TAG HIV Basic Science, Vaccines, and Cure Project Blog

Tracing HIV Reservoirs

Two new papers offer differing perspectives on the reservoirs of HIV that persist despite effective antiretroviral therapy. Nicolas Chomont and colleagues demonstrate that when memory CD4 T cells containing integrated HIV proliferate (as most memory CD4 T cells do occasionally in a process known as homeostatic self-renewal), they copy the HIV provirus along with their own genomes. When CD4 T cell numbers decline, homeostatic proliferation occurs more frequently and Chomont’s paper shows that this is associated with an increase in the number of latently infected memory CD4 T cells. The researchers describe the cells that undergo more frequent proliferation in this setting as “transitional memory” T cells. At earlier stages of infection when the CD4 T cell pool is relatively intact, the reservoir of infected memory CD4 T cells is found to be far smaller and integrated virus is primarily located in “central memory” cells that divide less frequently.

Based on these findings, the study authors suggest that anticancer drugs that interfere with memory T cell proliferation should be studied for their potential to deplete the HIV reservoir. However, given the potential toxicities associated with inhibiting T cell proliferation, the risk/benefit of such trials would need to be carefully evaluated. A more ideal therapy would be one that only targeted dividing CD4 T cells containing HIV DNA, but it is currently unclear whether such an approach is within the realm of possibility. 

The second paper - by Timothy Brennan and colleagues from Bob Siliciano’s laboratory - uses genetic analyses of HIV sequences to show that there is a reservoir of virus that seems to be coming from a cell type other than memory CD4 T cells. The study finds that in most cases, the residual virus detectable in individuals on suppressive ART is genetically distinct from the virus found in memory CD4 T cells. The authors note in their conclusion: “Numerous laboratories are actively pursuing various eradication strategies, most of which involve some aspect of targeting and purging the latent reservoir in resting memory CD4+ T cells.  If much of the residual viremia of patients undergoing HAART comes from another reservoir or compartment as suggested here, then eradication strategies will have to include ways to target and purge this additional reservoir to be successful.”

Nature Medicine

Published online: 21 June 2009 | doi:10.1038/nm.1972

HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation

Nicolas Chomont1,2,3, Mohamed El-Far1,2,3, Petronela Ancuta3, Lydie Trautmann1,2,3, Francesco A Procopio1,2,3, Bader Yassine-Diab1,2,3, Geneviève Boucher1, Mohamed-Rachid Boulassel4, Georges Ghattas5, Jason M Brenchley6, Timothy W Schacker7, Brenna J Hill8, Daniel C Douek8, Jean-Pierre Routy4,9, Elias K Haddad1,2,3,9 & Rafick-Pierre Sékaly1,2,3,9,10,11

1. Laboratoire d'Immunologie, Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM) Saint-Luc, Montréal, Québec, Canada. 2. Laboratoire d'Immunologie, Département de Microbiologie et d'Immunologie, Université de Montréal, Québec, Canada. 3. Institute National de la Santé et de la Recherche Médicale U743, CR-CHUM, Université de Montréal, Montréal, Québec, Canada. 4. Immunodeficiency Service and Division of Hematology, Royal Victoria Hospital, McGill University Health Centre (MUHC), McGill University, Montréal, Québec, Canada. 5. Department of Gastroenterology, MUHC, Montréal, Québec, Canada. 6. Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, US National Institutes of Health, Bethesda, Maryland, USA. 7. Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA. 8. Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, US National Institutes of Health, Bethesda, Maryland, USA. 9. Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada. 10. Department of Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon, USA. 11. Vaccine and Gene Therapy Institute, Port-Ste Lucy, Florida, USA.

Abstract

HIV persists in a reservoir of latently infected CD4+ T cells in individuals treated with highly active antiretroviral therapy (HAART). Here we identify central memory (TCM) and transitional memory (TTM) CD4+ T cells as the major cellular reservoirs for HIV and find that viral persistence is ensured by two different mechanisms. HIV primarily persists in TCM cells in subjects showing reconstitution of the CD4+ compartment upon HAART. This reservoir is maintained through T cell survival and low-level antigen-driven proliferation and is slowly depleted with time. In contrast, proviral DNA is preferentially detected in TTM cells from aviremic individuals with low CD4+ counts and higher amounts of interleukin-7–mediated homeostatic proliferation, a mechanism that ensures the persistence of these cells. Our results suggest that viral eradication might be achieved through the combined use of strategic interventions targeting viral replication and, as in cancer, drugs that interfere with the self renewal and persistence of proliferating memory T cells.

JVI Accepts, published online ahead of print on 17 June 2009

J. Virol. doi:10.1128/JVI.02568-08

Analysis of HIV-1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Timothy P. Brennan, John O. Woods, Ahmad R. Sedaghat, Janet D. Siliciano, Robert F. Siliciano, and Claus O. Wilke*

Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; Institute for Cell and Molecular Biology, The University of Texas at Austin, Austin, TX 78712; Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; Howard Hughes Medical Institute, Baltimore, MD 21205; Center for Computational Biology and Bioinformatics and Section of Integrative Biology, The University of Texas at Austin, Austin, TX 78712

Abstract

Highly active antiretroviral therapy (HAART) can reduce HIV-1 viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. Additionally, a long-lived latent reservoir for HIV-1 exists in resting memory CD4+ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we employ two measures of population structure, analysis of molecular variance and the Slatkin-Maddison test, to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4+ T cells, but that proviruses in resting and activated CD4+ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4+ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4+ T cells has implications for eradication efforts.

Illuminating Early Events in HIV Infection Using Single Genome Amplification

Two papers in the current Journal of Experimental Medicine offer unprecedented insight into the initial interactions between virus and host after HIV infection. An accompanying commentary by Zabrina Brumme and Bruce Walker eloquently articulates what these studies have achieved: “By identifying persons before seroconversion, pinpointing the transmitted virus, and assessing immune responses to that particular variant as it evolves, they provide a novel view of host and viral dynamics during the earliest stages of infection.”

In both studies the researchers use an optimized version of a technique called Single Genome Amplification (SGA), originally developed by Sarah Palmer and colleagues at the National Cancer Institute. While costly and labor-intensive, this technique allows sequencing of the HIV genome without many of the potential confounding errors that can occur with standard PCR. The researchers also used the sequences obtained by SGA to synthesize peptides for CD8 T cell response assays; this allowed detailed tracking of the impact of CD8 T cell responses on the virus genome.

The study results echo prior work from these groups suggesting that most HIV transmission events involve a single isolate; in 11 out of 12 cases SGA showed that all detected sequences were related to a single infecting virus. The remaining individual was infected with two viruses that could be unambiguously identified based on their sequences. In terms of viral evolution after infection, the researchers found that between transmission and peak viremia, diversification of HIV sequences was essentially random and showed no evidence of selection pressure from host immune responses. Subsequently, between 9-16 days later, the effects of selection became obvious, particularly effects attributable to HIV-specific CD8 T cell responses. By 32-45 days postinfection, almost the entire replicating virus population in each subject studied was replaced by viruses with mutations at two to five distinct loci in the genome, evincing selection pressure from both CD8 T cell and neutralizing antibody responses (and other unidentified sources also, perhaps innate and/or CD4 T cell immune responses).

The level of detail involved in the study also allowed the researchers to document virus escape from CD8 T cell responses earlier than has previously been reported. Mathematical modeling of the data indicated that HIV-specific CD8 T cells are more efficient at killing virus-infected cells during acute infection than prior estimates have suggested. Discussing the implications of their findings for T-cell-based vaccines, the authors state: “Modeling implied that a single T cell response was contributing as much as 15–35% of viral decline with multiple T cell responses. The implication of these observations is that vaccine-induced HIV-1–specific T cells will contribute to control of acute viremia if they are activated early in subsequent HIV-1 infection. However, because of the very rapid escape that occurs within the first few weeks of infection, T cell vaccines will need to stimulate a considerable breadth of T cell responses, clearly greater than the median of three epitopes induced by the Merck vaccine.”

Both papers are the work of researchers supported by the Center for HIV/AIDS Vaccine Immunology (CHAVI); without the substantial funding committed to this project by the National Institutes of Health, this work would not have been possible. 

Published online June 1, 2009

doi:10.1084/jem.20091094

The Journal of Experimental Medicine, Vol. 206, No. 6, 1215-1218

COMMENTARY

Tracking the culprit: HIV-1 evolution and immune selection revealed by single-genome amplification

Zabrina L. Brumme and Bruce D. Walker

Z.L. Brumme and B.D. Walker are at the Ragon Institute of MGH, MIT and Harvard, Charlestown MA 02129; Z.L. Brumme is at the Faculty of Health Sciences, Simon Fraser University, Burnaby BC V5A 1S6, Canada

ABSTRACT

Early control of HIV-1 infection is determined by a balance between the host immune response and the ability of the virus to escape this response. Studies using single-genome amplification now reveal new details about the kinetics and specificity of the CD8+ T cell response and the evolution of the virus during early HIV infection.

Published online June 1, 2009

doi:10.1084/jem.20090365

The Journal of Experimental Medicine, Vol. 206, No. 6, 1253-1272

ARTICLE

The first T cell response to transmitted/founder virus contributes to the control of acute viremia in HIV-1 infection

Nilu Goonetilleke1, Michael K.P. Liu1, Jesus F. Salazar-Gonzalez2, Guido Ferrari3, Elena Giorgi4, Vitaly V. Ganusov4, Brandon F. Keele2, Gerald H. Learn2, Emma L. Turnbull5, Maria G. Salazar2, Kent J. Weinhold3, Stephen Moore1, CHAVI Clinical Core B, Norman Letvin6, Barton F. Haynes3, Myron S. Cohen7, Peter Hraber4, Tanmoy Bhattacharya4,8, Persephone Borrow5, Alan S. Perelson4, Beatrice H. Hahn2, George M. Shaw2, Bette T. Korber4,8, and Andrew J. McMichael1

1 Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford OX3 9DS, England, UK

2 Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294

3 Duke University Medical Research, Duke University, Durham, NC 27710

4 Los Alamos National Laboratory, Theoretical Division, Los Alamos, NM 87545

5 The Jenner Institute, Oxford University, Compton RG20 7NN, England, UK

6 BIDMC, Harvard University, Boston, MA 02115

7 HIV Prevention Trials Unit, University of North Carolina, Chapel Hill, NC 27599

8 The Santa Fe Institute, Santa Fe, NM 87501

Identification of the transmitted/founder virus makes possible, for the first time, a genome-wide analysis of host immune responses against the infecting HIV-1 proteome. A complete dissection was made of the primary HIV-1–specific T cell response induced in three acutely infected patients. Cellular assays, together with new algorithms which identify sites of positive selection in the virus genome, showed that primary HIV-1–specific T cells rapidly select escape mutations concurrent with falling virus load in acute infection. Kinetic analysis and mathematical modeling of virus immune escape showed that the contribution of CD8 T cell–mediated killing of productively infected cells was earlier and much greater than previously recognized and that it contributed to the initial decline of plasma virus in acute infection. After virus escape, these first T cell responses often rapidly waned, leaving or being succeeded by T cell responses to epitopes which escaped more slowly or were invariant. These latter responses are likely to be important in maintaining the already established virus set point. In addition to mutations selected by T cells, there were other selected regions that accrued mutations more gradually but were not associated with a T cell response. These included clusters of mutations in envelope that were targeted by NAbs, a few isolated sites that reverted to the consensus sequence, and bystander mutations in linkage with T cell–driven escape.

Published online June 1, 2009

doi:10.1084/jem.20090378

The Journal of Experimental Medicine, Vol. 206, No. 6, 1273-1289

ARTICLE

Genetic identity, biological phenotype, and evolutionary pathways of transmitted/founder viruses in acute and early HIV-1 infection

Jesus F. Salazar-Gonzalez1, Maria G. Salazar1, Brandon F. Keele1, Gerald H. Learn1, Elena E. Giorgi2,3, Hui Li1, Julie M. Decker1, Shuyi Wang1, Joshua Baalwa1, Matthias H. Kraus1, Nicholas F. Parrish1, Katharina S. Shaw1, M. Brad Guffey1, Katharine J. Bar1, Katie L. Davis1, Christina Ochsenbauer-Jambor1, John C. Kappes1, Michael S. Saag1, Myron S. Cohen4, Joseph Mulenga5, Cynthia A. Derdeyn6, Susan Allen6, Eric Hunter6, Martin Markowitz7,8, Peter Hraber2, Alan S. Perelson2, Tanmoy Bhattacharya2,9, Barton F. Haynes10, Bette T. Korber2,9, Beatrice H. Hahn1, and George M. Shaw1

1 University of Alabama at Birmingham, Birmingham, AL 35294

2 Los Alamos National Laboratory, Los Alamos, NM 87545

3 University of Massachusetts, Amherst, MA 01002

4 The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599

5 Zambia-Emory HIV Research Project, Lusaka, Zambia

6 Emory University, Atlanta, GA 30329

7 Aaron Diamond AIDS Research Center, New York, NY 10016

8 The Rockefeller University, New York, NY 10065

9 Santa Fe Institute, Santa Fe, NM 87501

10 Duke University Medical Center, Durham, NC 27710

Identification of full-length transmitted HIV-1 genomes could be instrumental in HIV-1 pathogenesis, microbicide, and vaccine research by enabling the direct analysis of those viruses actually responsible for productive clinical infection. We show in 12 acutely infected subjects (9 clade B and 3 clade C) that complete HIV-1 genomes of transmitted/founder viruses can be inferred by single genome amplification and sequencing of plasma virion RNA. This allowed for the molecular cloning and biological analysis of transmitted/founder viruses and a comprehensive genome-wide assessment of the genetic imprint left on the evolving virus quasispecies by a composite of host selection pressures. Transmitted viruses encoded intact canonical genes (gag-pol-vif-vpr-tat-rev-vpu-env-nef) and replicated efficiently in primary human CD4+ T lymphocytes but much less so in monocyte-derived macrophages. Transmitted viruses were CD4 and CCR5 tropic and demonstrated concealment of coreceptor binding surfaces of the envelope bridging sheet and variable loop 3. 2 mo after infection, transmitted/founder viruses in three subjects were nearly completely replaced by viruses differing at two to five highly selected genomic loci; by 12–20 mo, viruses exhibited concentrated mutations at 17–34 discrete locations. These findings reveal viral properties associated with mucosal HIV-1 transmission and a limited set of rapidly evolving adaptive mutations driven primarily, but not exclusively, by early cytotoxic T cell responses.

Natural Neutralizing Antibodies

A number of recent studies have reported the detection of neutralizing antibodies in individuals with chronic HIV infection. In some rare cases, antibody responses capable of neutralizing a broad array of diverse HIV isolates have been documented. In a perspective piece just published by Nature Medicine, Leonidas Stamatatos and colleagues review these findings and suggest that they represent good news for the vaccine field. In particular, they note the data argue that B cells can make antibodies capable of neutralizing HIV; some scientists have been concerned that the antibody structure required to inhibit HIV cannot be created by the human immune system. Based on the new findings, the researchers recommend a series of steps for pursuing structure-based HIV vaccine design:

 

Figure 1 - Flow chart of structure-based HIV-1 vaccine design.

CREDIT:

Nature Medicine

Published online: 14 June 2009 | doi:10.1038/nm.1949

Neutralizing antibodies generated during natural HIV-1 infection: good news for an HIV-1 vaccine?

Leonidas Stamatatos1,2, Lynn Morris3,4, Dennis R Burton5 & John R Mascola6

1. Seattle Biomedical Research Institute, Seattle, Washington, USA.

2. Department of Global Health, University of Washington, Seattle, Washington, USA.

3. AIDS Virus Research Unit, National Institute for Communicable Diseases, Johannesburg, South Africa.

4. Centre for the AIDS Programme of Research in South Africa, Johannesburg, South Africa.

5. Department of Immunology and Microbial Science and the International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California, USA.

6. Vaccine Research Center, National Institute of Allergy and Infectious Diseases, US National Institutes of Health, Bethesda, Maryland, USA.

Abstract

Most existing viral vaccines generate antibodies that either block initial infection or help eradicate the virus before it can cause disease. For HIV-1, obstacles to eliciting protective neutralizing antibodies (NAbs) have often seemed insurmountable. The target of HIV-specific NAbs, the viral envelope glycoprotein (Env), is highly variable in amino acid sequence and glycosylation pattern. Conserved elements of HIV-1 Env seem to be poorly immunogenic, and previous attempts to generate broadly reactive NAbs by vaccination have proven ineffective. However, recent studies show that antibodies in the sera of some HIV-1–infected individuals can neutralize diverse HIV-1 isolates. Detailed analyses of these sera provide new insights into the viral epitopes targeted by broadly reactive NAbs. The findings discussed here suggest that the natural NAb response to HIV-1 can inform future vaccine design. A concerted effort of structure-based vaccine design will help guide the development of improved antibody-based vaccines for HIV-1.

Killer Helpers

CD4 T cells are typically portrayed as playing a supportive role in the generation and maintenance of immune responses, as their alternative name – helper cells – suggests. But over the past decade, studies have shown that there are a number of settings in which antigen-specific CD4 T cells exert direct cytotoxic activity (a capacity that was once believed to be an artifact of prolonged in vitro culture).

Two new papers describe the presence of cytotoxic virus-specific CD4 T cells in SIV and HIV infection, respectively. The first study, led by Jonah Sacha, obtained Gag- and Nef-specific CD4 T cells from a group of elite controller macaques infected with SIVmac239. The researchers had noticed that, after an experiment in which CD8 T cells were depleted, the animals experienced a rebound in viral load that was subsequently brought back down to undetectable levels. This regaining of control over viral replication was associated with an expansion of their Gag- and Nef-specific CD4 T cell responses, an observation that prompted the current study.

In Sacha’s in vitro analyses, cytotoxic Gag- and Nef-specific CD4 T cells were unable to recognize SIV-infected CD4 T cells, but efficiently recognized and killed infected macrophages. Recognition occurred very rapidly, within 1-2 hours, with activity peaking between 6 and 24 hours. The cytotoxic CD4 T cells consistently eliminated 30-40% of SIV-infected macrophages in culture. Sacha and colleagues conclude by stating “on the basis of the data presented here, we speculate that Gag- and Nef-specific CD4􏰀 T cells may play a more important role in the antiretroviral immune response than previously appreciated.”

The second paper, by Nan Zheng and colleagues from Kumamoto University, identifies cytotoxic Nef-specific CD4 T cells in 4/9 individuals analyzed. In this study, the Nef-specific CD4 T cells are reported to kill both HIV-infected macrophages and CD4 T cells, but the activity against macrophages is shown to be superior. 

Taken together, the findings suggest that it might be worth investigating whether cytotoxic virus-specific CD4 T cells can be induced by vaccination. Perhaps of note, one previous report from Ron Desrosiers laboratory has suggested that effector CD4 T cells with a cytotoxic phenotype (expressing perforin and the degranulation marker CD107) contribute to the protection obtained with live-attenuated SIV vaccines.

The PNAS paper is Open Access, click the title link for the full PDF. 

PNAS Published online before print May 28, 2009, doi: 10.1073/pnas.0813106106

Gag- and Nef-specific CD4+ T cells recognize and inhibit SIV replication in infected macrophages early after infection

Jonah B. Sacha a,1,2, Juan P. Giraldo-Vela a,1, Matthew B. Buechler a, Mauricio A. Martins a, Nicholas J. Maness a, Chungwon Chung a, Lyle T. Wallace a, Enrique J. León a, Thomas C. Friedrich b, Nancy A. Wilson a, Atsunobu Hiraoka  and David I. Watkins a

a Departments of aPathology and Laboratory Medicine andb  Pathobiological Sciences, University of Wisconsin, Madison, WI 53715; and c Osaka Dental University, Osaka 540-0008, Japan

1J.B.S. and J.P.G.-V. contributed equally to this work.

Abstract

The precise immunological role played by CD4+ T cells in retroviral infections is poorly defined. Here, we describe a new function of these cells, the elimination of retrovirus-infected macrophages. After experimental CD8+ cell depletion, elite controlling macaques with set-point viral loads ≤500 viral RNA copies/mL mounted robust Gag- and Nef-specific CD4+ T cell responses during reestablishment of control with ≥54% of all virus-specific CD4+ T cells targeting these 2 proteins. Ex vivo, these simian immunodeficiency virus (SIV)-specific CD4+ T cells neither recognized nor suppressed viral replication in SIV-infected CD4+ T cells. In contrast, they recognized SIV-infected macrophages as early as 2 h postinfection because of presentation of epitopes derived from virion-associated Gag and Nef proteins. Furthermore, virus-specific CD4+ T cells displayed direct effector function and eliminated SIV-infected macrophages. These results suggest that retrovirus-specific CD4+ T cells may contribute directly to elite control by inhibiting viral replication in macrophages.

JVI Accepts, published online ahead of print on 20 May 2009

J. Virol. doi:10.1128/JVI.00513-09

Strong ability of Nef-specific CD4+ cytotoxic T cells to suppress HIV-1 replication in HIV-1-infected CD4+ T cells and macrophages

Nan Zheng, Mamoru Fujiwara, Takamasa Ueno, Shinichi Oka, and Masafumi Takiguchi

Division of Viral Immunology and Division of Infectious Disease, Center for AIDS Research, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811; and AIDS Clinical Center, International Medical Center of Japan, 1-21-1, Toyama, Shinjuku, Tokyo 162-8655, Japan

Abstract

A restricted number of studies have shown that HIV-1-specific cytotoxic CD4+ T cells are present in HIV-1-infected individuals. However, the roles of this type of CD4+ T cell in the immune responses against an HIV-1 infection remain unclear. In this study, we identified novel Nef epitope-specific HLA-DRB1*0803-restricted cytotoxic CD4+ T cells. The CD4+ T cell clones specific for Nef187-203 showed strong IFN-production after having been stimulated with autologous B-LCLs infected with Nef recombinant vaccinia virus or pulsed with heat-inactivated virus particles, indicating the presentation of the epitope antigen through both exogenous and endogenous MHC class II processing pathways. Nef187-203-specific CD4+ T cell clones exhibited strong cytotoxic activity against both HIV-1-infected macrophages and CD4+ T cells from an HLA-DRB1*0803+ donor. In addition, these Nef-specific cytotoxic CD4+ T cell clones exhibited strong ability to suppress HIV-1 replication in both macrophages and in CD4+ T cells in vitro. Nef187-203-specific cytotoxic CD4+ T cells were detected in cultures of peptide-stimulated PBMC and in ex vivo PBMC from 40% and 20% of DRB1*0803+ donors, respectively. These results suggest that HIV-1-specific CD4+ T cells may directly control HIV-1-infection in vivo by suppressing virus replication in HIV-1 natural host cells.