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Effect of Nukes on HIV Resting Cells & Implication for Viral Suppression, Selecting Nukes: AZT, abacavir, 3TC
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"Zidovudine, Lamivudine, and Abacavir Have Different Effects on Resting Cells Infected with Human Immunodeficiency Virus In Vitro"
Antimicrobial Agents and Chemotherapy, August 2004, Vol. 48, No. 8
Jesús Saavedra-Lozano,1,2 Cynthia C. McCoig,1,2 Yanying Cao,1 Ellen S. Vitetta,1,3 and Octavio Ramilo1,2,4*
Cancer Immunobiology Center,1 Departments of Pediatrics,2 Microbiology, University of Texas Southwestern Medical Center,3 Children's Medical Center, Dallas, Texas4
"...our results suggest that in designing combinations of ARV, in addition to potency, different patterns of resistance, and the pharmacokinetic interactions among several agents, we could also consider combining drugs with activity in both productively (active) and latently (resting) infected cells, as suggested previously by other authors, to optimize the suppression of viral replication..."
ABSTRACT
We have previously described an in vitro model for the evaluation of the effects of different immunomodulatory agents and immunotoxins (ITs) on cells latently infected with human immunodeficiency virus (HIV). We demonstrated that latently infected, replication-competent cells can be generated in vitro after eliminating CD25+ cells with an IT.
Thus, by selectively killing the productively infected cells with an anti-CD25 IT we can generate a population of latently infected cells. CD25-- cells generated in this manner were treated with nucleoside analog reverse transcriptase inhibitors and subsequently activated with phytohemagglutinin in the presence of the drugs.
The antiviral activities of zidovudine (ZDV), lamivudine (3TC), and abacavir (ABC) were evaluated by using this model. 3TC and ABC demonstrated significant activity in decreasing HIV production from recently infected resting cells following their activation, whereas the effect of ZDV was more modest.
These results suggest that the differences in antiviral activity of nucleoside analogs on resting cells should be considered when designing drug combinations for the treatment of HIV infection. The model presented here offers a convenient alternative for evaluating the mechanism of action of new antiretroviral agents.
INTRODUCTION
Human immunodeficiency virus (HIV) replication in vivo involves the rapid turnover of CD4+ lymphocytes and the release of virions into the plasma. The majority of HIV detected in plasma is derived from these newly infected, short-lived, HIV-producing CD4+ T cells. Another population of HIV-infected cells consists of latently infected cells. These cells have become a major target of HIV research, since they are an important reservoir of virus and may be key to the persistence of infection. The success of highly active antiretroviral therapy in reducing circulating HIV in plasma to levels below the limit of detection in many infected individuals has encouraged investigators to initiate studies aimed at eradicating HIV. However, replication-competent HIV has been isolated from infected individuals after prolonged treatment with highly active antiretroviral therapy, and several studies have demonstrated that resting CD4+ T cells are a long-lived latent reservoir of the virus. The virus may remain viable in nonproducing resting cells in a latent form either as integrated or preintegrated DNA with the ability to integrate into host DNA after cell activation.
The persistence of HIV infection in patients with undetectable virus may also be secondary to ongoing low-level viral replication. This remaining viral replication could be due to protected sanctuaries and to variable tissue concentrations of antiretroviral drugs. Findings from two recent studies which used very sensitive techniques showed that, at least in aviremic individuals, latently infected cells do not allow viral replication (10, 23).
(10) Chun, T. W., J. S. Justement, R. A. Lempicki, J. Yang, G. Dennis, Jr., C. W. Hallahan, C. Sanford, P. Pandya, S. Liu, M. McLaughlin, L. A. Ehler, S. Moir, and A. S. Fauci. 2003. Gene expression and viral production in latently infected, resting CD4+ T cells in viremic versus aviremic HIV-infected individuals. Proc. Natl. Acad. Sci. USA 100:1908-1913
(23) Hermankova, M., J. D. Siliciano, Y. Zhou, D. Monie, K. Chadwick, J. B. Margolick, T. C. Quinn, and R. F. Siliciano. 2003. Analysis of human immunodeficiency virus type 1 gene expression in latently infected resting CD4+ T lymphocytes in vivo. J. Virol. 77:7383-7392
Several groups have underscored the need to design therapeutic interventions which target latently infected cells. One approach would be to use drugs that are more active in resting cells. If antiretroviral drugs (ARV) can be incorporated into resting cells and remain active for a period of time, these agents may reduce the dissemination of HIV to these cells following reactivation. As new ARV become available, it is therefore important to determine whether they are active in both productively infected and resting cells. This information should facilitate the design of drug combinations capable of suppressing viral replication in different cell populations and perhaps reduce the expansion of the pool of latently infected cells.
MATERIALS & METHODS
Drugs and IT.
The nucleoside analogs ZDV, 3TC, and ABC were provided by GlaxoSmithKline, Inc. (Research Triangle Park, N.C.). Stocks were prepared in 100% dimethyl sulfoxide at a 1 mM concentration and stored in 1-ml aliquots at --70°C. Drug dilutions were freshly prepared for each experiment. Initial dilutions were based on the 50% inhibitory concentrations for viral isolates, and based on preliminary experiments, we used 0.01 to 0.15 µM ZDV, 0.25 to 5 µM 3TC, and 2 to 15 µM ABC. The anti-CD25 IT, RFT5-dgA, was prepared and purified as previously described.
Virus and PBMCs.
Virus stocks and peripheral blood mononuclear cells (PBMCs) were prepared and infections were induced as described previously.
Activation with PHA.
The protocol for activation with phytohemagglutinin (PHA) is shown in Fig. 1. PBMCs were isolated from HIV-negative individuals and incubated with HIV. Cells were divided into two groups containing either complete medium (CM) (RPMI, glutamine, 15% fetal bovine serum, 10% interleukin-2, and penicillin-streptomycin) or CM plus 10 nM IT and cultured in 24-well tissue culture plates at a concentration of 106 cells/ml per well. After 3 days in culture, the media were replaced (either CM or CM plus IT). Three days later, cells were washed twice and the study drugs were added at different concentrations. The cells were cultured for 3 days. On the 10th day, both the media and the drugs were replaced and the cells were stimulated by adding PHA (1 µg/ml). Three days later, media were again replaced. Finally, on day 15 after infection, cell-free supernatants were collected and assayed for levels of p24. Cells from treatment groups were cultured in triplicate wells, and all experiments were repeated at least three times.
FIG. 1. Experimental protocol. PBMCs were isolated from HIV-negative individuals and incubated with HIV. They were divided into two groups and cultured in 24-well plates at 106 cells/well in CM or CM plus 10 nM IT. After 3 days in culture, the media were replaced (either CM or CM plus IT). Three days later, cells were washed twice and the study drugs (ZDV, 3TC, and ABC) were added at different concentrations. Cells were cultured for 3 days, both media and the drugs were replaced, and cells were stimulated by the addition of PHA (1 µg/ml). Three days later, media were again replaced. Finally, on day 15 after infection, cell-free supernatants were collected and assayed for p24 concentrations.
p24 antigen assay.
Concentrations of p24 antigen in cell-free supernatants were measured by using a commercially available enzyme-linked immunosorbent assay kit (NEN, New Life Science Products, Inc., Boston, Mass.).
Comparative effect of nucleosides on unfractionated (CD25+and CD25--) versus resting (CD25--) cells.
We analyzed the activity of these drugs in two different settings: (i) unfractionated cells containing both activated and latently infected cells (CM-nucleoside analog reverse transcriptase inhibitor [NRTI]) and (ii) resting cells (IT-NRTI). To assess the effect of treatment with IT alone, we included two groups of cells without NRTIs but incubated with either CM or CM plus IT. p24 production was determined after PHA activation, since CD25-- cells do not produce virus unless they are activated. Nevertheless, we compared each culture of resting CD25-- cells plus NRTI (IT-NRTI) with a control culture of CD25-- cells without NRTIs (IT alone). Hence, the additional reduction of p24 production observed in the IT-NRTI groups reflects the activity of the NRTIs on resting cells and their ability to suppress viral production from these cells following activation. Therefore, in each experiment, we measured the reductions of p24 production induced by the NRTIs at different concentrations under two conditions (unfractionated and resting cells) and calculated the subsequent ratio by comparing the effect in both settings. If the effect of the NRTI on resting cells is superior to that observed on unfractionated cells, it would demonstrate an additional effect at that particular drug concentration, since the NRTIs in the IT treatment groups were added to the cultures after the CD25-- resting cells were isolated.
Statistical analysis.
Because values did not follow a normal distribution, data are also presented with medians with ranges. Statistical analysis was performed by using Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks for nonparametric data with the SigmaStat 2000 software package.
AUTHOR DISCUSSION
The importance of latently infected cells in the pathogenesis of HIV infection has been underscored by several studies demonstrating the presence of HIV DNA and viable virus in purified resting memory T cells obtained from HIV type 1-infected individuals at different stages of the disease, including individuals with undetectable plasma viremia. Such studies have characterized a population of CD4+ DR-- CD45RO+ T lymphocytes which contain integrated and/or unintegrated HIV provirus.
In our in vitro model of acute infection, the CD4+ CD25-- latently infected cells contained predominantly incomplete and unintegrated HIV provirus but also small amounts of full-length HIV DNA. Thus, there are differences in the predominant species of HIV provirus present in in vitro latently infected cells compared with latently infected cells purified from HIV-infected individuals. Regardless of the predominant HIV DNA species, the consistent and clear differences observed among the different NRTIs evaluated indicate distinct antiviral activity of certain NRTIs on HIV-infected resting cells.
Our in vitro model has facilitated the direct assessment of the activity of nucleoside analogs on HIV-infected resting cells by directly isolating these cells after treatment with an IT. These cells were isolated, incubated with NRTIs, and then activated to induce virus production. The major findings to emerge from this study are as follows. (i) There are significant differences in antiviral activity among nucleoside analogs in resting cells infected with HIV in vitro. (ii) Among the three agents evaluated, ZDV did not show activity on resting cells infected in vitro, whereas 3TC and ABC had significant antiviral activity in these cells.
Few studies have examined the activity of antiretroviral agents on latently infected cells. Shirasaka et al. analyzed the effect of different NRTIs on resting and activated cells. Although they assessed antiviral activity by stimulating PBMCs and measuring p24 production, they used fresh PBMCs obtained from HIV-negative individuals not previously stimulated as resting cells. Thus, the activated CD25+ cells present in fresh PBMCs probably influenced their results. Our results are in agreement with their findings that some NRTIs had better anti-HIV activity on resting cells (ddC and ddI) than others (ZDV and stavudine [d4T]). Gao et al. studied the mechanisms of phosphorylation by using different dideoxynucleoside analogs (ddNs) and classified them into two groups: (i) cell activation-dependent ddNs, such as ZDV and d4T, which were preferentially phosphorylated and exhibited more potent anti-HIV a | | | | |
