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Previous Volume 329:1777-1782 December 9, 1993 Number 24 Next

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Acyclovir has been widely used over the past decade as an effective and safe drug for the treatment of infections with herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2)¹. Resistance to acyclovir was initially noted only in studies of HSV infection in vitro and in animal models,^2,3,4 then it was found in immunocompromised patients, and it has now become a well-documented clinical challenge to the care of patients infected with the human immunodeficiency virus (HIV)^{4,5,6,7,8,9,10,11,12}. Acyclovir resistance has not been a problem to date in the treatment of immunocompetent people with HSV infection. Although we identified acyclovir-resistant isolates in a study of long-term suppressive treatment for genital herpes in normal hosts and later studies documented occasional shedding of acyclovir-resistant virus by immunocompetent patients before, during, or after therapy, no correlation has been established between such isolates and clinical outcomes^{13,14,15,16,17}.

Acyclovir-resistant HSV is operationally defined by the need for a dose of more than 3 µg of drug per milliliter of culture medium to inhibit viral replication by 50 percent in vitro (ID₅₀)¹³. The antiviral activity of acyclovir requires that it first be converted to its monophosphate derivative by viral thymidine kinase. Cellular kinases then convert the drug to its active triphosphate form, which inhibits viral DNA polymerase and viral replication^18,19,20,21. Mutations in either the viral thymidine kinase or polymerase genes can lead to acyclovir resistance. The normal viral thymidine kinase (the TK⁺ phenotype) phosphorylates acyclovir, thymidine, and other related substrates. Mutations in the viral thymidine kinase gene leading to the formation of premature stop codons result in thymidine kinase-deficient (TK^-) virus strains, whereas other point mutations alter the specificity of thymidine kinase for some of its potential substrates, yielding the thymidine kinase-altered (TK^A) viral phenotype^2,22,23,24. Rare HSV strains maintain a TK⁺ phenotype but are rendered resistant to acyclovir by mutations in the viral DNA polymerase, the target of acyclovir triphosphate^3,5,21,25,26.

We describe an immunologically normal man with frequently recurring, symptomatic, culture-positive outbreaks of genital herpes that were not suppressed despite treatment with 4.8 g of oral acyclovir a day and sustained, appropriate serum drug levels. Isolates recovered from six of his sequential outbreaks during and after therapy were resistant to acyclovir (ID₅₀, 6 to 16.3 µg per milliliter) and were identical according to multiple biochemical and molecular criteria. The isolates tested displayed a predominantly TK^A phenotype and, in comparison with an acyclovir-sensitive HSV strain, contained the same four point mutations, including one that resulted in an alteration in the putative acyclovir-binding domain of the viral thymidine kinase.

Methods

HSV was isolated from genital lesions by culture in human embryonic lung cells (WI-38, BioWhittaker, Walkersville, Md.), as described elsewhere¹³. HSV-2 virion DNA was purified from infected cells as described elsewhere²⁷. Viral DNA was digested by the restriction endonucleases BamHI, BglII, EcoRI, HindIII, KpnI, and SalI (New England Biolabs, Beverly, Mass.). Virus type and genetic relatedness were determined by comparing the patterns of restriction-endonuclease digestion of the DNA from clinical isolates with those of a standard laboratory strain, HSV-2 333^28,29.

HSV-2 isolates were tested for susceptibility to antiviral drugs with standard plaque-reduction assays⁶. The ID₅₀ was calculated by plotting the log₁₀ concentration against the percentage of suppression of plaques formed.

HSV strains were tested for their ability to express thymidine kinase activity by [¹²⁵I]iododeoxycytidine ([¹²⁵I]dC) plaque autoradiography, as recently described and modified^30,31,32. This method enables one visually to distinguish virus with a TK^A phenotype, which results in faintly labeled plaques, from virus that is TK⁺ or TK^-; these respective phenotypes label plaques darkly or not all. Mixed populations of viruses that may include mutants or viruses that have reverted to the normal phenotype are easily identified by this method.

Extracts of HSV-infected 143B cells were assayed for thymidine kinase activity by methods described elsewhere³³. These cells lack thymidine kinase and provide a negative background on which to characterize the HSV thymidine kinase biochemically.

The polymerase chain reaction (PCR) was used to amplify the thymidine kinase gene from HSV-2 333 and from the clinical isolates with modifications of methods described previously³⁴. Two primers consisting of thymidine kinase-specific sequences were chosen from the published HSV-2 333 sequence³⁵. The reactions were carried out in Taq polymerase reaction buffer (Boehringer-Mannheim, Indianapolis) at standard concentrations. Thermal cycling was performed with an automated thermal cycler (Perkin-Elmer Cetus, Norwalk, Conn.) for 35 cycles consisting of 30 seconds at 97 °C, 2 minutes at 55 °C, and 4 minutes at 72 °C. Two modifications, the use of the 7-deaza-2'-deoxyguanosine triphosphate nucleotide and the hot-start method, were required for optimal amplification of specific products³⁴.

The PCR-amplified products from clinical isolate HSV-2 4365-9 were cloned into pGEM2 vector (Promega Corp., Madison, Wis.) by standard techniques²⁹. The resulting plasmid, pGTK9-2, contains the HSV-2 4365-9 thymidine kinase gene from bases -230 to +1275 relative to the start of transcription, as confirmed by sequencing. Sequencing primers were chosen to span the HSV-2 thymidine kinase gene at intervals of 200 to 250 bases and to provide serial overlapping sequences. The PCR-amplified products were sequenced according to a modified protocol using dimethylsulfoxide³⁶. Cloned plasmids were sequenced with the Sequenase Version 2.0 kit (United States Biochemical, Cleveland)¹⁵. Band compressions were resolved with Deaza T⁷ Sequencing Mixes (Pharmacia LKB Biotechnology, Piscataway, N.J.) as described elsewhere.

The patient gave informed consent for HIV-antibody testing, phlebotomy, and apheresis. Testing for HIV antibody was performed with the Western blot assay (Dupont, Wilmington, Del.), and testing for p24 antigen with an enzyme-linked immunosorbent assay (ELISA) (Coulter Source, Marietta, Ga.). Two-pass lymphocyte apheresis was performed with citrate-dextrose type A anticoagulant and the Haemonetics VSO cell separator (Haemonetics, Braintree, Mass.). Lymphocytes were purified by Ficoll-Hypaque centrifugation (Pharmacia LKB Biotechnology), cryopreserved in a controlled-rate freezer, and stored in liquid nitrogen until needed. Antigen-driven lymphocyte proliferation was assayed in quadruplicate cultures³⁷. The HSV antigens consisted of ultraviolet light-inactivated HSV-2 or HSV-1 (10⁵ plaque-forming-unit equivalents before irradiation) or recombinant HSV-2 glycoproteins D or B expressed in Chinese-hamster-ovary cells (1 µg per milliliter) (Chiron, Emeryville, Calif.). Tetanus toxoid (1 Lf unit per milliliter) and phytohemagglutinin A (1 µg per milliliter) were included as positive protein and mitogen controls, respectively.

Results

Case History

The patient was a 24-year-old man who presented in November 1990 with the onset of new, painful penile vesicles. Culture confirmed an HSV infection. Oral acyclovir was prescribed (200 mg five times daily) for five days, and the lesions resolved. The vesicles were first noted to recur in March 1991, and the patient began long-term therapy with acyclovir (200 mg twice daily). Despite this regimen, in June 1991 genital lesions began to recur every two to three weeks, yielding positive cultures for HSV. Lesions appeared only on the dorsum penis and consistently healed within seven days; over a period of months, however, escalating doses of oral acyclovir (up to 800 mg six times daily) failed to suppress recurrences (Figure 1).

View larger version (17K): [in this window] [in a new window]   Figure 1. Clinical Course of a Man with Acyclovir-Resistant Genital Herpes. After the primary infection in November 1990, recurrences were documented during office visits (tall arrows) and by the patient's diary entries or through telephone contact (short arrows). Viral cultures positive for HSV are indicated by plus signs; one negative culture is labeled with a minus sign. For viral isolates evaluated in detail, the assigned numbers are shown. The initiation and dose escalation of acyclovir treatment are indicated by the lines at the top, with the dosages drawn to scale in relation to the time line. The abbreviations bid, qid, 5id, and 6id denote two, four, five, and six times a day, respectively.

 
The patient reported no history of other recurrent infections or systemic illness. He was a sexually active homosexual who before November 1990 had only one partner. In the two weeks before his first outbreak he had three new sexual contacts, two of whom were positive for HIV antibody. One of these contacts acknowledged recurrent genital herpes that was suppressed with long-term acyclovir therapy; he had not noticed a recurrence in 18 months. The man declined further evaluation. Our patient reported his own antibody test for HIV as repeatedly negative over the past two years.

In May 1992, physical examination revealed only fresh, grouped vesicles on the dorsum penis. Acyclovir was withheld for six weeks, during which time three additional recurrences were documented, each healing within seven days and being no more severe or prolonged than those observed during drug treatment. Oral suppressive therapy was then resumed at a dose of 800 mg six times daily. In all, six positive viral cultures were obtained during and after acyclovir therapy over a six-month period (Figure 1).

Laboratory Evaluation

Laboratory studies of the patient's immune system were normal. The complete blood count, the differential count, and the results of a panel of 22 tests of blood chemistry and multiple serologic tests were normal. Immunoglobulin levels were normal. Serum antibodies to HSV-2 were detected by commercial enzyme immunoassay (BioWhittaker). The total CD4 lymphocyte count was 1001 cells per cubic millimeter. Enzyme immunoassay was negative for antibody to the human T-cell lymphotropic virus type I. ELISA and Western blotting for HIV antibody and ELISA for p24 antigen were all negative initially and again seven months later (in January 1993). Intradermal testing revealed normal delayed-type hypersensitivity responsiveness. Antigen-driven lymphocyte blastogenic responses to whole ultraviolet-inactivated HSV-2, ultraviolet-inactivated HSV-1, recombinant HSV-2 glycoproteins D and B, and tetanus toxoid, as well as the mitogenic response to phytohemagglutinin A, were normal as compared with the responses of 35 normal subjects with recurrent genital herpes. The serum level of acyclovir two hours after an oral dose of 800 mg (the peak level) was within the normally therapeutic range at 1.0 µg per milliliter, and the level immediately before a subsequent oral dose (the trough level) was 0.85 µg per milliliter.

Characterization of Virus Isolates

To confirm serial reactivations of the same strain of HSV-2, we performed restriction-endonuclease digestions with six enzymes²⁸. These analyses demonstrated that the six clinical isolates were identical strains of HSV-2 and were distinct from reference strain HSV-2 333 (data not shown).

The antiviral susceptibilities of the clinical isolates were assessed in parallel with those of several wild-type and mutant strains of HSV-1 and HSV-2 previously characterized in one of our laboratories (Table 1)^3,15,26. All six isolates obtained from the patient during or after treatment were moderately resistant to acyclovir (Table 1), with the ID₅₀ ranging from 6 to 16.3 µg per milliliter. The isolates were also resistant to ganciclovir, but they were sensitive to 1-beta-d-arabinofuranosyl thymine, which is phosphorylated by the viral thymidine kinase, and to two drugs that do not require activation by HSV thymidine kinase for viral inhibition, sodium phosphonoacetic acid and trisodium phosphonoformate (foscarnet) (Table 1)^38,39. This pattern of in vitro drug sensitivity indicates a normal viral polymerase enzyme and implies a mutation in the viral thymidine kinase gene.

View this table: [in this window] [in a new window]   Table 1. In Vitro Drug-Sensitivity Testing of Viral Strains from the Patient and Reference Strains.

 
[¹²⁵I]dC autoradiography revealed that the clinical isolates contained a mixed population of viruses in which an abnormal thymidine kinase phenotype dominated. Within the four isolates studied, 92 to 98 percent of plaques were due to virus with the TK^A phenotype, whereas 2 to 8 percent had the wild-type TK⁺ phenotype (Figure 2). To obtain pure stocks of TK^A virus from each of four clinical isolates, three cycles of plaque purification were performed and confirmed by [¹²⁵I]dC autoradiography.

View larger version (40K): [in this window] [in a new window]   Figure 2. [125I]dC Autoradiographs of Four Clinical Isolates Obtained from the Patient and Three Reference Strains. Reference strain HSV-1 Patton, its TKA derivative IUdRr, and the TK- strain HSV-1 B2006 have been described elsewhere30. TK+ denotes a wild-type thymidine kinase phenotype, TK- a thymidine kinase-deficient phenotype, and TKA a thymidine kinase-altered phenotype. The percentages of viruses with each phenotype were calculated directly from the plaque counts. For example, in the case of B2006, one virus that has reverted to the TK+ phenotype can be readily identified in the two fields of TK- plaques.

 
To characterize further the thymidine kinase activity of the isolates from our patient, extracts were made of 143B cells infected with the clinical isolates, their derivative plaque-pure TK^A viruses, or two previously described reference viruses, TK⁺ HSV-2 12927 and TK^A HSV-2 12720²³. The cell extracts were assayed for the ability to phosphorylate nucleosides. Phosphorylation of acyclovir by plaque-pure TK^A virus from the patient was depressed to 8 to 27 percent that of the TK⁺ HSV-2 12927 (data not shown). Although such phosphorylation by the patient's virus remained 54 times greater than that of the TK^A HSV 12720 control, the extent to which the patient's viruses phosphorylated acyclovir was not adequate to confer susceptibility to the drug on them. In addition the TK^A isolates from our patient demonstrated supranormal rates of thymidine phosphorylation (data not shown). Thus, the biochemical data support the theory that a thymidine kinase enzyme in the patient's virus was altered both in its ability to phosphorylate acyclovir and in its phosphorylation of its natural substrate thymidine.

We next defined the molecular basis for the thymidine kinase alterations in these clinical isolates. The cloned PCR-amplified product from isolate 4365-9 (plasmid pGTK9-2) was sequenced, and four discrete point mutations were identified in its thymidine kinase gene relative to the published sequence of HSV-2 strain 333 (Table 2). These mutations were confirmed by direct PCR sequencing, without intervening cloning, of the amplification products of the remaining clinical isolates, the plaque-purified viruses, and the amplified HSV-2 333 wild-type virus³⁵. The mutation at nucleotide 529 was critical in altering the domain of the viral thymidine kinase purported to interact with acyclovir, the nucleoside-binding site; this mutation probably underlies our patient's clinical resistance to acyclovir²².

View this table: [in this window] [in a new window]   Table 2. Mutations Identified in the Thymidine Kinase Gene of Acyclovir-Resistant HSV-2 Clinical Isolates as Compared with Reference Strain HSV-2 333.

 
Discussion

Over the past decade, acyclovir-resistant HSV has been documented in vitro, in animal models, and in diverse populations of immunocompromised patients. Acyclovir-resistant TK⁺ and TK^A strains of HSV have been isolated from immunocompetent patients, but when evaluated carefully they proved to be transient and clinically unimportant²³. We document here that an immunocompetent person can have repeated reactivations of drug-resistant HSV even when acyclovir is withdrawn. In the patient we describe, the resistant virus was clinically important because it was refractory to acyclovir treatment.

Acyclovir-resistant HSV is well documented in the HIV-infected community. It is possible that in the course of sexual contact our patient acquired a virus that was inherently resistant to acyclovir^7,8. Alternatively, resistance may have evolved over the course of his long-term, slowly escalating therapy. Without knowing what viruses were harbored by his sexual partners, it is impossible to discern exactly how acyclovir resistance developed in this patient.

Acyclovir-resistant pools of HSV typically represent heterogeneous mixtures of virus, and the antiviral-susceptibility curves and thymidine kinase biochemical assays of a given strain reflect the net composition of that pool⁴. Thus, the mixture of TK^A and TK⁺ viruses in the isolates from our patient (Figure 2) is not surprising. Both viral phenotypes are probably derived from the same strain, persisting in ganglia and emerging episodically to become reactivated in this patient. The moderate reductions in phosphorylation of acyclovir by the virus mixture were not reduced further after plaque purification, indicating that the TK⁺ viruses did not contribute substantially to the total phosphorylating activity of the pool. Unlike TK^- mutants, which usually show diminished pathogenicity and capacity for reactivation in animal models, TK^A viruses are fully pathogenic and capable of reactivation². It is precisely a TK^A strain that one would predict as most likely to underlie stable drug resistance in an immunocompetent person.

The conformation and active sites of the HSV-1 and HSV-2 thymidine kinase molecules have been proposed on the basis of amino acid homology with other known nucleoside-binding domains^24,39,40,41. Most of the acyclovir-resistant thymidine kinase mutants studied to date contained single mutations or premature stop codons^3,22,24. The virus shed by our patient was unusual in possessing four nonterminating mutations (Table 2). The amino acid substitutions at positions 78 and 220 are probably trivial, and the importance of the alteration in residue 140 is unknown. The mutation at nucleotide 529 lies in a location shown by Darby et al. to be sufficient when mutated to confer a TK^A phenotype in HSV-1,²² although in our patient's HSV-2 isolate the replacement of arginine by tryptophan presents a more drastic substitution than the glutamine substitution in Darby's strain Tr7. Thus, the substitution at base 529 in our patient's viral thymidine kinase may in itself be adequate to account for the decreased thymidine kinase phosphorylation of acyclovir.

Despite the occurrence of resistant HSV infections in severely immunocompromised patients, oral acyclovir has provided effective suppression of frequently recurring genital herpes in immunocompetent people for more than a decade^14,42,43,44. Our patient presents a new limitation to this therapeutic approach. He carries and has repeated reactivations of an HSV-2 strain bearing thymidine kinase mutations that confer in vitro and clinically important resistance to acyclovir. He continues to have symptomatic recurrences, although his intact immune responses limit the duration and severity of each outbreak. Although high-dose intravenous acyclovir or intravenous foscarnet might suppress his recurrences, they are expensive and carry potential risk, and any benefit would probably be temporary.

The carriage and reactivation of acyclovir-resistant HSV may still be rare and unrecognized in immunocompetent people. Vigilance is needed, as are new therapeutic and preventive strategies for HSV disease.

We are indebted to the late Mr. Holly Smith for excellent technical assistance and invaluable contributions over four decades, to Dr. Paulo de Miranda for determining acyclovir serum levels, to Dr. Scott Fritz of PRI/CynCorp for purifying and cryopreserving the patient's lymphocytes, to Dr. Sharon Safrin for review of the manuscript, to Dr. James Fyfe for helpful discussion and review of the manuscript, and to William Barrick, R.N., for referring this patient for study.

Source Information

From the Medical Virology Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. (R.G.K., S.E.S.); the Division of Virology, Burroughs Wellcome Company, Research Triangle Park, N.C. (E.L.H.); and Chiron Corporation, Emeryville, Calif. (M.T.).

Address reprint requests to Dr. Straus at the Laboratory of Clinical Investigation, Bldg. 10, Rm. 11N228, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892.

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