TAG HIV Basic Science, Vaccines, and Cure Project Blog

Plumbing HIV Pathogenesis in the Gut

A trio of recent papers delve into the gut as an important site of HIV pathogenesis. Two are from the research group of Joseph Wong at UCSF, and they involve the same small cohort individuals analyzed before (in the JID paper) and after (in the journal AIDS) they started a clinical trial involving intensification of their antiretroviral therapy. The third paper, led by Shari Gordon from Guido Silvestri’s laboratory, studies a larger group of 28 people with HIV (15 untreated and 13 on antiretroviral therapy) and 11 healthy controls.

One unifying theme of the results is that not all gut sites are equal; both the levels of HIV infection and the proportions of CD4 vs. CD8 T cells vary when samples from the duodenum, terminal ileum, right colon and rectum are compared in Steven Yukl’s JID paper. HIV DNA levels show a trend toward a stepwise increase as the sample sites descend, with the lowest levels in the terminal ileum and highest in the rectum. Conversely, unspliced HIV RNA – a potential indicator of active viral replication  - shows the opposite trend, with levels highest in the terminal ileum. Shari Gordon’s study samples a slightly different array of gut sites – terminal ileum, right colon, left colon, and sigmoid colon – but also finds variation in representation of CD4 T cell subsets; naïve CD4 T cell levels are highest in the terminal ileum but decrease progressively as the sample sites get lower down the GI tract.

The Yukl paper also reports some major differences between blood and gut samples. HIV DNA levels are consistently higher in all gut sites compared to peripheral blood mononuclear cells (PBMC) and the differences are highly statistically significant with the exception of the duodenum. And while there is a positive correlation between HIV DNA levels and markers of immune activation in PBMC, there is an inverse correlation in the gut, a novel finding that the researchers suggest may be due to infected cells in the gut being tolerant or anergic (the gut is known to be a site where T cell tolerance against commensal bacteria and food antigens is induced and maintained).

Shari Gordon’s study identifies several parallels between blood and gut. The levels of CD4 T cell depletion in blood correlate directly with the level of depletion measured in all gut sites. Levels of memory CD4 T cell proliferation (measured the Ki67 marker) also directly correlate in both compartments. Gordon finds that the magnitude of CD4 and CD8 T cell proliferation in the blood correlates with the severity of CD4 T cell depletion in the gut. This latter finding indicates that the loss of gut CD4 T cells impacts the normal balance (or “homeostasis”) of the immune system in a way that contributes to systemic immune activation. Encouragingly, the study reveals substantial repopulation of gut CCR5-expressing CD4 T cells in the cohort of individuals on ART and notes that: “in our hands the extent of depletion of intestinal CD4+CCR5+ T cells appeared to be less severe than what has been reported by others during HIV infection.”

The Steven Yukl paper in AIDS describes the impact of intensifying ART (with either raltegravir alone or raltegravir plus efavirenz or darunavir) for 12 weeks in seven of the eight individuals whose baseline values are reported in JID (one dropped out shortly after starting the trial for personal reasons). CD4 T cell counts and HIV DNA levels remained unchanged, but a significant drop in levels of unspliced HIV RNA was seen in the terminal ileum of 5/7 participants. There was also a slight decline in levels of activated CD4 and CD8 T cells in blood and gut, with the greatest change seen in the terminal ileum. The researchers conclude that the findings may be suggestive of some ongoing viral replication occurring in the terminal ileum that was impacted by ART intensification, but they stress that this interpretation needs to be confirmed by larger studies.

These papers add to the evidence that the gut plays an important role in the pathogenesis of HIV infection, but also highlight the need to better understand gut immunology in health as well as disease (an area of research that remains relatively obscure and under-supported). The suggestion that HIV may be establishing latency in tolerant CD4 T cells also has implications for cure research, because targeting this population may require different strategies than those being considered for other HIV reservoirs.

Published online October 12, 2010

The Journal of Infectious Diseases 2010;202:000–000

DOI: 10.1086/656722

MAJOR ARTICLE

Differences in HIV Burden and Immune Activation within the Gut of HIV‐Positive Patients Receiving Suppressive Antiretroviral Therapy

Steven A. Yukl,1,2 Sara Gianella,6,a Elizabeth Sinclair,3,2 Lorrie Epling,3,2 Qingsheng Li,5,a Lijie Duan,5 Alex L. M. Choi,1,2 Valerie Girling,3,2 Terence Ho,3,2 Peilin Li,1,2 Katsuya Fujimoto,1,2 Harry Lampiris,1,2 C. Bradley Hare,3,2 Mark Pandori,4 Ashley T. Haase,5 Huldrych F. Günthard,6 Marek Fischer,6 Amandeep K. Shergill,1,2 Kenneth McQuaid,1,2 Diane V. Havlir,3,2 and Joseph K. Wong1,2

1San Francisco Veterans Affairs Medical Center, 2University of California, San Francisco, 3San Francisco General Hospital, and 4Department of Public Health, San Francisco, California; 5University of Minnesota, Minneapolis; 6Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, Switzerland

Background. The gut is a major reservoir for human immunodeficiency virus (HIV) in patients receiving antiretroviral therapy (ART). We hypothesized that distinct immune environments within the gut may support varying levels of HIV.

Methods. In 8 HIV‐1‐positive adults who were receiving ART and had CD4+ T cell counts of >200 cells/μL and plasma viral loads of <40 copies/mL, levels of HIV and T cell activation were measured in blood samples and endoscopic biopsy specimens from the duodenum, ileum, ascending colon, and rectum.

Results. HIV DNA and RNA levels per CD4+ T cell were higher in all 4 gut sites compared with those in the blood. HIV DNA levels increased from the duodenum to the rectum, whereas the median HIV RNA level peaked in the ileum. HIV DNA levels correlated positively with T cell activation markers in peripheral blood mononuclear cells (PBMCs) but negatively with T cell activation markers in the gut. Multiply spliced RNA was infrequently detected in gut, and ratios of unspliced RNA to DNA were lower in the colon and rectum than in PBMCs, which reflects paradoxically low HIV transcription, given the higher level of T cell activation in the gut.

Conclusions. HIV DNA and RNA are both concentrated in the gut, but the inverse relationship between HIV DNA levels and T cell activation in the gut and the paradoxically low levels of HIV expression in the large bowel suggest that different processes drive HIV persistence in the blood and gut.

AIDS. 2010 Oct 23;24(16):2451-60.

Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy.

Yukl SA, Shergill AK, McQuaid K, Gianella S, Lampiris H, Hare CB, Pandori M, Sinclair E, Günthard HF, Fischer M, Wong JK, Havlir DV.

a San Francisco VA Medical Center (SFVAMC) and University of California, San Francisco (UCSF), San Francisco, California, USA b University Hospital Zurich, Division of Infectious Diseases and Hospital Epidemiology, University of Zurich, Zurich, Switzerland c San Francisco General Hospital and University of California, San Francisco (UCSF), USA d Department of Public Health, San Francisco, California, USA.

Abstract

OBJECTIVE: To determine whether raltegravir-containing antiretroviral therapy (ART) intensification reduces HIV levels in the gut.

DESIGN: Open-label study in HIV-positive adults on ART with plasma HIV RNA below 40 copies/ml.

METHODS: Seven HIV-positive adults received 12 weeks of ART intensification with raltegravir alone or in combination with efavirenz or darunavir. Gut cells were obtained by upper and lower endoscopy with biopsies from duodenum, ileum, colon, and rectum at baseline and 12 weeks. Study outcomes included plasma HIV RNA, HIV DNA and RNA from peripheral blood mononuclear cells (PBMC) and four gut sites, T-cell subsets, and activation markers.

RESULTS: Intensification produced no consistent decrease in HIV RNA in the plasma, PBMC, duodenum, colon, or rectum. However, five of seven participants had a decrease in unspliced HIV RNA per 10 CD4 T cells in the ileum. There was a trend towards decreased T-cell activation in all sites, which was greatest for CD8 T cells in the ileum and PBMC, and a trend towards increased CD4 T cells in the ileum.

CONCLUSION: Most HIV RNA and DNA in the blood and gut is not the result of ongoing replication that can be impacted by short-term intensification with raltegravir. However, the ileum may support ongoing productive infection in some patients on ART, even if the contribution to plasma RNA is not discernible.

Published online October 1, 2010

The Journal of Immunology, 2010, doi:10.4049/jimmunol.1001801

Disruption of Intestinal CD4+ T Cell Homeostasis Is a Key Marker of Systemic CD4+ T Cell Activation in HIV-Infected Individuals

Shari N. Gordon, Barbara Cervasi, Pamela Odorizzi, Randee Silverman, Faten Aberra, Gregory Ginsberg, Jacob D. Estes, Mirko Paiardini, Ian Frank, and Guido Silvestri

Department of Pathology and Laboratory Medicine and  Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104;  Animal Models and Vaccine Section and AIDS Vaccine Section, SAIC–Frederick, National Cancer Institute, National Institutes of Health, Frederick, MD, 20892; and  Yerkes National Primate Research Center, Emory University, Atlanta, GA 30329

HIV infection is associated with depletion of intestinal CD4+ T cells, resulting in mucosal immune dysfunction, microbial translocation, chronic immune activation, and progressive immunodeficiency. In this study, we examined HIV-infected individuals with active virus replication (n = 15), treated with antiretroviral therapy (n = 13), and healthy controls (n = 11) and conducted a comparative analysis of T cells derived from blood and four gastrointestinal (GI) sites (terminal ileum, right colon, left colon, and sigmoid colon). As expected, we found that HIV infection is associated with depletion of total CD4+ T cells as well as CD4+CCR5+ T cells in all GI sites, with higher levels of these cells found in ART-treated individuals than in those with active virus replication. While the levels of both CD4+ and CD8+ T cell proliferation were higher in the blood of untreated HIV-infected individuals, only CD4+ T cell proliferation was significantly increased in the gut of the same patients. We also noted that the levels of CD4+ T cells and the percentages of CD4+Ki67+ proliferating T cells are inversely correlated in both blood and intestinal tissues, thus suggesting that CD4+ T cell homeostasis is similarly affected by HIV infection in these distinct anatomic compartments. Importantly, the level of intestinal CD4+ T cells (both total and Th17 cells) was inversely correlated with the percentage of circulating CD4+Ki67+ T cells. Collectively, these data confirm that the GI tract is a key player in the immunopathogenesis of HIV infection, and they reveal a strong association between the destruction of intestinal CD4+ T cell homeostasis in the gut and the level of systemic CD4+ T cell activation.

Evidence for Sporadic Low-Level HIV Replication on ART

Una O’Doherty’s research group at the University of Pennsylvania has pioneered the development of tests to measure HIV DNA that is integrated into the genome of cells. The integration of HIV DNA presents a formidable obstacle to curing HIV infection, because it leads to the formation of a stable reservoir of infected cells that antiretroviral therapies cannot eradicate. HIV DNA can also exist in cells in an unintegrated form, but evidence indicates that this form degrades rapidly and does not persist. In a new paper in the journal Virology, O’Doherty and colleagues describe analyses designed to assess levels of unintegrated vs. integrated HIV DNA in a small group of individuals on ART with viral loads less than 75 copies for at least a year. The researchers report that three out of seven study participants showed sporadic excesses of the unintegrated form of HIV DNA compared to integrated DNA. They note that this result may indicate that a subset of individuals on ART experience sporadic low-level viral replication, which could contribute to the replenishment of their HIV reservoir. If confirmed, the findings will need to be taken into account by scientists working on approaches to curing HIV infection.

Virology. 2010 Oct 20. [Epub ahead of print]

Patients on HAART often have an excess of unintegrated HIV DNA: Implications for monitoring reservoirs.

Agosto LM, Liszewski MK, Mexas A, Graf E, Pace M, Yu JJ, Bhandoola A, O'Doherty U.

Graduate Program in Microbiology, Virology and Parasitology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

Abstract

HIV establishes a latent reservoir early in infection that is resistant to anti-retroviral therapy and has a slow rate of decay. It is thought that the majority of HIV DNA in treated patients is integrated since unintegrated HIV DNA appears to be unstable. Thus, to monitor the HIV latent reservoir, total HIV DNA is commonly measured in PBMC from infected individuals. We investigated how often total approaches integrated HIV DNA in treated patients. To do this, we first assessed how accurate our integration assay is and determined the error in our measurements of total and integrated HIV DNA. We demonstrated an excess of total over integrated HIV DNA was present in a subset of patients, suggesting that measurements of total HIV DNA do not always correlate to the level of integration. Determining the cause of this excess and its frequency may have important implications for understanding HIV latent reservoir maintenance.

Genetic Analyses Reveal Key Mechanism of HIV Control

A new study published online in Science Express last week reports a breakthrough in understanding the association between particular immune response genes and the ability of some individuals to control HIV replication without treatment. The research was conducted through the International HIV Controllers Study, a huge collaborative effort initiated by Bruce Walker from the Ragon Institute of Massachusetts General Hospital in Boston. The study collects samples from individuals who control viral load without treatment, either to less than 50 copies (elite controllers) or less than 2,000 copies (viremic controllers). A team led by Harvard geneticist Paul De Bakker conducted the analyses that led to the latest findings.

The findings relate to immunological alarms called class I HLA receptors, which display protein fragments (called peptides) from pathogens on the outside of infected cells in order to flag the cell for destruction by CD8 T cells (aka “killer” T cells). All cells in the human body except red blood cells are adorned with thousands of class I HLA receptors, and they are continually shuttling from the inside to the outside of the cell carrying peptides for passing CD8 T cells to inspect. Most of the time these peptides are from our own body’s proteins--essentially cellular trash--and CD8 T cells allow the cell to go about its business. But if a class I HLA receptor emerges with peptide from a pathogen, CD8 T cells will typically respond by destroying it.

There are a wide variety of different genes that make (or in genetics language “encode”) class I HLA receptors, all located in the MHC region of the human genome on chromosome six. The specific HLA genes an individual possesses are inherited from their parents. Different HLA genes make class I HLA receptors with slightly different structures, which determines the types of peptide structures the receptor can present, which in turn can influence the ability of CD8 T cells to recognize the presented peptide.

It has been known for more than a decade that certain HLA genes make receptors that seem particularly adept at presenting HIV peptides to CD8 T cells, and that possessing these HLA genes greatly increases the chances of becoming an elite controller. Conversely, some other HLA genes are associated with high HIV viral loads and rapid disease progression. However the detailed mechanisms underlying these associations had not been figured out.

The new study in Science drills down to specific structural features that are shared by class I HLA receptors encoded by the HLA genes associated with elite control. The paper includes a visual representation of a class I HLA receptor showing the locations of these features, which consist of different amino acids (the building blocks out of which proteins are made). The image is reproduced here, and as you can see the receptor looks a little like a pair of jaws; the mouthpart is referred to as a “binding pocket” that peptides slot into.

CREDIT: The International HIV Controllers Study, Science express November 4, 2010 10.1126/science.1195271 (reproduced with permission)

The crux of the new finding is that the type of amino acid located at the numbered positions has a huge impact on the likelihood of an HIV-infected person controlling their viral load without treatment. Exactly why this is has yet to be fully explained, but some previous evidence that suggests these class I HLA receptors are able to present parts of HIV that cannot mutate without compromising the ability of the virus to replicate. There are also studies showing that the functionality of CD8 T cells can be influenced by the specific viral epitope they recognize; in other words, CD8 T cells that recognize viral epitopes presented by these class I HLA receptors may simply work better than others. Gaining a better understanding of exactly how these specific amino acids confer benefit is now a priority for the research team.  

Another important aspect of the study is that it provides a unifying explanation for the associations between a several different HLA genes and HIV control that have been reported previously. What the genes all have in common is that they encode class I HLA receptors with one or more of the structural features De Bakker and colleagues identified.

The publication of the paper last week garnered a great deal of well-deserved coverage in the mainstream media. However, as is always the case, some stories are more accurate than others. One aspect that may not have come through clearly is that while possessing these class I HLA receptor variants massively increases the chances of becoming an elite controller, it does not mean that it is certain; some individuals who have them do not control viral load, and conversely they are absent in some elite controllers. This is not really surprising, as CD8 T cells work in concert with other components of the immune system such as CD4 T cells, and these other immune responses can also impact how well HIV is controlled. Another recent paper by Hendrick Streeck from Bruce Walker’s group--just published online in the Journal of Virology--provides an example by showing that HIV-specific CD4 T cells that produce IL-21 influence the function of HIV-specific CD8 T cell responses and are associated with lower viral loads.

What can be stated with certainty is that the International HIV Controllers Study is providing crucial insights into the mechanisms that underlie immune control of HIV. Close to four pages of the Science Express paper are taken up by the names of individuals and institutions that have referred people to the study, indicating the broad support for this research. Although they can’t be named in the paper, the many altruistic study participants who donated samples must be saluted also.

Published Online November 4, 2010

Science DOI: 10.1126/science.1195271

Science Express Index

REPORTS

The Major Genetic Determinants of HIV-1 Control Affect HLA Class I Peptide Presentation

The International HIV Controllers Study*

Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and analyzed the effects of individual amino acids within the classical HLA proteins. We identified >300 genome-wide significant SNPs within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA-viral peptide interaction as the major factor modulating durable control of HIV infection.

* All authors with their contributions and affiliations appear at the end of this paper.

JVI Accepts, published online ahead of print on 3 November 2010

J. Virol. doi:10.1128/JVI.02030-10

HIV-1-specific IL-21+ CD4+ T cell responses contribute to durable viral control through the modulation of HIV-specific CD8+ T cell function

Mathieu F. Chevalier, Boris Julg, Augustine Pyo, Michael Flanders, Srinika Ranasinghe, Damien Z. Soghoian, Douglas S. Kwon, Jenna Rychert, Jeffrey Lian, Matthias Mueller, Sam Cutler, Elizabeth McAndrew, Heiko Jessen, Florencia Pereyra, Eric S. Rosenberg, Marcus Altfeld, Bruce D. Walker, and Hendrik Streeck*

Ragon Institute of MGH, MIT and Harvard, Massachusetts General Hospital and Harvard University, Boston, Massachusetts, United States of America; Infectious Disease Unit, Department of Pathology, Massachusetts General Hospital, HIV clinic Jessen-Jessen-Stein, Berlin, Germany; Howard Hughes Medical Institute, Chevy Chase, Maryland, United States of America

Abstract

Functional defects in cytotoxic CD8+ T cell (CTL) responses arise in chronic human viral infections, but the mechanisms involved are not well understood. In mice, CD4 cell mediated IL-21 production is necessary for the maintenance of CD8+ T cell function and control of persistent viral infections. To investigate the potential role of IL-21 in a chronic human viral infection, we studied the rare subset of HIV-1 controllers, who are able to spontaneously control HIV-1 replication without treatment. HIV-specific triggering of IL-21 by CD4+ T-cells was significantly enriched in these persons (p=0.0007) while isolated loss of IL-21 secreting CD4+ T-cells was characteristic for subjects with persistent viremia and progressive disease. IL-21 responses were mediated by recognition of discrete epitopes largely in the Gag protein, and expansion of IL-21+CD4+ T cells in acute infection resulted in lower viral set-points (p=0.002). Moreover, IL-21 production by CD4+ T cells of HIV controllers enhanced perforin production by HIV-1-specific CD8+ T cells from chronic progressors even in late stages of disease, and HIV-1-specific effector CD8+ T cells showed enhanced ability to efficiently inhibit viral replication in-vitro after IL-21 binding. These data suggest that HIV-1-specific IL-21+CD4+ T cell responses might contribute to the control of viral replication in humans and are likely to be of great importance for vaccine design.