TAG HIV Basic Science, Vaccines, and Cure Project Blog

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

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  • Novel, Broadly Active HIV Inhibitor Shows Potential

    A paper published yesterday in Nature has stirred considerable excitement and widespread media coverage. Led by the laboratory of Michael Farzan at The Scripps Research Institute, the research involves a newly designed inhibitor of HIV named eCD4-Ig. The inhibitor is designed to bind the HIV envelope at sites that attach to CD4 and CCR5 molecules on CD4 T cells; blocking this interaction prevents HIV from gaining entry and infecting the cell. In laboratory experiments, eCD4-Ig showed activity against an unprecedentedly broad array of HIV and SIV variants, including viruses known to be resistant to even potent neutralizing antibodies. In addition to the greater breadth, activity was generally achieved at lower concentrations than has been documented with the broadly neutralizing antibodies (bNAbs) described to date. Due to similarities between HIV envelope binding to CCR5 and CXCR4 co-receptors, eCD4-Ig also proved capable of neutralizing CXCR4-dependent virus isolates. These results prompted the researchers to explore the preventive potential of eCD4-Ig in the rhesus macaque model, and it is the outcome of this part of the study that has particularly spurred the publicity.

    To deliver the eCD4-Ig (which was modified to match the rhesus macaque form of the protein), the researchers chose to incorporate the gene encoding eCD4-Ig into an AAV vector. AAV vectors are taken up by muscle tissue and act as a factory for producing the encoded protein; for this reason they have been widely used in gene therapy studies (to deliver factor IX to hemophiliacs, for example) and are also under evaluation as a method to deliver neutralizing antibodies against HIV. One small twist with the eCD4-Ig macaque study is that the researchers delivered an 80/20 mix of two AAV vectors: one encoding eCD4-Ig, the other encoding the gene for tyrosylprotein sulfotransferase 2 (TPST2). The role of the TPST2 is to alter the eCD4-Ig protein via a process called sulfation, and this is necessary to make the eCD4-Ig active. It’s not evident from the paper whether preliminary experiments indicated the addition of TPST2 was needed, or if the decision was based solely on biological considerations.

    Four macaques were administered the AAV vector mix and four served as controls. All animals were subsequently challenged intravenously with escalating doses of a hybrid SIV/HIV virus (SHIV-AD8). The four controls all eventually became infected, but the AAV recipients resisted infection despite multiple challenges over a 34-week period. Serum levels of eCD4-Ig were stable at the end of the 40-week study, and sera from the macaques neutralized HIV in vitro just as effectively as eCD4-Ig produced in the laboratory. Although antibody responses against the eCD4-Ig could be detected in the macaques, the levels were very low (much lower than were seen against the broadly neutralizing monoclonal antibodies 3BNC117, NIH45-45, 10-1074 and PGT121).

    The researchers cite several features of the approach that may make it suitable for advancing into human trials.

    • Protection was achieved at eCD4-Ig concentrations that are likely sustainable in humans, and the challenge dose of virus was higher than is the case in most human transmission events.
    • Previous macaque studies using AAV vectors to encode bNAbs have shown that protective titers can be sustained for at least several years.
    • A related protein that only targeted the CD4 binding site of HIV, CD4-Ig (also known as PRO 542), has been tested in human trials and found to be safe.
    • eCD4-Ig does not appear likely to provoke significant immune responses against itself in humans.

     But there are also some potential hurdles and caveats: 

    • The macaque experiment involved intravenous challenges and efficacy against a mucosal challenge has yet to be assessed.
    • Whether eCD4-Ig can safely be administered to humans remains to be established.
    • Advancing what is essentially a gene therapy approach into healthy individuals raises safety concerns that regulatory agencies will want to scrutinize very carefully (notably, the first ever phase I human clinical trial of an AAV vector encoding a bNAb against HIV began in the UK last year).
    • Further research is needed to establish if HIV can become resistant to eCD4-Ig, although the researchers believe that if the virus did develop the ability to bind CCR5 and CD4 in the presence of eCD4-Ig, the binding efficiency would be extremely impaired.
    • The safety of delivering the adjunctive TPST2 protein will need to be established. Rather than use a separate AAV vector to deliver TPST2, as was the case in the macaque study, the aim is to develop a single AAV vector that encodes both eCD4-Ig and TPST2. Human TPST2 is naturally expressed in multiple tissues, including muscle, giving some reason to hope that delivery via AAV vector would not be problematic.
    • There is evidence that the presence of AAV-specific memory CD8 T cell responses in humans can limit the ability of AAV vectors to deliver their protein cargo (a problem seen in hemophilia gene therapy trials). Whether the problem might affect AAV-based approaches for HIV is not yet known. Strategies to limit CD8 T cell recognition of AAV vectors are actively being pursued.  

    The paper does not explicitly discuss whether eCD4-Ig might have therapeutic potential (focusing instead on prevention) but notes that it is a potent inducer of antibody-dependent cell-mediated cytotoxicity (ADCC); it has been postulated that induction of ADCC against HIV-infected cells could be a component of a combination strategy to eliminate HIV reservoirs. A press release from the National Institute of Allergy and Infectious Diseases (NIAID) addresses the issue more directly and refers to eCD4-Ig as a “potential long-acting HIV therapeutic,” with a quote from Michael Farzan stating: “if one could inject either eCD4-Ig or our gene therapy tool into people with HIV infection, it might control HIV for extended periods in the absence of antiretroviral drugs.” The release also notes that therapeutic studies in macaques are getting underway, funded under NIAID’s recent Beyond HAART: Innovative Therapies to Control HIV-1 RFA.

    According to an article in the New York Times, the researchers are considering a stepwise development plan that would first test infusions of the eCD4-Ig protein in healthy volunteers, then evaluate AAV-delivered eCD4-Ig (and, presumably, TPST2) in HIV-positive individuals, then finally move the AAV-delivered version into trials in HIV-negative populations at high risk of HIV infection. In the Wall Street Journal, Farzan articulates the hope that “human trials could begin within a year” but this may be overly optimistic; given the additional work that needs to be done, two or three years might be more realistic.

    Links to several news articles about the research are below.

    Stopping HIV with an artificial protein – Jon Cohen, Science Magazine, February 18, 2015

    New Approach to Blocking H.I.V. Raises Hopes for an AIDS Vaccine – Donald G. McNeil Jr., New York Times, February 18, 2015

    Molecule Shows Ability to Block AIDS Virus – Betsy McKay, Wall Street Journal, Feb. 18, 2015

    Update 3/6/15: A webcast of Michael Farzan's presentation at CROI 2015 is now available. In the talk Farzan reveals that, since the publication of the Nature paper, the eCD4-Ig recipients have been challenged with two higher doses of SHIV-AD8 and remain uninfected.

    Nature (2015) doi:10.1038/nature14264

    Received 29 June 2013 Accepted 27 January 2015 Published online 18 February 2015

    AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges 

    Matthew R. Gardner,  Lisa M. Kattenhorn,  Hema R. Kondur,  Markus von Schaewen,  Tatyana Dorfman,  Jessica J. Chiang,  Kevin G. Haworth,  Julie M. Decker,  Michael D. Alpert,  Charles C. Bailey,  Ernest S. Neale,  Christoph H. Fellinger,  Vinita R. Joshi,  Sebastian P. Fuchs,  Jose M. Martinez-Navio,  Brian D. Quinlan,  Annie Y. Yao,  Hugo Mouquet,  Jason Gorman,  Baoshan Zhang,  Pascal Poignard,  Michel C. Nussenzweig,  Dennis R. Burton,  Peter D. Kwong,  Michael Piatak    Jeffrey D. Lifson,  Guangping Gao,  Ronald C. Desrosiers,  David T. Evans,  Beatrice H. Hahn,  Alexander Ploss,  Paula M. Cannon,  Michael S. Seaman  & Michael Farzan

    Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs)1, 2. However, even the best bNAbs neutralize 10–50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC80) > 5 μg ml−1), suggesting that high concentrations of these antibodies would be necessary to achieve general protection3, 4, 5, 6. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC50) < 0.05 μg ml−1). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17–77 μg ml−1 of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

    Nature (2015) doi:10.1038/nature14205

    Published online 18 February 2015

    HIV: Tied down by its own receptor

    Nancy L. Haigwood

  • Report of “Aggressive” HIV Variant in Cuba

    On January 28th, a paper reporting evidence of rapid progression associated with a recently identified HIV-1 recombinant circulating in Cuba (CRF19_cpx) was published online by the journal EBioMedicine. CRF stands for circulating recombinant form, and CRF19_cpx represents a combination of HIV-1 subtypes A, D and G. Last week, the authors issued a press release about the study, and since then media coverage has gradually ballooned. Most of the coverage takes its lead from the press release headline: “An aggressive form of HIV uncovered in Cuba.” The immediate problem is that the data are very preliminary, and it is way too soon to declare—as fact—that what has been uncovered is “an aggressive form of HIV.” It may or may not be, additional evidence is needed in order to make that determination. Many media stories also state that HIV CRF19_cpx causes AIDS within three years, which is potentially very misleading because what the study reports is that this rapid pace of progression was observed in a total of nine untreated people who were found to be infected with the strain; this does not prove that all people infected with HIV CRF19_cpx will progress at this rate. There is no evidence to suggest that HIV CRF19_cpx would not respond to treatment. 

    The study was prompted by anecdotal reports from clinicians in Cuba that they are seeing more cases of rapid progression from HIV infection to AIDS. Starting in November 2007, a total of 95 untreated HIV-positive individuals who were “attending the Institute for Tropical Medicine ‘Pedro Kourí’ (IPK) for medical care” were recruited to participate in the study. A total of 52 were defined as rapid progressors based on a diagnosis of AIDS within three years of the estimated date of seroconversion (estimated as the mid-point between last seronegative HIV test and the first HIV-positive test). Criteria for an AIDS diagnosis were a CD4 count <200 cells, CD4 percentage <14% or the presence of an opportunistic infection. For the purposes of the study, rapid progressors were required to have met the CD4 T cell criteria for an AIDS diagnosis and have an opportunistic infection, or met the CD4 T cell criteria for an AIDS diagnosis and had at least two prior CD4 T cell counts <350. Of the remaining 43 participants, 21 had been followed for more than three years after seroconversion and had not progressed to AIDS (categorized as “non-AIDS”) while 22 had received an AIDS diagnosis three years or more after seroconversion (categorized as “chronic-AIDS”; these individuals had been followed since around 2001 and had retrospective data available).

    Of the 52 cases of rapid disease progression, a total of nine were infected with HIV CRF19_cpx. The crux of the study is that this recombinant was not detected in any of the participants in the non-AIDS or chronic-AIDS groups, so it was overrepresented among rapid progressors. Using a Bayesian statistical model, the researchers demonstrate that infection with HIV CRF19_cpx was associated with rapid progression, as well as an increased likelihood of having oral thrush, higher levels of the chemokine RANTES, and greater prevalence of X4 tropism (as inferred from virus genotyping). There was also an association between HIV CRF19_cpx infection and having a higher viral load at diagnosis compared to other subtypes, but this analysis only involved six out of the nine participants. The fitness of HIV CRF19_cpx was estimated to be high, but based only on inference from genetic sequencing and not any assessments of virus replication.

    The study results certainly call for additional investigation of disease progression rates in larger numbers of individuals infected with HIV CRF19_cpx. In the discussion section of the paper, several limitations are noted, including the absence of any information on the genetics of the study participants. The fact that 43 out of the 52 rapid progressors had other strains of HIV is consistent with the literature showing that non-viral factors such as immune responses and immune response genes play a key role in determining progression rates. These factors might have contributed to the outcomes in the individuals infected with HIV CRF19_cpx. One issue the paper does not address explicitly is why so many study participants progressed to AIDS without receiving treatment; in some cases, presumably, HIV infection and AIDS were diagnosed simultaneously, but it seems unlikely that this occurred in all the individuals (update: the senior author Anne-Mieke Vandamme has since explained via email that "all patients were treated according to the prevailing Cuban/WHO treatment guidelines" and the final version of the paper is being edited to include this information). The study offers absolutely no reason to believe that HIV CRF19_cpx wouldn't be suppressed by antiretroviral therapy just as effectively as any other HIV strain. 

    Based on their press release, it appears the researchers wanted to highlight the relevance of their study to HIV prevention, as they begin by noting that acquisition of multiple strains of HIV can lead to the emergence of new recombinants. The release also expounds on a hypothesis offered in the paper that the apparently increased prevalence of X4 tropism in the nine HIV CRF19_cpx-infected participants was related to the high levels of RANTES and played a causative role in their rapid disease progression. The theory is that because RANTES binds to CCR5, the high levels seen in HIV CRF19_cpx infection favored the emergence of X4-tropism. However, although X4 tropism is associated with rapid CD4 T cell decline and disease progression, the cause and effect relationship remains uncertain. There are rare case reports of individuals genetically lacking CCR5 (CCR5Δ32 homozygotes) who have acquired infection with X4-tropic HIV strains, and while precipitous loss of CD4 T cells from the blood has been documented, this has not necessarily equated to the rapid onset of clinical disease. Also, there were concerns that CCR5-blocking drugs such as maraviroc might lead to rapid disease progression by selecting for X4 tropic HIV but this does not appear to have occurred in instances where X4 virus has emerged during treatment. Furthermore, no explanation is offered in the paper as to how HIV CRF19_cpx might cause increased production of the chemokine RANTES by immune cells.

    The bottom line is that the paper raises the possibility that HIV CRF19_cpx is a particularly pathogenic HIV variant, but lacks the evidence necessary to confirm that this is the case.

    EBioMedicine

    doi:10.1016/j.ebiom.2015.01.015

    Available online 28 January 2015

    In Press, Accepted Manuscript

    CRF19_cpx is an evolutionary fit HIV-1 variant strongly associated with rapid progression to AIDS in Cuba

    Vivian Kouria, 1, Ricardo Khourib, c, , 1, Yoan Alemána, Yeissel Abrahantesa, Jurgen Vercauterenb, Andrea-Clemencia Pineda-Peñab, d, Kristof Theysb, Sarah Megensb, Michel Moutschene, Nico Pfeiferf, Johan Van Weyenberghb, Ana B. Péreza, Jorge Péreza, Lissette Péreza, Kristel Van Laethemb, Anne-Mieke Vandammeb, g

    a Virology Department, Institute of Tropical Medicine Pedro Kourí, Autopista Novia del Mediodía Km 6, Marianao 13, Havana City, Cuba

    b KULeuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Clinical and Epidemiological Virology, B-3000 Leuven, Belgium

    c LIMI, Centro de Pesquisa Gonçalo Moniz, FIOCRUZ, Salvador-Bahia, Brasil

    d Clinical and Molecular Infectious Diseases Group, Faculty of Sciences and Mathematics, Universidad del Rosario, Bogotá, Colombia

    e AIDS Reference Center, Centre Hospitalier Universitaire de Liège, Liège, Belgium

    f Department of Computational Biology and Applied Algorithmics, Max Planck Institute for Informatics, Campus E1 4, 66123 Saarbrücken, Germany

    g Centro de Malária e outras Doenças Tropicais and Unidade de Microbiologia, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, Portugal 

    Received 17 October 2014, Revised 22 January 2015, Accepted 26 January 2015, Available online 28 January 2015 

    Abstract

    Background

    Clinicians reported an increasing trend of rapid progression (RP) (AIDS within 3 years of infection) in Cuba.

    Methods

    Recently infected patients were prospectively sampled, 52 RP at AIDS diagnosis (AIDS-RP) and 21 without AIDS in the same time frame (non-AIDS). 22 patients were sampled at AIDS diagnosis (chronic-AIDS) retrospectively assessed as > 3 years infected. Clinical, demographic, virological, epidemiological and immunological data were collected. Pol and env sequences were used for subtyping, transmission cluster analysis, and prediction of resistance, co-receptor use and evolutionary fitness. Host, immunological and viral predictors of RP were explored through data mining.

    Findings

    Subtyping revealed 25 subtype B strains, 6 C, 7 CRF18_cpx, 9 CRF19_cpx, 29 BG-recombinants and other subtypes/URFs. All patients infected with CRF19 belonged to the AIDS-RP group. Data mining identified CRF19, oral candidiasis and RANTES levels as strongest predictors of AIDS-RP. CRF19 was more frequently predicted to use the CXCR4 co-receptor, had higher fitness scores in the protease region, and patients had higher viral load at diagnosis.

    Interpretation

    CRF19 is a recombinant of subtype D (C-part of Gag,PR, RT and nef), subtype A (N-part of Gag, Integrase, Env) and subtype G (Vif, Vpr, Vpu and C-part of Env). Since subtypes D and A have been associated with respectively faster and slower disease progression, our findings might indicate a fit PR driving high viral load, which in combination with co-infections may boost RANTES levels and thus CXCR4 use, potentially explaining the fast progression. We propose that CRF19 is evolutionary very fit and causing rapid progression to AIDS in many newly infected patients in Cuba.

  • Lymphoid Tissue Fibrosis: New Studies and Candidate Therapies

    Research has documented that HIV infection is associated with significantly increased scarring damage to lymphoid tissue, termed fibrosis. Fibrosis can be quantified by measuring the deposition of collagen, and the amount of lymphoid tissue fibrosis in HIV-positive people has been shown to correlate directly with CD4 T cell depletion. A paper published toward the end of last year by Timothy Schacker’s group at the University of Minnesota describes results of an analysis of gut-associated lymphoid tissue (GALT) fibrosis in untreated HIV controllers and HIV-positive people on ART, compared to HIV-negative controls and the one person considered cured of HIV, Timothy Brown. The study was previously presented in part at CROI 2013. 

    On average, the amount of GALT fibrosis was significantly greater in all HIV-positive groups compared to HIV-negative controls. No significant differences were observed between the various groups of HIV-positive participants. Timothy Brown showed levels of fibrosis comparable to the study participants without HIV infection. The results indicate that, in most cases, HIV suppression—whether by ART or in untreated elite controllers—is insufficient to reverse or avert fibrosis. Although only very tentative conclusions can be drawn from a single individual, it appears that curing HIV infection has been more successful in reversing the problem in Timothy Brown. One caveat is that, although the difference is significant on average, there are some individuals in each of the HIV-positive groups in whom levels of collagen deposition in the GALT appear comparable to the HIV-negative control group (shown in figure 1b of the paper). 

    Because lymphoid tissue fibrosis is correlated with CD4 T cell depletion, Schacker’s group has also used the SIV/macaque model to assess whether antifibrotic drugs can improve CD4 T cell reconstitution. The results of these experiments were published in a separate paper last September. Pirfenidone, an antifibrotic drug approved for the treatment of idiopathic pulmonary fibrosis, was initially given prior to SIV challenge and then continued for 24 weeks in order to evaluate whether an effect could be demonstrated under idealized conditions. Results showed that fibrosis was prevented in treated macaques but not untreated controls, and the effect was independent of ART (which was administered to a subgroup of the pirfenidone recipients). CD4 T cell numbers were also preserved in peripheral blood and there was a 14% increase in lymphoid tissue CD4 T cells over the 24 weeks compared to an 8% decline in the untreated group. 

    In a follow up experiment, pirfenidone was started in combination with ART six weeks after SIV infection and a control group received ART alone. Treatment was again continued for 24 weeks. Lymphoid tissue fibrosis declined significantly in the pirfenidone group but continued to increase in animals receiving only ART. However no significant changes in blood or lymph node CD4 T cell numbers could be documented; the researchers suggest this might have been due to the relatively short duration of follow up.

    Based on these encouraging findings, Schacker’s group has now begun a clinical trial of losartan in HIV-positive people on ART. Losartan is approved for the treatment of hypertension but also has antifibrotic activity. The study will test if losartan can reduce fibrosis and improve immune reconstitution, and also look for any effect on the size of the HIV reservoir. The trial is currently enrolling and potential participants from Minneapolis and Madison are actively being sought. Participation does require some altruistic dedication to scientific research because tissue sampling is an important part of the protocol and biopsies of inguinal lymph node and GALT will be conducted at baseline, 12 months and 30 months after enrollment. Some participants will also receive HPV vaccine immunizations during the study in order to evaluate if antifibrotic therapy can improve immune function. Detailed information about the trial is available in the clinicaltrials.gov listing. 

    J Infect Dis. (2014) doi: 10.1093/infdis/jiu586

    First published online: October 24, 2014 

    Lymphoid fibrosis occurs in long-term nonprogressors and persists with antiretroviral therapy, but may be reversible with curative interventions

    Sanchez JL, Hunt PW, Reilly CS, Hatano H, Beilman GJ, Khoruts A, Jasurda JS, Somsouk M, Thorkelson A, Russ S, Anderson J, Deeks SG, Schacker TW.

    Abstract 

    HIV replication causes lymphoid tissue (LT) fibrosis which causes CD4 depletion. It is unknown if people who spontaneously control HIV replication have LT fibrosis. We measured LT fibrosis and CD4 cells in 25 HIV controllers, 10 non-controllers, 45 on therapy, and 10 HIV negative individuals. Controllers had significant LT fibrosis and CD4 depletion, similar to non-controllers, but the Berlin Patient (cured of HIV) had near normal LT. Thus, LT fibrosis occurs in all HIV-infected subjects and current therapy does not reverse it. Reversal of fibrosis during a curative intervention suggests that ongoing low-level virus production may maintain LT fibrosis. 

    J Infect Dis. (2014) doi: 10.1093/infdis/jiu519

    First published online: September 22, 2014

    Antifibrotic Therapy in Simian Immunodeficiency Virus Infection Preserves CD4+ T-Cell Populations and Improves Immune Reconstitution With Antiretroviral Therapy.

    Estes JD, Reilly C, Trubey CM, Fletcher CV, Cory TJ, Piatak M Jr, Russ S, Anderson J, Reimann TG, Star R, Smith A, Tracy RP, Berglund A, Schmidt T, Coalter V, Chertova E, Smedley J, Haase AT, Lifson JD, Schacker TW.

    Abstract

    Even with prolonged antiretroviral therapy (ART), many human immunodeficiency virus-infected individuals have <500 CD4+ T cells/µL, and CD4+ T cells in lymphoid tissues remain severely depleted, due in part to fibrosis of the paracortical T-cell zone (TZ) that impairs homeostatic mechanisms required for T-cell survival. We therefore used antifibrotic therapy in simian immunodeficiency virus-infected rhesus macaques to determine whether decreased TZ fibrosis would improve reconstitution of peripheral and lymphoid CD4+ T cells. Treatment with the antifibrotic drug pirfenidone preserved TZ architecture and was associated with significantly larger populations of CD4+ T cells in peripheral blood and lymphoid tissues. Combining pirfenidone with an ART regimen was associated with greater preservation of CD4+ T cells than ART alone and was also associated with higher pirfenidone concentrations. These data support a potential role for antifibrotic drug treatment as adjunctive therapy with ART to improve immune reconstitution.

  • Latency-Reversing and Immunomodulatory Activities of HDAC Inhibitors

    Results from the second clinical trial to test the HDAC inhibitor vorinostat as a latency-reversing agent, first presented at CROI 2013, have now been published in the open access journal PLoS Pathogens. The study design differed from the first trial, involving a longer 14-day dosing period. The results are broadly consistent, with a significant increase in cell-associated HIV RNA documented, but no change in multiple measures of the HIV reservoir or HIV RNA as assessed by the standard viral load test. Side effects experienced by participants were grade 1 or 2 (mild or moderate in severity) with lethargy, diarrhea and thrombocytopenia being most common; all resolved after the drug was stopped. The researchers noted long-term changes in expression of multiple cellular genes and caution that this could present a safety concern, stating: “the prolonged changes in host gene expression require careful long term follow up.”

    The paper adds to the evidence that HDAC inhibitors can activate at least some latent HIV, but do not shrink the HIV reservoir in most cases, suggesting additional interventions are needed to kill latently infected cells that are induced to produce virus. In terms of the latency-reversing potency of HDAC inhibitors, the results presented to date are consistent with in vitro studies indicating a hierarchy of vorinostat > panobinostat > romidepsin, with the latter mediating the greatest effect based on the detection of HIV RNA by standard viral load testing (these data were presented at the AIDS 2014 conference by Dr. Ole Schmeltz Søgaard and the presentation is available on YouTube). The safety concerns raised by the persistent alterations in host gene expression will require ongoing assessment in all the clinical trials that are being conducted.   

    HDAC inhibitors also have effects on the immune system, and this aspect is evaluated in study published recently in Blood. The research did not involve HIV-positive people but rather individuals undergoing hematopoietic cell transplantation who were receiving vorinostat to prevent graft-versus-host disease. The authors found increases in the numbers of immunosuppressive regulatory CD4 T cells (Tregs) and greater suppression by these cells on a per-cell basis, along with reduced inflammatory responses by peripheral blood mononuclear cells.

    Similar anti-inflammatory effects were reported in an analysis of HIV-positive participants in a clinical trial of panobinostat that was presented at AIDS 2014; the presentation was covered by Jules Levin of NATAP and the abstract and slides are posted to the NATAP website. There are reasons to believe that the anti-inflammatory activity of HDAC inhibitors could be of some benefit in HIV, but Tregs have also been linked to lower HIV-specific immunity, which could potentially be a negative (particularly if HIV-specific immunity needs to be boosted to eliminate latently infected cells). There is already some evidence that HDAC inhibitors can suppress the killing of HIV-infected cells by virus-specific CD8 T cells in laboratory assays (this paper was published in PLoS Pathogens in August).

    Complicating matters further, at the recent Strategies for an HIV Cure conference Jonathan Karn gave a presentation suggesting HDAC inhibitors may enhance natural killer (NK) cell responses by causing downregulation of MHC class I on target cells and upregulating NK cell receptors, possibly consistent with some preliminary evidence presented at the same conference indicating NK cell responses were associated with declines in HIV DNA levels in a few participants in the panobinostat trial.

    Additional research will be needed to investigate how the various possible consequences of HDAC inhibition balance out in HIV-positive people, and to assess the extent to which these effects persist after dosing is stopped.

    PLoS Pathog. 2014 Nov 13;10(10):e1004473. doi: 10.1371/journal.ppat.1004473. eCollection 2014.

    Activation of HIV Transcription with Short-Course Vorinostat in HIV-Infected Patients on Suppressive Antiretroviral Therapy.

    Elliott JH, Wightman F, Solomon A, Ghneim K, Ahlers J, Cameron MJ, Smith MZ, Spelman T, McMahon J, Velayudham P, Brown G, Roney J, Watson J, Prince MH, Hoy JF, Chomont N, Fromentin R, Procopio FA, Zeidan J, Palmer S, Odevall L, Johnstone RW, Martin BP, Sinclair E, Deeks SG, Hazuda DJ, Cameron PU, Sékaly RP, Lewin SR.

    Abstract

    Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells.

    Blood DOI: http://dx.doi.org/10.1182/blood-2014-10-605238

    Histone deacetylase inhibition regulates inflammation and enhances Tregs after allogeneic hematopoietic cell transplantation in humans

    Sung Won Choi, Erin Gatza, Guoqing Hou, Yaping Sun, Joel Whitfield, Yeohan Song, Katherine Oravecz-Wilson, Isao Tawara, Charles A. Dinarello, and Pavan Reddy

    Key Points

    • HDAC inhibition reduced pro-inflammatory cytokines and increased regulatory T cell number and function after allo-HCT.
    • HDAC inhibition enhanced STAT-3 acetylation and induced IDO after allo-HCT.

    Abstract

    We examined immunological responses in patients receiving histone deacetylase (HDAC) inhibition (vorinostat) for graft-versus-host disease (GVHD) prophylaxis following allogeneic hematopoietic cell transplant (allo-HCT). Vorinostat treatment increased histone acetylation in peripheral blood mononuclear cells (PBMC) from treated patients, confirming target HDAC inhibition. HDAC inhibition reduced pro-inflammatory cytokine levels in plasma and from PBMC, decreased ex vivo responses of PBMC to pro-inflammatory TLR-4 stimuli, but did not alter the number or response of conventional T cells (Tconv) to non-specific stimuli. However, the numbers of regulatory T cells (Tregs) were increased, which revealed greater demethylation of the Foxp3 T regulatory-specific demethylation region. Vorinostat-treated patients showed increased expression of CD45RA and CD31 on Tregs, and these Tregs demonstrated greater suppression on a per-cell basis. Consistent with preclinical findings, HDAC inhibition also increased STAT-3 acetylation and induced indoleamine-2,3-dioxygenase (IDO). Our data demonstrate that HDAC inhibition reduces inflammatory responses of PBMC but enhances Tregs after allo-HCT.

  • Is HIV Weakening Over Time?

    A little over nine years ago I wrote a blog post with this same title, about a widely publicized paper claiming that HIV had become less virulent. Although it's grim to be in the position of pouring cold water on optimistic-sounding scenarios, that paper was based on measuring HIV’s ability to replicate using a laboratory test, and other published data raised questions as to whether the test could actually predict differences in disease progression rates. Today, it’s déjà vu all over again because there has been an explosion of very similar media stories positing that HIV is evolving into a “milder form." And once again, the study prompting the coverage relies primarily on laboratory measurements of HIV replication capacity, despite the fact that a prior publication—by several of the same authors—reports that results from this test do not predict the rate of CD4 T cell decline over time.

    The new study, by Rebecca Payne and colleagues from the laboratory of Philip Goulder, was published by PNAS yesterday. Helpfully, the full text has been made freely available. Two populations of HIV-positive women are compared, from Gaborone, Botswana and Durban, South Africa. The researchers present evidence that in Botswana, where HIV has been circulating for a longer period, there has been greater virus adaptation to immune responses, leading to more escape mutations and a lower replication capacity. HIV replication capacity is compared using samples from 63 study participants in Gaborone and 16 in Durban, with these participants being closely matched for CD4 T cell counts (important because, as the paper reports, HIV replication capacity in a given individual increases as CD4 T cell counts decline). The average result of the replication capacity test was 0.72 in Gaborone and 0.81 in Durban, indicating that the virus in the former population is slightly less fit. The authors create a mathematical model based on these findings suggesting that both immune responses and the increasingly widespread use of antiretroviral therapy may be contributing to a decline in HIV virulence.

    The idea that HIV replication capacity can be linked to disease progression rate derives from a documented correlation between the results of the test and both viral load and CD4 T cell count. However, these correlations are based on measurements taken at single timepoints; in other words, cross-sectional analyses. The question of whether measurement of HIV replication capacity can predict subsequent disease progression rate was addressed in a prior study, which evaluated whether there was a correlation with the rate of CD4 T cell decline over time. No such correlation was found, nor even a hint of one. The discussion section of the Journal of Virology paper containing this result states:

    “Therefore, although there may be a benefit to decreased replication capacity (as supported by cross-sectional correlations with viral loads and CD4 counts), the data do not support an enduring benefit or a lasting significant impact of Gag-protease replication capacity on the rate of disease progression, at least once the chronic infection stage has been reached…the long-term clinical impact of immune-driven fitness costs requires further investigation, given the evidence for compensation and the observation that replication capacity does not correlate with the subsequent rate of CD4 decline in chronic infection.” 

    It is challenging to try and reconcile this prior result with the claim that the difference in replication capacity found in the new PNAS study would equate to an additional 2.5 years in the average time it takes to progress from HIV infection to AIDS (an estimate offered by Phillip Goulder in the BBC’s coverage). Before concluding that the virulence of HIV is declining, it would be prudent to wait to see if additional studies are able to correlate the apparent differences in HIV replication capacity with differences in CD4 T cell counts and health outcomes over time. 

    An additional wrinkle is that there are other studies arguing that HIV virulence is increasing over time; a meta-analysis with this finding was published in 2012 and a new analysis presented at CROI 2014 (and just published in the December issue of The Lancet HIV) reached the same conclusion. 

    There might be explanations for these very disparate results, but—pending additional evidence—it seems reasonable to maintain a healthy skepticism about all of them.  

    PNAS Published online before print December 1, 2014, doi: 10.1073/pnas.1413339111 

    Impact of HLA-driven HIV adaptation on virulence in populations of high HIV seroprevalence

    Rebecca Payne, Maximilian Muenchhoff, Jaclyn Mann, Hannah E. Roberts, Philippa Matthews, Emily Adland, Allison Hempenstall, Kuan-Hsiang Huang, Mark Brockman, Zabrina Brumme, Marc Sinclair, Toshiyuki Miura, John Frater, Myron Essex, Roger Shapiro, Bruce D. Walker, Thumbi Ndung’u, Angela R. McLean, Jonathan M. Carlson, and Philip J. R. Goulder

    Abstract

    It is widely believed that epidemics in new hosts diminish in virulence over time, with natural selection favoring pathogens that cause minimal disease. However, a tradeoff frequently exists between high virulence shortening host survival on the one hand but allowing faster transmission on the other. This is the case in HIV infection, where high viral loads increase transmission risk per coital act but reduce host longevity. We here investigate the impact on HIV virulence of HIV adaptation to HLA molecules that protect against disease progression, such as HLA-B*57 and HLA-B*58:01. We analyzed cohorts in Botswana and South Africa, two countries severely affected by the HIV epidemic. In Botswana, where the epidemic started earlier and adult seroprevalence has been higher, HIV adaptation to HLA including HLA-B*57/58:01 is greater compared with South Africa (P = 7 × 10−82), the protective effect of HLA-B*57/58:01 is absent (P = 0.0002), and population viral replicative capacity is lower (P = 0.03). These data suggest that viral evolution is occurring relatively rapidly, and that adaptation of HIV to the most protective HLA alleles may contribute to a lowering of viral replication capacity at the population level, and a consequent reduction in HIV virulence over time. The potential role in this process played by increasing antiretroviral therapy (ART) access is also explored. Models developed here suggest distinct benefits of ART, in addition to reducing HIV disease and transmission, in driving declines in HIV virulence over the course of the epidemic, thereby accelerating the effects of HLA-mediated viral adaptation.

  • TAG HIV Cure Research Media Monitor

    In recent months, Treatment Action Group has created a web page resource that will attempt to provide accurate information on HIV cure research news that makes a splash in the mainstream media, particularly those stories that get mangled in the translation or are possibly based on dubious scientific claims.

    So far the Cure Research Media Monitor page has covered four items:

  • Update on Early HIV Treatment in Infants: IMPAACT Trial Launched, Additional Case Reports Published

    On November 3rd, the National Institute of Allergy and Infectious Diseases (NIAID) and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) announced the launch of the IMPAACT P1115 trial, which will investigate very early initiation of HIV treatment in newborns. Much of the impetus for the trial came from the Mississippi baby case, and although that has turned out to involve an extended, 27-month remission from HIV replication rather than a cure, there remains a rationale for evaluating the feasibility, benefits and risks of early treatment in perinatal HIV infection.

    The news of the viral load rebound in the Mississippi case, which emerged this past summer, has led to some alterations to the P1115 protocol, including ongoing reviews of whether an interruption of antiretroviral therapy is justified when participants reach two years of age. Detailed information on the trial design is provided in an online Q&A document issued by NIAID and NICHD.

    Clustered around the time of this news release were several other publications related to pediatric HIV cure research. In a brief but informative viewpoint piece in the Journal of the International AIDS Society Jintanat Ananworanich and Merlin Robb review the Mississippi case and two other reports of early-treated infants in whom HIV viral load rebounded rapidly after treatment interruption. One occurred in Canada and was described in a prior blog post; the researcher Jason Brophy also presented details at the International AIDS Conference in Melbourne in July (a webcast of Brophy’s talk is available on the conference website). The second was from Milan, Italy and reported in a letter to The Lancet in October.

    Ananworanich and Robb outline some of the differences between these three cases that might underlie why an extensive period of remission occurred in one but not the others; these include higher baseline viral load (Milan) and evidence of low-level detectable HIV RNA after initial suppression (Canada). A third report from Ireland that was published recently is not mentioned in the review; this infant was treated from birth and had no detectable HIV-specific antibody responses or HIV DNA or RNA at age four, when treatment was interrupted. Viral load rebounded within seven days and climbed to over 100,000 copies before ART was successfully reinitiated. The authors of the case report note that, unlike in the Mississippi baby, the initial ART regimen was not fully suppressive (possibly due to an interaction between nevirapine and another needed medication) leading to an early viral load increase to over 3,000 copies while on treatment prior to an effective combination being instituted. 

    In discussing the outcome in this child, the researchers state: “This case unequivocally demonstrates that children on suppressive antiretroviral treatment from very early infancy can achieve a state of HIV persistence during which routine testing with standard HIV RNA and DNA assays can yield negative results. These may be falsely interpreted as absence of persistent replication-competent reservoirs. The prompt rebound in viremia emphasizes that the limitations in these markers to determine whether treatment should be stopped to assess viral remission. Cases such as this highlight the urgent need for more data and further research in this area.” Hopefully IMPAACT P1115 can help contribute to filling this knowledge gap.

    Also in the realm of pediatric HIV cure research, last month in JAMA Pediatrics Deborah Persaud and colleagues published results from a study that was presented at CROI earlier this year (a webcast of the presentation is online). In a cross-sectional analysis, markers of HIV persistence were compared in three groups of perinatally infected adolescents that had achieved viral load suppression prior to one year of age, between one and five years, or after five years. Levels of both replication-competent virus and proviral DNA were significantly lower in the first group, and earlier control of viral load was also associated with an increased likelihood of indeterminate or negative HIV antibody test results (proportions were 86%, 19%, and 3%, respectively). A significant correlation was documented between low proviral DNA and seronegative or indeterminate HIV antibody tests. Although not considered cured, adolescents with low or undetectable HIV reservoirs may be candidates for clinical trials of interventions that aim to clear remaining latently infected cells. 

    Journal of the International AIDS Society 2014, 17:19859, Published 20 November 2014

    Viewpoint

    The transient HIV remission in the Mississippi baby: why is this good news?

    Jintanat Ananworanich and Merlin L Robb 

    Lancet. 2014 Oct 4;384(9950):1320. doi: 10.1016/S0140-6736(14)61405-7.

    No cure of HIV infection in a child despite early treatment and apparent viral clearance.

    Giacomet V, Trabattoni D, Zanchetta N, Biasin M, Gismondo M, Clerici M, Zuccotti G. 

    Pediatr Infect Dis J. 2014 Sep 23. [Epub ahead of print]

    Rapid Viral Rebound after 4 Years of Suppressive Therapy in a Seronegative HIV-1 Infected Infant Treated from Birth.

    Butler KM, Gavin P, Coughlan S, Rochford A, Donagh SM, Cunningham O, Poulsom H, Watters S, Klein N.

    Abstract

    Attention has focused on the possibility of cure for HIV infected infants if treated promptly following delivery. The 'Mississippi baby', who had very prolonged remission following ARV discontinuation, may represent a unique situation. We report an infant treated from birth, who seroreverted, remained virologically suppressed, and had undetectable HIV-1 RNA and DNA at four years of age, yet experienced virologic rebound within days of discontinuation of antiretroviral therapy.

    JAMA Pediatr. Published online October 06, 2014. doi:10.1001/jamapediatrics.2014.1560

    Influence of Age at Virologic Control on Peripheral Blood Human Immunodeficiency Virus Reservoir Size and Serostatus in Perinatally Infected Adolescents

    Deborah Persaud, MD; Kunjal Patel, DSc, MPH; Brad Karalius, MPH; Kaitlin Rainwater-Lovett, PhD, MPH; Carrie Ziemniak, MS; Angela Ellis, BS; Ya Hui Chen, BA; Douglas Richman, MD; George K. Siberry, MD, MPH; Russell B. Van Dyke, MD; Sandra Burchett, MD; George R. Seage III, DSc, MPH; Katherine Luzuriaga, MD; for the Pediatric HIV/AIDS Cohort Study

    Importance: Combination antiretroviral therapy initiated within several weeks of human immunodeficiency virus (HIV) infection in adults limits proviral reservoirs that preclude HIV cure. Biomarkers of restricted proviral reservoirs may aid in the monitoring of HIV remission or cure.

    Objectives: To quantify peripheral blood proviral reservoir size in perinatally HIV-infected (PHIV+) adolescents and to identify correlates of limited proviral reservoirs.

    Design, Setting, and Participants: A cross-sectional study including 144 PHIV+ youths (median age, 14.3 years) enrolled in the United States–based Pediatric HIV/AIDS Cohort Study and receiving durable (median duration, 10.2 years) combination antiretroviral therapy, stratified by age at virologic control.

    Main Outcomes and Measures: The primary end point was peripheral blood mononuclear cell (PBMC) proviral load after virologic control at different ages. Correlations between proviral load and markers of active HIV production (ie, HIV-specific antibodies, 2–long terminal repeat circles) and markers of immune activation and inflammation were also assessed.

    Results: Proviral reservoir size was markedly reduced in the PHIV+ youth who achieved virologic control before 1 year of age (4.2 [interquartile range, 2.6-8.6] copies per 1 million PBMCs) compared with those who achieved virologic control at 1 to 5 years of age (19.4 [interquartile range, 5.5-99.8] copies per 1 million PBMCs) or after 5 years of age (70.7 [interquartile range, 23.2-209.4] copies per 1 million PBMCs; P < .001). A proviral burden of less than 10 copies per 1 million PBMCs in PHIV+ youth was measured in 11 (79%), 20 (40%), and 13 (18%) participants with virologic control before 1 year, at 1 to 5 years, and after 5 years of age, respectively (P < .001). Lower proviral load was associated with undetectable 2–long terminal repeat circles (P < .001) and HIV-negative or indeterminate serostatus (P < .001) but not with concentrations of soluble immune activation markers CD14 and CD163. 

    Conclusions and Relevance: Early effective combination antiretroviral therapy with prolonged virologic suppression after perinatal HIV infection leads to negligible peripheral blood proviral reservoirs in adolescence and is associated with negative or indeterminate HIV serostatus. These findings highlight the long-term effect of early effective control of HIV replication on biomarkers of HIV persistence in perinatal infection and the utility of HIV serostatus as a biomarker for small proviral reservoir size, although not necessarily for cure.

  • Three Papers on Macrophages and HIV Infection

    The question of whether HIV infection of macrophages plays an important role in pathogenesis remains controversial and unresolved. For cure researchers, the related question of whether macrophages contribute to viral persistence during antiretroviral therapy is crucial, but also unanswered. Three recent papers describe results from studies designed to shed light on this subject.

    In the journal Immunity, Jason Brenchley’s research group presents results from experiments in the SIV/macaque model using viruses that do and do not express the Vpx protein, which has previously been reported to be important for infection of myeloid cells (including macrophages). To their surprise, the researchers found that Vpx did not significantly influence the extent to which SIV was detectable in myeloid cells. Mucosal tissues showed little evidence of myeloid cell infection despite depletion of CD4 T cell targets, but in 40% of animals SIV DNA was detectable in myeloid cells in the mesenteric lymph nodes and spleen. However, further analyses revealed the presence of T cell receptor DNA in addition to the SIV DNA, suggesting that these cells were macrophages that had phagocytosed SIV-infected CD4 T cells. The researchers state: “we believe our data clearly suggest that viral RNA and DNA within myeloid cells can be attributed to clearance of virally infected cells and immune complexes and not bone fide SIV infection of myeloid cells in vivo.”

    An open access paper in Cell Host & Microbe offers a possible twist to this tale, reporting that phagocytosis of HIV-infected CD4 T cells by macrophages can lead to them becoming productively infected. The findings are derived from in vitro laboratory experiments. The data indicate that macrophages selectively target HIV-infected CD4 T cells for phagocytosis, although the researchers have yet to identify the exact mechanism for this selectivity. The online version of the paper is accompanied by three quicktime videos of macrophages phagocytosing multiple HIV-infected CD4 T cells (click the video tab under the Images/Data menu).

    Lastly, in PLoS Pathogens Luca Micci and colleagues show that artificial depletion of CD4 T cells in SIV-macaques (using a CD4-targeting monoclonal antibody) produces evidence of extensive infection of macrophages, as well as microglial cells in the brain. In lymphoid tissues of non-depleted animals, T cells made up more than 80% of the cells containing SIV RNA, whereas in the setting of CD4 T cell depletion more than 80% of the cells containing SIV RNA displayed macrophage markers. The macrophages showed evidence of high levels of activation and were shorter-lived than was expected based on previous studies. Since CD4 T cells were not completely depleted in the animals, it seems possible that phagocytosis of SIV-infected CD4 T cells might have contributed to the presence of SIV RNA in macrophages in this study, but the researchers do not specifically assess the possibility. 

    Unfortunately it appears that the publication of these papers within a short time period precluded the authors from being able to comment on each other’s work, which would have been helpful in understanding how the data fit together. Because it involves in vivo results from macaques without any manipulations, the Immunity paper may arguably represent the most reliable gauge of the contribution of macrophages in SIV infection. The results imply that previously reported studies documenting the presence of HIV DNA in macrophages are explained, at least in part, by phagocytosis of infected CD4 T cells. However, the study published in Cell Host & Microbe suggests that additional work might be needed to fully distinguish between macrophages containing viral DNA solely due to phagocytosis and those that are also productively infected.

    None of the papers offer a definitive answer to the question of whether infected macrophages make up part of the long-lived HIV reservoir that persists despite antiretroviral therapy. Brenchley and colleagues argue that, based on their findings, therapeutic interventions aiming to target latent HIV should continue to focus on CD4 T cells.

    Immunity. 2014 Sep 18;41(3):493-502. doi: 10.1016/j.immuni.2014.08.014.

    Tissue Myeloid Cells in SIV-Infected Primates Acquire Viral DNA through Phagocytosis of Infected T Cells.

    Calantone N, Wu F, Klase Z, Deleage C, Perkins M, Matsuda K, Thompson EA, Ortiz AM, Vinton CL, Ourmanov I, Loré K, Douek DC, Estes JD, Hirsch VM, Brenchley JM.

    Abstract

    The viral accessory protein Vpx, expressed by certain simian and human immunodeficiency viruses (SIVs and HIVs), is thought to improve viral infectivity of myeloid cells. We infected 35 Asian macaques and African green monkeys with viruses that do or do not express Vpx and examined viral targeting of cells in vivo. While lack of Vpx expression affected viral dynamics in vivo, with decreased viral loads and infection of CD4(+) T cells, Vpx expression had no detectable effect on infectivity of myeloid cells. Moreover, viral DNA was observed only within myeloid cells in tissues not massively depleted of CD4(+) T cells. Myeloid cells containing viral DNA also showed evidence of T cell phagocytosis in vivo, suggesting that their viral DNA may be attributed to phagocytosis of SIV-infected T cells. These data suggest that myeloid cells are not a major source of SIV in vivo, irrespective of Vpx expression.

    PLoS Pathog. 2014 Oct 30;10(10):e1004467. doi: 10.1371/journal.ppat.1004467. eCollection 2014.

    CD4 Depletion in SIV-Infected Macaques Results in Macrophage and Microglia Infection with Rapid Turnover of Infected Cells. (open access)

    Micci L, Alvarez X, Iriele RI, Ortiz AM, Ryan ES, McGary CS, Deleage C, McAtee BB, He T, Apetrei C, Easley K, Pahwa S, Collman RG, Derdeyn CA, Davenport MP, Estes JD, Silvestri G, Lackner AA, Paiardini M.

    Abstract

    In rhesus macaques (RMs), experimental depletion of CD4+ T-cells prior to SIV infection results in higher viremia and emergence of CD4-independent SIV-envelopes. In this study we used the rhesus recombinant anti-CD4 antibody CD4R1 to deplete RM CD4+ T-cells prior to SIVmac251 infection and investigate the sources of the increased viral burden and the lifespan of productively infected cells. CD4-depleted animals showed (i) set-point viral load two-logs higher than controls; (ii) macrophages constituting 80% of all SIV vRNA+ cells in lymph node and mucosal tissues; (iii) substantial expansion of pro-inflammatory monocytes; (iv) aberrant activation and infection of microglial cells; and (v) lifespan of productively infected cells significantly longer in comparison to controls, but markedly shorter than previously estimated for macrophages. The net effect of CD4+ T-cell depletion is an inability to control SIV replication and a shift in the tropism of infected cells to macrophages, microglia, and, potentially, other CD4-low cells which all appear to have a shortened in vivo lifespan. We believe these findings have important implications for HIV eradication studies.

    Cell Host & Microbe, Published Online: November 20, 2014

    Macrophage Infection via Selective Capture of HIV-1-Infected CD4+ T Cells (open access)

    Amy E. Baxter, Rebecca A. Russell, Christopher J.A. Duncan, Michael D. Moore, Christian B. Willberg, Jose L. Pablos, Andrés Finzi, Daniel E. Kaufmann, Christina Ochsenbauer, John C. Kappes, Fedde Groot, Quentin J. Sattentau

    DOI: http://dx.doi.org/10.1016/j.chom.2014.10.010 

    Summary

    Macrophages contribute to HIV-1 pathogenesis by forming a viral reservoir and mediating neurological disorders. Cell-free HIV-1 infection of macrophages is inefficient, in part due to low plasma membrane expression of viral entry receptors. We find that macrophages selectively capture and engulf HIV-1-infected CD4+ T cells leading to efficient macrophage infection. Infected T cells, both healthy and dead or dying, were taken up through viral envelope glycoprotein-receptor-independent interactions, implying a mechanism distinct from conventional virological synapse formation. Macrophages infected by this cell-to-cell route were highly permissive for both CCR5-using macrophage-tropic and otherwise weakly macrophage-tropic transmitted/founder viruses but restrictive for nonmacrophage-tropic CXCR4-using virus. These results have implications for establishment of the macrophage reservoir and HIV-1 dissemination in vivo.

  • HIV Reservoir Measurements and Viral Load Rebound after ART Interruption

    To assist in the search for therapies that might contribute to curing HIV infection, scientists are seeking to identify biomarkers that are associated with control of viral load after an antiretroviral therapy (ART) interruption. If reliable predictors of viral load control could be identified, the effect of candidate therapies on these biomarkers could be evaluated without necessarily having to ask clinical trial participants to undergo ART interruptions (which can pose risks to health). Two recent papers report that certain measurements of the HIV reservoir show statistically significant associations with viral load rebound, although the results are not entirely consistent.

    The SPARTAC trial studied the effects of a 48-week course of ART in people with acute HIV infection. The primary results were reported in the New England Journal of Medicine last year. In a paper in the journal Elife, the trial investigators describe assessments of whether the amount of HIV DNA in blood samples at ART interruption was linked to the time it took for HIV viral load to rebound to detectable levels, or the trial’s primary endpoints (CD4 T cell count less than 350 or restart of ART). The level of total HIV DNA was significantly correlated with time to a viral load above 400 copies; the higher the HIV DNA level, the quicker the time to rebound. Significant associations were also observed between HIV DNA levels and time to reach a CD4 T cell count of less than 350 or ART restart. However, when an assay with a lower viral load cut-off of 50 copies was used, HIV DNA levels were no longer significantly associated with time to rebound.

    The second paper, by Jonathan Li and colleagues, is based on analyses of participants in a randomized, placebo-controlled therapeutic HIV vaccine trial involving Merck’s Ad5-based vaccine candidate (since discontinued). The study design involved a 16-week analytical treatment interruption (ATI). The researchers were able to document statistically significant correlations between pre-ATI HIV RNA and DNA measurements and the viral load set point during the ATI. However, neither measure was significantly associated with time to viral load rebound above 200 copies.

    Taken together, the papers indicate that HIV reservoir measurements have some promise for predicting viral load rebound after ART interruption, but uncertainties remain. In both studies HIV DNA assays did not show significant correlations with time to rebound when a lower viral load cut off was used (<50 or <200 compared to <400).  The explanation for this finding is unclear, but could potentially relate to greater viral load test result variability at lower copy numbers. The AIDS Clinical Trials Group (ACTG) is now developing a research protocol with the specific aim of identifying biomarkers of viral load control, designated the PR 691 Monitored Antiretroviral Pause [MAP] Biomarker study.

    As part of the preparation for this protocol, Jonathan Li has performed an analysis of six prior ACTG trials involving ATIs to gain an understanding of what proportion of individuals are able to maintain undetectable viral loads for an extended period. The results were presented at the IDSA conference last month, showing that overall 6% of 263 participants had levels below 200 copies at week 12 of an ATI. The phenomenon was more common among those treated during acute or recent HIV infection compared to chronically infected individuals. Galit Alter has collaborated with Li to look at possible correlates of control in these study participants, identifying the presence of specific antibody subclasses that are associated with enhanced natural killer cell activity (this preliminary work was presented at the NIAID Strategies for an HIV Cure meeting the week following the IDSA conference). The forthcoming ACTG MAP biomarker study should hopefully be able to build on these findings. 

    Elife. 2014 Sep 12:e03821. doi: 10.7554/eLife.03821. [Epub ahead of print]

    HIV-1 DNA predicts disease progression and post-treatment virological control. (open access)

    Williams JP, Hurst J, Stöhr W, Robinson N, Brown H, Fisher M, Kinloch S, Cooper D, Schechter M, Tambussi G, Fidler S, Carrington M, Babiker A, Weber J, Koelsch KK, Kelleher AD, Phillips RE, Frater J; on behalf of the SPARTAC Trial Investigators.

    Abstract

    In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials.

    Elife. 2014 Oct 16;3:e04742. doi: 10.7554/eLife.04742.

    Biomarker reveals HIV's hidden reservoir. (open access)

    Cockerham LR, Deeks SG.

    Abstract

    Determining the total amount of HIV DNA in people undergoing antiretroviral therapy could accelerate the development of novel therapies and potential cures for HIV infection.

    AIDS. 2014 Sep 23. [Epub ahead of print]

    Relationship of HIV reservoir characteristics with immune status and viral rebound kinetics in an HIV therapeutic vaccine study.

    Li JZ, Heisey A, Ahmed H, Wang H, Zheng L, Carrington M, Wrin T, Schooley RT, Lederman MM, Kuritzkes DR; the ACTG A5197 Study Team.

    Abstract

    OBJECTIVES: The objective of this study is to evaluate the impact of therapeutic HIV vaccination on the HIV reservoir and assess the relationship of the viral reservoir with HIV-specific immune status and viral rebound kinetics.

    DESIGN: A retrospective analysis of ACTG A5197, a randomized, placebo-controlled trial of a therapeutic rAd5 HIV-1 gag vaccine.

    METHODS: Participants received vaccine/placebo at weeks 0, 4 and 26 prior to a 16-week analytic treatment interruption (ATI) at week 38. Cell-associated HIV-1 RNA and DNA (CA-RNA and CA-DNA) and HIV-1 residual viremia were quantified at weeks 0, 8 and 38. HIV-specific CD4/CD8 activity was assessed by an intracellular cytokine staining assay.

    RESULTS: At study entry, CA-RNA and CA-DNA levels were correlated inversely with the numbers of HIV-specific CD4 interferon-γ producing cells (CA-RNA: r = -0.23, P = 0.03 and CA-DNA: r = -0.28, P < 0.01, N = 93). Therapeutic HIV vaccination induced HIV-specific CD4 activity, but did not significantly affect levels of CA-RNA or CA-DNA. Vaccine recipients with undetectable residual viremia at week 8 had higher frequencies of HIV-specific CD4 and CD8 interferon-γ producing cells (undetectable versus detectable residual viremia: 277 versus 161 CD4 cells/10 lymphocytes, P = 0.03 and 1326 versus 669 CD8 cells/10 lymphocytes, P = 0.04). Pre-ATI CA-RNA and CA-DNA were associated with post-ATI plasma HIV set point (CA-RNA: r = 0.51, P < 0.01 and CA-DNA: r = 0.47, P < 0.01).

    CONCLUSION: Vaccine-induced T-cell responses were associated with a modest transient effect on residual viremia, but more potent immune responses and/or combination treatment with latency-reversing agents are needed to reduce the HIV reservoir. HIV reservoir measures may act as biomarkers of post-ATI viral rebound kinetics.

  • Panobinostat as a Latency-Reversing Agent: Phase I Trial Results Published

    In the spring of 2013, a wildly inaccurate Daily Telegraph story about HIV cure research claiming “there will be a breakthrough in finding a cure for HIV within months” was amplified around the Internet, understandably receiving a massive amount of attention and interest. To their credit, the researchers whose work prompted the piece published a statement addressing the inaccuracies, and eventually the Telegraph made extensive corrections. Recently, in the debut issue of The Lancet’s new online HIV journal, results from the trial at the center of the story were published – to no fanfare whatsoever.

    The drug being tested was the HDAC inhibitor panobinostat, which has been reported to reverse HIV latency in laboratory experiments. In the 15-person phase I trial, a small but significant increase of approximately 2-3 fold in cell-associated HIV RNA was documented, and plasma viremia was more frequently detected compared to baseline. A transient decrease in the amount of total HIV DNA in the blood was captured at day 14 of the study, but otherwise no measures of the HIV reservoir (including levels of replication-competent virus) were altered by panobinostat. A subset of nine study participants subsequently underwent an analytical treatment interruption, and HIV viral load rebounded to over 1,000 copies in all cases within a median of 17 days (range 14–56). The main side effect of the drug was fatigue, and no adverse events of greater than grade 1 occurred. However, as noted in an accompanying commentary by Stephen Kent and Miles Davenport, HDAC inhibitors have been shown to cause long-term changes in expression of multiple genes, so long-term safety will need to be carefully monitored.

    The results appear consistent with clinical trials of the HDAC inhibitors vorinostat and romidepsin, which also indicated that the drugs could activate at least some latent HIV. The romidepsin trial results were presented by Dr. Ole Søgaard at the International AIDS Society Towards an HIV Cure Symposium in July, and both the powerpoint presentation and an audio file are available online. The lack of a significant reduction in the HIV reservoir supports the proposal that additional interventions are needed to promote clearance of HIV-infected cells after latency is reversed, and Søgaard and colleagues are now exploring this possibility by combining romidepsin with therapeutic vaccination.

    The panobinostat paper does offer a hint of a greater effect of the drug in four participants, but these types of post-hoc (not planned in the original study protocol) subset analyses have to be interpreted with great caution. In these individuals, a decline in total HIV DNA levels of around 67–84% is reported to have persisted, and was correlated with a slightly longer time to viral load rebound during the analytical treatment interruption. At the recent Strategies for an HIV Cure meeting, which took place at the National Institutes of Health in Bethesda in October, Mathias Lichterfeld presented some preliminary data associating this delayed time to viral load rebound with interferon-stimulated gene expression and natural killer cell activity, suggesting a role for innate immunity. Given the small number of participants and the fact that the analyses are all post-hoc, these results can only be considered exploratory and require confirmation.

    Beyond the results obtained with individual candidates, there is a notable larger issue that looms over latency-reversing approaches generally. The experience of the Boston patients has demonstrated that significant HIV reservoir reductions of around 3 logs (1,000-fold) can lead to delayed viral load rebound after ART interruption, but not a cure. This is in line with mathematical modeling by Alison Hill (published recently in PNAS) which estimates that an HIV reservoir reduction more on the order of 5-6 logs (100,000-1 million-fold) would be needed to achieve a long-term cure in most people. In this context, the fact that no latency-reversing strategy has yet had any significant effect on HIV reservoir levels does not seem very encouraging. But, in case that sounds like doom saying, it’s also true that the work is at an early stage, and latency reversal is just one of many strategies being pursued in HIV cure research.

    The Lancet HIV, Volume 1, Issue 1, Pages e13 – e21, October 2014

    doi:10.1016/S2352-3018(14)70014-1

    Published Online: 16 September 2014 

    Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial

    Dr Thomas A Rasmussen MD a , Martin Tolstrup PhD a, Christel R Brinkmann PhD a, Rikke Olesen PhD a, Christian Erikstrup MD b, Ajantha Solomon c, Anni Winckelmann a, Sarah Palmer PhD d, Prof Charles Dinarello MD e, Maria Buzon PhD f g, Mathias Lichterfeld MD f g, Prof Sharon R Lewin PhD c h, Prof Lars Østergaard DMSc a, Ole S Søgaard MD a

    a Department of Infectious Diseases, Aarhus University Hospital, Denmark

    b Department of Clinical Immunology, Aarhus University Hospital, Denmark

    c Department of Infectious Diseases, Alfred Hospital and Monash University, Melbourne, VIC, Australia

    d Westmead Millennium Institute for Medical Research, University of Sydney, Westmead, NSW, Australia

    e Department of Medicine, Division of Infectious Diseases, University of Colorado Denver, Aurora, CO, USA

    f Infectious Disease Division, Massachusetts General Hospital, Boston, MA, USA

    g Ragon Institute of MGH, MIT and Harvard, Boston, MA, USA

    h Centre for Biomedical Research, Burnet Institute, Melbourne, VIC, Australia 

    Summary

    Background: Activating the expression of latent virus is an approach that might form part of an HIV cure. We assessed the ability of the histone deacetylase inhibitor panobinostat to disrupt HIV-1 latency and the safety of this strategy.

    Methods: In this phase 1/2 clinical trial, we included aviraemic adults with HIV treated at Aarhus University Hospital, Denmark. Participants received oral panobinostat (20 mg) three times per week every other week for 8 weeks while maintaining combination antiretroviral therapy. The primary outcome was change from baseline of cell-associated unspliced HIV RNA. Secondary endpoints were safety, plasma HIV RNA, total and integrated HIV DNA, infectious units per million CD4 T cells, and time to viral rebound during an optional analytical treatment interruption of antiretroviral therapy. This trial is registered with ClinicalTrial.gov, number NCT01680094.

    Findings: We enrolled 15 patients. The level of cell-associated unspliced HIV RNA increased significantly at all timepoints when patients were taking panobinostat (p<0·0001). The median maximum increase in cell-associated unspliced HIV RNA during panobinostat treatment was 3·5-fold (range 2·1—14·4). Panobinostat induced plasma viraemia with an odds ratio of 10·5 (95% CI 2·2—50·3; p=0·0002) compared with baseline. We recorded a transient decrease in total HIV DNA, but no cohort-wide reduction in total HIV DNA, integrated HIV DNA, or infectious units per million. Nine patients participated in the analytical treatment interruption, median time to viral rebound was 17 days (range 14—56). Panobinostat was well tolerated. 45 adverse events were reported, but only 16 (all grade 1) were presumed related to panobinostat.

    Interpretation: Panobinostat effectively disrupts HIV latency in vivo and is a promising candidate for future combination clinical trials aimed at HIV eradication. However, panobinostat did not reduce the number of latently infected cells and this approach may need to be combined with others to significantly affect the latent HIV reservoir.

    The Lancet HIV, Volume 1, Issue 1, Pages e2 – e3, October 2014

    doi:10.1016/S2352-3018(14)70015-3

    Published Online: 16 September 2014

    Tentative first steps to eradicate latent HIV

    Stephen J Kent, Miles P Davenport

    Purging latent HIV with reactivating drugs is one of the most promising approaches to curing HIV infection. In The Lancet HIV , Thomas Rasmussen and colleagues report results of a phase 1/2 trial 1 of panobinostat, a histone deacetylase inhibitor, to reactivate latent HIV. They show that the drug was well tolerated in the short term and the treatment increased both the amount of cell-associated HIV RNA and low-level transient HIV viraemia. There was, however, no overall change in measurements of the HIV reservoir after 8 weeks of panobinostat treatment.