Treatment intensification does not eliminate HIV in reservoir sites

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HIV continues to be released in small amounts from "reservoir" sites in the body despite suppressive antiretroviral therapy, and adding more drugs has not succeeded in eradicating the virus, researchers reported at the Sixteenth Conference on Retroviruses and Opportunistic Infections in Montreal last month.

HIV persistence and treatment intensification were themes running throughout this year's meeting, with an opening plenary talk by Robert Siliciano from the Howard Hughes Medical Institute at Johns Hopkins School of Medicine in Baltimore, US, and a dedicated poster discussion session.

Can HIV be eliminated?

Now that combination antiretroviral therapy can suppress HIV replication, researchers have begun to revisit the question of viral eradication. This exploration is aided by more sensitive viral load tests that can measure down to a single copy of HIV RNA.

Siliciano noted that there is debate about whether this small amount of residual HIV is due to low-level ongoing viral replication allowed by current antiretroviral drugs or whether it is released from stable reservoir sites.

Glossary

replication

The process of viral multiplication or reproduction. Viruses cannot replicate without the machinery and metabolism of cells (human cells, in the case of HIV), which is why viruses infect cells.

reservoir

The ‘HIV reservoir’ is a group of cells that are infected with HIV but have not produced new HIV (latent stage of infection) for many months or years. Latent HIV reservoirs are established during the earliest stage of HIV infection. Although antiretroviral therapy can reduce the level of HIV in the blood to an undetectable level, latent reservoirs of HIV continue to survive (a phenomenon called residual inflammation). Latently infected cells may be reawakened to begin actively reproducing HIV virions if antiretroviral therapy is stopped. 

deoxyribonucleic acid (DNA)

The material in the nucleus of a cell where genetic information is stored.

viraemia

The presence of virus in the blood.

 

peripheral blood mononuclear cells (PBMCs)

Any blood cell having a round nucleus (e.g., a lymphocyte, a monocyte or a macrophage). These blood cells are a critical component in the immune system. 

If residual HIV is due to continuing replication, adding more drugs – especially those that target different steps of the viral lifecycle – might be expected to further reduce or perhaps eliminate residual HIV. But studies in which a protease inhibitor was added to a suppressive NNRTI-based regimen did not see this effect, nor did more recent studies (described below) of intensification using raltegravir (Isentress).

"We have reached the theoretical limit of antiretroviral therapy," Siliciano said at a press conference preceding his talk. "In an adherent patient, the drugs we have now can completely stop the virus from replicating."

Given that residual virus is genetically similar and does not exhibit evolution, Siliciano argued that it is likely to come from stable genetic material in reservoir sites rather than ongoing replication. The existence of "transcriptionally silent" HIV genomes archived in latent cells, he said, "allows virus to persist as information".

It is well established that resting CD4 T-cells serve as one such site, but he presented evidence that a majority of residual virus is coming from an additional, not-yet-identified source – possibly a progenitor cell in the monocyte-macrophage lineage.

In summary, antiretroviral therapy stops viral evolution in adherent patients. The latent HIV reservoir is stable and is not replenished by ongoing viral replication. As such, treatment intensification has no effect on residual viraemia. Most residual viraemia results from release of virus from stable reservoirs sites, and at least two such reservoirs contribute.

"Progress toward eradication of the infection will require novel approaches to target the stable reservoirs that persist even when viral replication is completely halted," Siliciano concluded.

Residual HIV

Three studies presented in a poster discussion session on HIV persistence and eradication confirmed Siliciano's points.

Hiroyu Hatano and colleagues from the University of California at San Francisco measured HIV viral load using a highly sensitive assay (limit of detection 3 copies/ml) in 438 stored blood samples from 180 individuals enrolled in the SCOPE cohort, who had been HIV-infected for a median of twelve years and on suppressive antiretroviral therapy for a median of 13 months.

After at least one year of viral load suppression, approximately 80% of participants still had low-level detectable viraemia. The presence of residual HIV was similar in individuals who had been on suppressive treatment for 60 months, indicating that HIV RNA levels did not appear to decrease further after one year. As HIV antibody levels were also stable during long-term treatment, the researchers concluded, this suggests a "steady-state relationship" between the virus and the host response.

Veronique Avettand-Fenoel and colleagues from France undertook extensive analysis of the maturation state of resting CD4 T-cells taken from a patient infected with HIV for two years who received suppressive antiretroviral therapy during primary infection. These states include naive effector T-cells and primed memory T-cells. (For more on CD4 cell states, see this news article from 2004.)

Although the patient’s HIV RNA (the usual measure of viral load) remained below 20 copies/ml, HIV DNA was detected in his peripheral blood mononuclear cells (PBMCs). The investigators observed very stable HIV DNA levels amongst the different T-cell subsets over time, but found that intermediate effector memory T-cells contributed significantly more to the reservoir than other cell subsets.

Timothy Brennan from Siliciano's group at Johns Hopkins described a study in which ultrasensitive assays were used to compared the genetic structure of latent proviruses (HIV that has integrated itself into cells) in resting and activated CD4 cells from patients on suppressive therapy versus residual HIV that could still be detected in plasma.

In the majority of patients, proviruses in both resting and activated CD4 T-cells were genetically very similar, but residual plasma HIV was genetically distinct from the proviruses. Further analysis of one patient revealed that residual viraemia was genetically distinct from circulating activated CD4 T-cells, monocytes and PBMCs. These findings support Siliciano's argument that, in addition to the latent HIV reservoir in resting CD4 cells, there is an alternative cellular source of unknown origin that may be producing most of the residual virus seen in people on suppressive treatment.

Treatment intensification

Three other research teams presented findings from studies of treatment intensification.

In the first study, Maria Jose Buzon and colleagues from Spain, Sweden and the US randomly assigned 65 individuals on protease inhibitor- or NNRTI-based antiretroviral therapy with viral load below 50 copies/ml for at least one year to either stay on the same regimen (21 people) or intensify therapy by adding the integrase inhibitor raltegravir (44 people).

The researchers measured HIV DNA extracted from PBMCs at weeks 0, 2, 4 and 12. All participants maintained viral load below 50 copies/ml and a stable CD4 cell count. Although total and integrated (provirus) DNA levels remained stable, a transient increase in episomal (independent of a chromosome) DNA was seen at week two after raltegravir intensification, which disappeared by week four.

The investigators concluded that viral infection of cells continues despite antiretroviral therapy. The lack of any fluctuation in total or provirus DNA during treatment intensification indicates that "the majority of viral DNA in patients is archival and non-dynamic", they said. In the presence of raltegravir, they suggested new viral genomes are not able to integrate into a host cell's genetic material enabling complete replication, but instead are converted to episomes. It is not known whether this has any clinical relevance.

In a related study, Joseph Jones and colleagues from the University of Pittsburgh and the US National Cancer Institute enrolled nine men and one woman on protease inhibitor- or NNRTI-based antiretroviral therapy who had been treated for an average of about nine years and had achieved viral load below 50 copies/ml. Using a sensitive single-copy assay, HIV RNA was measured before, during and after 400mg twice-daily raltegravir was added to their current regimen for 30 days.

Drug intensification was well tolerated with no reported adverse events. Median viral load during intensification was 8.9 copies/ml, which was not significantly different from the median of 5.0 copies/ml at baseline. Presenter Frank Maldarelli said there was "no detectable effect of intensification," suggesting that residual HIV viraemia is not due to continued replication.

Finally, Rajesh Gandhi presenting findings from ACTG A5173, a collaboration of seven US hospitals and institutions including the National Institutes of Health, Johns Hopkins and the Harvard School of Public Health. The investigators enrolled 19 treatment-naive individuals with a CD4 count above 100 cells/mm3 and viral load above 1000 copies/ml.

In this non-randomised exploratory study, all participants started an intensive four-drug, triple-class regimen consisting of ritonavir-boosted saquinavir (Invirase), Truvada (tenofovir/emtricitabine) and the injectable fusion inhibitor enfuvirtide (T-20, Fuzeon).

One participant dropped out early due to enfuvirtide-related injection pain. Although 17 participants (89%) achieved a viral load below 50 copies/ml by 48 weeks, only nine continued to take enfuvirtide. Of these nine, four experienced a slight decrease in the number of latent resting memory CD4 T-cells whilst five had a slight increase.

Overall, the investigators found no evidence of decay in the latent HIV reservoir over 96 weeks, and the study was halted prematurely after an interim analysis. "This stability of the latent reservoir, which is similar to that seen in previous studies of less intensive antiretroviral therapy, suggests that new strategies are needed to eradicate HIV," they concluded.

References

Siliciano R New approaches for understanding and evaluating the efficacy of ARVs Sixteenth Conference on Retroviruses and Opportunistic Infections, Montreal, abstract 16, 2009.

Hatano H et al. Evidence of persistent low-level viremia in long-term HAART-suppressed individuals. Sixteenth Conference on Retroviruses and Opportunistic Infections, Montreal, abstract 425, 2009.

Avettand-Fenoel V et al. Stability of HIV reservoir resting CD4 T cell subsets under effective HAART. Sixteenth Conference on Retroviruses and Opportunistic Infections, Montreal, abstract 426, 2009.

Brennan T et al. Population structure analysis of HIV-1 in plasma and integrated provirus in resting CD4+ T cells suggests a novel source may be the dominant contributor to residual viremia in patients on HAART. Sixteenth Conference on Retroviruses and Opportunistic Infections, Montreal, abstract 427, 2009.

Buzon M et al. Transient increase in episomal viral cDNA following raltegravir intensification of a stable HAART regimen. Sixteenth Conference on Retroviruses and Opportunistic Infections, Montreal, abstract 423a, 2009.

Jones J et al. No decrease in residual viremia during raltegravir intensification in patients on standard ART. Sixteenth Conference on Retroviruses and Opportunistic Infections, Montreal, abstract 423b, 2009.

Gandhi R et al. No evidence for decay in the latent reservoir in HIV-infected patients receiving intensive enfuvirtide-containing ART. Sixteenth Conference on Retroviruses and Opportunistic Infections, Montreal, abstract 424, 2009.

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