Basic Science: Concise Communication
Effect of target cell availability on HIV-1 production in vitro
Schwartz, Elissa J.a; Neumann, Avidan U.b; Teixeira, Avelino V.a; Bruggeman, Leslie A.a; Rappaport, Jayd; Perelson, Alan S.c; Klotman, Paul E.a
From the aDivision of Nephrology, Mount Sinai Medical Center, New York, NY 10029, USA; bDepartment of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel; cTheoretical Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
dPresent address: Center for NeuroVirology and Cancer Biology, Temple University, Philadelphia, PA 19140, USA.
Correspondence to: Elissa J. Schwartz, Box 1243, Division of Nephrology, Mount Sinai Medical Center, One Gustave L. Levy Place, New York, NY 10029, USA. Tel: +1 212 241 8007; fax: +1 212 987 0389; e-mail: email@example.com
Requests for reprints to: Paul E. Klotman, Box 1243, Division of Nephrology, Mount Sinai Medical Center, One Gustave L. Levy Place, New York, NY 10029, USA. Tel: +1 212 241 8007; fax: +1 212 987 0389; e-mail: firstname.lastname@example.org
This work was performed under the auspices of the U.S. Department of Energy. Los Alamos National Laboratory strongly supports academic freedom and a researcher's right to publish; as an institution, however, the Laboratory does not endorse the viewpoint of a publication or guarantee its technical correctness.
Received: 3 October 2000;
revised: 13 July 2001; accepted: 24 July 2001.
Sponsorship: This work was supported by Public Health Service Grants DK50795 (to P.E.K.) and RR06555 (to A.S.P.). A.U.N was supported by the Gonda-Goldschmied Medical Diagnostic Center and the Committee for the Advancement of Research of Bar-Ilan University.
Objective: The recovery of CD4 target cells following antiretroviral therapy may facilitate virus production and escape from antiretroviral suppression. To address this hypothesis, we directly examined whether the CD4 target cell number increases viral production in the presence of suboptimal therapy.
Design: The effect of the CD4 T cell number on HIV-1 replication with a suboptimal dose of zidovudine was studied in vitro.
Methods: Varying numbers of CD4 T cells were infected with HIV-1 and treated with 1 nM zidovudine. Virus production was measured by p24 antigen capture enzyme-linked immunosorbent assay. Partial sequencing of HIV-1 pol was performed to assess zidovudine-resistant mutations.
Results: Wild type virus production was found to increase eightfold in cultures with 100 × 104 cells compared with cultures with 10 × 104 cells. The IC90 of zidovudine was 4 logs higher in cultures with 16 × 104 cells compared with cultures with 1 × 104 cells. No zidovudine-resistant mutations were found.
Conclusion: Target cell availability may play a direct role in wild type HIV-1 resurgence following therapy.
Reducing the number of infectable target cells, in combination with antiretroviral therapy, has been suggested as a novel approach to control HIV-1 infection [1–5]. Several treatment strategies that result in CD4 cell lymphopenia, such as hydroxyurea–didanosine and mycophenolic acid–abacavir, have shown beneficial effects [6–13]. An explanation for this therapeutic advantage has been provided by mathematical models of viral dynamics in which HIV is limited by the number of available target cells [1,14–18]. The models suggest that the suppression of CD4 target cells by such agents can lead to viral load reduction, and conversely that the recovery of CD4 target cells after the initiation of antiviral therapy can support virus production if the potency of therapy is below a threshold. Demonstration of this mechanism in vivo is challenging, due to many confounding variables such as host immune response and drug availability. Several studies [19,20] have, however, fitted mathematical models to clinical data and demonstrated that target cell recovery could be responsible for the wild type viral rebound seen in patients on therapy before drug-resistant strains emerge. Furthermore, target cell recovery correlated with the loss of viral suppression in patients switching from triple combination therapy to less intensive maintenance regimens , as was mathematically predicted . Here, we used an in vitro system to study this mechanism directly.
The present study aimed to determine directly whether wild type HIV-1 production increases when more target cells are available in the continued presence of antiviral therapy. We show that zidovudine was less effective at inhibiting virus production when more CD4 T cells were present in culture. Furthermore, greater target cell availability resulted in more virus production when the zidovudine concentration was below a determined threshold. These in vitro data support the theory [1,18,19] that the availability of target cells plays a significant role in wild type viral production in the presence of a suboptimal drug regimen.
Materials and methods
Cells and virus
Human T lymphoblastoid CEM cells were cultured in RPMI 1640 containing 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine. Inoculating virus was the T cell tropic, laboratory strain HIV-1 IIIB clone HXB2, obtained as a supernatant from acutely infected CEM cells. The TCID50/ml was 4.5 × 103, as determined by endpoint titration with CEM cells.
For zidovudine dosage studies, CD4 T cells (8 × 104) were incubated for 2 h with 400 TCID50 of HIV-1, washed with phosphate-buffered saline, and seeded in triplicate in 24-well plates (2 ml/well) with media alone or media with 1 fM to 100 mM zidovudine (Sigma Chemical Co., St Louis, MO, USA). Cell viability was monitored using trypan blue dye exclusion, and zidovudine toxicity was measured on uninfected, zidovudine-treated cells using the CellTiter 96 Cell Proliferation Assay (Promega Biotech, Madison, WI, USA). The drug was found to be non-toxic at the administered doses. Cell-free supernatants (200 μl) were harvested on day 7 or 8, and virus production was quantified by p24 antigen capture enzyme-linked immunosorbent assay (DuPont NEN, Boston, MA, USA) as per manufacturer's protocol.
For CD4 T cell availability studies, varying numbers of cells (1 × 104 to 100 × 104) were infected for 2 h, washed, and seeded in triplicate in 24-well plates with media with or without zidovudine. Each culture was infected with the same amount of inoculating virus. Cultures with a greater number of cells thus had a lower multiplicity of infection (MOI). The volume of all cultures was 2 ml. To maintain a constant viable cell number throughout the experiment (as determined by hemocytometric counting after trypan blue dye exclusion), cultures were mixed daily, depleted of 500 μl of cells and media, and replenished with 500 μl fresh media with or without zidovudine. Cell-free supernatants (200 μl) were harvested on day 7 or 8, and virus production was quantified using p24 antigen capture enzyme-linked immunosorbent assay as described.
HIV-1 RNA samples [from cell-free supernatants harvested from day 7 cultures of 100 × 104 initial cells with 1 nM zidovudine and from inoculating viral supernatant (10 μl) diluted in 130 μl water] were sequenced as described previously . Here, 250 ng primer was used in reverse transcription, the first polymerase chain reaction product (5 μl) served as a template for the second polymerase chain reaction (25 cycles), and amplification products were sequenced automatically (ABI 377, Applied Biosystems, Foster City, CA, USA) with overlapping primers. Sequence alignments were performed by comparing sequences to HXB2 (GenBank accession no. K03455), looking for amino acid substitutions M41L, D67N, K70R, T215F/Y, and K219E/Q, which confer zidovudine resistance .
Simulation of virus production as a function of target cell availability
HIV-1 infection with zidovudine treatment was simulated using the mathematical model of McLean and colleagues [15,16]. This model, as well as others , suggests that antiviral suppression is lost when the drug efficacy is below a threshold and target cell availability increases. For specified viral and target cell parameters , the simulation showed that zidovudine inhibited virus production only when drug efficacy was more than 90%, and below this drug threshold, virus production increased as the source of target cells increased (Fig. 1). The simulation thus showed that virus production is dependent on target cell availability, below the drug threshold.
Determination of zidovudine threshold in vitro
We then investigated whether HIV-1 is dependent on target cell availability in vitro, independent of the host immune response. Initially, the zidovudine dose that reduces virus production by 90% (IC90) was determined. CD4 T cells were infected with HIV-1 and incubated with media alone or media with 1 fM to 100 mM zidovudine. Virus production was measured at the time of maximum syncytia formation in untreated cultures, i.e., on days 7 and 8.
Zidovudine inhibited HIV-1 production in a dose-dependent fashion. Cultures treated with 1 pM zidovudine were inhibited to 35% of untreated control values, and complete inhibition was observed with 1 mM zidovudine (Fig. 2). Cultures treated with 1 nM zidovudine were inhibited to 10% of control values (i.e. 90% inhibition). These results are similar to previously published data , in which different methods and infection parameters were used. On the basis of these results, the zidovudine dose used in subsequent experiments was 1 nM.
Increased virus production with greater target cell availability in the presence of zidovudine
Virus production was then measured in cultures treated with 1 nM zidovudine but with different initial densities of target CD4 T cells. Control cultures were not given zidovudine. Virus production was quantified on day 7.
As shown in Fig. 3, virus production was greater in cultures with more target cells. HIV-1 production increased eightfold (P < 0.001) in cultures with a high target cell number (100 × 104) and sixfold (P < 0.01) in cultures with an intermediate target cell number (20 × 104) compared with those with a low target cell number (10 × 104). This trend correlated with that shown in Fig. 1. Therefore, when more target cells were available for viral replication, 1 nM zidovudine inhibited HIV-1 production less effectively. Cultures with a lower MOI were less sensitive to 1 nM zidovudine.
HIV-1 escape from inhibitory effect of zidovudine with greater target cell availability when drug concentration was below threshold
Virus production was then measured in cultures with different numbers of target CD4 T cells as well as different zidovudine doses to determine the role of the drug concentration on viral production. Control cultures were not given zidovudine. Virus production was quantified on day 8.
HIV-1 production was greater with more target CD4 T cells when the zidovudine concentration was low (10 fM, 1 pM, and 1 nM) but not when it was high (10 nM and 100 nM) (Fig. 4). With 10 fM, 1 pM, and 1 nM zidovudine, virus production increased eightfold (P < 0.001, P < 0.01, and P < 0.01, respectively) in cultures with a high target cell number (16 × 104) compared with those with a low target cell number (1 × 104). The differences in virus production between high and low target cell numbers with 10 nM and 100 nM zidovudine were not significant. HIV-1 thus overcame inhibition by zidovudine with greater target cell availability (i.e. lower MOI) when the drug treatment was suboptimal, but not when it was above the 90% inhibition level. Furthermore, the concentration at which zidovudine inhibited virus production by 90% (IC90) on day 8 was 1 pM in cultures with a low target cell number and 10 nM in cultures with a high target cell number. A 4 log increase in drug concentration was thus necessary to inhibit virus production by 90% with a high target cell number.
Wild type genotype
To investigate the possibility that the increased virus production was caused by the emergence of zidovudine-resistant viral strains, virus from treated cultures with a high target cell number was sequenced and examined for zidovudine-resistant mutations. Both inoculating virus as well as virus harvested from day 7 cultures were found to be wild type genotype (Table 1). The increased virus production in cultures with a high target cell number was thus caused by the greater availability of target cells and not the emergence of zidovudine-resistant viral strains.
These studies demonstrate that zidovudine inhibited HIV-1 less effectively in vitro when a greater number of target cells was available (Fig. 3). Viral production was limited by the availability of target cells. These data support the hypothesis [1,18,19] that a threshold or critical drug efficacy exists, below which an increase in the target cell pool causes a loss of wild type viral suppression by zidovudine (Fig. 4). The specific drug threshold is expected to vary in individual patients, depending upon parameters such as the growth and death rates of target cells and virus infectivity .
The limitations inherent in in vitro systems should be taken into account with these findings. In culture, all cells are potential targets of infection, whereas only a fraction of cells become infected in vivo. Furthermore, these studies cannot exclude other mechanisms that may also have contributed to these findings, such as the differential metabolism of zidovudine and cell viability at high cell density. To address these concerns, zidovudine was replenished daily to maintain consistent intracellular zidovudine concentrations, and cell viability was optimized to maintain a constant viable cell number throughout the experiment.
Our results support the suggestion  that the therapeutic benefit of hydroxyurea–didanosine treatment [6,8,10] may be a result of antiviral effects as well as the depletion of the target cell pool. Furthermore, these results support the hypothesis that the increase in wild type virus seen in patients treated with zidovudine but who have not yet developed zidovudine resistance may result from an expanded target cell pool . Similarly, the loss of viral suppression seen in patients who switch to maintenance therapy may be caused by target cell recovery [21,25]. In each case, target cell expansion may raise the drug threshold above the effective concentration in these patients. Developing drug strategies that take into account the increase in potential target cells during therapy may be an important consideration in long-term antiretroviral therapy.
The authors would like to thank Drs Mary Klotman, Arevik Mosoian, Michael Miller, and Wendy W-Y. Lou for helpful comments.
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