Two overlapping groups of investigators attempted to determine if there was evidence of persisting replication competent HIV in patients receiving highly active antiretroviral therapy (HAART) who had had undetectable plasma HIV RNA by sensitive methods for prolonged periods of time.
Wong and colleagues obtained peripheral blood mononuclear cells from six patients treated with ZDV, 3TC, and indinavir for approximately two years and whose plasma HIV RNA had remained below 50 copies/mL. After in vitro depletion of CD8+ cells, the remaining CD4+ lymphocytes were stimulated with immobilized antibodies to CD3 (the T cell receptor) and CD28 (an important costimulatory molecule). The CD4+ lymphocytes were then cocultured with healthy donor lymphocytes that had been "prestimulated" with phytohemagglutinin (PHA); IL-2 was added to the coculture.
HIV was recovered by this coculture technique from the lymphocytes of all six patients. Partial sequencing of their HIV pol genes, however, revealed no new drug resistance mutations and no evidence of molecular evolution, indicating that the virus had not been replicating and was truly latent.
Finzi and colleagues examined lymphocytes from 22 patients who had received various HAART regimens for as long as 30 months and whose viral load was consistently less than 200 copies of HIV RNA per mL of plasma. Peripheral blood mononuclear cells were depleted of monocytes, CD8+ lymphocytes, B cells, and NK cells. In addition, activated CD4+ T cells were removed by bead depletion with antibodies to CD25 (the alpha chain of the IL-2 receptor), CD69 (an early activation marker), and HLA-DR (expressed on activated T cells), with further purification by flow cytometric sorting of small lymphocytes expressing CD4 but not HLA-DR. These steps yielded a cell population, 97% of which was comprised of resting CD4+ T cells with less than 1% contamination with activated CD4+ T cells.
These resting CD4+ T cells were then activated with PHA and irradiated peripheral blood mononuclear cells from a healthy donor who was not HIV infected. Subsequent restimulation with CD8 depleted PHA blasts from an HIV-negative donor was performed twice.
Sufficient numbers of resting CD4+ T cells were obtained from 18 patients, including the individual who had been treated for 30 months; replicating virus was isolated from each of them. In some cases, cytopathicity was demonstrated. The frequency of carriage of replication competent provirus ranged from 0.2 to 16.2 per 106 resting CD4+ T cells. Cross-sectional analysis found no evidence of decrease in this frequency with increasing duration of HAART.
As with the study by Wong and colleagues, analysis of the pol gene of viral isolates found little evidence of evolution of drug resistance.
COMMENT BY STAN DERESINSKI, MD, FACP
I apologize for the complexity of the methodology described as well as the immunologic jargon, but these are important studies and it is necessary to understand the lengths to which the investigators had to go to demonstrate the presence of replication competent virus in these patients. Previous investigators had suggested that, to the extent proviral DNA was present in patients with a prolonged period during which HIV RNA was undetectable in plasma, it was not capable of replicating. This study demonstrates that this virus is, in fact, replication competent under the appropriate conditions. Thus, the suggestion that as little as two years of HAART may be sufficient to effect cure of HIV infection is not correct, at least in the one patient studied who reached this duration of therapy.
Vila and colleagues previously reported two patients who started treatment with ddI and hydroxyurea for one year beginning three and 12 months after HIV infection, one year after discontinuation of treatment, had no apparent evidence of of viral replication in peripheral blood or in lymph nodes (Vila J, et al. Lancet 1997;350:635-636). However, proviral DNA remained detectable in both patients. Based on the observations reviewed here, it seems likely that both patients also probably still had replication competent virus present. A similar case, treated with indinavir, ddI, and hydroxyurea, was reported by Franco Lori (Cohen J. Science 1997;277:1927).
The life span of memory CD4+ T cells is not well defined but may, in some cases, be years in duration. It is also uncertain whether other cell types infectable with HIV, such as macrophages and glial cells, may live even longer. At any rate, the life span of latently HIV-infected memory CD4+ lymphocytes may define the minimum duration of HAART necessary for viral eradication. Cross-sectional analysis of the frequencies of latently infected CD4+ lymphocytes in the study by Finzi and colleagues failed, however, to demonstrate any evidence of decline of this frequency over time, suggesting that this compartment may be very long-lived, making "cure" a very long-term endeavor.
That’s the bad news. The good news is that HAART is capable of freezing in time, for all practical purposes, the evolutionary history of the virus by effectively stopping viral replication. In the absence of replication, no mutations occur, and, consequently, the virus cannot escape the immune response and cannot develop further antiretroviral resistance. Thus, HAART has the capability of prolonged efficacy, although not that of short-term cure.
Which of the following is correct?
a. The cell reservoir of latent replication competent HIV appears to be long-lived resting memory CD4+ T cells.
b. Despite the presence of undetectable plasma HIV RNA for prolonged periods of time as a result of ongoing HAART, HIV present in peripheral blood lymphocytes continues to replicate as evidenced by the continued accumulation of antiretroviral resistance mutations.
c. The frequency of CD4+ T lymphocytes latently infected with replication competent HIV appears to decrease rapidly after several months of HAART.
d. The frequency of CD4+ T lymphocytes latently infected with replication competent HIV is approximately 1%.
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