The Cancer Institute of New Jersey, Robert Wood Johnson School of Medicine, 195 Little Albany Street, New Brunswick, New Jersey 08901, USA1
The Genesee Hospital and the University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA2
Author for correspondence: Roger Strair.Fax +1 732 235 8098. e-mail strairrk{at}umdnj.edu
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Abstract |
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Introduction |
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Similar clinical and laboratory studies analysing early HIV infection in the presence of 3'-azido-3'-deoxythymidine (AZT) have also been undertaken (Loveday et al., 1995 ). In contrast to the clinical studies with nevirapine, early HIV infection in the presence of AZT does not appear to be predominated by the outgrowth of AZT-resistant HIV. While the amount of virus circulating in plasma shortly after the initiation of AZT rapidly declines, the remaining circulating virus early after this decline does not contain mutations known to encode resistance to AZT (Loveday et al., 1995
). A component of this genetically AZT-sensitive virus may be derived from longer-lived cells with more latent infection (Perelson et al., 1996
); however, the amount of virus present and the ultimate emergence of genetically resistant virus containing multiple mutations associated with AZT-resistance implies that there may also be virological, cellular or pharmacological features which allow HIV-1 that is initially genotypically and phenotypically sensitive to AZT to replicate in the presence of AZT. These latter features may impact on the kinetics of virus production in the presence of AZT and allow ongoing replication that supports the development of variants with the multiple mutations known to be associated with high level genetic resistance to AZT.
In prior studies, a quantitative in vitro model system has been used to analyse a single cycle of recombinant HIV infection in the presence of antiviral drugs (Strair et al., 1993 ; Medina et al., 1995
, 1998
). This model system has demonstrated a pattern of virus infection that shares some important characteristics with the pattern of infection seen in the initial clinical studies analysing virus dynamics: (i) early breakthrough infection in the presence of an NNRTI that is dependent on genetic drug resistance; and (ii) early breakthrough infection in the presence of AZT that is, at least partially, independent of the presence of genetic resistance (Strair et al., 1993
; Medina et al., 1995
). Furthermore, this recombinant virus system has allowed an estimate of the prevalence of unselected NNRTI-resistant variants in a population of unselected replication-competent HIV-1 that has subsequently been approximated by mathematical modelling of HIV-1 kinetics after the initiation of an NNRTI (Havlir et al., 1996
; Strair et al., 1993
). Therefore, some features of early recombinant virus infection in the presence of antiviral drugs in vitro are similar to those described in clinical studies of HIV dynamics after the initiation of an NNRTI (Wei et al., 1995
) or AZT (Loveday et al., 1995
) and further analyses of this in vitro recombinant system may allow a characterization of some of the mechanisms that allow HIV replication in the presence of antiviral drugs.
To determine the predominant mechanisms responsible for early HIV infection in the presence of another nucleoside reverse transcriptase inhibitor, stavudine (d4T), we utilized a similar in vitro recombinant system. The results of this analysis indicate that a significant component of early infection in the presence of d4T is a consequence of infection by genetically sensitive virus that has infected a subset of the host cell population which is refractory to the antiviral effects of d4T.
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Methods |
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Virus.
Production of recombinant HIVgpt has been described elsewhere (Strair et al., 1993 ; Page et al., 1990
). The amphotropic cell line PA317 was transfected with the recombinant murine retrovirus pLXSN (Miller & Rosman, 1989
) and was used as the source of the recombinant MLVneo virus. Stock preparations of HIV-1 IIIB were harvested from H9 cells by the `shake off method'. Stock virus infectivity was determined by end-point dilution in MT-2 cells. Virus-induced cytopathic effect was scored 7 days post-infection and the TCID50 was calculated with the Reed and Muench equation (Reed & Muench, 1938
).
Compounds.
Stavudine (d4T) and floxuridine (5-fluoro-2-deoxyuridine, FUdR) were purchased from Sigma and were dissolved in PBS, sterile-filtered and stored at -20 °C.
HIV-1 RT assay.
HIV-1 production in infected cells was determined by a 32P-based assay as described by Willey et al. (1988) . RT activity was determined by quantification of 32P bound to DE81 paper by using a Molecular Dynamics phosphorimager. The results are reported as pixel units/µl of the reaction mixture.
Cytotoxicity assay.
A checkerboard analysis of the cytotoxicity of d4T and FUdR alone and in combination was assayed. Triplicate wells of 24-well plates containing 1x105 cells were cultured in the absence or presence of various concentrations of each drug alone or in combination. Samples were taken every 2 days for 810 days. Drug cytotoxicity was quantified by the MTT reduction assay (Mosman, 1983 ). The amount of formazan produced in 4 h was determined by dissolving the product in 0·1 M HCl made with 2-propanol and then measuring the absorbance at 570 nm.
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Results |
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Isolation and characterization of cells infected in the presence of d4T
The presence of a selectable marker gene in the recombinant retroviruses depicted in Fig. 1 allowed the direct isolation of cells infected by these viruses in the presence of d4T (Fig. 2
) and the demonstration that a component of the infection was a consequence of infection by viruses that were not genetically resistant to d4T. HeLa-T4 cells infected with MLVneo (Fig. 1C)
in the presence of d4T were isolated, cloned and expanded into cell lines that were subsequently infected with recombinant HIV, recombinant MLV (MLVLacZ; see Strair et al., 1991
, 1993
) or replication-competent HIV-1. As demonstrated in Table 2
, approximately 37% of the isolated HeLa-T4 cells were repeatedly refractory to the antiviral effects of d4T (they could readily be infected with recombinant MLVLacZ in the presence of 50 µM or 100 µM d4T). The same cells readily infected with recombinant MLV in the presence of d4T were also readily infected with recombinant HIV or replication-competent HIV-1 in the presence of d4T (data not shown). Similar experiments with AZT, ddI and ddC indicate that refractoriness to the antiviral effects of each of these nucleoside analogues contributes to early breakthrough infection in vitro in the presence of the drug (Table 2
). A comparison of the prevalence of cells refractory and persistently refractory to the antiviral effects of these drugs is shown in Table 2
. The prevalence of cells persistently refractory to the antiviral effects of d4T is higher than the prevalence of cells persistently refractory to the antiviral effects of AZT, ddI or ddC. These studies directly demonstrate a subpopulation of host cells that are infected with recombinant viruses in the presence of d4T and can be infected repeatedly with other recombinant viruses or replication-competent HIV in the presence of d4T. These cells were not pre-selected by exposure to antiviral drugs.
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These results provide support to the concept that a component of early infection of these cell lines in vitro occurs as a consequence of mechanisms independent of genetic drug resistance. The phenomenon of early virus breakthrough in the presence of these nucleoside analogues has been demonstrated with replication-defective recombinant HIV, replication-competent HIV and two replication-defective recombinant murine retroviruses. In addition, early infection as a consequence of cellular features has been seen with two different host cell types (HeLa-T4 and Jurkat 6.1).
As has been demonstrated previously for AZT (Medina et al., 1996 ), a component of the refractoriness to the antiviral effects of d4T can be reversed by the addition of FUdR (Table 4
). The antiviral efficacy of the combination therapy, as measured by the d4T IC50, is markedly improved with combination therapy. Prior studies of the antiviral efficacy of FUdR have demonstrated limited antiviral efficacy of FUdR alone, but marked antiviral efficacy of combined AZT and FUdR (Medina et al., 1996
). Table 4
shows the capacity of the FUdRd4T combination to reverse some of the cellular refractoriness to d4T described above. The FUdRd4T combination has marked antiviral activity in cells demonstrated to be persistently refractory to the antiviral effects of d4T. To assure that FUdR did not inhibit the antiviral activity of d4T in normal unmanipulated PBMC, HIV-1 was used to infect PBMC in the presence of various concentrations of d4T and FUdR. As demonstrated in Fig. 4
, the d4TFUdR combination resulted in markedly enhanced antiviral activity (compared to d4T alone) in PBMC. FUdR alone resulted in minimal antiviral activity.
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Discussion |
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In this report we utilized an in vitro model of HIV infection to provide two lines of evidence that early HIV breakthrough infection in the presence of d4T is not solely a consequence of infection by HIV with genetic drug resistance. Initial studies demonstrated that the frequency of HIV infection in the presence of d4T was very similar with several stocks of virus predicted to have significant differences in genetic heterogeneity. These studies suggested that any pre-existing unselected d4T-resistant HIV in the population of HIV used to rescue the replication-defective HIV was not detected above the very high level of infection occurring with the other virus populations. The fact that nearly 50% of the Jurkat cells infected with HIV in the presence of d4T are readily re-infected in the presence of high concentrations of d4T provides definitive evidence of a high level of infection in the absence of genetic d4T resistance. These results are not limited to recombinant HIV and were also demonstrated with MLV-based viruses as well as replication-competent HIV.
A subset of the cells infected with HIV in the presence of d4T does not have a persistent phenotype of being refractory to d4T. These cells may have been infected as a consequence of cell cycle related phenomena, intravirion reverse transcription (Zhang et al., 1994 ) or other viral, cellular or pharmacological features that are not characterized by persistent cellular phenotypic change.
Previous studies have demonstrated that the combination of FUdR with AZT or d4T has significant antiretroviral activity (Medina et al., 1996 ; Ahluwalia et al., 1996
). Furthermore, the combination of FUdR and AZT has potent antiretroviral activity in cells refractory to the antiretroviral activity of AZT (Medina et al., 1996
). These studies demonstrate the capacity to improve the antiviral efficacy of d4T by the addition of drugs such as FUdR with the capacity of interacting with the biochemical mechanisms responsible for AZT and/or d4T metabolic activation. This increased antiviral efficacy in cells refractory to the antiretroviral effects of AZT or d4T may have therapeutic implications for the treatment of early HIV infection in the presence of d4T (e.g. cells infected with d4T-sensitive HIV in the presence of d4T). As this early replication may contribute to the ultimate emergence of genetic resistance, determination of the mechanisms contributing to the presence of virus after initiation of therapy may impact on efforts to achieve long term suppression of the virus.
In summary, we have demonstrated that early HIV breakthrough infection in vitro in the presence of d4T is not solely a consequence of infection with virus that is genetically resistant to d4T. As with AZT, infection with drug-sensitive virus is a major mechanism of infection early after the introduction of the drug in vitro. Further analyses of the mechanisms responsible for HIV breakthrough in the presence of antiviral drugs are essential to efforts to define drug combinations that provide durable suppression of HIV infection and viraemia.
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Received 15 March 1999;
accepted 25 May 1999.
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