Studies of PI-Resistant Virus Offer Tantalizing Possibilities for Immune Reconstitution
“The 1,500 Copy Cut-Off”
Every year, Keystone Symposia sponsor two parallel conferences on HIV pathogenesis and vaccines. The meetings take place at the Keystone Resort high in the Rocky Mountains, allowing researchers to mix the latest data with a daytime trip to the ski slopes. This year’s event offered no earth-shattering new insights, but provided some glimpses of future directions in HIV research. Richard Jeffreys was there to digest and report it all. Read the full report here.
Mike McCune and the Goldilocks Hypothesis: “Just enough virus, of just the right type, might be good.”
Many individuals who appear to be failing protease inhibitor-based antiretroviral drug combinations — based on detectable viral load and the presence of drug-resistance mutations — continue to do well both clinically and immunologically. At Keystone, Mike McCune from the Gladstone Institute in San Francisco reviewed efforts to explain this type of discordant response, which poses a challenge to traditional definitions of “treatment failure.” In collaboration with clinician Steve Deeks from San Francisco General Hospital, McCune started out by generating a series of hypotheses:
- Protease inhibitor-resistant virus may have reduced replicative capacity compared to wild-type virus.
- Reduced replicative capacity may be associated with reduced T-cell activation and turnover.
- Reduced replicative capacity in thymic tissue may spare T-cell production by the thymus.
- Reduced replicative capacity may facilitate the generation of stronger HIV-specific CD4 and CD8 T-cell responses.
The first hypothesis, suggested initially by test-tube data, was confirmed by Steve Deeks in a treatment interruption trial. In this study, individuals with resistant virus who were stable on a protease inhibitor-containing combination stopped treatment for twelve weeks. Over the first few weeks after interruption resistant virus remained detectable and CD4 T-cell counts stayed stable, but then wild-type (non-resistant) HIV rapidly emerged and entirely replaced the resistant virus population. This emergence of wild-type virus was associated with a significant increase in viral load and a decline in CD4 T-cell counts.
Moving onto the second hypothesis, McCune measured markers of T-cell activation and turnover in three groups of individuals: seventeen untreated, 36 with virological failure (defined as a viral load >2,500 copies/ml for >24 weeks despite being on protease inhibitor-containing treatment for at least eighteen months) and eighteen with virological success (defined as a viral load of <50 copies/ml for >24 weeks after receiving protease inhibitor-containing treatment for at least eighteen months). Comparing expression of the activation markers CD38 and HLA-DR on CD4 T cells between groups, McCune reported that they were significantly reduced in the virological failure group compared to the untreated controls. Activation markers were also significantly lower in the virological success group compared to the virological failure group.
A similar picture emerged when the same activation markers on CD8 T cells were measured. The proliferation marker Ki67 exhibited a somewhat different pattern: in CD4 T cells, expression was significantly higher in untreated individuals compared to those with virological failure or success, but there was no significant difference between the latter two groups.
McCune also measured T-cell turnover in seventeen individuals with virological failure using the deuterated glucose technique pioneered by Marc Hellerstein from the University of California at Berkeley. In this case, data were compared to historical controls (both untreated and those with virological success) from a previous study using the same technique. CD4 T-cell turnover was significantly reduced in the virological failure cohort compared to untreated individuals, but, as seen with Ki67, the difference between the virological failure and success groups was not significant.
The median CD4+ T cell half-lives for the three groups were 22 days (untreated), 68 days (virologic failure) and 82 days (virologic success). McCune emphasized that in untreated HIV infection, viral load correlates with the degree of T-cell activation and turnover, whereas in individuals with protease inhibitor-resistant virus this correlation is lost. He speculated that this situation may be analogous to that of sooty mangabey monkeys, which sustain high SIV viral loads but manifest little evidence of T-cell activation and no disease progression.
Addressing the third hypothesis, McCune noted that direct viral effects on thymic production are tough to prove in people. The thymus is not easily sampled, and direct measures of T-cell output from this organ are still lacking. The strongest support for the idea that protease inhibitor-resistant virus may be easier on the thymus comes from a study using a culture system.
Both protease inhibitor-resistant and wild-type HIV isolates were taken from participants in Steve Deeks’ treatment interruption study. The ability of these isolates to replicate in thymic organ culture was then compared and, as predicted, protease inhibitor-resistant viruses reproduced poorly compared to their wild-type counterparts.
McCune was able to produce a larger body of data in support of his fourth hypothesis. The Gladstone team employed intracellular cytokine staining to measure the magnitude of the HIV-specific CD4 and CD8 T-cell response (this technique captures HIV-specific T cells based on their ability to produce the cytokine interferon-gamma when stimulated with viral antigens).
This analysis included 28 untreated individuals, 66 experiencing virological failure on protease inhibitor-based treatment and 87 categorized as a virological success on protease inhibitor-based treatment. McCune reported that the total gag-specific CD4 T-cell response was significantly higher in the virological failure group than either the untreated or virological success groups. There was a significant correlation between gag-specific CD4 and CD8 T-cell responses in all groups.
McCune highlighted the fact that the magnitude of the HIV-specific CD8 T-cell response correlated with maintenance of a stable viral load over four months of follow-up. Looking at the magnitude of the virus-specific T-cell response and viral load from a single timepoint, McCune found that the data fitted a “bell-shaped curve”: individuals with viral loads less than 1,500 copies showed a positive correlation between the size of the gag-specific T-cell response and viral load, whereas those above the 1,500 copy cut-off exhibited a negative correlation.
McCune concluded his presentation with the provocative “Goldilocks Hypothesis:” that in some cases, the persistent presence of protease inhibitor-resistant virus may be beneficial in terms of preventing immune activation, sparing thymic output and fostering functional anti-HIV CD4 and CD8 responses. This appears to account for the “disconnect” seen in the Deeks cohort and other virologic “failures” who maintain or increase CD4 cell counts and do not progress clinically despite continually measurable viral load. Understanding the underlying cause of this change in viral pathogenicity could potentially shed light on the long-standing mysteries of the benign SIV infections seen in sooty mangabey and African green monkeys and thus open up new avenues of HIV research.