TAG HIV Basic Science, Vaccines, and Cure Project Blog

Assessing Antiretroviral Therapy Interruptions in HIV Cure Research

Last month, researchers from the Laboratory of Immunoregulation at the National Institute of Allergy and Infectious Diseases (NIAID) published a paper in PLoS Pathogens addressing the use of antiretroviral therapy (ART) interruptions in HIV cure research. The joint lead authors were Katherine E. Clarridge and Jana Blazkova, and the focus of the study was on the effects of an analytical treatment interruption (ATI) performed during a clinical trial of the broadly neutralizing antibody VRC01. The main results offer reassurance that the ATI had no long-term negative effects for participants—as articulated in an accompanying NIAID press release titled NIH Study Supports Use of Short-Term HIV Treatment Interruption in Clinical Trials—but there are some possible safety concerns that the data does not address.

Results of the VRC01 trial were published in 2016 in the New England Journal of Medicine. The protocol design involved an initial VRC01 infusion upon entering the study, followed by an ATI three days later. VRC01 infusions were subsequently administered every four weeks until week 24. Criteria for restarting ART included:

• A confirmed >30% decline in baseline CD4 cell count or an absolute CD4 cell count <350 cells/mm³
• A sustained (≥4 weeks) HIV RNA level of >5,000 copies/mL
• Any HIV-related symptoms

Ten participants were enrolled. VRC01 only led to a slight delay in viral load rebound, so ART was restarted in all cases with the duration of the ATI ranging from 22 to 115 days (median 57 days). Nine of the individuals restarted ART due to meeting the criteria for a sustained viral load increase to over 5,000 copies/mL, while the remaining participant experienced a confirmed >30% decline in baseline CD4 cell count. No participant had a CD4 T cell drop to less than 350 cells/mm³.

The researchers evaluated multiple HIV reservoir measures as well as biomarkers of immune activation and inflammation, comparing results obtained prior to the ATI with those observed after viral load was re-suppressed by ART. In almost all cases, there were no significant differences pre- and post-ATI. The lone exception was the chemokine RANTES, a potential inflammatory biomarker, which remained significantly elevated after ART resumption (at least at the time point measured, which was a median of 363 days after restarting).

Several parameters were evaluated during the ATI, revealing that there were increases in measures of the HIV reservoir and immune activation that subsequently declined to baseline levels after ART was resumed. However, data on inflammatory biomarkers during the ATI are not reported, making it unclear to what extent inflammation may have been temporarily elevated—it seems likely that it was, given the transient spike in markers of immune activation. The relationship between short-term inflammation and risk of clinical events is uncertain but potentially of concern, so this is an aspect of ATIs that deserves additional study. Among the steps taken to maximize safety in the trial, the exclusion criteria included evidence of heart disease, which might render an individual particularly susceptible to inflammation-related risks.

Another question that has been raised regarded the safety of ATIs—particularly by longtime treatment activist Jules Levin of the National AIDS Treatment Advocacy Project (NATAP)—is whether HIV levels in the central nervous system (CNS) might increase and have the potential to cause harm. CNS samples were not taken in this NIAID study.

Overall, the trial results are consistent with the idea that short-term ATIs can be performed safely in the context of HIV cure research, but they do not necessarily represent the last word on the topic. There was a robust dialogue regarding the use of ATIs at the recent Regulation of Clinical Research Related to HIV Cure meeting that took place in Bethesda on January 25, and a webcast of the session should soon be available on the Forum for Collaborative Research website. TAG has also received support from the Elizabeth Taylor AIDS Foundation to survey community-based treatment activists regarding their views on ATIs, and a report will be published in the fall of 2018.

The Laboratory of Immunoregulation at NIAID has recently published a different study involving an ATI that highlights another important consideration in this type of HIV cure research: the inclusion of a placebo arm. The researchers conducted a clinical trial of a combination therapeutic HIV vaccine approach (DNA primes followed by a vesicular stomatitis virus vector boost) administered to individuals who initiated ART soon after infection. The vaccines were found to induce HIV-specific T cell responses, but this did not lead to superior control of viral load rebound during an ATI compared to placebo immunization.

The researchers noted that, perhaps surprisingly, there were four cases of post-ATI control of viral load to low levels that occurred in the placebo arm of the trial. The finding emphasizes that a subset of early-treated individuals can exhibit prolonged viral load suppression after an ATI, and that this needs to be considered when designing trials of therapeutic interventions. In the absence of a placebo arm, spontaneous control of HIV replication could be misinterpreted as evidence of a therapeutic effect. The authors specifically cite the open-label trial of therapeutic vaccination plus romidepsin that was presented by Beatriz Mothe at CROI 2017, pointing out that a similar proportion of participants in the placebo arm of their study maintained viral loads below 2000 copies/ml for at least 16 weeks.

The inclusion of placebo control arms in trials is not necessarily as simple as it might sound, because there are cost and logistical issues that are prohibitive for some research groups. However, it is clearly a necessary step for establishing that a candidate intervention has had a genuine effect, and results from open-label trials should be interpreted with caution until confirmed in a randomized controlled context. 

Estimating How Frequently Latent HIV Reactivates

The primary barrier to curing HIV infection is the persistence of the virus in a latent form in long-lived resting memory CD4 T cells. The number of latently infected resting memory CD4 T cells in a typical individual on ART is estimated to be in the range of 1-60 million. An important strand of HIV cure research involves attempting to reduce the size of this persistent HIV reservoir, in hopes of delaying or—better yet—preventing viral load rebound when ART is interrupted. In order to gain insight into how feasible this might be, researchers have employed mathematical modeling to estimate how the size of the HIV reservoir relates to time to viral load rebound.

A widely cited model created Alison Hill and colleagues has suggested that reservoir reductions on the order of 5-6 logs (100,000-1 million fold) would be necessary to delay viral load rebound for 30 years or more in most individuals (an outcome that would approach a lifelong cure), while a yearlong delay would likely require a drop of at least 3 logs (1,000 fold). Last month in PLoS Pathogens, a new model was published that argues that significant delays in viral load rebound might be achieved with far more modest declines in HIV reservoir size.

The study draws on data from several different clinical trials in which ART was interrupted and time to viral load rebound assessed. A series of calculations are used to generate an estimate of how often—on average—a latently infected resting memory CD4 T cell would have to start producing virus to explain the kinetics of the rise in viral load observed after ART interruption. The math is complex and opaque to a non-mathematician, but produces a result of one successful reactivation of latent HIV every six days, considerably less frequent than a previous estimate of five times per day based on studies involving drug resistance mutations (this prior estimate is used in the Alison Hill model). A separate analysis in the new paper, using different methodology based on the genetic characteristics of rebounding HIV, arrives at a reasonably similar estimate of once every 3.6 days.

The researchers extrapolate that a yearlong delay in viral load rebound might therefore be achievable with a reduction in the HIV reservoir of 60-70 fold, a more optimistic scenario than proposed previously by Alison Hill et al. But, on the surface at least, the Hill model appears more consistent with the well-publicized cases of the Boston patients, two individuals who were reported to have experienced HIV reservoir reductions of at least 3 logs as a result of receiving stem cell transplants to treat cancers. After a carefully conducted ART interruption, viral load rebound occurred after around three months in one case and eight months in the other. The authors of the new model suggest that this apparent discrepancy might be explained by the presence of a larger, unmeasured HIV reservoir in the tissues of the Boston patients.

The Alison Hill et al PNAS paper also cites a case report by Tae-Wook Chun and colleagues describing an individual with an HIV reservoir approximately 1,500 fold lower than a typical person on ART in whom viral load rebound occurred 50 days after ART interruption. Although it would be premature to draw conclusions from so few case reports, it has to be noted that a 60-70 fold decline in the HIV reservoir doesn’t appear to have delayed viral load rebound for a year in anybody as yet. And in three examples where greater reservoir reductions appear to have occurred, viral load rebound was not delayed for a year.

The cautious interpretation would be that additional data are needed to help refine the mathematical modeling and ascertain which models most closely approximate the biological reality. An intervention (beyond stem cell transplantation, which cannot be studied on a large scale) that significantly lowered HIV reservoir levels would also allow for a more direct assessment of the impact on time to viral load rebound.

Remission Definitions

A final comment on the use of the term  “remission” in this context: it’s important to note the distinction between the type remission being described in this work and the “virological remission” reported in the recent case of the teenage post-treatment controller and the similar VISCONTI cohort participants. The former involves a complete absence of HIV activity due to remaining latently infected cells staying in a non-activated resting state for the duration of the remission, and any eventual viral load rebound occurring as a result of a latently infected cell becoming activated and producing infectious virus (as best as anyone can tell with available technology, this appears to be the type of remission that occurred in the Boston patients and Mississippi baby). The latter scenario of post-treatment control of viral load is different because it involves ongoing limitation of HIV replication by immune responses; in other words, low-level HIV replication activity that is kept in check by the immune system. Current evidence implies that the former type of remission is more likely to be associated with a state of health comparable to being HIV-negative (and therefore consistent with most people’s understanding of the term remission), whereas post-treatment control might be associated with some degree of inflammation-mediated risk of disease, making it perhaps questionable as to whether the term remission should be applied. 

PLoS Pathog. 2015 Jul 2;11(7):e1005000. doi: 10.1371/journal.ppat.1005000. eCollection 2015.

HIV Reactivation from Latency after Treatment Interruption Occurs on Average Every 5-8 Days-Implications for HIV Remission.

Pinkevych M, Cromer D, Tolstrup M, Grimm AJ, Cooper DA, Lewin SR, Søgaard OS, Rasmussen TA, Kent SJ, Kelleher AD, Davenport MP.

Abstract

HIV infection can be effectively controlled by anti-retroviral therapy (ART) in most patients. However therapy must be continued for life, because interruption of ART leads to rapid recrudescence of infection from long-lived latently infected cells. A number of approaches are currently being developed to 'purge' the reservoir of latently infected cells in order to either eliminate infection completely, or significantly delay the time to viral recrudescence after therapy interruption. A fundamental question in HIV research is how frequently the virus reactivates from latency, and thus how much the reservoir might need to be reduced to produce a prolonged antiretroviral-free HIV remission. Here we provide the first direct estimates of the frequency of viral recrudescence after ART interruption, combining data from four independent cohorts of patients undergoing treatment interruption, comprising 100 patients in total. We estimate that viral replication is initiated on average once every ≈6 days (range 5.1- 7.6 days). This rate is around 24 times lower than previous thought, and is very similar across the cohorts. In addition, we analyse data on the ratios of different 'reactivation founder' viruses in a separate cohort of patients undergoing ART-interruption, and estimate the frequency of successful reactivation to be once every 3.6 days. This suggests that a reduction in the reservoir size of around 50-70-fold would be required to increase the average time-to-recrudescence to about one year, and thus achieve at least a short period of anti-retroviral free HIV remission. Our analyses suggests that time-to-recrudescence studies will need to be large in order to detect modest changes in the reservoir, and that macaque models of SIV latency may have much higher frequencies of viral recrudescence after ART interruption than seen in human HIV infection. Understanding the mean frequency of recrudescence from latency is an important first step in approaches to prolong antiretroviral-free viral remission in HIV.

HIV Reservoir Measurements and Viral Load Rebound after ART Interruption

To assist in the search for therapies that might contribute to curing HIV infection, scientists are seeking to identify biomarkers that are associated with control of viral load after an antiretroviral therapy (ART) interruption. If reliable predictors of viral load control could be identified, the effect of candidate therapies on these biomarkers could be evaluated without necessarily having to ask clinical trial participants to undergo ART interruptions (which can pose risks to health). Two recent papers report that certain measurements of the HIV reservoir show statistically significant associations with viral load rebound, although the results are not entirely consistent.

The SPARTAC trial studied the effects of a 48-week course of ART in people with acute HIV infection. The primary results were reported in the New England Journal of Medicine last year. In a paper in the journal Elife, the trial investigators describe assessments of whether the amount of HIV DNA in blood samples at ART interruption was linked to the time it took for HIV viral load to rebound to detectable levels, or the trial’s primary endpoints (CD4 T cell count less than 350 or restart of ART). The level of total HIV DNA was significantly correlated with time to a viral load above 400 copies; the higher the HIV DNA level, the quicker the time to rebound. Significant associations were also observed between HIV DNA levels and time to reach a CD4 T cell count of less than 350 or ART restart. However, when an assay with a lower viral load cut-off of 50 copies was used, HIV DNA levels were no longer significantly associated with time to rebound.

The second paper, by Jonathan Li and colleagues, is based on analyses of participants in a randomized, placebo-controlled therapeutic HIV vaccine trial involving Merck’s Ad5-based vaccine candidate (since discontinued). The study design involved a 16-week analytical treatment interruption (ATI). The researchers were able to document statistically significant correlations between pre-ATI HIV RNA and DNA measurements and the viral load set point during the ATI. However, neither measure was significantly associated with time to viral load rebound above 200 copies.

Taken together, the papers indicate that HIV reservoir measurements have some promise for predicting viral load rebound after ART interruption, but uncertainties remain. In both studies HIV DNA assays did not show significant correlations with time to rebound when a lower viral load cut off was used (<50 or <200 compared to <400).  The explanation for this finding is unclear, but could potentially relate to greater viral load test result variability at lower copy numbers. The AIDS Clinical Trials Group (ACTG) is now developing a research protocol with the specific aim of identifying biomarkers of viral load control, designated the PR 691 Monitored Antiretroviral Pause [MAP] Biomarker study.

As part of the preparation for this protocol, Jonathan Li has performed an analysis of six prior ACTG trials involving ATIs to gain an understanding of what proportion of individuals are able to maintain undetectable viral loads for an extended period. The results were presented at the IDSA conference last month, showing that overall 6% of 263 participants had levels below 200 copies at week 12 of an ATI. The phenomenon was more common among those treated during acute or recent HIV infection compared to chronically infected individuals. Galit Alter has collaborated with Li to look at possible correlates of control in these study participants, identifying the presence of specific antibody subclasses that are associated with enhanced natural killer cell activity (this preliminary work was presented at the NIAID Strategies for an HIV Cure meeting the week following the IDSA conference). The forthcoming ACTG MAP biomarker study should hopefully be able to build on these findings. 

Elife. 2014 Sep 12:e03821. doi: 10.7554/eLife.03821. [Epub ahead of print]

HIV-1 DNA predicts disease progression and post-treatment virological control. (open access)

Williams JP, Hurst J, Stöhr W, Robinson N, Brown H, Fisher M, Kinloch S, Cooper D, Schechter M, Tambussi G, Fidler S, Carrington M, Babiker A, Weber J, Koelsch KK, Kelleher AD, Phillips RE, Frater J; on behalf of the SPARTAC Trial Investigators.

Abstract

In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials.

Elife. 2014 Oct 16;3:e04742. doi: 10.7554/eLife.04742.

Biomarker reveals HIV's hidden reservoir. (open access)

Cockerham LR, Deeks SG.

Abstract

Determining the total amount of HIV DNA in people undergoing antiretroviral therapy could accelerate the development of novel therapies and potential cures for HIV infection.

AIDS. 2014 Sep 23. [Epub ahead of print]

Relationship of HIV reservoir characteristics with immune status and viral rebound kinetics in an HIV therapeutic vaccine study.

Li JZ, Heisey A, Ahmed H, Wang H, Zheng L, Carrington M, Wrin T, Schooley RT, Lederman MM, Kuritzkes DR; the ACTG A5197 Study Team.

Abstract

OBJECTIVES: The objective of this study is to evaluate the impact of therapeutic HIV vaccination on the HIV reservoir and assess the relationship of the viral reservoir with HIV-specific immune status and viral rebound kinetics.

DESIGN: A retrospective analysis of ACTG A5197, a randomized, placebo-controlled trial of a therapeutic rAd5 HIV-1 gag vaccine.

METHODS: Participants received vaccine/placebo at weeks 0, 4 and 26 prior to a 16-week analytic treatment interruption (ATI) at week 38. Cell-associated HIV-1 RNA and DNA (CA-RNA and CA-DNA) and HIV-1 residual viremia were quantified at weeks 0, 8 and 38. HIV-specific CD4/CD8 activity was assessed by an intracellular cytokine staining assay.

RESULTS: At study entry, CA-RNA and CA-DNA levels were correlated inversely with the numbers of HIV-specific CD4 interferon-γ producing cells (CA-RNA: r = -0.23, P = 0.03 and CA-DNA: r = -0.28, P < 0.01, N = 93). Therapeutic HIV vaccination induced HIV-specific CD4 activity, but did not significantly affect levels of CA-RNA or CA-DNA. Vaccine recipients with undetectable residual viremia at week 8 had higher frequencies of HIV-specific CD4 and CD8 interferon-γ producing cells (undetectable versus detectable residual viremia: 277 versus 161 CD4 cells/10 lymphocytes, P = 0.03 and 1326 versus 669 CD8 cells/10 lymphocytes, P = 0.04). Pre-ATI CA-RNA and CA-DNA were associated with post-ATI plasma HIV set point (CA-RNA: r = 0.51, P < 0.01 and CA-DNA: r = 0.47, P < 0.01).

CONCLUSION: Vaccine-induced T-cell responses were associated with a modest transient effect on residual viremia, but more potent immune responses and/or combination treatment with latency-reversing agents are needed to reduce the HIV reservoir. HIV reservoir measures may act as biomarkers of post-ATI viral rebound kinetics.

Panobinostat as a Latency-Reversing Agent: Phase I Trial Results Published

In the spring of 2013, a wildly inaccurate Daily Telegraph story about HIV cure research claiming “there will be a breakthrough in finding a cure for HIV within months” was amplified around the Internet, understandably receiving a massive amount of attention and interest. To their credit, the researchers whose work prompted the piece published a statement addressing the inaccuracies, and eventually the Telegraph made extensive corrections. Recently, in the debut issue of The Lancet’s new online HIV journal, results from the trial at the center of the story were published - to no fanfare whatsoever.

The drug being tested was the HDAC inhibitor panobinostat, which has been reported to reverse HIV latency in laboratory experiments. In the 15-person phase I trial, a small but significant increase of approximately 2-3 fold in cell-associated HIV RNA was documented, and plasma viremia was more frequently detected compared to baseline. A transient decrease in the amount of total HIV DNA in the blood was captured at day 14 of the study, but otherwise no measures of the HIV reservoir (including levels of replication-competent virus) were altered by panobinostat. A subset of nine study participants subsequently underwent an analytical treatment interruption, and HIV viral load rebounded to over 1,000 copies in all cases within a median of 17 days (range 14–56). The main side effect of the drug was fatigue, and no adverse events of greater than grade 1 occurred. However, as noted in an accompanying commentary by Stephen Kent and Miles Davenport, HDAC inhibitors have been shown to cause long-term changes in expression of multiple genes, so long-term safety will need to be carefully monitored.

The results appear consistent with clinical trials of the HDAC inhibitors vorinostat and romidepsin, which also indicated that the drugs could activate at least some latent HIV. The romidepsin trial results were presented by Dr. Ole Søgaard at the International AIDS Society Towards an HIV Cure Symposium in July, and both the powerpoint presentation and an audio file are available online. The lack of a significant reduction in the HIV reservoir supports the proposal that additional interventions are needed to promote clearance of HIV-infected cells after latency is reversed, and Søgaard and colleagues are now exploring this possibility by combining romidepsin with therapeutic vaccination.

The panobinostat paper does offer a hint of a greater effect of the drug in four participants, but these types of post-hoc (not planned in the original study protocol) subset analyses have to be interpreted with great caution. In these individuals, a decline in total HIV DNA levels of around 67–84% is reported to have persisted, and was correlated with a slightly longer time to viral load rebound during the analytical treatment interruption. At the recent Strategies for an HIV Cure meeting, which took place at the National Institutes of Health in Bethesda in October, Mathias Lichterfeld presented some preliminary data associating this delayed time to viral load rebound with interferon-stimulated gene expression and natural killer cell activity, suggesting a role for innate immunity. Given the small number of participants and the fact that the analyses are all post-hoc, these results can only be considered exploratory and require confirmation.

Beyond the results obtained with individual candidates, there is a notable larger issue that looms over latency-reversing approaches generally. The experience of the Boston patients has demonstrated that significant HIV reservoir reductions of around 3 logs (1,000-fold) can lead to delayed viral load rebound after ART interruption, but not a cure. This is in line with mathematical modeling by Alison Hill (published recently in PNAS) which estimates that an HIV reservoir reduction more on the order of 5-6 logs (100,000-1 million-fold) would be needed to achieve a long-term cure in most people. In this context, the fact that no latency-reversing strategy has yet had any significant effect on HIV reservoir levels does not seem very encouraging. But, in case that sounds like doom saying, it’s also true that the work is at an early stage, and latency reversal is just one of many strategies being pursued in HIV cure research.

The Lancet HIV, Volume 1, Issue 1, Pages e13 - e21, October 2014

doi:10.1016/S2352-3018(14)70014-1

Published Online: 16 September 2014 

Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial

Dr Thomas A Rasmussen MD a , Martin Tolstrup PhD a, Christel R Brinkmann PhD a, Rikke Olesen PhD a, Christian Erikstrup MD b, Ajantha Solomon c, Anni Winckelmann a, Sarah Palmer PhD d, Prof Charles Dinarello MD e, Maria Buzon PhD f g, Mathias Lichterfeld MD f g, Prof Sharon R Lewin PhD c h, Prof Lars Østergaard DMSc a, Ole S Søgaard MD a

a Department of Infectious Diseases, Aarhus University Hospital, Denmark

b Department of Clinical Immunology, Aarhus University Hospital, Denmark

c Department of Infectious Diseases, Alfred Hospital and Monash University, Melbourne, VIC, Australia

d Westmead Millennium Institute for Medical Research, University of Sydney, Westmead, NSW, Australia

e Department of Medicine, Division of Infectious Diseases, University of Colorado Denver, Aurora, CO, USA

f Infectious Disease Division, Massachusetts General Hospital, Boston, MA, USA

g Ragon Institute of MGH, MIT and Harvard, Boston, MA, USA

h Centre for Biomedical Research, Burnet Institute, Melbourne, VIC, Australia 

Summary

Background: Activating the expression of latent virus is an approach that might form part of an HIV cure. We assessed the ability of the histone deacetylase inhibitor panobinostat to disrupt HIV-1 latency and the safety of this strategy.

Methods: In this phase 1/2 clinical trial, we included aviraemic adults with HIV treated at Aarhus University Hospital, Denmark. Participants received oral panobinostat (20 mg) three times per week every other week for 8 weeks while maintaining combination antiretroviral therapy. The primary outcome was change from baseline of cell-associated unspliced HIV RNA. Secondary endpoints were safety, plasma HIV RNA, total and integrated HIV DNA, infectious units per million CD4 T cells, and time to viral rebound during an optional analytical treatment interruption of antiretroviral therapy. This trial is registered with ClinicalTrial.gov, number NCT01680094.

Findings: We enrolled 15 patients. The level of cell-associated unspliced HIV RNA increased significantly at all timepoints when patients were taking panobinostat (p<0·0001). The median maximum increase in cell-associated unspliced HIV RNA during panobinostat treatment was 3·5-fold (range 2·1—14·4). Panobinostat induced plasma viraemia with an odds ratio of 10·5 (95% CI 2·2—50·3; p=0·0002) compared with baseline. We recorded a transient decrease in total HIV DNA, but no cohort-wide reduction in total HIV DNA, integrated HIV DNA, or infectious units per million. Nine patients participated in the analytical treatment interruption, median time to viral rebound was 17 days (range 14—56). Panobinostat was well tolerated. 45 adverse events were reported, but only 16 (all grade 1) were presumed related to panobinostat.

Interpretation: Panobinostat effectively disrupts HIV latency in vivo and is a promising candidate for future combination clinical trials aimed at HIV eradication. However, panobinostat did not reduce the number of latently infected cells and this approach may need to be combined with others to significantly affect the latent HIV reservoir.

The Lancet HIV, Volume 1, Issue 1, Pages e2 - e3, October 2014

doi:10.1016/S2352-3018(14)70015-3

Published Online: 16 September 2014

Tentative first steps to eradicate latent HIV

Stephen J Kent, Miles P Davenport

Purging latent HIV with reactivating drugs is one of the most promising approaches to curing HIV infection. In The Lancet HIV , Thomas Rasmussen and colleagues report results of a phase 1/2 trial 1 of panobinostat, a histone deacetylase inhibitor, to reactivate latent HIV. They show that the drug was well tolerated in the short term and the treatment increased both the amount of cell-associated HIV RNA and low-level transient HIV viraemia. There was, however, no overall change in measurements of the HIV reservoir after 8 weeks of panobinostat treatment.

Update Published on the First Berlin Patient

In the late 1990s, a single case report about an individual displaying control of HIV viral load for nearly two years after interrupting antiretroviral therapy (ART) drew a huge amount of publicity, including a lengthy story in the New York Times magazine by Mark Schoofs in June of 1998. The case report was ultimately published in the New England Journal of Medicine on May 27, 1999. The man was dubbed “the Berlin patient” long before the moniker was also applied to Timothy Brown, the only adult considered cured of HIV infection. This first Berlin patient was under the care of Dr. Heiko Jessen, and he was treated with the unusual combination of ddI (Videx), indinavir (Crixivan) and the cancer drug hydroxyurea (which at the time was sometimes used both to potentiate ddI and for possible immune-modulating activity). Treatment was started during acute infection and subsequently interrupted twice for short periods before being completely discontinued. During the second interruption and after discontinuation, HIV viral load levels remained undetectable based on the assay in use, which had a cut-off of 500 copies/ml. The individual was reported to have strong HIV-specific CD4 T cell and CD8 T cell responses, suggesting cellular immunity might be responsible for the salutary outcome.

The case was influential in generating the hypothesis that treatment of acute infection followed by structured treatment interruptions might boost HIV-specific CD4 T cell and CD8 T cell responses and lead to post treatment control of HIV replication. The hypothesis was tested by the research group of Bruce Walker at Massachusetts General Hospital but—after early indications of promise—the hoped for result of prolonged control of HIV after ART withdrawal was not achieved.  Since that time, the fate of the original Berlin patient has been somewhat mysterious, with some published papers suggesting he had been lost to follow up. One of the last public updates given was by Bruce Walker at the 2003 IAS conference in Paris; Walker noted he had visited Jessen en route to the meeting, and learned that the individual was maintaining good control of viral load, but also that it had turned out that he possessed the HLA B*57 allele that is strongly associated with elite control of HIV replication in the absence of treatment.

Yesterday the New England Journal of Medicine published a letter from Heiko Jessen, Todd Allen and Hendrik Streeck that provides an update on the case. Titled “How a Single Patient Influenced HIV Research - 15-Year Follow-up,” it reveals that the individual has mostly maintained a low viral load off ART in the intervening years, with a mean level of 2,812 copies and only one blip to 25,000 copies. CD4 T cell count has remained relatively stable with a mean of 729 cells although a figure accompanying the paper indicates there have been a couple of dips below 500 (one accompanying the viral load blip some time ago, and one quite recently). The letter also notes that the individual has the HLA B*57 allele and concludes: “Although the early initiation of treatment may have long-term benefits for certain patients, a likely explanation for control of viral replication in this patient is genetic background, regardless of intervention. Thus, this case represents a cautionary tale of drawing broad conclusions from a single patient.”

Since not every HIV-positive person with the HLA B*57 allele becomes an elite controller, this conclusion is perhaps debatable, but it is impossible to know whether or not this individual would have ultimately maintained a low viral load even without the short course of ART. What is certain is that he is not cured of HIV infection. Unfortunately there may be some confusion about this because a forthcoming book is promoting the idea that, along with Timothy Brown, the original Berlin patient is, in fact, cured. Although there is some uncertainty about the appropriate application of the term “functional cure,” there is no definition that allows for persistently detectable HIV viral load. The commonly used terminology for cases like that of the first Berlin patient (as has been applied to members of the VISCONTI cohort) is "post treatment controller" or "virological remission." Extended follow up will be required to define to what extent individuals in this situation are protected from disease progression. 

Dendritic Cell-Based Therapeutic HIV Vaccine Transiently Modulates Viral Load

A study published in the January 2 issue of Science Translational Medicine reports that a dendritic cell (DC)-based therapeutic HIV vaccine was able to significantly lower viral load after an antiretroviral therapy (ART) interruption, albeit only transiently. The publication drew considerable press coverage, most of it reasonably accurate although some headline writers got carried away (while potentially representing a step forward for therapeutic HIV vaccine research, the results are not a “huge breakthrough” as one headline stated).

A number of prior trials have offered hints that therapeutic vaccination can mildly improve immune control of HIV replication, but they have tended to be small and uncontrolled (lacking a placebo comparator). A randomized, placebo-controlled trial of the Merck Ad5-based HIV vaccine conducted by the ACTG did find a trend toward lower viral load during a treatment interruption, but the result did not quite reach statistical significance. The major strengths of this new study, led by Felipe García from the University of Barcelona, are that it was randomized and placebo-controlled, and that the viral-load difference between vaccine- and placebo recipients was statistically significant at the 24-week timepoint after ART interruption (−0.80 vs. −0.19 logs, P = 0.01). However, after 48 weeks of follow-up, the difference was no longer significant (P = 0.66).

The researchers also report the following number of participants who displayed viral-load decreases of greater than 1 log (a reduction of 90 percent or more compared to baseline) in the vaccine versus placebo groups at different timepoints during follow-up:

• Week 12: 12/22 (55%) versus 1/11 (9%)
• Week 24: 7/20 (35%) versus 0/10 (0%)
• Week 48: 3/17 (18%) versus 1/6 (17%)

These differences were statistically significant in favor of the vaccine group at weeks 12 and 24, but not at week 48. There were no significant differences in CD4 T-cell counts between the two groups at any time over the 48-week period. Analyses of HIV-specific T-cell responses (using an ELISPOT assay measuring interferon-gamma production in response to HIV Gag, Nef, and Env gp41 peptides) revealed a statistical trend toward an inverse association with viral load in the vaccine group (P = 0.08), while in the placebo group there was a significant positive association with viral load. This result suggests that HIV-specific T-cell responses in vaccine recipients were at least partly effective, whereas in placebo recipients the detected HIV-specific T cells were flailing away ineffectively.

The researchers manufactured the vaccine by harvesting monocyte-derived DCs from study participants and exposing them to heat-inactivated HIV isolated from the same individuals. The placebo consisted of DCs unexposed to HIV. A relatively high dose of ~10 million DCs was administered three times, at two-week intervals (either before or immediately after ART interruption). In discussing the results, the researchers point out that several DC-based vaccines have previously shown hints of promise, with the use of HIV or HIV antigens from individual study participants (autologous HIV) appearing to be important for achieving significant viral-load effects. They acknowledge that the degree of virological control achieved in their study is far from the optimal goal of achieving a functional cure, which is defined as a long-lasting reduction in viral load to undetectable levels after ART interruption. Despite this caveat, they conclude: “This is a proof of concept justifying further investigation of new candidates and/or new optimized strategies of vaccination with the final objective of obtaining a functional cure as an alternative to ART for life.”

The publication of this study occurs at a time of renewed interest in therapeutic HIV vaccines in the context of cure research, prompted by a study suggesting that HIV-specific immunity needs to be enhanced in order to help eradicate HIV reservoirs. Furthermore, there is some evidence that a proportion—perhaps even the majority—of the latent HIV reservoir resides in HIV-specific CD4 T cells, leading to the hypothesis that therapeutic HIV vaccines could also help activate these CD4 T cells and thus awaken the virus they contain, making it visible to the immune system. Studies are now being planned that will further investigate these issues. 

Sci Transl Med 2 January 2013: Vol. 5, Issue 166, p. 166ra2 Sci. Transl. Med. DOI: 10.1126/scitranslmed.3004682

RESEARCH ARTICLE

A Dendritic Cell–Based Vaccine Elicits T Cell Responses Associated with Control of HIV-1 Replication

Felipe García1,*,†, Nuria Climent1,*, Alberto C. Guardo1, Cristina Gil1, Agathe León1, Brigitte Autran2, Jeffrey D. Lifson3, Javier Martínez-Picado4,5, Judit Dalmau4, Bonaventura Clotet4, Josep M. Gatell1, Montserrat Plana1,*, Teresa Gallart1,*, For the DCV2/MANON07-ORVACS Study Group

1Hospital Clinic-IDIBAPS, HIVACAT, University of Barcelona, 08036 Barcelona, Spain. 2INSERM UMR-S-945, Université Paris-VI Pierre-et-Marie-Curie, Hôpital Pitié-Salpêtrière, 75013 Paris, France. 3AIDS and Cancer Virus Program, SAIC-Frederick Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA. 4AIDS Research Institute IrsiCaixa, Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain. 5Institució Catalana de Recerca i Estudis Avançats, 08010 Barcelona, Spain.

*These authors contributed equally to this work.

ABSTRACT

Combination antiretroviral therapy (cART) greatly improves survival and quality of life of HIV-1–infected patients; however, cART must be continued indefinitely to prevent viral rebound and associated disease progression. Inducing HIV-1–specific immune responses with a therapeutic immunization has been proposed to control viral replication after discontinuation of cART as an alternative to “cART for life.” We report safety, tolerability, and immunogenicity results associated with a control of viral replication for a therapeutic vaccine using autologous monocyte-derived dendritic cells (MD-DCs) pulsed with autologous heat-inactivated whole HIV. Patients on cART with CD4+ >450 cells/mm3 were randomized to receive three immunizations with MD-DCs or with nonpulsed MD-DCs. Vaccination was feasible, safe, and well tolerated and shifted the virus/host balance. At weeks 12 and 24 after cART interruption, a decrease of plasma viral load setpoint ≥1 log was observed in 12 of 22 (55%) versus 1 of 11 (9%) and in 7 of 20 (35%) versus 0 of 10 (0%) patients in the DC–HIV-1 and DC-control groups, respectively. This significant decrease in plasma viral load observed in immunized recipients was associated with a consistent increase in HIV-1–specific T cell responses. These data suggest that HIV-1–specific immune responses elicited by therapeutic DC vaccines could significantly change plasma viral load setpoint after cART interruption in chronic HIV-1–infected patients treated in early stages. This proof of concept supports further investigation of new candidates and/or new optimized strategies of vaccination with the final objective of obtaining a functional cure as an alternative to cART for life.

Inflammation Literature Accumulates

For everyone who lived through the period prior to the advent of combination antiretroviral therapy (ART), the vanquishing of the opportunistic diseases of severe immune deficiency – such as PML, MAC, CMV retinitis and other horrors – is a source of profound relief. But as concerns about OIs have diminished, another longer-term worry has begun to emerge in the form of inflammation. At the time combination ART first became available, the evidence of the role of immune activation in driving progression to AIDS was just beginning to gain acceptance among the research community. Understandably, however, the main focus was on the damage that persistent activation was doing to the immune system (e.g. T cell loss and dysfunction) and relatively little attention was paid to the potential impact on other organ systems and health outcomes. There were exceptions, such as dementia, where the contribution of inflammation in the brain was well described, and HIV-associated nephropathy (HIVAN), which had been linked to inflammation in the kidneys, but problems such as arteriosclerosis and cardiovascular disease were largely off the radar screen.

This all changed radically a few years ago when results of the SMART (Strategic Timing of AntiRetroviral Therapy) trial were announced and published. The goal of SMART was to evaluate whether ART could be used intermittently to keep CD4 T-cell counts out of the danger zone for opportunistic infections, as opposed to using ART continuously. Based on individual anecdotes and smaller studies, many hoped that intermittent ART would at least be equivalent to continuous, and might even be superior in terms of quality of life due to reduced side effects. But the SMART results were unequivocal, showing that intermittent ART was associated with a doubling of the risk of serious illness and death compared to continuous ART. The absolute risk of illness and death was relatively low in both arms (around 3% and 1.5% respectively) but the difference was highly statistically significant and the large sample size of 5,472 people meant the findings were unusually robust. Differences between the arms were also seen at all CD4 strata and not just the lowest. Most of the illnesses that occurred were not AIDS-defining events based on the CDC criteria, but cardiovascular, kidney and liver disease and cancers.

The researchers who conducted SMART collaborated with specialists from outside of the HIV research field to analyze factors linked to illness and mortality in the trial. One such expert, Lewis Kuller, led a study that showed that biomarkers of inflammation (specifically IL-6 and D-dimer) were strongly linked to mortality in SMART, with odds ratios that dwarfed the associations Kuller had previously documented in non-HIV-infected elderly people.

These findings have led to an explosion of research into inflammatory biomarkers and health outcomes in HIV infection. Four new studies are appended below. The first paper from the Journal of Infectious Diseases, by Jacqueline Neuhaus and colleagues, shows that biomarkers of inflammation, coagulation, and kidney function were significantly higher among SMART participants (on or off ART) compared to a control group comprised of two large population-based studies named MESA and CARDIA. Even when the analysis was restricted to participants on ART with viral load levels less than 400 copies/ml, all the measured biomarkers (high‐sensitivity C‐reactive protein, IL-6, D-dimer and cystatin C) were significantly elevated compared to controls. Of note, the laboratory measurements were all done in the same centralized laboratory using the same methods. The second JID paper takes a different tack and looks at correlations between baseline levels of several immune activation markers and clinical events after starting ART. Several markers were associated with illness and mortality even when analysis was restricted to events that only occurred after 51 weeks or more of ART.

Two additional papers look at C-reactive protein in different settings: in a study just published in the Journal of AIDS, researchers in Rakai, Uganda show that CRP levels increase during disease progression in their cohort of HIV-positive people despite a lack of evidence of microbial translocation. The second study investigates high sensitivity CRP (hsCRP) and other inflammatory biomarkers among HIV-positive children at the Rainbow Babies and Children's Hospital in Cleveland. The researchers report that hsCRP levels were significantly higher in children with HIV compared to matched uninfected controls, but other biomarkers did not show significant differences. The majority of the children in the study were on ART with viral loads less than 50 copies/ml. A measurement of the health of the carotid artery called intima-media thickness (IMT) was associated with hsCRP levels (as has been reported in adults), raising the concern that HIV infection may increase risk of cardiovascular disease across all age groups.  

On a related note, the Forum for Collaborative HIV Research is sponsoring a public meeting on the subject of HIV infection and cardiovascular disease at the Doubletree Hotel, Washington, DC from 10am to 5pm on June 23. Registration is free and available online although the information on the meeting only seems to have gone out via email and it is not yet on the forum’s website. For those unable to attend in person the meeting is also slated to be webcast.

The Journal of Infectious Diseases 2010;201:1783–1785

DOI: 10.1086/652751

EDITORIAL COMMENTARY

Inflammation and Complications of HIV Disease

Michael P. Dubé and Fred R. Sattler

Department of Medicine and Division of Infectious Diseases, Keck School of Medicine, University of Southern California, Los Angeles

The Journal of Infectious Diseases 2010;201:1788–1795

DOI: 10.1086/652749

Markers of Inflammation, Coagulation, and Renal Function Are Elevated in Adults with HIV Infection

Jacqueline Neuhaus,1 David R. Jacobs, Jr,1 Jason V. Baker,1,2 Alexandra Calmy,7 Daniel Duprez,1 Alberto La Rosa,8 Lewis H. Kuller,3 Sarah L. Pett,9 Matti Ristola,10 Michael J. Ross,4 Michael G. Shlipak,5 Russell Tracy,6 and James D. Neaton,1 for the International Network for Strategic Initiatives in Global HIV Trials (INSIGHT) Strategies for Management of Anti‐Retroviral Therapy (SMART), Multi‐Ethnic Study of Atherosclerosis (MESA), and Coronary Artery Development in Young Adults (CARDIA) Research Groupsa

1University of Minnesota and 2Hennepin County Medical Center, Minneapolis, Minnesota; 3University of Pittsburgh, Pittsburgh, Pennsylvania; 4Mount Sinai School of Medicine, New York, New York; 5University of California, San Francisco; 6University of Vermont, Burlington; 7Geneva University Hospital, Geneva, Switzerland; 8Asociacion Civil Impacta Salud y Educacion, Lima, Peru; 9University of New South Wales, Sydney, Australia; 10Helsinki University Central Hospital, Helsinki, Finland

Background. Human immunodeficiency virus (HIV) replication and immune activation may increase inflammation and coagulation biomarkers. Limited data exist comparing such biomarkers in persons with and without HIV infection.

Methods. For persons 45–76 years of age, levels of high‐sensitivity C‐reactive protein (hsCRP), interleukin (IL)–6, D‐dimer, and cystatin C were compared in 494 HIV‐infected individuals in the Strategies for Management of Anti‐Retroviral Therapy (SMART) study and 5386 participants in the Multi‐Ethnic Study of Atherosclerosis (MESA) study. For persons 33–44 years of age, hsCRP and IL‐6 levels were compared in 287 participants in the SMART study and 3231 participants in the Coronary Artery Development in Young Adults (CARDIA) study.

Results. hsCRP and IL‐6 levels were 55% (P<.001) and 62% (P<.001) higher among HIV‐infected participants than among CARDIA study participants. Compared with levels noted in MESA study participants, hsCRP, IL‐6, D‐dimer, and cystatin C levels were 50%, 152%, 94%, and 27% higher, respectively (P<.001, for each), among HIV‐infected participants. HIV‐infected participants receiving antiretroviral therapy who had HIV RNA levels 400 copies/mL had levels higher (by 21% to 60%) (P<.001) than those in the general population, for all biomarkers.

Conclusions. hsCRP, IL‐6, D‐dimer, and cystatin C levels are elevated in persons with HIV infection and remain so even after HIV RNA levels are suppressed with antiretroviral therapy. Additional research is needed on the pathophysiology of HIV‐induced activation of inflammatory and coagulation pathways, to guide potential interventions.

The Journal of Infectious Diseases 2010;201:1796–1805

DOI: 10.1086/652750

Pretreatment Levels of Soluble Cellular Receptors and Interleukin‐6 Are Associated with HIV Disease Progression in Subjects Treated with Highly Active Antiretroviral Therapy

Robert C. Kalayjian,1 Rhoderick N. Machekano,1 Nesrine Rizk,1 Gregory K. Robbins,3 Rajesh T. Gandhi,3 Benigno A. Rodriguez,2 Richard B. Pollard,4 Michael M. Lederman,2 and Alan Landay5

1MetroHealth Medical Center and 2University Hospitals–Case Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio; 3Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts;4University of California–Davis Medical Center, Sacramento; 5Rush Medical College, Chicago, Illinois

Background. To identify inflammatory pathways that may contribute to the pathogenesis of human immunodeficiency virus (HIV) disease, we explored associations between AIDS or death and different inflammatory markers, including selected soluble tumor necrosis factor superfamily receptors (sTNFRs) and ligands, interleukin (IL)–6, and CD8 T cell activation, in individuals treated with highly active antiretroviral therapy (HAART).

Methods. A case‐control study of subjects in AIDS Clinical Trials Group (ACTG) protocols 384 and 5015, who were matched according to the CD4 cell count and plasma viral load at baseline, was performed using conditional logistic regression.

Results. Higher pretreatment concentrations of sTNFR‐1, sCD27, sCD40L, and plasma IL‐6 were associated with a new AIDS‐defining illness or death in separate models adjusted for age, sex, hemoglobin, and the latest CD4 cell counts. In additional models that excluded case patients with opportunistic infections, sTNFR‐1, sCD27, and sCD40L were each associated with a new AIDS‐defining malignancy or death that developed at a median of 51 weeks after initiation of HAART, by which time the majority of subjects had a CD4 cell count of >200 cells/cm3 and had achieved a plasma viral load of <50 copies/mL.

Conclusion. These data are compatible with a model in which these soluble inflammatory markers identify pathways that may contribute to the pathogenesis of HIV disease progression, pathways that might not be a direct consequence of ongoing HIV type 1 replication.

J Acquir Immune Defic Syndr. 2010 May 12. [Epub ahead of print]

C-Reactive Protein Levels Increase During HIV-1 Disease Progression in Rakai, Uganda, Despite the Absence of Microbial Translocation.

Redd AD, Eaton KP, Kong X, Laeyendecker O, Lutalo T, Wawer MJ, Gray RH, Serwadda D, Quinn TC; on behalf of the Rakai Health Sciences Program.

From the Laboratory of Immunoregulation, Division of Intramural research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD; Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD; Department of Population, Family, and Reproductive Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD; Rakai Health Sciences Program, Kalisizo, Uganda; and School of Public Health, Makerere University, Kampala, Uganda.

Abstract

INTRODUCTION: Microbial translocation has been implicated as a contributing factor to the heightened immune activation observed during HIV-1 disease progression. When examined in a longitudinal study of HIV-1 seroconverters in Rakai, Uganda, microbial translocation was not associated with HIV-1 disease progression. However, the role of general immune activation in HIV disease progression in this population was not fully examined. METHODS: Longitudinal serum samples of HIV-1 seroconverters in three HIV-1 disease progression groups [long-term nonprogressors (LTNP), standard progressors (SP), and rapid progressors (RP)] from Rakai, Uganda, were tested for levels of C-reactive protein (CRP), a marker for immune activation. RESULTS: CRP levels significantly increased in the SP group (P < 0.0001) but not in the RP group or the LTNP group. CRP levels during the first year post-HIV seroconversion in the RP group were significantly higher than those observed in the LTNP group (P < 0.05). For the entire population, CRP levels negatively correlated with lipopolysaccharide levels (P < 0.05) and were not associated with endotoxin antibody levels. CONCLUSIONS: This study suggests that in this population, increased immune activation is significantly associated with HIV-1 disease progression but not microbial translocation.

Atherosclerosis. 2010 Apr 24. [Epub ahead of print]

Heightened inflammation is linked to carotid intima-media thickness and endothelial activation in HIV-infected children.

Ross AC, O'Riordan MA, Storer N, Dogra V, McComsey GA.

Rainbow Babies and Children's Hospital, Case Western Reserve University, Cleveland, OH, United States.

Abstract

OBJECTIVES: HIV+ patients are at increased risk of cardiovascular disease (CVD). Inflammation plays a role in adults, but has not yet been assessed in HIV+ children. We compared proinflammatory cytokines and adhesion molecules in HIV+ children versus healthy controls, and assessed their relationship to carotid intima-media thickness (IMT). METHODS: Evaluations were performed on 27 HIV+ children and 30 HIV-healthy controls (2-21 years) who were prospectively enrolled in our pediatric cohort. Measurements included internal carotid artery (ICA) and common carotid artery (CCA) IMT, fasting lipids, insulin, proinflammatory markers (TNF-alpha, soluble TNF receptors (sTNFR-I, -II), IL-6, high sensitivity C-reactive protein (hsCRP), myeloperoxidase (MPO)), and adhesion molecules (soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-1 (sVCAM-1), von Willebrand factor). RESULTS: Among HIV+, mean age was 11 years, 33% males, 70% black. 96% acquired HIV vertically; median CD4 count (CD4%) was 1058 (35%) cells/ml; 96% were on antiretroviral therapy (ART); 70% had HIV-1 RNA <50copies/ml. Groups were similar in age, race, sex, BMI, proinflammatory cytokines, adhesion markers, and carotid IMT except for hsCRP which was higher in HIV+ (P<0.001). In multiple regression analyses, hCRP, age, and female sex were positively associated with IMT. ART duration and sTNFR-II were positively associated with sVCAM-1. CONCLUSIONS: This study shows increased hsCRP in HIV+ children compared to healthy controls. As seen in adults, hCRP was associated with carotid IMT, which support a role for inflammation in CVD risk of HIV+ children

Low Level HIV Replication Versus Latency: Identifying the Source of Viral Rebounds During Treatment Interruption

In HIV research, there is a persistent and vigorous debate around the question of whether or not viral replication persists in the face of successful antiretroviral therapy. During a plenary session at the International AIDS Conference in Mexico City back in August, Bob Siliciano made a compelling argument that, in most cases, antiretroviral therapy completely shuts down virus production. Now, a new paper in PNAS provides additional support for this view. Beda Joos and colleagues evaluated a staggering 1,753 genetic sequences from the envelope region of HIV, sampled over the course of a treatment interruption trial known as SSITT (Swiss-Spanish Intermittent Treatment Trial). The study design involved a series of two-week treatment breaks followed by a prolonged interruption (therapy was subsequently reinitiated according to the CD4 and viral load thresholds used in current treatment guidelines). 

The researchers used the sequence data to plot the relationships between the different viruses, using a technique called phylogenetic analyses. For each study participant analyzed, the sequences were used to define “the most recent common ancestor” (MRCA), which is an approximation of the ancestral virus sequence from which all the others derived. Viruses that appeared during treatment interruptions (TIs) were then compared to the MRCA, to see if the sequences suggested that there had been ongoing replication and evolution while the study participants were on ART. The results showed that the rebounding viruses during TI were actually more distant from the MRCA than the viruses detected when the participants first entered the study. The researchers conclude: “the striking lack of a temporal relationship between rebounding virus and pretreatment viruses strongly suggests that rebounding virus originates from reactivated, latently infected cells rather than from a cellular pool or compartment engaged in low-level replication.” 

PNAS October 28, 2008 vol. 105 no. 43 16725-16730

Published online before print October 20, 2008, doi: 10.1073/pnas.0804192105

HIV rebounds from latently infected cells, rather than from continuing low-level replication

Beda Joosa,1, Marek Fischera, Herbert Kustera, Satish K. Pillaib, Joseph K. Wongb, Jürg Bönic, Bernard Hirscheld, Rainer Webera, Alexandra Trkolaa, Huldrych F. Güntharda,1, and The Swiss HIV Cohort Study2

aDepartment of Medicine, Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zürich, CH-8091 Zurich, Switzerland;

bSan Francisco Veterans Affairs Medical Center, University of California, San Francisco, CA 94121;

cNational Center for Retroviruses, University of Zurich, CH-8028 Zurich, Switzerland; and

dDivision of Infectious Disease, University Hospital Geneva, 1211 Geneva, Switzerland

Rapid rebound of plasma viremia in patients after interruption of long-term combination antiretroviral therapy (cART) suggests persistence of low-level replicating cells or rapid reactivation of latently infected cells. To further characterize rebounding virus, we performed extensive longitudinal clonal evolutionary studies of HIV env C2-V3-C3 regions and exploited the temporal relationships of rebounding plasma viruses with regard to pretreatment sequences in 20 chronically HIV-1-infected patients having undergone multiple 2-week structured treatment interruptions (STI). Rebounding virus during the short STI was homogeneous, suggesting mono- or oligoclonal origin during reactivation. No evidence for a temporal structure of rebounding virus in regard to pretreatment sequences was found. Furthermore, expansion of distinct lineages at different STI cycles emerged. Together, these findings imply stochastic reactivation of different clones from long-lived latently infected cells rather than expansion of viral populations replicating at low levels. After treatment was stopped, diversity increased steadily, but pretreatment diversity was, on average, achieved only >2.5 years after the start of STI when marked divergence from preexisting quasispecies also emerged. In summary, our results argue against persistence of ongoing low-level replication in patients on suppressive cART. Furthermore, a prolonged delay in restoration of pretreatment viral diversity after treatment interruption demonstrates a surprisingly sustained evolutionary bottleneck induced by punctuated antiretroviral therapy.

Inflammatory and Coagulation Biomarkers Linked to Mortality in Large Treatment Interruption Trial

The strong association between immune activation and disease progression in HIV infection has been demonstrated and confirmed by multiple studies over the past 25 years. In several reports, expression of the CD38 activation marker by CD8 T cells has correlated more closely with progression than viral load or CD4 T cell counts. Elevated levels of immune activation were even noted in the first AIDS case reports published in December 1981 in the New England Journal of Medicine (NEJM); at that time, CD38 was known as the thymocyte-associated antigen T10. Research has also shown that immune activation significantly increases the levels of pro-inflammatory cytokines in people with HIV infection compared to uninfected controls. Suppression of HIV replication by antiretroviral therapy (ART) typically causes an immediate and precipitous decline in immune activation, but levels tend to remain slightly higher than in controls even after several years of treatment.

What has been relatively unclear until recently is the degree to which the immune activation and inflammation caused by HIV infection can impact health. This under-appreciated aspect of disease pathogenesis was highlighted by the outcome of a large, 5,472-person randomized trial known as SMART (Strategies for the Management of AntiRetroviral Therapy), which compared continuous ART with an intermittent, CD4-guided ART strategy. Smaller studies had suggested this approach might be a safe and potentially toxicity- and cost-sparing approach to treating HIV and the primary risks were thought to involve drug resistance, increased possibility of transmission and acute infection symptoms after interruption. Only a few clinicians, such as Diane Havlir, voiced concern that the inflammatory consequences of HIV replication might cause treatment interruptions to be harmful. It was thus a surprise to many when in late 2006, it was announced that the intermittent therapy arm of SMART was being halted due to a doubling of the risk of illness and death compared to the continuous ART arm. The results, published subsequently in NEJM, showed that the intermittent arm did worse on every endpoint, and the increased risk of clinical events and death was seen across all CD4 strata, not just among individuals with low baseline counts or nadirs. The majority of the events involved cardiovascular, liver and kidney disease and not opportunistic infections, suggesting that immune activation and inflammation had played a larger role in the outcomes than immunodeficiency. Now, in the latest PLoS Medicine, the SMART researchers report that analyses of biomarkers of inflammation and blood coagulation offer strong evidence that this was indeed the case.

The study authors begin by noting that prior studies have demonstrated links between HIV replication and endothelial cell dysfunction (endothelial cells line the interior surface of blood vessels), and that this was associated with elevations in markers of hypercoagulability (a tendency for the blood to clot inappropriately) which were reduced by ART. Levels of the pro-inflammatory cytokine IL-6 have also been shown to be elevated in cross-sectional studies of HIV-infected individuals compared to uninfected controls, and IL-6 levels correlate with viral load in advanced disease. Similarly, levels of the pro-inflammatory marker C-reactive protein have been shown to increase over the course of HIV infection, with the highest levels reported among individuals progressing to AIDS.

In the SMART trial, a pre-planned substudy regularly measured four inflammatory biomarkers: high sensitivity C-reactive protein (hsCRP), IL-6, amyloid A, and amyloid P, and two coagulation markers: D-dimer and prothrombin fragment 1+2. To evaluate the impact of these biomarkers on the risk of death among SMART participants, the researchers conducted a case-control study in which two controls were matched with each of the 85 participants who died during the trial in order to compare biomarker levels using the samples taken prior to death. A second analysis was also conducted to look at levels in a larger random sample of 250 participants from each trial arm. Additional analyses looking at links between these biomarkers and specific clinical events are also being conducted, but results will be published separately.

The results showed that IL-6, D-Dimer and C-reactive protein levels were significantly higher among individuals that died compared to the matched controls (p= <0.0001, <0.0001 and 0.005, respectively). Comparisons between individuals with levels in the highest versus lowest quartiles showed that the strongest associations were with IL-6 and D-Dimer, with odds ratios of 8.3 and 12.4, respectively. C-reactive protein also showed a significant association but with a lower odds ratio of 2.0.

In the larger analysis of a random sample of 250 participants from each arm after one month on study, the researchers found that individuals randomized to interrupt ART showed significant increases in IL-6 and D-Dimer levels: the median increase in IL-6 was 0.60 pg/ml (a 30% increase) and for D-Dimer 0.05 μg/ml (a 16% increase). In comparison, the continuous ART group showed median increases of 0.0 μg/ml and 0.12 pg/ml (a 5% increase) for D-dimer and IL-6, respectively. The researchers report that changes in hsCRP and amyloid A were in the same direction but did not differ significantly between the two groups. The largest increases were seen among individuals in the interruption group who entered the study on ART with viral loads below 400 copies/ml (a 27% increase in D-Dimer and 43% increase in IL-6).

In discussing their results, the authors note that elevated IL-6 and D-Dimer levels have been associated with all-cause mortality in studies of uninfected elderly and critically ill individuals, lending plausibility to the associations observed in SMART. They also point out that while inflammation is the most likely factor driving these biomarker elevations, the study does not formally prove this, and other potential contributors could involve microbial translocation (which elevates LPS, which in turn can elevate D-Dimer and provoke IL-6 production by monocytes) and direct effects of HIV proteins or HIV replication. One implication of the data highlighted by the researchers is that “specific therapies that reduce the inflammatory response to HIV and decrease hsCRP, IL-6, and D-dimer levels may warrant investigation as an approach for reducing risk of death among HIV-infected individuals.”

A potentially important and worrying issue raised by these results which isn’t discussed in the paper is that studies of treatment interruptions are likely more dangerous to people’s health than has previously been appreciated. The general consensus has been that restricting this type of research to individuals with high CD4 counts and regularly monitoring CD4 counts and viral load during ART interruption is sufficient to ensure safety. These new data suggest that IL-6 and D-Dimer levels should be evaluated in order to exclude those at highest risk from treatment interruption trials. Other factors that were significantly associated with mortality in SMART also need to be taken into consideration, these included age (p = 0.02), smoking (p = 0.01), prior cardiovascular disease (p = 0.04), and co-infection with hepatitis B or C (p = 0.03). Clear information about these findings also needs to be added to the informed consent of any ART interruption protocol. One example is a recently initiated trial of a therapeutic vaccine made by Bionor Immuno; this study involves a six month ART interruption and is enrolling people with CD4 counts over 400 and nadirs over 200. The SMART data show that, absent additional safeguards, this type of trial may put participants at unnecessary risk of illness and death.

PLoS Medicine Vol. 5, No. 10, e203 doi:10.1371/journal.pmed.0050203

Inflammatory and Coagulation Biomarkers and Mortality in Patients with HIV Infection (free access to full text)

Lewis H. Kuller1, Russell Tracy2, Waldo Belloso3, Stephane De Wit4, Fraser Drummond5, H. Clifford Lane6, Bruno Ledergerber7, Jens Lundgren8, Jacqueline Neuhaus9, Daniel Nixon10, Nicholas I. Paton11, James D. Neaton9*, for the INSIGHT SMART Study Group

1 University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 2 University of Vermont, Burlington, Vermont, United States of America, 3 Hospital Italiano de Buenos Aires, Buenos Aires, Argentina, 4 Saint-Pierre Hospital, Brussels, Belgium, 5 National Centre in HIV Epidemiology and Clinical Research, University of New South Wales, Sydney, Australia, 6 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America, 7 University Hospital, Zurich, Switzerland, 8 University of Copenhagen, Copenhagen, Denmark, 9 University of Minnesota, Minneapolis, Minnesota, United States of America, 10 Virginia Commonwealth University, Richmond, Virginia, United States of America, 11 Medical Research Council Clinical Trials Unit, London, United Kingdom

Background
In the Strategies for Management of Anti-Retroviral Therapy trial, all-cause mortality was higher for participants randomized to intermittent, CD4-guided antiretroviral treatment (ART) (drug conservation [DC]) than continuous ART (viral suppression [VS]).
We hypothesized that increased HIV-RNA levels following ART interruption induced activation of tissue factor pathways, thrombosis, and fibrinolysis.

Methods and Findings
Stored samples were used to measure six biomarkers: high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), amyloid A, amyloid P, D-dimer, and prothrombin fragment 1+2. Two studies were conducted: (1) a nested case–control study for studying biomarker associations with mortality, and (2) a study to compare DC and VS participants for biomarker changes. For (1), markers were determined at study entry and before death (latest level) for 85 deaths and for two controls (n = 170) matched on country, age, sex, and date of randomization. Odds ratios (ORs) were estimated with logistic regression. For each biomarker, each of the three upper quartiles was compared to the lowest quartile. For (2), the biomarkers were assessed for 249 DC and 250 VS participants at study entry and 1 mo following randomization. Higher levels of hsCRP, IL-6, and D-dimer at study entry were significantly associated with an increased risk of all-cause mortality. Unadjusted ORs (highest versus lowest quartile) were 2.0 (95% confidence interval [CI], 1.0–4.1; p = 0.05), 8.3 (95% CI, 3.3–20.8; p < 0.0001), and 12.4 (95% CI, 4.2–37.0; p < 0.0001), respectively. Associations were significant after adjustment, when the DC and VS groups were analyzed separately, and when latest levels were assessed. IL-6 and D-dimer increased at 1 mo by 30% and 16% in the DC group and by 0% and 5% in the VS group (p < 0.0001 for treatment difference for both biomarkers); increases in the DC group were related to HIV-RNA levels at 1 mo (p < 0.0001). In an expanded case–control analysis (four controls per case), the OR (DC/VS) for mortality was reduced from 1.8 (95% CI, 1.1–3.1; p = 0.02) to 1.5 (95% CI, 0.8–2.8) and 1.4 (95% CI, 0.8–2.5) after adjustment for latest levels of IL-6 and D-dimer, respectively.

Conclusions
IL-6 and D-dimer were strongly related to all-cause mortality. Interrupting ART may further increase the risk of death by raising IL-6 and D-dimer levels. Therapies that reduce the inflammatory response to HIV and decrease IL-6 and D-dimer levels may warrant investigation.

SMART: What a Difference Two Months Make

The results of the Data Safety Monitoring Board (DSMB) review of the SMART study that took place November 1-2, 2005 are available online: http://www.communityconsortium.org/research/nov05-summary.pdf

The salient quote:

"Conclusion: No consideration of early stopping based on safety or efficacy is indicated.”

Two months later, it's all over.