Sensitivity of Herpes Virus Isolates from

Acyclovir Clinical Trials

PETER COLLINS, Ph.D. GEOFFREY APPLEYARD, M.A., B.Sc.,

B.M. NICHOLAS M. OLIVER

Beckenham, United Kingdom

From the Antiviral Chemotherapy Section, Well- come Research Laboratories, Beckenham, Kent, United Kingdom. Requests for reprints should be addressed to Dr. Peter Collins, Antiviral Chemo- therapy Section, Wellcome Research Laborato- ries, Langley Court, Beckenham, Kent, BR3 3BS, United Kingdom.

Clinical isolates of herpes simplex virus and varicella zoster virus have been examined for changes in drug sensitivity after treatment of patients with acyclovir. Some preliminary sensitivity results de- termined by plaque reduction assays in Vero cells for herpes simplex virus and MRC-5 cells for varicella zoster virus are presented. Where both pretreatment and posttreatment virus isolates were available, no significant changes in sensitivity to acyclovir were observed. Similarly, isolates taken after treatment was begun, without a pre- treatment sample, gave IC50 values no greater than expected when compared with other pretreatment isolates.

Acyclovir” (9-[2-hydroxyethoxymethyl]guanine) is a highly effective antiviral agent specifically inhibiting the replication of members of the herpes virus group, in particular herpes simplex virus types 1 and 2 and varicella zoster virus [l-4]. The mechanism by which acyclovir inhibits herpes virus replication involves two virus-specific enzymes. Firstly, a herpes virus thymidine kinase is responsible for phos- phorylation of acyclovir to its monophosphate; further phosphorylation to the triphosphate form is mediated by cellular kinases. The acyclovir triphosphate then interferes with the DNA polymerase of herpes virus, thus preventing DNA replication [5-71.

Alterations of either the herpes virus thymidine kinase or the DNA polymerase could therefore confer resistance to acyclovir. Resistant strains of herpes virus have been produced in the laboratory and their possible development in nature must be considered after repeated use of acyclovir [8,9].

To determine whether such mutants are found in nature or mutate after acyclovir therapy, we have monitored clinical isolates of herpes simplex virus and varicella zoster virus from acyclovir clinical trials in the United Kingdom.

MATERIALS AND METHODS

Cell Cultures. Vero cells (Flow Laboratories, Irvine, Scotland) were main- tained in Eagle minimal essential medium containing 5 percent fetal calf serum, 0.11 percent sodium bicarbonate, and 0.25 percent crystamycin (50,000 units/ml sodium penicillin G and 50 mg/ml streptomycin sulphate; Glaxo Laboratories, Greenford, Middlesex, England). Stock cultures were split routinely 1:4 twice weekly.

MRC-5 human fibroblast cells (Cell Culture Department, Wellcome Re- search Laboratories, Beckenham, Kent, England) were maintained in Eagle

basal medium containing 10 percent fetal calf serum, 0.11 percent sodium

bicarbonate, and 0.25 percent crystamycin. Stock cultures were split routinely 1:2 every five days and were maintained between passes 25 and 40.

l Zovirax@, Burroughs Wellcome.

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RESISTANCE-COLLINS ET AL.

Figure 1. Sensitivities of herpes sim- plex virus types 1 and 2 isolates to acy- clovir.

Virus isolates. Isolates of herpes simplex virus and varicella zoster virus were received from four clinical studies in the U.K.: (1) U.K. trial 49, labial herpes simplex versus 5 percent acyclovir ointment; (2) U.K. trial 49a, labial herpes simplex virus versus 5 percent acyclovir ointment, treatment begun during the prodromal period; (3) U.K. trial 62, genital herpes simplex virus versus 5 percent acyclovir ointment; and (4) the emergency treatment study: intravenous acyclovir ad- ministered on a named patient basis.

Isolates of herpes simplex virus received from the trial centers were stored at -7OOC. Stock cultures were prepared in Vero cells and 1 ml aliquots of cell-extracted virus sus- pension were stored at -7O’C.

Varicella zoster virus isolates were cultured in MRC-5 cells, and stocks of infected cells suspended in maintenance medium containing 2 percent fetal calf serum, 0.22 percent sodium bicarbonate, and 7.5 percent dimethylsulphoxide were stored in 1 ml aliquots in liquid nitrogen. Acyclovir. Acyclovir was supplied by Dr. H. J. Schaeffer, Burroughs Wellcome Co., (Research Triangle Park, NC). Stock solutions of 1 mM and 100 PM were prepared in deionized water and stored frozen in 1 ml aliquots at -7OOC. Plaque Reduction Assay. The sensitivities of herpes sim- plex virus isolates were measured using the plaque-reduction assay in Vero cells. Multiwell plates (Linbro, Flow Labora- tories, Irvine. Scotland) were seeded with 200,000 cells in 1 ml growth medium/well and incubated at 37OC in 5 percent COP overnight. The monolayer cultures were then infected with 0.2 ml volumes virus suspension containing 100 to 200 plaque-forming units. After adsorption for one hour the in- ocula were removed and the cultures overlaid with a main- tenance medium overlay containing oarboxymethylcellulose and doubling micromolar concentrations of acyclovir. The cultures were returned to the incubator for another 72 hours, then fixed and stained with methyl violet to visualize the

plaques. Plaque counts were expressed as the percent of the virus control cultures from which dose-response lines were constructed. ICsO (50 percent inhibitory concentration yA4) values were then read from the resultant curves.

Plaque reduction assays against varioelta zoster virus were performed in MPG5 cells in closed cultures and under a liquid maintenance medium overlay. Cultures were incubated for seven days at 37OC before being fixed, stained, and assessed as described for herpes simplex virus.

RESULTS

Herpes Simplex Virus. ICso values (PM) for 100 iso- lates of herpes simplex virus were determined and are represented in Figure 1. The isolates were described

as either type 1 or type 2; this classification has not

been confirmed in our laboratory, but was based on the evidence provided by the trial center. Although an overlap between the sensitivity of type 1 and type 2 strains was observed, the latter were found to be gen- erally less sensitive. It was evident from the histograms that no shift in sensitivity to acyclovir occurred after treatment. This was confirmed by comparing mean and median I&o values for each group. The mean lCso value for pretreatment type 1 isolates was 0.084 FM (median 0.076 PM), and the corresponding posttreatment value was 0.087 PM (median 0.066 PM). Similarly, the mean lCs0 value for pretreatment type 2 isolates was 0.34 p M (median 0.32 PAI) as compared with 0.37 pM(median 0.33 PUM) for isolates taken after treatment.

Figure 1 represents all available data irrespective of whether pretreatment or posttreatment isolates were available from each patient. In Figure 2 we eliminated nonmatched isolates and then plotted the loglo I&, values obtained from isolates taken from patients after

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Figure 2. Comparison of the acyclovir sensitivities of pretreatment and posttreatment isolates of herpes simplex virus.

treatment was begun against the corresponding pre- treatment value. Posttreatment specimens were found not to be any more or less sensitive than the original pretreatment specimen in this comparison. Varicella Zoster Virus. Specimens of varicella zoster virus from 19 patients who had been treated with in- travenous acyclovir on an emergency basis were re- ceived for drug sensitivity testing in MPG-5 cells. Pre- and posttreatment isolates from only two patients were

matched; the remaining isolates were 15 pretreatment and 11 posttreatment.

The matched isolates gave pretreatment I& values of 7.8 and 10.7 PM, compared with 4.1 and 13.4 yM,

respectively, for isolates taken after treatment was begun. The mean ICsO value for the nonmatched pre- treatment specimens was 6.3 pM (median 3.9 PM), compared with 6.9 pM (median 7.7 yM) for the post- treatment specimens. These results again suggest that no shift in sensitivity occurs after acyclovir therapy.

COMMENTS

This preliminary study of the U.K. clinical trials of acy- clovir for herpes simplex virus and varicella zoster virus infections has been of great use for validating the test system used to monitor acyclovir sensitivity. The re- producibility of I(&, values for virus isolates has been good, and we remain confident that we would detect resistant virus should it appear.

The results do not show any significant shifts in acyclovir sensitivity to suggest the acquisition of re- sistance. This conclusion is made on the basis of a relatively small number of observations, particularly if samples for which there are no pretreatment specimens are excluded. The importance of pretreatment samples is emphasized by the ranges of sensitivities observed, which may vary up to 10 times; for example, type 1 I& values ranged from 0.02 PM to 0.21 PM, type 2 0.13 /J M to 0.77 FM, and for varicella zoster virus 1.7 p M

to 17.5 p M. A shift in sensitivity of five to lo-times for a posttreatment specimen could be missed if a pre- treatment value was not available and the I&, value fell within the expected range, albeit towards the upper limit of that range.

REFERENCES

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