial of the candidate latency reversing agent panobinostat were published last year in The Lancet HIV (see blog post from November 2014). The paper notes that in an exploratory analysis, a subset of four (out of a total of 15) participants experienced a significant decline in cell-associated HIV DNA levels of around 70-80%, although there was no detectable change in the trial cohort overall. Furthermore, the HIV DNA reduction in these four individuals was associated with a slightly longer time to viral load rebound during an analytical ART interruption. At the NIAID-sponsored Strategies for an HIV Cure meeting in October 2014, Matthias Lichterfeld reported that several measures of innate immunity correlated with the observed diminution of HIV DNA, and these analyses have now been published online in the Journal of Virology.

    The most prominent finding is an association between natural killer (NK) cells expressing markers of enhanced functionality (including CD57, associated with cytotoxic activity) and reductions in HIV DNA during the trial. The researchers suggest the effect might be due to reversal of HIV latency causing up-regulatiion of receptors that activate NK cells on infected CD4 T cells and/or causing down-regulation of HLA class I receptors on these cells (both phenomena would promote NK cell-mediated killing of the CD4 T cells). Complementary correlations were seen with additional markers of innate immunity, including expression of interferon-stimulated genes and plasmacytoid dendritic cell frequencies. The IL28B “CC” genotype, which has been linked to superior innate immune function against hepatitis C, was also associated with a greater reduction of HIV DNA during panobinostat treatment.

    HIV-specific CD8 T cell responses, which have been suggested to be important for mediating clearance of the HIV reservoir after latency reversal, did not correlate with HIV DNA levels. The researchers note that this could be due to the HIV-specific CD8 T cell dysfunction that is known to occur in chronic infection and the presence of escape mutations in the latent HIV reservoir that abrogate CD8 T cell recognition. No evidence of an inhibitory effect of panobinostat on HIV-specific CD8 T cells (a possibility raised by a prior laboratory study) was found. Interestingly, a larger HIV-specific CD4 T cell response at baseline (but not at any other timepoint) was associated with a greater HIV DNA decline during the trial; the explanation for this finding is not known but will be probed in future work.

    The discussion section of the paper emphasizes that the research involves small numbers and exploratory subset analyses, so the results must be interpreted very cautiously. But the idea that promoting innate immunity may aid in the clearance of the HIV reservoir after latency reversal is encouraging, and will be explored further in ongoing and new clinical trials. For example, toll-like receptor (TLR) agonists may have the capacity to stimulate innate immunity, and several are now being tested in HIV-positive people on ART to assess any effects on the latent reservoir (see TAG’s cure-related clinical trials page under latency-reversing agents).

    J Virol. 2015 Jul 29. pii: JVI.01484-15. [Epub ahead of print]

    Innate immune activity correlates with CD4 T cell-associated HIV-1 DNA decline during latency-reversing treatment with panobinostat.

    Olesen R, Vigano S, Rasmussen T, Søgaard OS, Ouyang Z, Buzon M, Bashirova A, Carrington M, Palmer S, Brinkmann CR, Yu XG, Østergaard L, Tolstrup M, Lichterfeld M.

    Abstract

    Pharmaceutical reactivation of dormant HIV-1 proviruses by histone deacetylase inhibitors (HDACi) represents a possible strategy to reduce the reservoir of HIV-1 infected cells in individuals treated with suppressive antiretroviral combination therapy (cART). However, effects of such latency-reversing agents on the viral reservoir size are likely to be influenced by host immune responses. Here, we analyzed immune factors associated with changes in proviral HIV-1 DNA levels during treatment with the potent HDACi panobinostat in a human clinical trial involving 15 cART-treated HIV-1 patients. We observed that the magnitude, breadth, and cytokine-secretion profile of HIV-1-specific CD8 T cell responses were unrelated to HIV-1 DNA changes in CD4 T cells during panobinostat treatment. In contrast, proportions of CD3- CD56+ total NK cells and CD16+ CD56dim NK cells were inversely correlated with HIV-1 DNA throughout the study, and changes of HIV-1 DNA during panobinostat treatment were negatively associated with corresponding changes in CD69+ NK cells. Decreasing levels of HIV-1 DNA during latency-reversing treatment were also related to proportions of plasmacytoid dendritic cells, to distinct gene expression patterns of interferon-stimulated genes and to expression of the IL28B "CC" genotype. Together, these data suggest that innate immune activity can critically modulate effects of latency-reversing agents on the viral reservoir and may represent a target for future immunotherapeutic interventions in HIV-1 eradication studies.

    IMPORTANCE: Currently available antiretroviral drugs are highly effective in suppressing HIV-1 replication, but the virus persists despite treatment in a latent form that does not actively express HIV-1 gene products. One approach to eliminate these cells, colloquially termed as the "shock and kill" strategy, focuses on the use of latency-reversing agents that induce active viral gene expression in latently infected cells, followed by immune-mediated killing. Panobinostat, a histone deacetylase inhibitor, demonstrated potent activities in reversing HIV-1 latency in a recent pilot clinical trial, and reduce HIV-1 DNA in a subset of patients. Interestingly, we found that innate immune factors, such as natural killer cells, plasmacytoid dendritic cells and expression patterns of Interferon-stimulated genes, were most closely linked to a decline of HIV-1 DNA during treatment with panobinostat. These data suggest that innate immune activity may play an important role in reducing the residual reservoir of HIV-1 infected cells.

  • News and Notes from IAS 2015

    Last week saw a slew of news emerge from the IAS 2015 conference and related satellite meetings in Vancouver. Below are links to some presentations of possible interest and information on how to find materials online.

    2015 Towards an HIV Cure Symposium 

    On July 18 & 19, immediately preceding the main conference, the fifth IAS Towards an HIV Cure Symposium took place. Most of the presentations (divided into day 1 and day 2) and posters are now posted, along with the abstract book, with audio pending.

    Among the most talked about presentations (beyond Asier Sáez-Cirión’s widely publicized description of long-term virological remission in a teenager, covered on the blog previously) was Jonathan Karn’s report that estrogen receptors appear to play a role in HIV latency in CD4 T cells. Karn showed results from laboratory studies indicating sex differences in responses to latency-reversing agents; specifically, reversal of HIV latency by a variety of different compounds was profoundly inhibited by the hormone estradiol in women but not men. Conversely, compounds that antagonize the estrogen receptor—such as the breast cancer treatments tamoxifen and fulvestrant—enhanced latency reversal. Karn’s research is part of an ongoing amfAR-funded effort to better understand HIV latency and reservoirs in women. During the Q&A after Karn spoke, Brigitte Autran mentioned a French study (by Lise Cuzin and colleagues) of men and women on long-term ART that found that HIV DNA levels were significantly lower in women; these results have just been published in the journal AIDS. Karn also presented his results at IAS 2015 and a webcast of the talk is available.

    John Mascola from the Vaccine Research Center at the National Institutes of Health offered a glimpse at the viral load reductions obtained in a phase I trial of the broadly neutralizing antibody (bNAb) VRC01 in HIV-positive individuals not yet on ART; the results looked generally compatible with those obtained with the bNAb 3BNC117. After a single VRC01 dose viral load fell by around 1-1.5 logs, with six out of eight participants experiencing a 10-fold or greater drop. Two individuals who did not see a viral load decline turned out to have VRC01-resistant virus at baseline, suggesting that bNAbs will need to be combined to maximize their effects. These data are due to be published soon and are not included in the version of Mascola’s presentation that has been posted online.

    Andrés Finzi discussed the possibility of exploiting the antibodies that are already present in HIV-positive people as a means to eliminate virus-infected cells. Finzi’s group has identified compounds known as CD4 mimetics that can alter the state of the HIV envelope in a way that allows common non-neutralizing antibodies to induce antibody-mediated cellular cytotoxicity (ADCC) and thereby mediate killing of HIV-infected CD4 T cells. Unfortunately Finzi’s powerpoint presentation seems to have been mangled by the translation into PDF form but the work was published recently in PNAS. Finzi believes the CD4 mimetics will be amenable to testing as oral drugs, and might provide an alternative to injectable bNAbs for the purposes of inducing ADCC against HIV-infected cells after latency reversal. 

    Christopher Peterson won the IAS young investigator award for his work demonstrating that stem cells genetically modified to abrogate expression of the CCR5 receptor can be successfully transplanted in the pigtailed macaque model. Peterson showed that the approach gave rise to gene-modified immune cells of multiple lineages, both in normal pigtailed macaques and SHIV-infected animals on ART. Results of studies evaluating the anti-SHIV effects of the transplants will be available soon.

    IAS 2015

    The main conference website does not have the most user-friendly system for accessing presentations and abstracts. The portal is called the programme-at-a-glance, but you may find yourself doing more squinty-eyed poring and clicking than glancing. There is a helpful section called roadmaps that lists conference sessions by topic area, such as “HIV Pathogenesis,” “Towards an HIV Cure” and “Vaccine.” Slides and webcasts (where available) are accessed via the conference session where the presentation occurred. The abstracts page allows browsing of different categories and the filtering option can be used to limit browsing to just posters. As far as I can tell, there is no way of searching abstracts using keywords. An IAS 2015 abstract book has been published by the Journal of the International AIDS Society (both in PDF and HTML format), but it only includes the few posters presented in oral discussion sessions.

    Additional cure research presentations at IAS 2015 included an excellent overview of the field by Nicolas Chomont, available as a webcast. 

    Jake Estes described a potentially important new technology capable of visually identifying SIV-infected cells in macaque tissues. Both viral RNA and DNA can be visualized, which has the potential to shed light on the locations of the latent virus reservoir in ART-treated animals. The technology is called RNAscope and DNAscope in situ hybridization. Estes's slides can be downloaded (be warned that due to the many images it is a huge 64MB file).

    Susana Valente discussed how Tat inhibition may lockdown latent HIV, impeding its ability to reactivate—the opposite strategy to latency reversal (Valente describes it as “silencing the HIV reservoir”). The work was published recently in the open access journal mBio and Valente’s talk is webcast.

    On the HIV vaccine front, two excellent overview presentations are available via webcast: Tony Fauci discussed the state of the field in a talk titled “Progress and challenges in HIV prevention,” while Glenda Gray delivered a plenary talk that focused on plans for the next round of efficacy trials. 

    Community-based news sources providing coverage from IAS 2015 include the National AIDS Treatment Advocacy Project, HIV i-Base, AVAC and AIDSMap.