Identification of a Reservoir for HIV-1 in Patients on Highly Active Antiretroviral Therapy

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The frequency of resting CD4 + T cells carrying replication-competent HIV-1 was determined as described (11). Briefly highly purified resting cells were plated in duplicate fivefold serial dilutions beginning at 5 × 10 6 cells per well in culture medium consisting of RPMI with 10% heat-inactivated fetal bovine serum penicillin (50 U/ml) streptomycin (50 μg/ml) and 4 mM l -glutamine supplemented with recombinant interleukin-2 (100 U/ml). Resting CD4 + T cells were activated by the addition to each well of highly purified PHA (0.25 μg/ml; Wellcome Diagnostics Research Triangle Park NC) and a >10-fold excess of freshly isolated PBMCs from one or more HIV-1 seronegative donors. These PBMCs were inactivated by γ irradiation with 5000 roentgens in a cesium source irradiator and then washed twice. The next day PHA was removed and CD8-depleted PBMCs which had been stimulated with PHA for 3 days from an HIV-1–negative donor were added to each well. Cultures were fed and split as needed and freshly prepared CD8-depleted PHA blasts were added on days 7 to 9. Supernatants collected on day 14 were analyzed for p24 antigen by enzyme-linked immunosorbent assay (ELISA). Preculturing purified resting CD4 + T cells before activation did not consistently decrease the infected cell frequencies indicating that stable peristent forms of latent virus predominate in these patients.

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If the initial levels of latently infected cells before treatment are positively correlated with the time on therapy at which a given patient is studied then the actual decay rate may be more rapid than the rate suggested by a cross-sectional plot of IUPM versus time. However there is no reason to believe that the initial levels are correlated with time on therapy. The initial levels of latently infected cells before treatment in these patients are unknown. In a previous study (11) of untreated patients and patients on nucleoside analog therapy the frequency of cells carrying persistent replication-competent forms of virus ranged from 1 to 81 per 10 6 resting CD4 + T cells. The frequency of cells carrying any replication-competent form of virus ranged from 1 to 420 per 10 6 resting CD4 + T cells in the peripheral blood. These values for the frequencies of latently infected cells were not correlated with CD4 count or viral load. Therefore there is no reason to expect a correlation between the pretreament level of latently infected T cells and the time at which a given patient was evaluated in our study.

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Single-letter abbreviations for the amino acid residues are as follows: A Ala; E Glu; F Phe; G Gly; I Ile; K Lys; L Leu; M Met; N Asn; Q Gln; R Arg; S Ser; T Thr; and V Val.

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Some of the patients had received antiretroviral therapy with the following drugs before starting a regimen that suppressed viral load to undetectable levels: AZT (patients 1 2 4 7 10 13 14 15 18 21 and 22) zalcitabine (ddC) (patients 4 10 12 and 18) didanosine (ddI) (patients 1 4 and 8) 3TC (patients 1 2 4 6 7 and 13) d4T (patients 2 6 12 and 15) IDV (patient 2) and RTV (patient 9).

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We thank J. Keruly P. Pentz and C. Raines for coordinating patient recruitment; E. Pritchard and K. Cooper for help with collection of the samples; M. Castro for help with the culture experiments; Y.-H. Kuo for assistance with the statistical analysis and C. Ignacio for help with resistance genotyping. We express special thanks to the patients who participated in this study. Supported by NIH grants AI28108 and AI23871 (R.F.S.); AI27670 AI38858 and AI36214 (Center for AIDS Research); and AI29164 (D.R.) and by the Research Center for AIDS and HIV Infection of the San Diego Veterans Affairs Medical Center.