REPORT
Notes on HIV pathogenesis, therapy and eradication
Apuntes sobre la patógenesis del VIH, su terapia y eliminación
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Celia Fernández-Ortega, Anna Ramírez-Suárez, Taimí Paneque-Guerrero, Dionne Casillas-Casanova
Centro de Ingeniería Genética y Biotecnología, CIGB. Ave. 31 e/ 158 y 190, Cubanacán, Playa, CP 11 600, La Habana, Cuba.
Abstract
This report summarizes the main topics delivered as lectures at the meeting Frontiers in HIV pathogenesis, Therapy and Eradication, held at the Whistler Conference Centre, in Whistler British Columbia, Canada, in March 26-31. The meeting was organized as part of the Keystone Symposia on Molecular and Cell Biology joined with the conference Cell Biology of Virus Entry. The current status on research for eradicating HIV-1 latent reservoirs and ways to reactivate them for destruction with current or novel antiretroviral therapies were the key topics. Targeted inhibition of virus entry, new classes of inhibitors, animal models for testing antiretroviral therapies and virus cell-to-cell contact-mediated transmission, together with structural analysis of HIV-1 proteins and replication processes, were also presented. The useful discussions at both the oral and poster sessions paved the way for understanding processes essential for future therapeutic attempts to effectively inhibit virus replication and transmission, including its eradication.
Keywords: HIV-1, antiretroviral therapy, viral latency, viral entry, HIV persistence.
Resumen
]]> Este reporte resume los tópicos fundamentales de las conferencias impartidas en el congreso Fronteras en la patogénesis del VIH, su terapia y erradicación, que se celebró en el Centro de Congresos de Whistler, Whistler British Columbia, Canadá, del 26 al 31 de marzo de este año. El congreso se organizó de conjunto con el evento Biología Celular de la Entrada Viral, como parte de los Simposios de Keystone sobre Biología Molecular y Celular. Los tópicos fundamentales abarcaron el estado actual de las investigaciones para erradicar los reservorios latentes del VIH-1, y las estrategias para activarlos mediante terapia antirretroviral convencional o de nueva generación, y de esa forma promover su destrucción. También se discutieron temas relacionados con la entrada viral como blanco terapéutico, nuevas clases de inhibidores, los modelos animales disponibles para evaluar las terapias antirretrovirales y la transmisión del VIH-1 mediante el contacto entre células. Las discusiones tanto en las sesiones orales como en la presentación de carteles favorecieron una mejor comprensión de los procesos esenciales para intentar en un futuro desarrollar terapias no solo para inhibir la replicación y transmisión del VIH-1, sino también para eliminarlo.Palabras clave: VIH-1, terapia antiretroviral, latencia viral, entrada viral, persistencia del VIH.
INTRODUCTION
Up to now, antiretroviral therapy (ART) has been unable to eradicate the human immunodeficiency virus type 1 (HIV-1) infection [1], while providing sustained suppression of plasma viral load in most HIV-infected individuals. This is currently considered by the research community as dependent on persistent viral reservoirs [2, 3]. Therefore, considerable efforts have been made on therapies to reactivate the latent cellular viral reservoirs by using various agents, such as cytokines, histone deacetylase inhibitors (HDAC) and mitogens, assuming that those cells would die of HIV-induced cytopathic effects and antiretroviral drugs would prevent the spread of infection [4].
The best studied reservoir of latent HIV-1 is resting CD4+ T cells [5, 6]. Disrupting latency via the induction HIV-1 expression in these cells is a potential strategy to facilitate the clearance of this reservoir. This has been attained in vitro with HDACs, but still not proven in patients [7, 8] and this is only the first step down an uncertain path towards viable eradication therapies. Regimens, safety and doses required to fully purge persistent viral genomes remain to be established, without exceeding the capability of ART to contain the spread of infection [9, 10]. Nevertheless, agents that disrupt latency may not be sufficient to clear the replication-competent virus, and strategies to enhance the immune response or to induce death in infected cells death need to be developed. Others reservoirs such as the long term, non-replicating myeloid or astroglial cells may require different novel approaches beyond.
On the other hand, assessing and optimizing therapies that activate the latent virus requires animal models. The most advanced models available are the SIV non-human primates and the humanized bone marrow-liver-thymus (BLT) mouse [11]. In this sense, validated animals models that recapitulate key aspects of the human condition are critical to develop and enforce novel approaches for treatment, prevention and an eventual cure for HIV-1 infection [12].
The only clinical strategy implemented to cure the HIV-1 infection derives from the single documented case of HIV-1 cure to date [13]. It comprises subsequent myeloablative chemotherapy, followed by total body irradiation, anti-thymocyte globulin infusion and allogenic hematopoietic stem cell transplantation (HSCT) from a CCR5∆32 homozygous donor. It is unclear which components of the many and complex therapies that patient went through will be required to eradicate HIV-1 reservoirs in others and whether a similar outcome can be achieved [14]. Clinical trials are planned to further elucidate these issues. Other groups propose engineering cells to resist HIV-1 infection, by modifying them as CCR5went through will be required to eradicate HIV-1 reservoirs in others and whether a similar outcome can be achieved [14]. Clinical trials are planned to further elucidate these issues. Other groups propose engineering cells to resist HIV-1 infection, by modifying them as CCR5went through will be required to eradicate HIV-1 reservoirs in others and whether a similar outcome can be achieved [14]. Clinical trials are planned to further elucidate these issues. Other groups propose engineering cells to resist HIV-1 infection, by modifying them as CCR5
All these topics were concern of the Frontiers in HIV Pathogenesis, Therapy and Eradication conference, held at the Whistler Conference Centre, in Whistler, Canada, last March 26-31th.
]]>Frontiers in HIV pathogenesis, therapy and eradication
The meeting was organized as part of the Keystone Symposia on Molecular and Cell Biology joined with the conference Cell Biology of Virus Entry. The convention was structured in several sessions: Keynote session, Retroviral entry mechanisms, Inhibition of virus entry, Mechanisms of HIV latency, HIV therapy: State of the art, Targeting latency for eradication, Cell biology of HIV infection, New approaches to antiviral treatment, Models for therapy and eradication, and Host factors and HIV replication. Alan N. Engelman, Eric O. Freed and John M. Coffin were the scientific organizers of the meeting.
A pre-meeting workshop was organized for those granted the Global Health Travel Award. Lectures on Molecular virology of HIV-1 replication, Molecular mechanisms of HIV latency and Clinical aspects of HAART therapy were dictated by Eric Freed from the National Center Cancer, USA; Melanie Ott from the University of California (San Francisco, USA), and Robert F. Siliciano from the Johns Hopkins University School of Medicine, USA respectively. The workshop was moderated by Jeff Lehman, Scholarship coordinator of the Keystone Symposia. It was an opportunity to debate with these prestigious researchers in the field of HIV.
Robert F. Siliciano delivered a remarkable lecture on how antiretroviral therapy for HIV infection really works. He stated that residual viremia after ART intensification is not due to ongoing replication but rather to release from stable reservoirs. Some classes of drugs have steep, highly cooperative dose response curves, reflecting a unique form of intermolecular cooperation. A biophysical model for the cooperative inhibition of infectivity was described, also explaining the effects of resistance mutations that alter not only the half maximal inhibitory concentration (IC50) but also the shape of dose response curves. Dr. Siliciano also explained on methods for calculating the combined inhibitory potential of drug combinations, permitting a comparison of antiviral activity of different drug regimes. This quantitative approach to understand ART may be useful for developing simpler ART regimes for patients [17-20].
Another lecture was delivered by Thomas J. Hope from the Northwestern University of Chicago, USA, on how early events of HIV-1 infection can influence late outcomes. After fusion of HIV-1 with the plasma membrane of a target cell, it must complete a series of steps before its genome can enter the nucleus and integrate into the cell´s genome. Hope’s group demonstrated that reverse transcription facilitates the process of uncoating. They showed that Q63/67A, E45A and N74D mutations in the p24CA protein slow the rate of uncoating of the viral capsid. Viruses showing these three mutations were also shown to use an alternative nuclear import pathway and influence integration site selection. They also studied the mechanism of TRIM5 alpha-mediated restriction by using green fluorescent protein (GFP) as a fluid phase marker incorporated into mature virions. The authors stated that early steps of HIV-1 replication are intertwined to a great extent and that the process of uncoating can influence later events such as nuclear import and even integration site preference [21-23].
Michel J. Root from the Thomas Jefferson University, Philadelphia, USA spoke about the HIV-1 gp41 structural changes associated with viral membrane fusion. He explained that despite the wide disparity in the native state structures of fusion glycoproteins of enveloped viruses, the final conformational transitions that drive membrane fusion show a remarkable degree of topological conservation. These proteins assume a homotrimeric extended intermediate state, bridging the viral and cellular membranes before collapsing into a symmetric trimer of hairpin conformation that juxtaposes the two membranes. Dr. Root’s group determined that the HIV-1 fusion glycoprotein Env (gp120/gp41) function requires folding of all three hairpins per gp41 trimer and that these hairpins fold in a highly cooperative manner. Their results suggested that the function of a single Env trimer is sufficient to promote viral entry. That is difficult to reconcile with the current N-ethylmaleimide–sensitive factor attachment protein receptor-like model of viral membrane fusion and suggest an alternate mechanism for the entry of HIV-1 [24].
Dr. Clare Jolly from the University College of London, UK, dictated a lecture in the Retroviral Entry Mechanisms Session on cell-cell transfer of HIV-1. This transmission takes place in virological synapses between an HIV-1 infected cell and a neighboring receptor-expressing target cells. The active, cytoskeleton-driven recruitment of viral and cellular proteins and the dynamic nature of virological synapses suggest that these structures are highly regulated, although the molecular effectors are mostly unknown [25]. This group showed that T cell-T cell HIV-1 virological synapses polarize the microtubule organizing center and associated organelles within the HIV-1 infected cells towards the engaged target T cell. Currently, they are identifying factors that regulate the cell-cell spread at the virological synapses as potential therapeutic targets for antiviral therapy [26].
Peter D. Kwong from the National Institute of Allergy and Infectious Diseases in Bethesda, MD, USA, talked on antibody-mediated inhibition of HIV-1 entry. He mentioned that monoclonal antibodies from HIV-1 infected individuals (15-25% of HIV-1 infected individuals generate neutralizing antibodies) fall into two distinct categories for an effective neutralization of the HIV-1 gp120 regarding the target site: 1) at the site of CD4 receptor binding; and 2) at conserved glycans. He proposes the use CD4-binding-site-directed and glycan-directed monoclonal antibodies as templates to design immunogens to re-elicit such antibodies [27]. This also reinforces a possible active role of glycans for immune control of the virus, beyond the glycan-shield theory.
Another interesting lecture was delivered by Michael Farzan from the Harvard Medical School, Massachusetts, USA. He focused on sulfotyrosine-binding pockets of gp120 as a therapeutic target. HIV-1 co-receptors are modified at their amino-terminal tyrosines by sulfate groups. These sulfotyrosines directly contact HIV-1 gp120 and contribute significantly to the binding energy required for the interaction of gp120 and the CCR5 co-receptor. The sulfotyrosine-binding pockets are cross-clade conserved and bear co-receptor preference. The author elaborated that sulfopeptides derived from neutralizing antibodies function as coreceptor-mimetics and can induce or stabilize the CD4-bound conformation of gp120. Fusions of these peptides with CD4-Ig or CD4-mimetic peptides are highly potent at neutralizing a range of HIV-1 isolates [28].
]]> Melanie Ott from the University of California (San Francisco, USA), delivered an outstanding lecture on regulation of HIV-1 transcription by Tat post-translational modifications. The HIV-1 Tat protein is a critical activator of transcription elongation via its interactions with TAR RNA and the positive transcription elongation factor b (P-TEFb). Tat also interacts with many chromatin-modifying enzymes, being also targeted by some of them. The authors focused on two critical modifications within Tat: monomethylation of lysine 51 (K51) and acetylation of lysine 50 (K50). They identified the lysine-specific demethylase 1 (LSD1/KDM1) as targeting Tat K51 and as new coactivator of HIV transcription in latently infected T cells [29].A very comprehensive lecture was delivered by Tae-Wook Chun from the National Institute of Allergy and Infectious Diseases in Bethesda, MD, USA. He analyzed the perspectives for eradicating the cellular HIV-1 reservoirs and the development of therapeutic strategies. The existence of latently-infected, resting CD4+ T cells carrying replication-competent HIV-1 provirus has posed one of the greatest challenges to the long-term control or eradication of HIV-1 in infected individuals on ART. There has been considerable work on therapies to reactivate the latent viral reservoir. The author pinpointed that such approaches have shown no clinical benefit to date and that it has been suggested that low levels of HIV-1 replication may persist in subsets of CD4+ T cells in blood and lymphoid tissues of infected individuals receiving ART but showing not detectable plasma viral load. He explained the potential mechanisms of HIV persistence and prospects for eradication and new therapeutic approaches in HIV-infected individuals under effective ART [4].
David M. Margolis (University of North Carolina at Chapel Hill, USA) subsequently focused on disrupting latency for eradication via the induction HIV-1 expression in latently infected resting CD4+ T cells. He pointed out the in vitro effectiveness of HDAC inhibitors such as suberoylanilide hydroxamic acid (vorinostat, VOR) for this purpose, pending to be tested in patients [30]. A larger and more complex scenario needs to be developed for eradication studies than that resembled by current in vitro models. Dr. Margolis mentioned that although the challenges can scarcely be underestimated, exciting emerging tolls and advances suggest that focused and collaborative efforts may succeed in eradicating HIV infection in mid-to near term [7].
Zeger Debyser from the Catholic University of Leuven, Belgium, elaborated on a novel class of integration inhibitors (LEDGF/p75 inhibitors, LEDGINs). He pointed that unlike clinically approved integrase strand transfer inhibitors, LEDGINs do not bind to the catalytic site of the HIV-1 integrase but to its LEDGF/p75 binding pocket. A broad activity spectrum against multiple clades and clinical isolates, and the synergism between LEDGIns and integrase strand transfer inhibitors in combination reveal the potential of these compounds for clinical use [31].
J. Víctor García-Martínez from the University of North Carolina at Chapel Hill, USA, examined the humanized BLT mice as an outstanding in vivo model to study HIV infection. His team demonstrated that a combination of well-characterized human retroviral drugs is capable of effectively controlling viral replication in BLT mice, as well as the presence of latently-infected resting human CD4+ T cells in ART-treated BLT which can be induced ex vivo to produce HIV-1. These authors observed a frequency of infected resting human CD4+ T cells in tissues from BLT mice in the same range observed in patients undergoing suppressive ART [12].
Alan Engelman from the Dana-Farber Cancer Institute in Boston, USA talked on HIV-host interactions in nuclear import and integration. They have investigated HIV-host interactions relevant for preintegration complex (PIC) nuclear import and integration. Genetic analyses identified cellular transportin 3 (TNPO3) and nucleoporin 153 (NUP153) and HIV-1 capsid (CA) at the PIC nuclear import though, interestingly, TNPO3 and NUP153 each bind to the viral integrase in vitro. They have determined that TNPO3 and NUP153 can separately bind to the CA protein in vitro, the latter preferentially binding to CA over IN [32, 33].
At the end of the talk, Eric O. Freed from the National Cancer Institute in Frederick, USA, delivered a remarkable lecture on host cell factors relevant for retrovirus assembly and release. The HIV-1 Gag polyprotein precursor (Pr55Gag) domains responsible for the assembly/release process are largely known but the role of host cell factors and pathways for particle production remain to be fully unraveled. The authors identified novel candidate cellular cofactors that are potentially involved in the late retroviral replication events [34-37]. Growth arrest-specific 7 (GAS7) and proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1) are sequence-related proteins that harbor the conserved F-BAR domain that deforms membranes. It was shown that GAS7 interacts directly with Tsg101 and the capsid region of HIV-1Gag via its F-BAR domain, being incorporated into virions and further cleaved by the viral protease.
CONCLUDING REMARKS
In spite of having reduced plasma viral RNA to undetectable levels and, consequently, HIV-related morbidity and mortality and its perinatal and behavior-associated transmission [38], ART is unable to eradicate the virus. A comprehensive understanding of HIV pathogenesis will be necessary to achieve a cure for HIV infection, especially its persistence and latency. The scientific community is working to develop better in vitro and in vivo models for that purpose. Some drugs are currently proposed for effective virus control and eradication. Clinical trials should be carefully designed. The HIV Pathogenesis, Therapy and Eradication Conference organized by the Keystone Symposia this year in Whistler, Canada, was a great opportunity to exchange knowledge between research groups working in all these topics. More than 50 lectures and very extensive sessions of poster discussion were carried out. Only a representative number of results discussed during the conference are included in this report.
]]>ACKNOWLEDGEMENTS
The authors acknowledge to Global Health Travel Award the opportunity to attend to the HIV Pathogenesis, Therapy and Eradication Conference organized by Keystone Symposia. Authors also acknowledge to Jeff Lehman, Scholarship coordinator from Keystone Symposia, by his opportune and efficient assistance.
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Celia Fernández-Ortega. Centro de Ingeniería Genética y Biotecnología, CIGB. Ave. 31 e/ 158 y 190, Cubanacán, Playa, CP 11 600, La Habana, Cuba. E-mail: celia.fernandez@cigb.edu.cu.
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