TAG HIV Basic Science, Vaccines, and Cure Project Blog

Jostling Latent HIV from Slumber

A study in the new issue of the Journal of Immunology suggests that triggering a cell surface molecule called toll-like receptor 8 (TLR8) may be a means to activating latent HIV infection. Erika Schlaepfer and Roberto Speck report that, in vitro, targeting TLR8 with the drug resiquimod (aka R-848) prompted HIV activity in latently infected cells of myeloid-monocytic origin (which include monocytes, macrophages, dendritic cells, microglial cells, and hematopoietic stem cells) and also had an activating effect on HIV in latently infected CD4 T cells, by causing production of the cytokine TNF-alpha. The researchers took a very preliminary look at whether individuals on HAART might be able to respond to such an approach, and found that--solely based on TNF-alpha production--their monocytes reacted to TLR8 stimulation comparably to those from HIV negative controls. 

The conclusion from the findings is that “TLR8 agonists, in combination with HAART, are intriguing compounds for purging HIV from its latent reservoirs and sanctuary sites.” Schlaepfer and Speck caution, however, that “we believe that compounds, and, in particular, TLR8 agonists, acting on the latent reservoir should be given in cycles, because a longer-term administration of any such compound might be too toxic.” Resiquimod has been studied in humans with hepatitis C infection and appeared quite potent in terms of inducing alpha interferon production (and associated side effects). The manufacturer, 3M, does not appear to be developing it further but has recently offered it to the pharmaceutical industry for license. Development of a TLR7 agonist as a hepatitis C treatment was stopped in 2007 due to toxicology studies showing "intense immune stimulation in animals."

The Journal of Immunology, April 1, 2011, vol. 186 no. 7 4314-4324

TLR8 Activates HIV from Latently Infected Cells of Myeloid-Monocytic Origin Directly via the MAPK Pathway and from Latently Infected CD4+ T Cells Indirectly via TNF-α

Erika Schlaepfer and Roberto F. Speck

Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, 8091 Zurich, Switzerland

Abstract

We previously showed that the TLR7/8 agonist, R-848, activated HIV from cells of myeloid-monocytic origin. In this work, we show that this effect was solely due to triggering TLR8 and that NF-κB was involved in the TLR8-mediated activation of HIV from latently infected cells of myeloid-monocytic origin. Inhibition of Erk1/2 or p38α resulted in attenuation of TLR8-mediated activation of NF-κB. Western blots confirmed that TLR8 triggering activated Erk1/2 and p38α but, surprisingly, not JNK. Although the Erk1/2 inhibitors resulted in a less attenuated TLR8-mediated NF-κB response than did p38α inhibitors, they had a more pronounced effect on blocking TLR8-mediated HIV replication, indicating that other transcription factors controlled by Erk1/2 are involved in TLR8-mediated HIV activation from latently infected cells. TNF-α, which was secreted subsequent to TLR8 triggering, contributed to the activation of HIV from the latently infected cells in an autocrine manner, revealing a bimodal mechanism by which the effect of TLR8 triggering can be sustained. We also found that TNF-α secreted by myeloid dendritic cells acted in a paracrine manner in the activation of HIV from neighboring latently infected CD4+ T cells, which do not express TLR8. Notably, monocytes from highly active antiretroviral therapy-treated HIV+ patients with suppressed HIV RNA showed a robust TNF-α secretion in response to TLR8 agonists, pointing to a functional TLR8 signaling axis in HIV infection. Thus, triggering TLR8 represents a very promising strategy for attacking the silent HIV from its reservoir in HIV+ patients treated successfully with highly active antiretroviral therapy.

CD8 T Cells Targeting HIV Can Be Killers After All

In a prior post from last year, I reported on some controversy regarding the mechanisms by which CD8 T cells inhibit HIV and SIV replication. Two studies had been published suggesting that the main mechanism is not direct killing of infected cells--as generally believed--but rather the release of soluble antiviral factors such as chemokines. But as mentioned at the time, the authors of both papers noted there was evidence indicating that CD8 T cells can kill virus-infected cells prior to the release of new virions, which would render their direct cytotoxic activity invisible in the particular experimental systems that were used in these studies.

In a paper published last month in PLoS One, Christian Althaus and Rob De Boer explicitly model this scenario mathematically. The model shows that, indeed, early CD8 T cell killing is entirely compatible with the previously reported results. The authors write:

“Here we have formally demonstrated that when CTL-mediated killing occurs early during the intracellular eclipse phase it can control HIV replication very efficiently, while remaining consistent with current observations on the up-slopes and down-slopes of the viral load observed during CD8+ T cell depletion experiments and antiretroviral treatment.”

PLoS One. 2011 Feb 7;6(2):e16468.

Implications of CTL-mediated killing of HIV-infected cells during the non-productive stage of infection.

Althaus CL, De Boer RJ.

Theoretical Biology, Utrecht University, Utrecht, The Netherlands.

Abstract

Patients infected with HIV exhibit orders of magnitude differences in their set-point levels of the plasma viral load. As to what extent this variation is due to differences in the efficacy of the cytotoxic T lymphocyte (CTL) response in these patients is unclear. Several studies have shown that HIV-infected CD4+ T cells also present viral epitopes that are recognized by CTLs before the productive stage of infection, i.e., during the intracellular eclipse phase before the infected cell starts to produce new viral particles. Here, we use mathematical modeling to investigate the potential impact of early killing of HIV-infected cells on viral replication. We suggest that the majority of CTL-mediated killing could occur during the viral eclipse phase, and that the killing of virus-producing cells could be substantially lower at later stages due to MHC-I-down-regulation. Such a mechanism is in agreement with several experimental observations that include CD8+ T cell depletion and antiretroviral drug treatment. This indicates a potentially important role of CTL-mediated killing during the non-productive stage of HIV-infected cells.

Reduced HIV Replication in CD4 T Cells from Elite Controllers

A number of studies have investigated whether control of HIV replication in the absence of treatment (referred to as elite control if viral load is <50 copies and viremic control if <2,000) is associated with having CD4 T cells that are intrinsically resistant to infection. Results to date have shown that elite and viremic controllers have CD4 T cells that support viral replication just as well as those sampled from individuals with progressive disease. However, Mathias Lichterfeld’s laboratory at the Ragon Institute of Massachussetts General Hospital wondered if perhaps the typical conditions for measuring HIV replication in CD4 T cells in the lab—which involve the use of strong activation stimuli such as PHA—were masking subtler differences. They now report on their exploration of this possibility in the Journal of Clinical Investigation.

The study finds that CD4 T cells from elite controllers resist HIV infection and viral replication significantly better than those from individuals with progressing disease and HIV-negative controls. The same is true for viremic controllers, but in their case the differences are smaller. The pattern remains consistent using a variety of different approaches for measuring HIV infection and replication in vitro, and holds for both R5- and X4-tropic HIV isolates. The researchers, led jointly by Huabiao Chen, Chun Li, Jinghe Huang, and Thai Cung from the lab, go on to uncover that increased expression of a host cell protein called p21 in CD4 T cells from controllers correlates with reduced susceptibility to HIV. This is further confirmed by experiments showing that inhibition of p21 expression using a short interfering RNA substantially abrogates the apparent protective effect. In terms of the causative pathway, the researchers offer evidence that p21 blocks another host cell protein, cyclin-dependent kinase 9 (CDK9), which according to prior studies plays an important role in HIV transcription.

The discussion section of the paper points out that p21 expression has previously been connected to inhibition of HIV in macrophages and hematopoietic stem cells. The authors speculate that the reduced susceptibility of controller CD4 T cells synergizes with other reported mechanisms of immunological control, noting specifically that “a highly functional CD8+ T cell response against HIV-1 may only develop in the setting of a CD4+ T cell compartment that is less capable of supporting highly replicative HIV-1 infection.” Broadly similar findings were also reported at the recent CROI conference in a poster by an independent group at the Institut Pasteur in France.

The discovery of p21's role in controllers may open new avenues for inhibiting HIV by targeting its interactions with host cell proteins. It also provides encouragement for strategies aiming to induce CD4 T cell resistance to the virus (such as the CCR5-deletion approach being developed by Sangamo Biosciences), because it implies that these cells would then be better able to support the function of CD8 T cells and possibly other components of the immune response against HIV.

J Clin Invest. doi:10.1172/JCI44539.

Research Article

CD4+ T cells from elite controllers resist HIV-1 infection by selective upregulation of p21

Huabiao Chen1,2, Chun Li1,3, Jinghe Huang1, Thai Cung1, Katherine Seiss1, Jill Beamon1, Mary F. Carrington1, Lindsay C. Porter1, Patrick S. Burke1, Yue Yang1, Bethany J. Ryan1, Ruiwu Liu4, Robert H. Weiss5, Florencia Pereyra1, William D. Cress6, Abraham L. Brass1, Eric S. Rosenberg7, Bruce D. Walker1,2, Xu G. Yu1 and Mathias Lichterfeld7

1Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Boston, Massachusetts, USA.

2Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.

3Program of Biological Sciences in Dental Medicine, Harvard University, Cambridge, Massachusetts, USA.

4Department of Biochemistry and Molecular Medicine and

5Division of Nephrology, Department of Medicine, UCD, Davis, California, USA.

6Department of Molecular Oncology, Lee Moffitt Cancer Center, Tampa, Florida, USA.

7Infectious Disease Division, Massachusetts General Hospital, Boston, Massachusetts, USA.

Authorship note: Huabiao Chen, Chun Li, Jinghe Huang, and Thai Cung contributed equally to this work.

Elite controllers represent a unique group of HIV-1–infected persons with undetectable HIV-1 replication in the absence of antiretroviral therapy. However, the mechanisms contributing to effective viral immune defense in these patients remain unclear. Here, we show that compared with HIV-1 progressors and HIV-1–negative persons, CD4+ T cells from elite controllers are less susceptible to HIV-1 infection. This partial resistance to HIV-1 infection involved less effective reverse transcription and mRNA transcription from proviral DNA and was associated with strong and selective upregulation of the cyclin-dependent kinase inhibitor p21 (also known as cip-1 and waf-1). Experimental blockade of p21 in CD4+ T cells from elite controllers resulted in a marked increase of viral reverse transcripts and mRNA production and led to higher enzymatic activities of cyclin-dependent kinase 9 (CDK9), which serves as a transcriptional coactivator of HIV-1 gene expression. This suggests that p21 acts as a barrier against HIV-1 infection in CD4+ T cells from elite controllers by inhibiting a cyclin-dependent kinase required for effective HIV-1 replication. These data demonstrate a mechanism of host resistance to HIV-1 in elite controllers and may open novel perspectives for clinical strategies to prevent or treat HIV-1 infection.

Intensifying Treatment Does Not Reduce HIV Reservoirs, but Gut Immune Responses May Have a Role to Play

A study led by Hiroyu Hatano from Steve Deeks’s group at UCSF reports that adding the integrase inhibitor raltegravir (Isentress) to a standard antiretroviral (ART) regimen failed to reduce residual HIV replication, or the HIV reservoir, in a group of individuals with suboptimal CD4 count increases despite prolonged HIV suppression. The intensification approach also did not significantly increase CD4 T cell counts or reduce levels of immune activation, which the authors note adds to the evidence that low-level HIV replication is unlikely to be a major cause of suboptimal CD4 T cell gains in people on ART.

Interestingly, a secondary analysis--conducted in collaboration with Barbara Shacklett’s laboratory at UC Davis--revealed a novel association between the magnitude of HIV Gag-specific CD8 T cell responses in gut-associated lymphoid tissue (GALT) and the size of the HIV reservoir (as measured by proviral DNA in peripheral blood mononuclear cells). Hatano and colleagues caution that the findings are preliminary and require confirmation by larger studies, but also write: “approaches aimed at expanding HIV-specific CD4+ and CD8+ T cell responses in the gut mucosa may accelerate clearance of the viral reservoir. The next logical step would be to pursue therapeutic vaccine studies using HIV vaccines that elicit strong mucosal T cell responses in HAART-treated patients.”

Coincidentally, a paper that appeared online today in the journal Blood describes an approach to augmenting HIV-specific CD8 T cell immunity in GALT. The researchers sampled HIV-specific CD8 T cells from individuals on ART, expanded them in the laboratory, and then re-infused them. Persistence up to 84 days was documented, as well as trafficking to GALT. Many of the cells displayed a “central memory” phenotype, which is believed to be important due to the ability of these cells to self-renew and proliferate robustly upon encountering antigen. Although the infusion approach has practical limitations, the study authors suggest that their results imply that vaccination strategies aiming to boost systemic and GALT HIV-specific CD8 T cell responses are worth pursuing.

J Infect Dis. (2011) 203 (7): 894-897. doi: 10.1093/infdis/jiq150

Hide and Seek… Can We Eradicate HIV by Treatment Intensification?

Julian Schulze zur Wiesch1,2 and Jan van Lunzen1,2 1University Medical Center, Hamburg-Eppendorf, Infectious Diseases Unit 2Heinrich-Pette-Institute Leibniz Institute for Experimental Virology, Hamburg, Germany 

(See the article by Hatano et al., on pages 960–68.)

J Infect Dis. (2011) 203 (7): 960-968. doi: 10.1093/infdis/jiq138

A Randomized, Controlled Trial of Raltegravir Intensification in Antiretroviral-treated, HIV-infected Patients with a Suboptimal CD4+ T Cell Response

Hiroyu Hatano1, Timothy L. Hayes2, Viktor Dahl3, Elizabeth Sinclair1, Tzong-Hae Lee4, Rebecca Hoh1, Harry Lampiris1,5, Peter W. Hunt1, Sarah Palmer3, Joseph M. McCune1, Jeffrey N. Martin1, Michael P. Busch1,4, Barbara L. Shacklett2 and Steven G. Deeks1

1University of California, San Francisco 2University of California, Davis 3Karolinska Institutet and Swedish Institute for Infectious Disease Control, Solna, Sweden 4Blood Systems Research Institute, San Francisco 5San Francisco VA Medical Center, California Reprints or correspondence: Hiroyu Hatano, MD, San Francisco General Hospital, Bldg 80, Ward 84, 995 Potrero Ave, San Francisco, CA 94110.

Abstract

Background. Some human immunodeficiency virus (HIV)–infected individuals are not able to achieve a normal CD4+ T cell count despite prolonged, treatment-mediated viral suppression. We conducted an intensification study to assess whether residual viral replication contributes to replenishment of the latent reservoir and whether mucosal HIV-specific T cell responses limit the reservoir size.

Methods. Thirty treated subjects with CD4+ T cell counts of <350 cells/mm3 despite viral suppression for ≥1 year were randomized to add raltegravir (400 mg twice daily) or matching placebo for 24 weeks. The primary end points were the proportion of subjects with undetectable plasma viremia (determined using an ultrasensitive assay with a lower limit of detection of <.3 copy/mL) and a change in the percentage of CD38+HLA-DR+CD8+ T cells in peripheral blood mononuclear cells (PBMCs).

Results. The proportion of subjects with undetectable plasma viremia did not differ between the 2 groups (P = .42). Raltegravir intensification did not have a significant effect on immune activation or HIV-specific responses in PBMCs or gut-associated lymphoid tissue.

Conclusions. Low-level viremia is not likely to be a significant cause of suboptimal CD4+ T cell gains during HIV treatment.

Blood First Edition Paper, prepublished online March 21, 2011; DOI 10.1182/blood-2010-11-320226.

HIV-specific CD8+ T cells from HIV+ individuals receiving HAART can be expanded ex vivo to augment systemic and mucosal immunity in vivo

Aude G. Chapuis1, Corey Casper2, Steve Kuntz2, Jia Zhu2, Annelie Tjernlund3, Kurt Diem2, Cameron J. Turtle1, Melinda L. Cigal1, Roxanne Velez1, Stanley Riddell1, Lawrence Corey2 and Philip D. Greenberg1

1 Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA, United States; 2 Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, United States; 3 Department of Medicine, Solna, Infectious Disease Unit, Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden

Abstract

Most HIV+ individuals require lifelong highly active anti-retroviral therapy (HAART) to suppress HIV replication, but fail to eliminate the virus in part because of residual replication in gut-associated lymphoid tissues (GALT). Naturally-elicited HIV-specific CD8+T-cells generated in the acute and chronic infectious phases exhibit anti-viral activity, but decrease in number following HAART. Therapeutic vaccines represent a potential strategy to expand cellular responses, although previous efforts have been largely unsuccessful, conceivably due to a lack of responding HIV-specific central-memory CD8+T-cells (Tcm). To determine if patients receiving HAART possess CD8+T-cells with Tcm qualities that are amenable to augmentation, HIV-specific CD8+T-cell clones were derived from HIV-reactive CD28+CD8+T-cell lines isolated from 7 HIV+ HAART-treated patients, expanded ex vivo, and re-infused into their autologous host. Tracking of the cells in vivo revealed that clones could persist for 84 days, maintain expression and/or re-express CD28, up-regulate CD62L, secrete IL-2, proliferate upon cognate antigen encounter and localize to the rectal mucosa. These results suggest some infused cells exhibited phenotypic and functional characteristics shared with Tcm in vivo, and imply that more effective therapeutic vaccination strategies targeting CD8+Tcm in patients on HAART might provide hosts with expanded, long-lasting immune responses not only systemically but also in GALT.

Revisiting the Statistics of the RV144 HIV Vaccine Efficacy Trial in Thailand

In the new issue of the Journal of Infectious Diseases, a group of researchers headed by Peter Gilbert take a statistical tour around the results of the RV144 HIV vaccine efficacy trial in Thailand, in order to draw lessons for future research. When the trial results were first announced, there was some controversy over whether the analysis presented – called the modified intent-to-treat (MITT) – was appropriate. The Gilbert paper echoes many commentators at the time by concluding that this is, in fact, the standard and appropriate primary analysis for a vaccine efficacy trial, as it excludes individuals who were subsequently found to be HIV-infected at baseline (who are included in the strict intent-to-treat analysis).

However, using Bayesian statistics, the researchers show that there is considerably more uncertainty about the robustness of the 31% efficacy estimate than has been cited to date in public discourse about the trial. They write: “these considerations lead to our conclusion that the RV144 data provide moderate evidence of low-level positive VE [vaccine efficacy]– with ≥22% chance remaining for no efficacy under a range of prior assumptions− an inference that reflects greater uncertainty than has much of the discussion about this trial.” Because Bayesian statistics can consider the impact of prior beliefs, the paper is able to also note that “the skeptic who assigns prior chance of 90% that the vaccine is ineffective will conclude after seeing the RV144 data that there remains a 70% chance that the vaccine is ineffective.”

Despite the uncertainty about the results, the researchers take pains to emphasize that the evidence of efficacy is sufficiently compelling to justify confirmatory trials. They suggest that the use of Bayesian statistical analyses be considered in these trials, and also argue for additional efforts to improve adherence to vaccine regimens, noting that the per-protocol analyses of RV144 – which is limited to only those participants who received immunizations on the exact schedule (or very close to it) – was underpowered and difficult to interpret because of a dramatic 24% reduction in the analyzed population.

J Infect Dis. (2011) 203 (7): 969-975.

doi: 10.1093/infdis/jiq152

Statistical Interpretation of the RV144 HIV Vaccine Efficacy Trial in Thailand: A Case Study for Statistical Issues in Efficacy Trials

Peter B. Gilbert1,2, James O. Berger4, Donald Stablein5, Stephen Becker3, Max Essex7, Scott M. Hammer9, Jerome H. Kim6 and Victor G. DeGruttola8

1Vaccine Infectious Disease Division, Fred Hutchinson Cancer Research Center

2Department of Biostatistics, University of Washington

3Bill and Melinda Gates Foundation, Seattle, Washington

4Department of Statistical Science, Duke University, Durham, North Carolina

5The EMMES Corporation

6Department of Molecular Virology and Pathogenesis, Division of Retrovirology, Walter Reed Army Institute of Research, US Military HIV Research Program, Rockville, Maryland

7Harvard School of Public Health AIDS Initiative, Department of Immunology and Infectious Diseases

8Department of Biostatistics, Harvard School of Public Health, Boston, Massachusetts

9Division of Infectious Diseases, Columbia University, College of Physicians and Surgeons, New York, New York

Reprints or correspondence: Peter Gilbert, PhD, Vaccine Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, PO Box 19024, Seattle, WA 98109.

Abstract

Recently, the RV144 randomized, double-blind, efficacy trial in Thailand reported that a prime-boost human immunodeficiency virus (HIV) vaccine regimen conferred ∼30% protection against HIV acquisition. However, different analyses seemed to give conflicting results, and a heated debate ensued as scientists and the broader public struggled with their interpretation. The lack of accounting for statistical principles helped flame the debate, and we leverage these principles to provide a more scientific interpretation. We first address interpretation of frequentist results, including interpretation of P values, synthesis of results from multiple analyses (ie, intention-to-treat versus per-protocol/fully immunized), and accounting for external efficacy trials. Second, we address how Bayesian statistics, which provide clearly interpretable statements about probabilities that the vaccine efficacy takes certain values, provide more information for weighing the evidence about efficacy than do frequentist statistics alone. Third, we evaluate RV144 for completeness of end point ascertainment and integrity of blinding, necessary tasks for establishing robustly interpretable results.

The Immunological Effects of Old Age and Anti-CMV Immunity in HIV Infection

Following on the heels of yesterday’s post, a study has just been published by the journal AIDS that echoes the same themes. The researchers, led by Victor Appay, find that depletion of naïve CD4 and CD8 T cells represents the major parallel between HIV infection and aging and, like Beth Jamieson’s group, they note that the effects are additive. The researchers also report that immune responses to another chronic infection – CMV – are associated with reduced naïve T cell reconstitution in people with HIV on antiretroviral therapy, consistent with the idea that chronic infections can place a drain on naïve T cell resources (CMV infection has also been associated with reduced naïve T cell levels in individuals not infected with HIV). Additionally, Appay and colleagues look at markers of T cell senescence in HIV and aging, but find that the parallels are not as clear as those documented for naïve T cells.

AIDS. 2011 Mar 15. [Epub ahead of print]

Old age and anti-CMV immunity are associated with altered T cell reconstitution in HIV-1 infected patients.

Appay V, Fastenackels S, Katlama C, Ait-Mohand H, Schneider L, Guihot A, Keller M, Grubeck-Loebenstein B, Simon A, Lambotte O, Hunt PW, Deeks SG, Costagliola D, Autran B, Sauce D.

a INSERM UMR S 945, Infections and Immunity, Avenir Group, Université Pierre et Marie Curie-Paris6, Hôpital Pitié-Salpêtrière, 75013 Paris, France b AP-HP, Groupe hospitalier Pitié-Salpêtrière, Laboratoire d'Immunologie Cellulaire et Tissulaire, 75013 Paris, France c INSERM UMR S 943, Université Pierre et Marie Curie-Paris6, Hôpital Pitié-Salpêtrière, 75013 Paris, France d AP-HP, Groupe hospitalier Pitié-Salpêtrière, Service des Maladies Infectieuses et Tropicales, 75013 Paris, France e Institute for Biomedical Aging Research, Austrian Academy of Sciences, A-6020 Innsbruck, Austria f AP-HP, Groupe hospitalier Pitié-Salpêtrière, Service de Médecine Interne, 75013 Paris, France g AP-HP, Department of Internal Medicine and Infectious Diseases, Bicêtre Hospital, Bicêtre, France h Université Paris-Sud, INSERM U802, Bicêtre, France i HIV/AIDS Division, Department of Internal Medicine, University of California San Francisco, CA, USA.

Abstract

OBJECTIVE AND DESIGN: Increasing evidence support a parallel between HIV-1 infection and immune aging, which is particularly apparent with common changes in naïve versus memory T cell proportions. Here, we aimed at refining the value of common T cell associated markers of immunosenescence during HIV disease progression or aging, and at exploring further the impact in this context of old age as well as CMV co-infection, which is predominant in HIV-1 infected individuals.

METHODS: Frequencies of naïve or CD57 memory T cells as well as the magnitude of CMV-pp65 T cells were measured in HIV-1 infected patients grouped according to disease progression status, treatment and age.

RESULTS: Our results indicate that the decline in naïve T cell levels rather than the accumulation of CD57 senescent T cells identifies best the premature development of an immunosenescence phenotype with HIV disease progression. Moreover, advanced age or mounting of strong CMV specific responses impact independently on CD4 T cell counts and recovery with antiretroviral therapy.

CONCLUSIONS: The present findings indicate that HIV-1 infection amplifies the effect of age on naive T cell levels, and highlight the constraint on the capacity of treated patients to reconstitute their CD4+ T cell compartment due to age and CMV co-infection.

More Studies on the Loss of Naïve T cells

I wrote recently about a review by Beth Jamieson and Tammy Rickabaugh describing the parallel effects of HIV infection and aging on the pool of naïve T cells in humans. Three recent papers address different aspects of naïve T cell loss, including the first study to document a decrease in this population in people with chronic hepatitis C infection.

In PLoS One, Beth Jamieson’s group reports on a study of naïve CD4 T cell levels in younger (20-32 years) and older (39-58 years) individuals with untreated HIV infection, compared to age-matched HIV-negative controls.  The researchers use a cell surface marker named CD31 to discriminate between naïve CD4 T cells that have recently been produced by the thymus (CD31+) and those that have proliferated in the circulation (CD31-). Consistent with previous studies, HIV infection had a strong effect on naïve CD4 T cell levels that was additive to that seen in aging; the absolute number of CD31+ naïve CD4 T cells in the younger individuals mirrored those measured in HIV-negative controls who were 17-28 years older. While both HIV infection and aging were associated with declines in CD31+ naïve CD4 T cell numbers, loss of CD31- naïve CD4 T cells was only observed HIV infection; in this case the effect was independent of aging as the absolute loss was similar in both the younger and older HIV-positive participants. In a separate longitudinal analysis of the effects of antiretroviral therapy, CD31+ naïve CD4 T cells achieved levels comparable to age-matched controls after two years of treatment. However, CD31- naïve CD4 T cell levels remained significantly reduced.

The researchers also evaluate telomere lengths in both naïve CD4 T cell subsets, finding them to be reduced both as a result of HIV infection and aging; as was seen for CD31+ naïve CD4 T cell numbers, the effects were additive. Jamieson and colleagues conclude by suggesting that their results likely explain why disease progression occurs more rapidly among HIV-positive individuals over the age of 50, because this older population already has reduced numbers of naïve CD4 T cells, making the impact of HIV infection more severe. They also note that incomplete recovery of naïve CD4 T cells may play a role in increasing the risk of aging-associated diseases in people with HIV.

One commonly cited causative mechanism of naïve T cell depletion in HIV is the persistent activation of these cells, which leads to their differentiation into memory cells. Another contributing factor is lymphoid tissue fibrosis (a type of scarring damage associated with immune activation & inflammation). Naïve T cells continually recirculate through lymphoid tissue and depend on signals received in that environment for their survival. A recent study by Ming Zeng and colleagues delves into this link between lymphoid tissue fibrosis and naïve T cell loss in both SIV and HIV infection. 

The researchers find that fibroblastic reticular cells (FRC)--which form the pathways along which T cells travel in lymph nodes--are the major source of IL-7, a cytokine essential for naïve T cell survival. Fibrotic damage (measured by the accumulation of collagen) is shown to disrupt the FRC network and therefore impede the ability of T cells to access IL-7, causing an increase in T cell apoptosis.  Both naïve CD4 and CD8 T cells are affected. Additional studies reveal that the loss of T cells in turn exacerbates the damage to FRCs by reducing the production of a cytokine called lymphotoxin-β, which is vital for maintaining FRC networks. The results suggest that there is a vicious cycle in which fibrosis damages FRCs, which causes T cell loss, which then further exacerbates FRC loss.

Continuing their investigative work, Zeng et al look for a source of collagen and find that production of the cytokine TGF-beta by regulatory T cells is increased in HIV, and TGF-beta induces collagen production by fibroblasts. In lab experiments, the antifibrotic drug pirfenidone blocks TGF-beta signaling and reduces collagen production, leading the researchers to conclude that this drug may deserve consideration as an adjunctive therapy for promoting immune reconstitution in HIV.

Lastly, a study published in the March 1st issue of the Journal of Infectious Diseases demonstrates that another persistent chronic infection, hepatitis C, can accelerate naïve CD4 T cell loss. The authors conclude that their findings provide an explanation for the reduced response to vaccinations observed in people with chronic HCV.

PLoS One. 2011 Jan 26;6(1):e16459.

The Dual Impact of HIV-1 Infection and Aging on Naïve CD4 T-Cells: Additive and Distinct Patterns of Impairment.

Rickabaugh TM, Kilpatrick RD, Hultin LE, Hultin PM, Hausner MA, Sugar CA, Althoff KN, Margolick JB, Rinaldo CR, Detels R, Phair J, Effros RB, Jamieson BD.

Department of Medicine, UCLA AIDS Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

Abstract

HIV-1-infected adults over the age of 50 years progress to AIDS more rapidly than adults in their twenties or thirties. In addition, HIV-1-infected individuals receiving antiretroviral therapy (ART) present with clinical diseases, such as various cancers and liver disease, more commonly seen in older uninfected adults. These observations suggest that HIV-1 infection in older persons can have detrimental immunological effects that are not completely reversed by ART. As naïve T-cells are critically important in responses to neoantigens, we first analyzed two subsets (CD45RA(+)CD31(+) and CD45RA(+)CD31(-)) within the naïve CD4(+) T-cell compartment in young (20-32 years old) and older (39-58 years old), ART-naïve, HIV-1 seropositive individuals within 1-3 years of infection and in age-matched seronegative controls. HIV-1 infection in the young cohort was associated with lower absolute numbers of, and shorter telomere lengths within, both CD45RA(+)CD31(+)CD4(+) and CD45RA(+)CD31(-)CD4(+) T-cell subsets in comparison to age-matched seronegative controls, changes that resembled seronegative individuals who were decades older. Longitudinal analysis provided evidence of thymic emigration and reconstitution of CD45RA(+)CD31(+)CD4(+) T-cells two years post-ART, but minimal reconstitution of the CD45RA(+)CD31(-)CD4(+) subset, which could impair de novo immune responses. For both ART-naïve and ART-treated HIV-1-infected adults, a renewable pool of thymic emigrants is necessary to maintain CD4(+) T-cell homeostasis. Overall, these results offer a partial explanation both for the faster disease progression of older adults and the observation that viral responders to ART present with clinical diseases associated with older adults.

J Clin Invest. doi:10.1172/JCI45157.

Cumulative mechanisms of lymphoid tissue fibrosis and T cell depletion in HIV-1 and SIV infections

Ming Zeng1, Anthony J. Smith1, Stephen W. Wietgrefe1, Peter J. Southern1, Timothy W. Schacker2, Cavan S. Reilly3, Jacob D. Estes4, Gregory F. Burton5, Guido Silvestri6, Jeffrey D. Lifson4, John V. Carlis7 and Ashley T. Haase1

1Department of Microbiology and

2Department of Medicine, Medical School, University of Minnesota, Minneapolis, Minnesota, USA.

3Division of Biostatistics, School of Public Health, University of Minnesota, Minneapolis, Minnesota, USA.

4AIDS and Cancer Virus Program, Science Applications International Corporation–Frederick Inc., National Cancer Institute, Frederick, Maryland, USA.

5Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah, USA.

6Yerkes National Primate Research Center, and Emory University, Atlanta, Georgia, USA.

7Department of Computer Science and Engineering, Institute of Technology, University of Minnesota, Minneapolis, Minnesota, USA.

Authorship note: Ming Zeng and Anthony J. Smith contributed equally to this work.

The hallmark of HIV-1 and SIV infections is CD4+ T cell depletion. Both direct cell killing and indirect mechanisms related to immune activation have been suggested to cause the depletion of T cells. We have now identified a mechanism by which immune activation-induced fibrosis of lymphoid tissues leads to depletion of naive T cells in HIV-1 infected patients and SIV-infected rhesus macaques. The T regulatory cell response to immune activation increased procollagen production and subsequent deposition as fibrils via the TGF-β1 signaling pathway and chitinase 3-like-1 activity in fibroblasts in lymphoid tissues from patients infected with HIV-1. Collagen deposition restricted T cell access to the survival factor IL-7 on the fibroblastic reticular cell (FRC) network, resulting in apoptosis and depletion of T cells, which, in turn, removed a major source of lymphotoxin-β, a survival factor for FRCs during SIV infection in rhesus macaques. The resulting loss of FRCs and the loss of IL-7 produced by FRCs may thus perpetuate a vicious cycle of depletion of T cells and the FRC network. Because this process is cumulative, early treatment and antifibrotic therapies may offer approaches to moderate T cell depletion and improve immune reconstitution during HIV-1 infection.

J Infect Dis. 2011 Mar;203(5):635-45. Epub 2011 Jan 10.

Reduced Naive CD4 T Cell Numbers and Impaired Induction of CD27 in Response to T Cell Receptor Stimulation Reflect a State of Immune Activation in Chronic Hepatitis C Virus Infection.

Yonkers NL, Sieg S, Rodriguez B, Anthony DD.

Department of Pathology.

Abstract

Background. Chronic hepatitis C virus (HCV) infection is characterized by reduced numbers of functional HCV-specific T cells. In addition, chronically HCV-infected individuals have reduced response to vaccine. Alterations in naive CD4 T cell phenotype or function may contribute to these immune impairments. Methods. Using flow cytometric analysis and enzyme-linked immunospot assay, we examined peripheral naive CD4 T cell phenotype and function in chronically HCV-infected patients and control subjects. Results. We observed significantly lower absolute cell numbers of naive CD4 T cells in HCV-infected patients, localized to the CD127(+)CD25(low/-) and CD31(+) (RTE) subsets. Moreover, we found greater percentages of naive cells expressing CD25 and KI67 in HCV-infected patients, consistent with immune activation, further supported by higher plasma sCD27 levels. Functional analysis revealed an intact interferon-γ response to allogeneic B cell stimulus. However, after direct TCR stimulation, naive CD4 T cells from HCV-infected patients had altered up-regulation of KI67 and CD25 and less CD27 expression. The latter was associated with elevated baseline activation state. In addition, naive CD4 T cells from HCV-infected patients were more susceptible to cell death. Conclusions. These numerical and functional defects may contribute to inadequate formation of virus and neoantigen-specific T cell responses during chronic HCV infection.

Sifting Through STEP: New Data from the Merck HIV Vaccine Efficacy Trial

Several new papers about the STEP trial - the efficacy study of Merck’s Ad5-based HIV vaccine candidate - have recently been published. As was widely publicized when STEP was stopped in 2007, the vaccine did not show efficacy in preventing HIV infection or slowing disease progression in vaccinees who became infected, and the Ad5 vector actually enhanced the risk of HIV acquisition in a subset of uncircumcised participants with high baseline titers of antibodies against Ad5.

Despite this failure, the newly published analyses offer evidence that the rationale behind the vaccine strategy – the induction of T cell responses targeting HIV – was not entirely flawed. Rather, it appears that in order to have a better chance of offering benefit, vaccine-induced T cells need to target a broader array of HIV components and have a superior ability to kill HIV-infected cells compared to the HIV-specific T cells created by Merck’s candidate.

In a paper in Nature Medicine, Rolland Tovanabutra and colleagues describe their studies of 68 STEP participants who became infected with HIV, 40 from the vaccine group and 28 placebo recpients. By looking at the genetic sequences of the infecting viruses, they find evidence that vaccine-induced CD8 T cell responses did lead to HIV developing mutations associated with immune escape (most significantly in the Gag protein). In other words, the CD8 T cells inhibited viral replication sufficiently to cause the outgrowth of variants able to avoid CD8 T cell recognition. However, as the researchers note, this occurred too rapidly to cause any significant lowering of HIV viral load. On average, Merck’s vaccine only induced a CD8 T cell response to one epitope in each of the proteins it contained (Gag, Pol and Nef), leading the researchers to suggest that a much broader response is needed. In individuals who control viral load well in the absence of treatment, broadly targeted CD8 T cells force HIV to develop multiple escape mutations that impair the ability of the virus to replicate; based on their analyses, Tovanabutra and colleagues propose that this sort of “cornering” strategy needs to be pursued by future T cell-based vaccines.

In the March 15^th issue of the Journal of Infectious Diseases, a paper by Daniel Fitzgerald and colleagues offers a detailed description of post-infection outcomes among STEP participants (52 men from the vaccine arm and 35 placebo). The study confirms that receipt of the Merck vaccine had no effect overall on viral load, CD4 T cell counts or time to initiation of antiretroviral therapy (ART). There is one interesting subset analyses, which finds that participants with HLA gene variants known to be associated with slow progression (HLA B27, B57 & B5801) had significantly lower viral loads if they received vaccine compared to placebo. The mean difference was -0.86 log (95% CI -1.52 to -0.20, p=.03). Although subset analyses always need to be interpreted with great caution, this finding is consistent with results obtained in the SIV/macaque model where comparable MHC gene variants have been linked to improved responses to T cell-based vaccines. Human recipients of the ALVAC vaccine possessing HLA B27 and B57 have also been shown to develop superior CD8 T cell responses to HIV antigens. In a commentary accompanying the Fitzgerald paper, Marcus Altfeld and Philip Goulder note that the findings may offer a clue that the right kind of CD8 T cell responses can offer benefit in terms of viral load control.

Bolstering this view is a paper in PLoS Pathogens from Mark Connors research group at the National Institute of Allergy and Infectious Diseases. The study, led by Stephen Migueles, investigated how efficiently CD8 T cells induced by the Merck vaccine could kill HIV-infected CD4 T cells in vitro. Overall, the killing capacity of vaccine-induced CD8 T cells was comparable to that of individuals with progressing HIV infection, and fell short of that observed in long-term non-progressors. But the CD8 T cell responses of vaccine recipients with the B27, B57 and B58 HLA gene variants were significantly better at killing HIV-infected cells compared to individuals lacking those variants, consistent with the subset analysis reported by Daniel Fitzgerald et al. The Migueles study only included a relatively small number of uninfected recipients of the Merck vaccine, but in discussing their results the authors report that “our assay will be applied to participants in the Step study who subsequently acquired HIV infection post-vaccination, including a group of individuals who are restricting HIV replication to the limits of detection in currently available viral load assays. These types of analyses will begin to determine the utility of measurements of cytotoxic capacity in predicting vaccine efficacy, and provide further insight into the mechanisms of immunologic control of HIV.”

Nat Med. 2011 Feb 27. [Epub ahead of print]

Genetic impact of vaccination on breakthrough HIV-1 sequences from the STEP trial.

Rolland M, Tovanabutra S, Decamp AC, Frahm N, Gilbert PB, Sanders-Buell E, Heath L, Magaret CA, Bose M, Bradfield A, O'Sullivan A, Crossler J, Jones T, Nau M, Wong K, Zhao H, Raugi DN, Sorensen S, Stoddard JN, Maust BS, Deng W, Hural J, Dubey S, Michael NL, Shiver J, Corey L, Li F, Self SG, Kim J, Buchbinder S, Casimiro DR, Robertson MN, Duerr A, McElrath MJ, McCutchan FE, Mullins JI.

Abstract

We analyzed HIV-1 genome sequences from 68 newly infected volunteers in the STEP HIV-1 vaccine trial. To determine whether the vaccine exerted selective T cell pressure on breakthrough viruses, we identified potential T cell epitopes in the founder sequences and compared them to epitopes in the vaccine. We found greater distances to the vaccine sequence for sequences from vaccine recipients than from placebo recipients. The most significant signature site distinguishing vaccine from placebo recipients was Gag amino acid 84, a site encompassed by several epitopes contained in the vaccine and restricted by human leukocyte antigen (HLA) alleles common in the study cohort. Moreover, the extended divergence was confined to the vaccine components of the virus (HIV-1 Gag, Pol and Nef) and not found in other HIV-1 proteins. These results represent what is to our knowledge the first evidence of selective pressure from vaccine-induced T cell responses on HIV-1 infection in humans.

J Infect Dis. 2011 Mar;203(6):753-5.

The STEP Study Provides a Hint That Vaccine Induction of the Right CD8+ T Cell Responses Can Facilitate Immune Control of HIV.

Altfeld M, Goulder PJ.

Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard, Boston, Massachusetts.

J Infect Dis. 2011 Mar;203(6):765-772.

An Ad5-Vectored HIV-1 Vaccine Elicits Cell-mediated Immunity but does not Affect Disease Progression in HIV-1-infected Male Subjects: Results From a Randomized Placebo-Controlled Trial (The Step Study).

Fitzgerald DW, Janes H, Robertson M, Coombs R, Frank I, Gilbert P, Loufty M, Mehrotra D, Duerr A; for the Step Study Protocol Team.

Center for Global Health, Weill Cornell Medical College, New York, New York.

Abstract

Background. The Step study was a randomized trial to determine whether an adenovirus type 5 (Ad5) vector vaccine, which elicits T cell immunity, can lead to control of human immunodeficiency virus (HIV) replication in participants who became HIV-infected after vaccination. Methods. We evaluated the effect of the vaccine on trends in HIV viral load, CD4+ T cell counts, time to initiation of antiretroviral therapy (ART), and AIDS-free survival in 87 male participants who became infected with HIV during the Step study and who had a median of 24 months of post-infection follow-up. Results. There was no overall effect of vaccine on mean log(10) viral load (estimated difference between groups, -0.11; P = .47). In a subset of subjects with protective HLA types (B27, B57, B58), mean HIV-1 RNA level over time was lower among vaccine recipients. There was no significant difference in CD4+ T cell counts, time to ART initiation, or in AIDS-free survival between HIV-1-infected subjects who received vaccine versus those who received placebo. Conclusions. HIV RNA levels, CD4+ T cell counts, time to initiation of ART, and AIDS-free survival were similar in vaccine and placebo recipients. There may have been a favorable effect of vaccine on HIV-1 RNA levels in participants with HLA types associated with better control of HIV-1.

PLoS Pathog 7(2): e1002002. doi:10.1371/journal.ppat.1002002

Trivalent Adenovirus Type 5 HIV Recombinant Vaccine Primes for Modest Cytotoxic Capacity That Is Greatest in Humans with Protective HLA Class I Alleles

Stephen A. Migueles1, Julia E. Rood1, Amy M. Berkley1, Tiffany Guo1, Daniel Mendoza1, Andy Patamawenu1, Claire W. Hallahan3, Nancy A. Cogliano1, Nicole Frahm2, Ann Duerr2, M. Juliana McElrath2, Mark Connors1*

1 Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America, 2 Vaccine and Infectious Disease Division and the HIV Vaccine Trials Network, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 3 Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America

If future HIV vaccine design strategies are to succeed, improved understanding of the mechanisms underlying protection from infection or immune control over HIV replication remains essential. Increased cytotoxic capacity of HIV-specific CD8+ T-cells associated with efficient elimination of HIV-infected CD4+ T-cell targets has been shown to distinguish long-term nonprogressors (LTNP), patients with durable control over HIV replication, from those experiencing progressive disease. Here, measurements of granzyme B target cell activity and HIV-1-infected CD4+ T-cell elimination were applied for the first time to identify antiviral activities in recipients of a replication incompetent adenovirus serotype 5 (Ad5) HIV-1 recombinant vaccine and were compared with HIV-negative individuals and chronically infected patients, including a group of LTNP. We observed readily detectable HIV-specific CD8+ T-cell recall cytotoxic responses in vaccinees at a median of 331 days following the last immunization. The magnitude of these responses was not related to the number of vaccinations, nor did it correlate with the percentages of cytokine-secreting T-cells determined by ICS assays. Although the recall cytotoxic capacity of the CD8+ T-cells of the vaccinee group was significantly less than that of LTNP and overlapped with that of progressors, we observed significantly higher cytotoxic responses in vaccine recipients carrying the HLA class I alleles B*27, B*57 or B*58, which have been associated with immune control over HIV replication in chronic infection. These findings suggest protective HLA class I alleles might lead to better outcomes in both chronic infection and following immunization due to more efficient priming of HIV-specific CD8+ T-cell cytotoxic responses.

2011 Conference on Retroviruses & Opportunistic Infections: Webcasts & Abstracts Online

The 2011 CROI just wrapped up in Boston, and abstracts and webcasts of all sessions – including poster discussions – are now available on the conference website. Among the most publicized presentations were two describing preliminary results of a gene therapy approach developed by Sangamo Biosciences; the first was given by Jay Lalezari and the webcast can be found at the start of the Monday oral abstract session entitled “HIV: Innovative Therapeutic Approaches, ART, and Drug Resistance.” The second was by Carl June on Wednesday in the symposium “Obstacles to a Cure.” The latter session also includes a presentation by Paula Cannon on plans to use the Sangamo technology to modify stem cells, potentially circumventing the need to use CD4 T cell extraction and reinfusion (this idea is earlier in development and not yet in human trials).

Other major topics at the meeting included updates from the iPrEx pre-exposure prophylaxis trial and encouraging data from long term follow up of a study in Uganda assessing the efficacy of circumcision for HIV prevention. Excellent coverage of these and other CROI 2011 presentations can be found on sites such as AIDSMap, AIDSMeds, The Body, HIVandHepatitis.com and NATAP. 

The links below are to some studies that caught my attention.

Paper #: 158

Title: Cytolytic CD4+ T Cells in Individuals Spontaneously Controlling Viral Replication following Acute HIV Infection

Authors and Affiliations: Damien Soghoian*1, S Ranasinghe1, S Cutler1, K Axten1, H Jessen2, B Walker1, and H Streeck1

1Ragon Inst of MGH, MIT, and Harvard, Boston, MA, US and 2Jessen-Jessen-Stein, Berlin, Germany

Paper #: 331

Title: Induction of Cytolytic, V2-specific, Polyfunctional CD4+ T Cells in the Thai Phase III HIV Vaccine Trial

Authors and Affiliations: Jeffrey Currier*1, M de Souza1, S Ratto-Kim1, G Kijak1, C Andrews1, S Rerks-Ngerm2, M Robb1, N Michael1, M Marovich1, and J Kim1

1US Military HIV Res Prgm, Rockville, MD and 2Ministry of Publ Hlth, Nonthaburi, Thailand

- these two papers suggest that CD4 T cells with the capacity to kill virus-infected cells may be an important component of an effective immune response against HIV.

Paper #: 991

Title: Genital Tract Inflammation in Women Participating in the CAPRISA TFV Microbicide Trial Who Became Infected with HIV: A Mechanism for Breakthrough Infection?

Authors and Affiliations: Lindi Roberts*1, J-A Passmore1,2, C Williamson1,3, F Little1, V Naranbhai3, S Sibeko3, G Walzl4, Q Abdool Karim3,5, and S Abdool Karim3,5

1Univ of Cape Town, South Africa; 2Natl Hlth Lab Svc, Cape Town, South Africa; 3Ctr for the AIDS Prgm of Res in South Africa, Durban; 4Stellenbosch Univ, Cape Town, South Africa; and 5Columbia Univ, New York, NY, US

- I’ve written before on the blog about the potential role of genital tract immune activation in increasing susceptibility to HIV infection, and this study of women participating in the CAPRISA 004 tenofovir gel trial adds to the evidence that this is a crucially important factor driving transmission risk.

Paper #: 349

Title: Immune Correlates of Unusual Control of Viral Replication after Cessation of HAART

Authors and Affiliations: Ellen Van Gulck*, L Heyndrickx, C Merlin, S Coppens, D Atkinson, E Florence, A Buvé, and G Vanham

Inst of Tropical Med, Antwerp, Belgium

- Over the years, many studies involving ART interruption have been presented and published, and occasionally they have described rare individuals who experience viral rebound but then control HIV to undetectable levels for some time without restarting treatment. This study describes four such cases, and for the first time ascribes them a name: “secondary controllers” (SC). Hopefully this bestowing of an acronym will lead to a more systematic evaluation of the phenomenon, as happened with elite controllers (the name given to individuals who control HIV to undetectable levels without ever receiving ART).