Researchers claim to find HIV sanctuaries - Controvery
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Science 29 Jan 2016
For all the progress made in HIV treatment, a depressing fact stands in the way of a cure. Even after powerful antiretroviral drugs (ARVs) have driven the virus down to undetectable levels in the blood, it isn't gone. It lurks in "reservoirs," out of reach of treatment, and if drugs are stopped it almost always rebounds. But exactly how these reservoirs stick around for a person's entire life is contentious.
A new study supports a controversial proposal about why HIV persists in spite of aggressive treatment. The standard view is that the virus lies dormant inside of human chromosomes, out of reach of treatment because it is not replicating. The new study, published online in the 27 January issue of Nature by a prominent group of HIV/AIDS researchers, suggests instead that despite the barrage of drugs, HIV continues to replicate, sheltered in the lymph nodes, which ARVs have trouble reaching.
"The debate is over: There is ongoing replication" in people on ARVs who have no detectable virus in their blood, contends the Nature study's lead author, virologist Steven Wolinsky of Northwestern University Feinberg School of Medicine in Chicago, Illinois. If he's right, the current best hope of curing HIV infection-"kicking" latently infected cells to produce virus, which would lead to their destruction-will need rethinking. Instead, a cure might also have to up the dose of existing ARVs or develop others that better target tissues like the lymph nodes.
The debate is actually far from over. Daria Hazuda, who heads antiviral research at Merck in West Point, Pennsylvania, calls the new data "intriguing and compelling" and says the study is "very consistent with other pieces of data that have been percolating for a decade." But other HIV experts say the data don't support the study's bold conclusions. "It's quite upsetting," says virologist John Mellors of the University of Pittsburgh in Pennsylvania. "The authors should be much more cautious." The study "just doesn't agree with results that we have," says John Coffin, a retrovirologist at Tufts University in Boston.
More than 4 years ago, two of the Nature paper's co-authors reported that concentration of ARVs in lymph nodes and other tissues may be far lower than in blood-not high enough to stop all viral replication (Science, 23 December 2011, p. 1614). To test that possibility, the team analyzed HIV genetic sequences in difficult-to-obtain samples of lymph nodes taken over 6 months in three patients. All had started ARVs and fully suppressed the virus on standard blood tests. HIV mutates each time it copies itself, so if the virus were replicating in the lymph nodes, the sequences should show signs of evolution. The researchers found just that: sequence changes showing that HIV had evolved in the lymph node of each patient. A slow trickle of new virus was apparently being produced.
Coffin counters that he and others have seen no signs of evolution in several studies that examined virus found in the blood of patients who take powerful ARVs for longer than a decade. He and Mellors fault the machine Wolinsky and his colleagues relied on-a Roche 454 DNA Sequencer-which is no longer used by most labs. "The reason it's obsolete is it had unacceptably high error rates," Mellors says.
"He's categorically wrong," Wolinsky counters. "As long as you know the errors introduced by the technique, you can control for it." He points out the group analyzed each sample twice and obtained the same results. What's more, Wolinsky says the viral family trees created from sequences in each person match the mutation rates that would be expected if new viruses were made. "I think the data are real," says Douglas Richman, a virologist at the University of California, San Diego, who has long been in Coffin and Mellors's camp. Richman adds that the study's authors are "the best in the world" at analyzing HIV in tissues and viral evolution.
Wolinsky and his co-authors enlisted leading viral evolutionary biologist and modeler Andrew Rambaut from the United Kingdom's University of Oxford to address another argument made by those who contend ARVs stop all new virus production: If replication is occurring because of insufficient concentrations of the drugs in the tissues, then drug-resistant mutants should emerge. Rambaut's model suggests that when ARV concentrations are low, wild-type virus isn't hampered much and handily "outcompetes" resistant variants.
"You can use a model to support anything you want, but you can prove nothing," Mellors says. "You can model that the sun orbits the Earth."
Sharon Lewin, an HIV cure researcher at the University of Melbourne in Australia, isn't totally convinced, either. She says she'd like to see data from more than three people and also from those suppressed for longer than 6 months. But she adds that "the work raises lots of new questions and avenues for research."
Wolinsky and co-authors agree. They hope their study will spark clinical experiments that attempt to increase ARV concentration in lymph nodes and then assess the impact on HIV reservoirs. "It's a spectacular debate," Wolinsky says. "And if indeed we have ongoing replication in these drug sanctuaries, we now have a new path to a cure."
Persistent HIV-1 replication maintains the tissue reservoir during therapy
"Our results, which reconstruct the dynamics of HIV-1 spread within the body, imply that in patients with no detectable viral RNA in plasma, the virus reservoir is constantly replenished by low-level virus replication in lymphoid tissue. Distinguishing between low amounts of viral replication and pools of latently infected cells that may persist and reactivate HIV-1 infection is methodologically difficult."
"Our results reveal how dynamic and spatial processes act together to permit HIV-1 to persist within the infected host and avoid development of resistance despite antiretroviral therapy. From these temporally and compartmentally structured sequence data, we conclude that continued virus production from infected cells in lymphoid tissue sanctuary sites, where drug concentrations are not fully suppressive, can continue to replenish the viral reservoir and traffic to blood or lymphoid tissue18. We further show that the virus does not inevitably develop resistance to antiretroviral drugs because the lower concentration of drugs in the sanctuary sites is not sufficient to confer a competitive advantage upon drug-resistant strains. Our findings explain the failure of treatment intensification to fully suppress de novo infection and highlight issues surrounding the barriers to delivering antiretroviral drugs at clinically effective concentrations in the infectious viral reservoir. The state-of-the-art sequencing approach, innovative time-calibrated phyloanatomic tree construction, and a novel model of compartmentalized intra-host population dynamics provide a new perspective on the persistence of HIV-1 in the body. Achieving optimal cellular pharmacokinetics and spatial distribution of antiretroviral drugs in lymphoid tissue to fully suppress viral replication and preserve immune function would be a prerequisite to the elimination of the viral reservoir and ultimately a step towards a cure for HIV-1 infection."
Ongoing HIV Replication Replenishes Viral Reservoirs During Therapy
NIH, For Immediate Release: Wednesday, Jan. 27, 2016
NIH-Funded Study Provides Insights Into HIV Evolution and Persistence
In HIV-infected patients undergoing antiretroviral therapy (ART), ongoing HIV replication in lymphoid tissues such as the lymph nodes helps maintain stores, or reservoirs, of the virus, a new study funded by the National Institutes of Health suggests. A better understanding of how HIV persists in the body is essential for developing strategies to eliminate viral reservoirs-a prerequisite to achieving a cure for HIV infection.
Current ART regimens quickly suppress HIV to levels undetectable in the blood in most patients, but cannot eliminate persistent viral reservoirs in the tissues. Scientists have debated whether these reservoirs are maintained because latently infected cells are long-lived, because low-level HIV replication persists or for both reasons.
To help address this question, Northwestern University's Steven Wolinsky, M.D., and colleagues sequenced viral DNA from lymph-node and blood cells collected from three HIV-infected patients before and during the first six months of ART. In these patients, the virus evolved over time, indicating ongoing replication, but did not accumulate mutations conferring drug resistance. Previous work had suggested that antiretroviral drug concentrations are lower in lymphoid tissue than in blood, and that HIV can hide in sanctuaries that drugs do not penetrate well. In this study, researchers demonstrated that continued HIV replication in lymphoid tissue sanctuaries refills viral reservoirs in patients on ART who have achieved undetectable blood levels of HIV.
Next, the investigators constructed a mathematical model to explain how the virus evolves during ART without the emergence of highly drug-resistant strains. According to their calculations, drug-sensitive HIV strains tend to dominate over drug-resistant strains when the effective drug concentration is low. At intermediate drug concentrations, drug-resistant strains start to dominate, and at high concentrations, HIV cannot grow. These observations suggest the importance of devising strategies to deliver clinically effective drug concentrations throughout the lymphoid tissue compartment, the investigators note.
Future studies using drugs that better penetrate the entire lymphoid tissue compartment should provide a more complete picture of how viral reservoirs are maintained and help pave a promising path to a cure, according to the authors.
The researchers found that cells in the lymph node tissue can still produce new virus and infect new target cells.
The groundbreaking idea of reservoirs of HIV made headlines in July 2014 when a Mississippi girl born with HIV, who was believed to be cured after early treatment, tested positive for the virus after stopping therapy. In her case, doctors think the infection reemerged from a viral reservoir containing cells in a resting state that were not proliferating.
The latest study appears to show a different type of "sanctuary," as the researchers called it, harboring cells with low levels of HIV replication that move into the blood. Researchers used a mathematical model to track the amount of virus and the amount of infected cells as they grew and circulated through the body.
This suggests that virus growth could occur in a place where drug concentrations are very low.
"These findings are important as it is critical for the field of HIV cure research to know whether new infectious cycles are indeed continuing in patients on seemingly effective treatment," Deborah Persaud, a professor of infectious diseases at Johns Hopkins University School of Medicine, told The Washington Post.
R Lorenzo-Redondo et al. Persistent HIV-1 replication maintains the tissue reservoir during therapy. Nature DOI: 10.1038/nature16933 (2016).
Persistent HIV-1 replication maintains the tissue reservoir during therapy
Ramon Lorenzo-Redondo1*, Helen R. Fryer2*, Trevor Bedford3, Eun-Young Kim1, John Archer4, Sergei L. Kosakovsky Pond5 ,
Yoon-Seok Chung6, Sudhir Penugonda1, Jeffrey G. Chipman7, Courtney V. Fletcher8, Timothy W. Schacker9, Michael H. Malim10,
Andrew Rambaut11, Ashley T. Haase12, Angela R. McLean2 & Steven M. Wolinsky1
Lymphoid tissue is a key reservoir established by HIV-1 during acute infection. It is a site associated with viral production, storage of viral particles in immune complexes, and viral persistence. Although combinations of antiretroviral drugs usually suppress viral replication and reduce viral RNA to undetectable levels in blood, it is unclear whether treatment fully suppresses viral replication in lymphoid tissue reservoirs. Here we show that virus evolution and trafficking between tissue compartments continues in patients with undetectable levels of virus in their bloodstream. We present a spatial and dynamic model of persistent viral replication and spread that indicates why the development of drug resistance is not a foregone conclusion under conditions in which drug concentrations are insufficient to completely block virus replication. These data provide new insights into the evolutionary and infection dynamics of the virus population within the host, revealing that HIV-1 can continue to replicate and replenish the viral reservoir despite potent antiretroviral therapy.
Combinations of antiretroviral drugs routinely impair HIV-1 production and replication to levels that are undetectable in the blood within weeks of starting treatment1. None of the current treatments, however, is capable of eradicating the virus from a long-lived reservoir in resting memory CD4+ T cells and other cell types that potentially protect the virus from antiretroviral drugs or immune surveillance2, 3, 4, 5. Intermittent virus production from reactivation of a small proportion of latently infected CD4+ T cells (rather than low levels of ongoing replication) is thought to drive viral rebound detected in blood of treated patients with well-suppressed infection6, 7, 8. Ongoing replication is considered unlikely because neither viral genetic divergence over time, nor the emergence of drug resistance mutations have been convincingly documented9, 10. As earlier studies only examined viral sequences derived from the blood of patients who continued to suppress viral replication in that anatomic compartment11, the conclusions are not necessarily generalizable to other compartments in the body, particularly to lymphoid tissue where the frequency of infection per cell is mostly higher12 and the intracellular drug concentrations are much lower than in blood13. Under low drug concentrations, the virus may continue to replicate and evolve in 'sanctuary sites' within the reservoir of cells in lymphoid tissue, and remain undetectable in the bloodstream for a time depending on viral population migration dynamics between the two compartments. Here we use a multi-pronged strategy of deep-sequencing, time-calibrated phylogenetic analysis, and mathematical modelling to characterize the distinct temporal structure and divergence of compartmentally sampled viral sequences. We discover ongoing replication in lymphoid tissue sanctuary sites of patients despite undetectable blood levels of virus. Our sampling approach differs fundamentally from those of previous studies14, 15, 16, which do not address evolutionary dynamics within lymphoid tissue, and better suits investigation of the dynamic nature of the viral reservoir during treatment with potent antiretroviral drugs.
Our results, which reconstruct the dynamics of HIV-1 spread within the body, imply that in patients with no detectable viral RNA in plasma, the virus reservoir is constantly replenished by low-level virus replication in lymphoid tissue. Distinguishing between low amounts of viral replication and pools of latently infected cells that may persist and reactivate HIV-1 infection is methodologically difficult. A small number of HIV-1 sequences isolated at consecutive time points that persisted without evidence of genetic change might result from long-lived central memory cells, a fraction of which may have reverted to a resting state, or latently infected transitional memory cells that persist by clonal expansion (driven by homeostatic proliferation) or survival of long-lived infected CD4+ T cells that contain replication-competent virus8, 15, 22, 31, 32, 33, 34. Two of the subjects (1774 and 1679) showed that some haplotypes persist as a single tree branch through time (Fig. 1d and f), consistent with proliferation of HIV-1-infected cells or long-term cell survival33, 34. Regardless of the different mechanisms for self-renewal/persistence by which some of these quite similar latent or defective viral lineages may have persisted, these quiescent viruses differ from others that have evolved and trafficked between compartments. The temporally and compartmentally sampled data show that viral lineages continue to diverge in well-suppressed patients and help to explain the persistence of infectious viral reservoirs with little reduction of the virus pool35. The dynamic nature of the viral population in lymphoid tissue sanctuaries-where infected cells can still produce new viruses, infect new target cells, and replenish the pool-undermines previous estimates of the time necessary to purge the reservoir of latently infected cells and achieve virus eradication3.
Our results reveal how dynamic and spatial processes act together to permit HIV-1 to persist within the infected host and avoid development of resistance despite antiretroviral therapy. From these temporally and compartmentally structured sequence data, we conclude that continued virus production from infected cells in lymphoid tissue sanctuary sites, where drug concentrations are not fully suppressive, can continue to replenish the viral reservoir and traffic to blood or lymphoid tissue18. We further show that the virus does not inevitably develop resistance to antiretroviral drugs because the lower concentration of drugs in the sanctuary sites is not sufficient to confer a competitive advantage upon drug-resistant strains. Our findings explain the failure of treatment intensification to fully suppress de novo infection and highlight issues surrounding the barriers to delivering antiretroviral drugs at clinically effective concentrations in the infectious viral reservoir. The state-of-the-art sequencing approach, innovative time-calibrated phyloanatomic tree construction, and a novel model of compartmentalized intra-host population dynamics provide a new perspective on the persistence of HIV-1 in the body. Achieving optimal cellular pharmacokinetics and spatial distribution of antiretroviral drugs in lymphoid tissue to fully suppress viral replication and preserve immune function would be a prerequisite to the elimination of the viral reservoir and ultimately a step towards a cure for HIV-1 infection.