TAG HIV Basic Science, Vaccines, and Cure Project Blog

Lymphocyte Production Capacity in HIV: Links to Immune Activation & Immune Reconstitution on ART

A number of studies have documented that the body’s machinery for producing new immune system cells is impaired by HIV infection. In a new paper in the journal Blood, Delphine Sauce and colleagues delve into the issue further by analyzing circulating CD34+ hematopoietic progenitor cells (HPC) in over one hundred HIV positive individuals at various stages of disease, compared to both age-matched and elderly (75-96 years old) uninfected controls. The research reveals significant correlations between the number of HPC and multiple types of immune cells including CD4 T cells, CD8 T cells, B cells, natural killer cells and neutrophils (the strongest correlation being between circulating HPC and CD4 T cells). The number of HPC is found to decline over the course of HIV disease progression such that middle-aged HIV-positive individuals with CD4 T cell counts less than 200 had similar levels of circulating HPCs to the elderly uninfected controls.

Additional analyses show that markers of immune activation such as CD38 expression on CD8 T cells are inversely associated with HPCs; the higher the levels of immune activation, the lower the HPC counts. The researchers demonstrate that among elite controllers with declining CD4 T cell counts, HPC numbers and functional capacity are diminished despite persistently low viral load, consistent with the link between immune activation and CD4 T cell loss that has been reported in this population. HIV-positive individuals with poor CD4 T cell recovery despite antiretroviral therapy (ART) also show reduced HPC numbers and functional capacity compared to individuals with better immune reconstitution.

The study authors conclude that preservation of lymphocyte production capacity (lymphopoiesis) is important for preventing HIV disease progression and that strategies for enhancing lymphopoiesis should be evaluated in the setting of poor CD4 T cell recovery on ART. One approach that is currently under evaluation in a clinical trial for people on ART with CD4 T cell counts less than 250 is TXA127 (angiotensin 1-7), a drug formulation of a naturally occurring substance that may stimulate production of HPCs.

Published online before print March 24, 2011, doi: 10.1182/blood-2011-01-331306

Blood March 24, 2011 blood-2011-01-331306

HIV disease progression despite suppression of viral replication is associated with exhaustion of lymphopoiesis

Delphine Sauce1, Martin Larsen2, Solene Fastenackels1, Michele Pauchard3, Hocine Ait-Mohand4, Luminita Schneider3, Amelie Guihot1, Faroudy Boufassa5, John Zaunders6, Malika Iguertsira7, Michelle Bailey6, Guy Gorochov2, Claudine Duvivier8, Guislaine Carcelain9, Anthony D Kelleher10, Anne Simon7, Laurence Meyer11, Dominique Costagliola3, Steven G Deeks12, Olivier Lambotte13, Brigitte Autran1, Peter W Hunt12, Christine Katlama3, and Victor Appay1,*

1 INSERM UMR S 945, Infections and Immunity, Avenir Group, Universite Pierre et Marie Curie-Paris6, Hopital Pitie-Salpetriere, Paris, France;

2 Immunoregulation and Immunotherapy, INSERM UMR S 945, Universite Pierre et Marie Curie-Paris6, Hopital Pitie-Salpetriere, Paris, France;

3 INSERM UMR S 943, Universite Pierre et Marie Curie-Paris6, Hopital Pitie-Salpetriere, Paris, France;

4 AP-HP, Groupe hospitalier Pitie-Salpetriere, Service des Maladies Infectieuses et Tropicales, Paris, France;

5 INSERM U81018, University Paris-Sud, Hopital Bicetre, Le Kremlin-Bicetre, France;

6 St Vincent's Centre for Applied Medical Research and University of New South Wales, Sydney, NSW, Australia;

7 AP-HP, Groupe hospitalier Pitie-Salpetriere, Service de Medecine Interne, Paris, France;

8 Institut Pasteur, Centre Medical de l'Institut Pasteur; Centre d'Infectiologie Necker-Pasteur, Departement Infection et Epidemiologie, Paris, France;

9 AP-HP, Groupe hospitalier Pitie-Salpetriere, Laboratoire d'Immunologie Cellulaire et Tissulaire, Paris, France;

10 St. Vincent's Centre for Applied Medical Research and University of New South Wales, Sydney, NSW, Australia;

11 AP-HP, Service d'Epidemiologie et de Sante Publique, Hopital Bicetre, Le Kremlin-Bicetre, France;

12 HIV/AIDS Division, Department of Internal Medicine, University of California, San Francisco, San Francisco, CA, United States;

13 AP-HP, Department of Internal Medicine and Infectious Diseases, Hopital Bicetre, Le Kremlin-Bicetre, France

Abstract

The mechanisms of CD4+ T cell count decline, the hallmark of HIV disease progression, and its relationship to elevated levels of immune activation are not fully understood. Massive depletion of CD4+ T cells occurs during the course of HIV-1 infection, so that maintenance of adequate CD4+ T cell levels likely depends primarily on the capacity to renew depleted lymphocytes, i.e. the lymphopoiesis. We performed here a comprehensive study of quantitative and qualitative attributes of CD34+ hematopoietic progenitor cells directly from the blood of a large set of HIV infected individuals compared with uninfected donors, in particular elderly. Our analyses underline a marked impairment of primary immune resources with the failure to maintain adequate lymphocyte counts. Systemic immune activation emerges as a major correlate of altered lymphopoiesis, which can be partially reversed with prolonged antiretroviral therapy. Importantly, HIV disease progression despite elite control of HIV replication or virological success on antiretroviral treatment is associated with persistent damage to the lymphopoietic system, or exhaustion of lymphopoiesis. These findings highlight the importance of primary hematopoietic resources in HIV pathogenesis and the response to antiretroviral treatments.

Eating Envelope to Improve Vaccines

Inducing antibodies capable of neutralizing a broad array of HIV isolates is the holy grail for vaccine research. One of the key obstacles is the virus’s outer envelope, which is densely cloaked in sugary molecules that shield the parts that are vulnerable to antibody-mediated attack. When unmodified HIV envelopes are used to make vaccines, the antibody responses that result are largely useless because they target these irrelevant decoys. But Ema Crooks from James Binley’s laboratory at the Torrey Pines Institute for Molecular Studies may have come up with a way to address the problem. The strategy involves using specific enzymes to digest the “junk” envelope, leaving behind the envelope structures (trimers) that are more likely to induce effective antibodies. A similar strategy was described at the recent Keystone HIV vaccines meeting by John Moore’s research group at Cornell University. The next step will be to test these new junk-depleted HIV envelopes as vaccine immunogens.

JVI Accepts, published online ahead of print on 6 April 2011

J. Virol. doi:10.1128/JVI.00154-11

Enzyme Digests Eliminate Non-Functional Env from HIV-1 Particle Surfaces Leaving Native Env Trimers Intact and Viral Infectivity Unaffected.

Ema T. Crooks, Tommy Tong, Keiko Osawa, and James M. Binley

Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121, USA

Abstract

HIV-1 viruses and virus-like particles (VLPs) bear non-native "junk" forms of Envelope glycoprotein (Env) that may undermine the development of antibody responses against functional gp120/gp41 trimers, thereby blunting the ability of particles to elicit neutralizing antibodies. Here, we sought to better understand the nature of junk Env with a view to devising strategies for its removal. Initial studies revealed that native trimers were surprisingly stable in the face of harsh conditions, suggesting that junk Env is unlikely to arise by trimer dissociation or gp120 shedding. Furthermore, the limited gp120 shedding that occurs immediately after synthesis of primary HIV-1 isolate Envs is not caused by aberrant cleavage at the tandem gp120/gp41 cleavage sites, which were found to cleave in a co-dependent manner. A major VLP contaminant was found to consist of an early, monomeric form of gp160, termed "gp160ER" that bypasses protein maturation and trafficks directly into particles. Gp160ER was found to bind two copies of mAb 2G12, consistent with its exclusively high mannose glycan profile. These findings prompted us to evaluate enzyme digests as a way to remove aberrant Env. Remarkably, sequential glycosidase-protease digests led to a complete or near-complete removal of junk Env from many viral strains, leaving trimers and viral infectivity largely intact. "Trimer-VLPs" may be useful neutralizing antibody immunogens.

Keystone Symposia: HIV Evolution, Genomics and Pathogenesis (X7), Whistler Conference Centre, Whistler, British Columbia, Canada, March 20–25, 2011

Abstract 154

Generation of stable, deglycosylated HIV-1 Env trimers for crystallization and immunogenicity studies

Depetris, R.S.1, Julien, J.P.2, Pejchal, R.2, Rabidou, D.1, Kachare, M.1, Olson, W.3, Wilson, I.2, Sanders, R.1 & Moore, J.P.1

1 Dept. of Microbiology and Immunology, Weill Cornell Medical College, 2 The Scripps Research Institute, 3 Progenics Pharmaceuticals

The envelope glycoproteins (Env) are the focus of HIV-1 vaccine development programs aimed at eliciting humoral responses. The extensive glycosylation of the Env trimer hinders immunogenicity and crystallography studies, because glycans both shield conserved neutralization epitopes and create structural heterogeneity. We have evaluated different ways to obtain stable, properly folded, fully deglycosylated gp140 trimers. This goal can be achieved by inhibiting the generation of complex carbohydrates by expressing the proteins in mutant cell lines, then treating them with appropriate glycosidases under mild conditions compatible with trimer integrity. The deglycosylated Env proteins are trimeric, with stability comparable to wild type. Their antigenic structure is generally conserved, except for glycan-dependent epitopes. The deglycosylated trimers do not aggregate significantly, and have no increased sensitivity to proteolytic digestion. Deglycosylated Env trimers are promising candidates for crystallization studies and are worth evaluating as immunogens. 

Disulfiram Evaluated for Reversing HIV Latency

Back in October 2009, Bob Siliciano’s laboratory at Johns Hopkins described a new in vitro system for screening drugs with the potential to reverse HIV latency. At the time of that publication, only one such compound had been identified using the screen, 5HN, which was too toxic to be considered for clinical evaluation. In a new paper just published online by the Journal of Virology, the researchers report the identification of another drug, disulfiram, which is already FDA approved as a treatment for alcoholism. At concentrations that are known to be achieved in vivo, disulfiram induced HIV gene expression in latently infected CD4 T cells without causing cellular activation or cytokine release. However, in the case of resting CD4 T cells isolated directly from HIV positive individuals on ART, an effect of disulfiram treatment on virus production could not be clearly demonstrated. The authors therefore stress that it is uncertain whether the drug can deplete HIV reservoirs in vivo. To try and address this question, the research group of Steve Deeks at UCSF is now conducting a pilot clinical trial. The collaboration between Deeks and Siliciano has been facilitated and supported by the recently launched amfAR Research Consortium on HIV Eradication (ARCHE).

In another paper from Siliciano’s lab published a few weeks ago, the same in vitro model of latent HIV infection of CD4 T cells is used to probe how viral latency is established. One theory posits that latency ensues when HIV integrates into inactive parts of the CD4 T-cell’s genetic code--essentially the genetic equivalent of driving down a one-way street and not being able to hit reverse. The new study finds little evidence to support this theory, but rather shows that the virus preferentially integrates into genes that are active and busily going about their protein-making business. This result supports an alternate theory that latency results from something called transcriptional interference. In this scenario, the machinery used to make proteins from the gene sort of sticks a wheel clamp on the virus and, while there’s plenty of genetic traffic, HIV’s genes are immobilized in the midst of it.

JVI Accepts, published online ahead of print on 6 April 2011

J. Virol. doi:10.1128/JVI.02033-10

Disulfiram reactivates latent HIV-1 in a Bcl-2 transduced primary CD4+ T cell model without inducing global T cell activation

Sifei Xing, Cynthia K. Bullen, Neeta S. Shroff, Liang Shan, Hung-Chih Yang, Jordyn L. Manucci, Shridhar Bhat, Thomas C. Quinn, David M. Margolis, Janet D. Siliciano, and Robert F. Siliciano

Department of Medicine, and Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Howard Hughes Medical Institute, Baltimore, Maryland, USA; Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA; University of North Carolina, Chapel Hill, North Carolina, USA

Abstract

Highly active antiretroviral therapy (HAART) can reduce plasma HIV-1 levels to below the detection limit. However, due to the latent reservoir in resting CD4+ cells, HAART is not curative. Elimination of this reservoir is critical to curing HIV-1 infection. Agents that reactivate latent HIV-1 through nonspecific T cell activation are toxic. Here we demonstrate in a primary CD4+ T cell model that the FDA-approved drug disulfiram reactivates latent HIV-1 without global T cell activation. The extent to which disulfiram reactivates latent HIV-1 in patient cells is unclear, but the drug alone or in combination may be useful in future eradication strategies.

JVI Accepts, published online ahead of print on 23 March 2011

J. Virol. doi:10.1128/JVI.02536-10

Influence of host gene transcription level and orientation on HIV-1 latency in a primary cell model

Liang Shan, Hung-Chih Yang, S. Alireza Rabi, Hector C. Bravo, Rafael A. Irizarry, Hao Zhang, Joseph B. Margolick, Janet D. Siliciano, and Robert F. Siliciano*

Departments of Medicine and Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Department of Biostatistics and Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Howard Hughes Medical Institute, Baltimore, Maryland 21205, USA

Abstract

Human Immunodeficiency Virus Type 1 (HIV-1) establishes a latent reservoir in resting memory CD4+ T cells. This latent reservoir is a major barrier to the eradication of HIV-1 in infected individuals and is not affected by highly active antiretroviral therapy (HAART). Reactivation of latent HIV-1 is a possible strategy for elimination of this reservoir. The mechanisms with which latency is maintained are unclear. In the analysis of the regulation of HIV-1 gene expression, it is important to consider the nature of HIV-1 integration sites. In this study, we analyzed the integration and transcription of latent HIV-1 in a primary CD4+ T cell model of latency. The majority of integration sites in latently infected cells were in introns of transcription units. Serial analysis of gene expression (SAGE) demonstrated that more than 90% of those host genes harboring a latent integrated provirus were transcriptionally active, mostly at high level. For latent, we observed a modest preference for integration in the same transcriptional orientation as the host gene (63.8% vs. 36.2%). In contrast, this orientation preference was not observed in acutely infected or persistently infected cells. These results suggest that transcriptional interference may be one of the important factors in the establishment and maintenance of HIV-1 latency. Our findings suggest that disrupting the negative control of HIV-1 transcription by upstream host promoters could facilitate the reactivation of latent HIV-1 in some resting CD4+ T cells.