At NIH-Sponsored ‘Principles’ Panel, A First Peek at Second Phase Decay Data — and at Lymphoid Architecture
FDCs as storage depots
In mid-November, an NIH panel convened under the auspices of the Office of AIDS Research (OAR) to discuss scientific and clinical data which might help produce new state-of-the-art guidelines for the treatment of HIV infection. Mark Harrington represented TAG at the meeting. Other AIDS research luminaries who shared new data ran the gamut from Ashley Haase (University of Minnesota) and David Ho (Aaron Diamond) to David Chernoff (Chiron Corp.) and Steven Herman (Roche Molecular Systems).
Dr. Ho reviewed the current state of knowledge regarding the kinetics of HIV replication: the viral life-cycle lasts about 2.6; the half-life of a productively infected CD4+ T cell is about 1.6 days; the half-life of virus production is approximately 5.7 hours. As has been shown many times over the year, on potent treatment viral load falls some 2 logs (99%) within 3 weeks (first-phase viral decay) of initiation of treatment and then falls more slowly as other infected cellular components (macrophages and latently-infected T cells) become activated, produce virus and are killed or die of apoptosis (cell suicide). The second-phase decay half-life is believed to be between 8-28 days. Using these numbers as guides, Ho estimates that 93-99% of virus is produced by actively-infected CD4+ T lymphocytes with a half-life of one day, 1-7% is produced by actively-infected macrophages with a half-life of 14.4 days and < 1% is produced by latently-infected T cells with a half-life of 8.5 days. Much of the second-phase decay, then, reflects loss of infected macrophages.
Proviral DNA (that is, reverse transcribed and integrated viral RNA which has incorporated itself into the nucleus of an infected cell), however, is unlikely to disappear as quickly. In fact, at 120 days 1,000 provirus copies could be detected per 1 million cells. The possible existence of unforeseen slower third-phase decay or viral persistence in sanctuary sites inaccessible to treatments cannot be ruled out. Many fixed tissue macrophages (e.g., in the spleen) which are HIV DNA+ are HIV RNA-negative. And while many of these latently infected cells probably contain defective proviral DNA, even defective provirus could still result in cell death if cells bearing them were detected and killed by CTLs (cytotoxic T lymphocytes).
Follicular dendritic cells (FDCs) in lymphoid tissue act as a storage depot for infectious HIV in the lymph nodes, where viral particles are bound to FDCs by antibodies, but may remain infectious. Lymphoid tissue amounts to about 1% of the total body weight — 700 grams in a 70 kilogram person. University of Minnesota’s Dr. Ashley Haase, using new imaging techniques, showed that the FDC viral pool is much larger than the pool of newly-produced RNA inside of infected cells. Productively infected cells amount to only a fraction of total lymphocyte turnover (just 200 million per day), implying that other, indirect mechanisms of lymphocyte destruction must be at work — but what? Haase explains, “We’re looking at proliferation and apoptosis in HIV-positive and negative persons,” he answers. “We’re seeing a lot of additional proliferation and apoptosis in CD4s from infected persons, which drop in accordance with treatment. Replenishment of the CD4 reservoir is complicated; it includes cell expansion, retrafficking and other factors.”
Dr. Haase showed that after six months of potent triple-drug therapy, the CD4 cell count begins to rise in lymphoid tissue, but what’s going on with the lymphoid architecture? In Haase’s experiments, not only are the CD4s returning in lymphoid tissue (and FDC-trapped antigen levels falling) but the lymphoid architecture “appears to be returning to a healthier, more functional state,” he explains.
In contrast to disappointing antiviral effects in lymphoid tissue seen with AZT and ddI (Cohen et al.), researchers are seeing encouraging reductions in viral RNA in all compartments of lymphoid tissue. At 24 weeks they observed a two-fold decrease in the magnitude of the FDC-associated HIV pool. David Ho commented that this suggests that the half-life of FDC-trapped HIV is about three weeks. Four (unpublished) studies at Johns Hopkins, Boston City Hospital, Chiron Corp. and New Jersey Medical Center, have shown that, on potent therapy, viral load is dropping precipitously in lymphoid tissues. While potent combination antiretroviral therapy clearly affects these reservoirs, viral residues remain. Moreover, virus could emerge from immunologically privileged sites such as the central nervous system to reinfect peripheral cells.
In a separate presentation, Chiron’s David Chernoff discussed improvements in the sensitivity of the bDNA (Quantiplex) assay. The newer-generation (version 3.0) bDNA assay has greater photoluminescence, providing greater sensitivity, down to about 50 copies/ml of plasma.
To resolve (only recently appreciated) uncertainties about the Mellors numbers (published in mid-1996 to great fanfare) and to help laboratories prepare specimens properly, Chiron tested a known amount of virus in three different standard tubes: EDTA, ACD, and heparin. In EDTA tubes the average RNA value was 171,000 copies/ml; in ACD tubes it was 150,000 copies/ml; and in heparin, 106,000. EDTA tubes are the most sensitive, and so the highest viral copy numbers are are recorded when blood is stored in them. Heparinized tubes are always the least sensitive. ACD tubes are somewhere in between. Using heparin as the anticoagulant (as was done in John Mellors’ acclaimed study) reduces the RNA levels to 65% of EDTA levels after 2 hours-and to 45% after 30 hours. (If Mellors’ plasma samples had been stored in EDTA or ACD tubes instead of heparin, his cut-off plasma RNA values would have been significantly higher.) The relationship between the Mellors numbers and PCR and NASBA remains unknown.
Chernoff showed that samples can vary by as much as two- to three-fold due to operator and assay differences, as well as intrapatient biological variability, although there does not appear to be a diurnal periodicity to HIV plasma viral load as there is with CD4 counts.
When Roche’s Steven Herman looked at HIV RNA plasma viral load variability using the Roche (Amplicor) assay in 20 asymptomatic antiretroviral naive patients with CD4 > 400, he found the maximum variation to be three-fold or less in 15 patients, three- to four-fold in 18 patients and four- to six-fold in one. David Chernoff noted that many people become concerned if their viral load increases from, for example, 5,000 to 10,000. “But that’s well within the normal variation of the assay,” he cautions. Roche’s Herman concurred, “A two-fold change in HIV RNA is not significant. A significant change is larger than a three-to-five-fold change in RNA, e.g., a rise from 10,000 to over 50,000 copies.” And this sort of variability is especially high in patients with very low numbers.