 |
|
|
| |
Back to Basics:
the science at CROI focuses on the tough questions
that remain in HIV pathogenesis
|
| |
| |
CROI 2009 Feb 8-12 Montreal
David Margolis, MD, University of North Carolina
Is replication completely stopped by ART?:
As the Bernard Fields lecturer in virology, Bob Siliciano opened the 16th CROI with a discussion of HIV persistence. Siliciano (abstr. #16) began by reviewing the decay of plasma viremia upon the initiation of therapy: 1) the rapid initial decay that is thought to reflect the interruption of viral infection and production by activated cells of short lifespan that produce most of the plasma virus, which is also short-lived, 2) a second slower phase of decay which was thought to originate in long-lived cells such as macrophages, but is not apparent when antiretroviral therapy (ART) includes an integrase inhibitor, perhaps due to the efficacy of this new class of drug in both resting and activated cells, and 3) the slow or negligible decay of infection in persistently infected resting CD4 T cells.
Siliciano then summarized evidence that current ART completely interrupts ongoing cycles of replication. As first demonstrated by Roger Pomerantz with an assay that detected 5 copies/ml of HIV RNA, and more recently confirmed by Sarah Palmer and John Coffin using a linear assay that can measure a single copy of HIV RNA in plasma, at least 80% of patients on ART are actually viremic at a low level. ART therefore reduces the level of viremia to a new plateau that is a half-log to one log lower than the limit of detection of current, clinical assays.
One explanation for this would be ongoing cycles of viral replication, and infection of new cells, despite ART. However, evidence from a detailed analysis of the residual viremia, suggests that it is archival and non-evolving. No evidence of new drug-resistance mutations has been found, despite continued viremia in the presence of ART.
This would mean that although low levels of virus might be detected due to intermittent or continuous release from cells that had previously been infected, once full suppression is achieved on ART no cells in the body become newly infected. As evidence for this, Siliciano displayed a phylogenetic tree of viral sequences from a single patient. Viral sequences derived from cells which reflect the contents of the latent reservoir, and sequences detected in these free virus particles obtained over time in the plasma were genetically intermingled, and often identical. And this is consistent with the idea that at least some of the residual viremia is derived from this latent reservoir, but more importantly does not evolve despite the selective pressure of ART over many, many months. As resistance develops by the combined forces of random errors of reverse transcription, and the selective pressure of drugs to increase the replicative advantage of genomes that by chance contain drug-resistance mutations, the fact that new drug resistance mutations cannot be detected shows that although there may be low levels of virus expression there are not was is termed "effective rounds of replication" that would inevitably select for drug-resistant genomes. Importantly, in this situation ART should never fail if drug therapy can be maintained.
Another prediction of the view that current ART is fully suppressive of new rounds of replication, and that low-level viremia reflects release of viral particles from infected cells is that intensification of ART should not affect low-level viremia. Studies by Maldarelli, Coffin, and Mellors performed at the NCI showed no change in viremia when measured by the single-copy assay in patient with <50 copies of HIV RNA/ml of plasma in whom therapy was intensified by either lopinavir/ritonavir, atazanavir/ritonavir, or efavirenz.
Siliciano concluded this part of his discussion in saying that a small contribution from ongoing replication was difficult to absolutely exclude. He predicted that skeptics would always exist in this long-running debate, but that the major problem in the era of current ART is not ongoing rounds of low-level replication, but the release of virus from stable reservoirs. Ironically, a preliminary finding presented a bit later at this very meeting (abstr. 423a, discussed below) immediately challenged this assertion by providing evidence of very low-level, ongoing viral replication.
The effect of ART on persistent HIV:
Maldarelli and colleagues at the NCI performed an additional intensification study to probe the ability of ART to affect low-level viremia (abstr. 423b). As they have with lopinavir/ritonavir, atazanavir/ritonavir, and efavirenz, they conducted a trial of treatment intensification with raltegravir in patients with plasma HIV-1 RNA <50 copies/mL but residual viremia ≥1 copies/mL. Five patients added raltegravir (RAL) 400 mg twice daily to their therapy for a 30-day intensification period. The level of residual viremia before intensification (median, 1.9 copies/mL plasma) was similar to that found in prior studies of patients on standard combination therapy. Overall, the median plasma HIV-1 RNA level during RAL intensification (3.2 copies/mL) was not different from the median pre-intensification level (p = 0.72), but ranged from 0.1 copies/ml to 10 copies/ml.
No significant decreases in HIV-1 RNA levels were observed during drug intensification in individual patient analyses. So short-term intensification (ie. 30 days) with a new (to the patient) and potent RT inhibitor, protease inhibitor, or integrase inhibitor does not affect low-level viremia. It seems unlikely that drug compartment issues could explain the lack of effect of drugs of three different classes. In small published and unpublished studies, my research group has also seen no such effect using an entry inhibitor (enfuvirtide). Therefore, the cells from which this viremia emanates must have a half-life of much longer than 30 days.
In another study related to this issue, Ghandi, Eron, and other ACTG 5174 investigators studied the effect of an intensive ART on the stability of infection within resting CD4+ T cells using a regimen that simultaneously targeted entry, reverse transcription, and viral production (abstr. #424). Treatment-naive patients initiated therapy with enfuvirtide, tenofovir, emtricitibine (FTC), and ritonavir-boosted saquinivir. Resting cell infection frequency was measured in subjects who attained a VL <50 and remained on enfuvirtide and other ART for at least 48 weeks. Resting cell infection assays were done every 24 weeks, and nine volunteers contributed a median of 4 latent reservoir measurements each. Of these, four had a slight decay in the number of latently infected cells and five subjects had a slight increase over 96 weeks. Overall, there was no evidence for decay in the latent reservoir (95% confidence interval for half-life: 11 months to infinity). Of interest, the frequency of resting cell infection appeared to increase as CD4 count at initiation of therapy decreased. Also, although not independent of CD4 count, the frequency of resting cell infection increased as viral load setpoint increased, and decreased as the level of the activation marker CD38 increased.
Ghandi and Eron studied the initiation of therapy in patients with chronic infection. Although current ART can reliably ablate plasma viremia over time, it does not appear to alter the number of cells from which replication-competent virus can be recovered, once this persistent infection is established. The group's finding that the frequency of resting cell infection was associated with biological markers such as CD4 count (and potentially viral load or immune activation) is consistent with observations made by our group in patients studied within the CHAVI program (abstr. #514). We also found that the frequency of resting cell infection in a group of patients in whom therapy was initiated during acute infection was related to the duration of exposure to unchecked viremia.
Together, these findings create a relatively simple model of the equilibrium state of HIV replication and persistence on current, successful ART. Active replication is fully extinguished by ART, and while newer, better drugs may prevent or combat drug-resistance or drug toxicities, there is no need for better inhibitors of viral replication. And as said repeatedly at CROI and elsewhere, once reservoirs of persistent infection are (rapidly) established in resting CD4 T cells or other cell populations "...new approaches will be needed..." to eliminate persistent infection.
However, this simple view was provocatively challenged by a study from the Barcelona group presented by Buzon (abstr. #423a). By using a different assay than the other intensification studies presented, this group uncovered a different result. The group randomized 65 patients with <50 HIV-1 RNA copies/mL for at least 1 year either to intensify their HAART with RAL (n=44), or to continue their HAART (n = 21) for 48 weeks. They measured levels of episomal (circular), integrated, and total proviral DNA at weeks 0, 2, 4, and 12 by PCR techniques.
As you know, HIV RNA is reverse transcribed into DNA copies. Most of the double-stranded copies that are produced are defective or incomplete, and can be degraded by cellular enzymes over time. A few complete copies can be packaged in pre-integration complexes with cellular proteins and viral integrase, enter the nucleus and become viral integrants. Some complete HIV DNA genomes fail to integrate and host repair enzymes knit the double strands into DNA circles. So HIV DNA in a cell can exist as linear DNA, circular DNA, or integrated DNA. Linear or circular DNA will be diluted if the cell divides, or as cellular nucleases act on linear molecules. So in the absence of new infection of uninfected cells, a state thought to be achieved by current fully suppressive ART, no new HIV DNAs should be created. Over long periods of ART, total proviral DNA (the sum of all the DNA forms) has been observed to decline, but integrated copies have not been observed to decline during ART, and circular DNA declines after ART is initiated.
However, the Barcelona group used RAL as a new drug that intervened at a novel step of the replicative cycle, adding an additional obstacle for viral DNA to leave the pool of unintegrated linear DNA and enter the pool of proviral DNA. The 65 patients studied were suppressed on ART regimens that included 2 NRTI and either a PI or a NNRTI.
All subjects remained aviremic (<50 copies/mL) and had stable CD4 counts during the study period. In only 5 patients, all of whom were on PI-containing ART, could episomal, 2-LTR HIV DNA circles be detected at week 0, prior to RAL. There was a transient and significant increase (p = 0.0391) in episomal HIV-1 DNA at week 2 in the group of patients intensified with RAL as a whole, comparing to baseline. The abstract stated that this was not associated with ART composition.
However, it appeared that all 5 patients with detectable circles at entry were on PIs, and that 13 patients had detectable circles at week 2. All 5 patients detectable at week 0 were also detectable at week 2, although it was unclear if the level of circle detection had increased in these 5, or if an increase was measurably significant given assay variability. Of the 13 patients with detectable circles at week 2, 8 had been undetectable at week 0, and all but one or two of them were on PI-containing therapy. In contrast, total and proviral DNA levels remained stable during intensification.
If the LTR circle assay proves to be reproducible and reliable within patients over time, then this would provide strong evidence that de novo viral infection continues in the face of ART, but does not prove that complete rounds of replication occur. Given the knowledge that most patients have ongoing low-level viremia, the discovery that some of these particles can enter cells and complete reverse transcription is credible. RAL intensification may decrease the number of nascent viral genomes that integrate, and increase the number that are converted to episomal circles. The lack of any fluctuation in total and proviral DNA upon intensification reinforces the notion that the majority of viral DNA in patients is archival and non-dynamic.
Further, when re-measured at week 4 of RAL intensification, circles were only detectable in the five patients in whom circles had been originally seen at week 0. That this increase in circles above the assay limit of detection is transient in the 8 patients newly detected at week 2 is understandable only in part, at least to me. Recently infected, and likely proliferating, cells may divide or die, later reducing the number of circles below the level of detection. So the cells that contain the newly increased level of circles at week 2 may not survive very long, or may be dividing.
However, RAL intensification was still ongoing at week 4. One would therefore think that circle level would only decrease if new infection rate also decreased, suggesting that RAL had an effect on virus production as well, or perhaps that the circle assay was simply intermittently positive at the low end of the detectable range. However, the Maldarelli group did not see any effect of RAL on low-level viremia. These observations are difficult to reconcile; obviously further study and validation is warranted.
Where and how does HIV persist:
Wightman and colleagues (abstr. 416) measured the effect of ART on HIV DNA persistence within naïve CD4 T cells that had recently left the thymus, and distinguished by the surface marker CD31. It has been previously established that most latently, persistently infected CD4 cells are within the memory subset, but infection of naïve cells may occur. 10 patients were followed after ART initiation over 18 months. HIV DNA within cell populations was measured by real time PCR, adjusting for cell equivalents by the parallel quantitation of CCR5.
All patients sustained HIV RNA <50 copies/mL within 6 months. CD4+ cells increased overall, with naive cells increasing by 8% and the memory population decreasing by 12%. The percentage of CD31+ naive cells did not change, consistent with both a thymic and peripheral contribution to the naive CD4 T cell increase. As expected, HIV DNA content was higher in memory CD4 T cells than in CD31+ or CD31- naive cells. However, after ART HIV DNA declined significantly in memory cells but was stable in naïve cells. The authors concluded that "infected" naive CD31+ or CD31- CD4 T cells represent a very stable reservoir on ART. However, as mentioned above, the presence of HIV DNA is not equivalent to the presence of latent, replication-competent HIV. It remains to be proven if naïve cells are a more stable site of persistent HIV infection.
However, Chomont (abstr. 417) focused on which specific subtypes of memory CD4 T cells contain latent, replication-competent HIV. They found, both by HIV DNA quantitation, and by viral outgrowth assays, that central memory and transitional memory CD4 T cells constitute the major reservoirs for the virus and their relative contribution to this reservoir is a direct function of CD4 count and proliferation. Christine Rouzioux's group (abstr. 426) found very similar results in a smaller patient group using DNA PCR, although in their study there was a predominance of HIV DNA in effector memory T cells, rather than central memory T cells. Uniquely, Chomont and colleagues suggested that natural homeostatic proliferation, an interleukin-7-driven mechanism for maintaining the appropriate number of CD4 cells, could allow infected cells to replicate and maintain the size of the infected pool. These conclusions led them to suggest a unique strategy for eradication of persistent HIV infection: to inhibit the function of IL-7 and block homeostatic maintenance of CD4 cell proliferation.
Joe Wong at UCSF, with a group of collaborators, looked at viral sequences recovered from cells to ask if random defects in the ability of viruses to respond to the Tat viral transactivator could result in more of these crippled viruses establishing latency (abstr. 419). Using samples from 5 individuals who were started on ART within 6 months of infection and achieving a viral load <50 copies/mL, they found mutations in the tat gene and the TAR sequence (where Tat acts). Therefore, such "wimpy viruses" might end up trapped in latency within resting cells. This group has recently published a nice study suggesting that virus rebounds from resting cells after ART is stopped (Joos et al., Proc Natl Acad Sci U S A 2008). It would be of interest to know if it is possible to detect Tat or TAR mutants in rebounding viruses before reversion mutations to fully active Tat predominate.
A new strategy to purge persistent HIV:
Gisslen and colleagues (abstr. 88) presented provocative findings suggesting the IV immunoglobulin therapy could reduce the frequency of latent HIV-1 infection. Like many great ideas in medicine, this trial was sparked by a clinical observation: a patient on ART was treated with IV Ig for an autoimmune disease, stopped ART, and remained aviremic for several months. The idea was that the mild immune and complement activation induced by IV Ig might ignite replication of latent HIV, and its clearance.
9 subjects on ART were treated with 30 gm IV Ig for 5 days. Highly purified resting memory CD4+ T cells were isolated and activated, and replication-competent HIV-1 was quantified. Replication-competent HIV-1 was detected in resting T cells in 7 of the 9 subjects. IVIG was said to decrease the frequency of resting CD4 cell infection by more than 68% during the study period (8 to 12 weeks) in 5 of 9 subjects. The investigators also observed a transitory increase in plasma HIV-1 RNA using a sensitive assay (LOD 2 copies/ml), and in increase in serum IL-7 levels.
This study was provocative and interesting, especially given the safety record of IV Ig. However, the frequency of resting cell infection appeared to vary over a range of 50 to 0.1 per million in the patients studied, and the quantitative measurements of infection could not be explained by the presenter. It was not clear how many replicate cultures were studied, and what quantitative change in viral recovery led to the conclusion that resting cell infection had decreased three-fold. These findings would need to be clarified, but if shown to be credible the further testing and validation of this pilot study might be worthwhile.
Overall, these presentations at CROI offered an exciting and detailed new picture of persistent HIV infection in patients on ART. The picture requires further clarification, as by some measures current ART has achieved its functional limit, but by others improvements in antiviral activity, either in hidden cells or hidden anatomic compartments, might improve its effectiveness. It appears to be more and more practical to achieve and maintain full suppression of HIV viremia in more and more people across the globe. But further advances in therapy that might achieve eradication of persistent HIV infection remains an elusive goal.
|
| |
|
|
|
|
|
