Antiviral Therapy: Current Concepts and Practices

Transcription

1 CLINICAL MICROBIOLOGY REVIEWS, Apr. 1992, p Vol. 5, No /92/ $02.00/0 Copyright 1992, American Society for Microbiology Antiviral Therapy: Current Concepts and Practices BONNIE BEAN Departments of Pathology and Medicine, Humana Hospital-Michael Reese, Chicago, Illinois INTRODUCTION CELLULAR AND VIRAL REPLICATION ANTIVIRAL AGENTS Amantadine and Rimantadine Ribavirin Vidarabine Acyclovir Ganciclovir Foscarnet Zidovudine Didanosine Investigational Antiretroviral Agents IMMUNOGLOBULINS IMMUNOMODULATORS PROBLEMS OF ANTWIRAL THERAPY Resistance Latency Immunosuppression by Antiviral Agents PROSPECTS FOR THE FUTURE Liposomes Combination Therapy Computer-Aided Drug Design Role of the Clinical Microbiology Laboratory CONCLUSIONS ACKNOWLEDGMENTS REFERENCES INTRODUCTION Interest in antiviral chemotherapy began in the 1950s, when the search for antitumor agents generated a great deal of interest in DNA synthesis inhibitors and produced a number of compounds capable of inhibiting viral DNA synthesis. Antiviral agents were first successfully administered to patients in the 1960s, when Bauer prevented disease by giving thiosemicarbazone (methisazone) to patients exposed to smallpox (20) and Kaufman greatly improved the healing of herpes keratitis by treating patients with topical idoxuridine (233). Progress was slow, however, because of the difficulty in finding compounds capable of inhibiting viruses while at the same time leaving host cell functions intact. With the late 1970s and early 1980s came development and marketing of acyclovir, the first antiviral agent nontoxic enough to be of value in treating a wide range of herpesvirus infections in ambulatory as well as seriously ill patients. The late 1980s and early 1990s are seeing an explosion in antiviral agents and in approaches to antiviral therapy that is fueled, in part, by the AIDS epidemic. This article reviews the basis of antiviral therapy, the agents themselves, the problems to be solved, and prospects for the future. CELLULAR AND VIRAL REPLICATION Like antibacterial agents, useful antiviral agents must have certain properties. They must reach their target organs, be active intracellularly as well as extracellularly, and be 146 metabolically stable. Most important, they must inhibit virus replication without disturbing host cell function. Because viruses reproduce intracellularly and use host cell metabolic machinery in doing so, it was thought for many years that specific interference with viral replication was impossible. Experience with early antiviral compounds corroborated this view; the drugs were either too toxic or insufficiently potent to be of use (460). Two drugs that illustrate this well are idoxuridine (5-iodo-2'-deoxyuridine) and trifluorothymidine (5-trifluoromethyl-2'-deoxyuridine) (Fig. 1). Idoxuridine was first synthesized by Prusoff in 1959 (363) and was subsequently shown to inhibit a number of DNA viruses (for a review, see reference 364). When administered systemically to patients, however, it did not decrease the risk of death from herpes encephalitis and it caused myelosuppression in almost all patients who received it (40). On the other hand, the drug was effective and much less toxic when administered topically to patients with herpes keratitis (233). It is still used for this purpose. Recently, there has been a resurgence of interest in topical use of idoxuridine coupled with dimethyl sulfoxide for treatment of herpes labialis (450). Trifluorothymidine, first developed as an antitumor agent, was found to inhibit herpes simplex virus (HSV) in vitro, but its selectivity index (ratio of drug concentration causing cellular toxicity to that needed for viral inhibition) was very low compared with those of other agents (99) and it was not developed for systemic use. Like idoxuridine, it is used for topical treatment of herpes keratitis. Despite their shortcomings, these drugs were important in

2 VOL. 5, 1992 ANTIVIRAL THERAPY 147 OH IDOXURIDINE TRIFLUOROTHYMIDINE VIl FIG. 1. Some nucleoside analogs first used as antiviral agents. that they demonstrated the feasibility of antiviral therapy in patients. They also sustained interest in a continuing search for more selective agents. By the late 1970s, it was known that most human pathogenic viruses possess enzymes coded by the viruses themselves and not present in uninfected cells (327). Most of these enzymes are involved in viral nucleic acid synthesis, as discussed below. Their discovery represented a major advance in antiviral therapy by making it possible to direct efforts at finding specific inhibitors of these enzymes rather than nonspecific inhibitors of viral growth in cell culture. Replication of viruses can be divided into several steps: (i) attachment to the cell, (ii) penetration, (iii) uncoating of nucleic acid, (iv) transcription and translation of early (regulatory) proteins, (v) nucleic acid synthesis, (vi) synthesis of late (structural) proteins, (vii) assembly of mature virions, and (viii) release from the cell (for an example, see Fig. 2). All of these steps are potential targets for interference, although viral attachment, penetration, uncoating, assembly, and release closely resemble normal cellular processes and are thought to be carried out, in many instances, by cellular enzymes (210). It is at the point of nucleic acid synthesis that viral processes diverge most from their cellular counterparts and are most likely to require virus-specified enzymes. This is because eukaryotic cells contain doublestranded DNA and, when they replicate, make new DNA from the parental DNA template in the cell nucleus (Fig. 3). In addition, they transcribe mrna from DNA and transport it into the cytoplasm, where proteins are translated. Viruses, on the other hand, may contain DNA or RNA as their genomic material, and the nucleic acid may be double or single stranded, circular or linear, and segmented or in one continuous piece. RNA may be either positive or negative stranded (having the same sense or direction as mrna, and thus directly translatable, or having the opposite sense and not directly translatable). Viruses may replicate in the nucleus, in the cytoplasm, or in both. Despite these differences from their cellular hosts, viruses must be able to fit into host cell synthetic pathways if they are to replicate successfully. They must present to the cell either a form of DNA that can be transcribed directly into mrna or a form of mrna that the cell can recognize and translate into proteins (390). Many different pathways have evolved by which viruses accomplish this; some examples are shown in Fig. 3. Retroviruses, for example, carry single-stranded duplex RNA as their genomic material and, when infecting cells, must supply a virus-encoded reverse transcriptase to transcribe this RNA OH DARABINE into cdna which is then inserted into the host cell DNA (Fig. 2). Picornaviruses such as rhinoviruses and enteroviruses carry a positive single-stranded RNA that can be directly translated by host cell enzymes, but they must encode an RNA replicase which allows use of the genomic RNA as a template for new negative-stranded RNA. Orthomyxoviruses (influenza viruses), on the other hand, carry a negative-stranded RNA, and thus must supply an RNAdependent RNA polymerase from which a plus-stranded mrna can be made. Such unique enzymes, critical to viral replication but unnecessary for cellular function, offer very good targets for selective inhibition by antiviral agents. They are not the only enzyme targets, however. Other enzymes such as nucleoside kinases and DNA polymerases are encoded by both cells and viruses (Fig. 4). The properties of such enzymes can differ greatly with respect to substrate specificity, binding affinity for substrates, and susceptibility to inhibition by various compounds. These differences can be exploited in developing antiviral compounds. For example, thymidine kinases catalyze the first step in the preparation of pyrimidine nucleosides for incorporation into DNA, phosphorylation of the 5' carbon atom of the pentose ring (Fig. 4). Thymidine kinase specified by HSV binds acyclovir much better than cellular thymidine kinase does and phosphorylates it 3 million times faster (235). DNA polymerases are responsible for incorporating nucleotide triphosphates into growing DNA chains, and again, herpesvirus-specified DNA polymerases differ from their cellular counterparts: they are 30 times more susceptible to inhibition by acyclovir triphosphate than are the alpha DNA polymerases of human origin (122). Differences like these make it possible to identify compounds that selectively inhibit viral functions. What advantage is there to viruses in encoding and carrying enzymes also made by cells? The answer is survival. Viruses carrying the extra enzymes have a wider host range and can infect cells that do not encode a critical enzyme or that express it only at a certain point in the cell cycle. HSV mutants that do not encode thymidine kinase, for example, are less capable of establishing reactivatable latent infections in neurons (which are nondividing cells) than are thymidine kinase-encoding strains (121, 142, 471). Many antiviral agents are nucleoside analogs, structures that closely resemble the natural nucleosides used as building blocks for DNA synthesis (Fig. 1 and 5). As such, these drugs are phosphorylated by cellular nucleoside kinases and incorporated into growing DNA chains by DNA polymer-

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4 VOL. 5, 1992 ANTIVIRAL THERAPY 149 RETROVIRUSES as du RNA ORTHOMYXOVIRUSES as RNA (-) cdna * REOVIRUSES ds RNA ; % // * PICORNAVIRUSES a / t ss RNA(+) *J crdsia mrna 4 proteins" DNA supercolled HEPATITIS B VIRUS FIG. 3. Protein and nucleic acid synthetic pathways for eukaryotic cells (bold) and some representative human viruses (dashed lines). *, need for a virus-specified enzyme; -, negative stranded; +, positive stranded. ss, single stranded; du, duplex; ds, double stranded. ases, competing with the natural nucleosides as substrates for both enzymes (Fig. 4). Many of the early antiviral agents, such as idoxuridine and trifluorothymidine, were equally good substrates for both viral and cellular kinases. Even though they were slightly better inhibitors of viral than of cellular polymerases (194, 365), they were activated and capable of decreasing DNA synthesis in both infected and uninfected cells. Many of the newer nucleosides are less toxic, in part because there is at least one point in their functional pathways which is specific for virus-infected cells. For example, acyclovir is activated only in virus-infected cells, and zidovudine inhibits an enzyme, human immunodeficiency virus (HIV) reverse transcriptase, not found in normal cells. Although the greatest success has been achieved by using inhibitors of nucleic acid synthesis as antiviral agents, additional enzymes are necessary for other steps in the viral replication cycle and should be amenable to inhibition by putative antiviral compounds. Special interest in such compounds has been generated by the AIDS epidemic and the increasingly obvious need to treat HIV infection with combinations of drugs active at different sites of viral replication. Such combination therapy, it is hoped, will decrease drug toxicity and reduce the chances of antiviral resistance developing during treatment. Unfortunately, steps such as viral attachment and penetration and assembly and release are catalyzed by cellular enzymes (210), and it has been difficult to find compounds that interfere specifically with these events. Nevertheless, some success has been achieved with amantadine (an inhibitor of uncoating) for management of influenza, and recombinant soluble CD4 may yet prove effective at inhibiting attachment of HIV to host lymphocytes. Interferons, which inhibit viral mrna transcription and protein synthesis, are also useful as antiviral agents. In addition, HIV proteases, which cleave precursor polypeptides to form functional reverse transcriptases, are very promising targets for selective inhibition. ANTIVIRAL AGENTS Amantadine and Rimantadine The adamantanes, amantadine (1-aminoadamantane hydrochloride) and its alpha-methyl derivative, rimantadine, are used for management of influenza A virus infections. They are cyclic amines with bulky, cagelike structures unlike those of other known antiviral agents (Fig. 6). Their mechanisms of action and spectra of antiviral activity are identical, but rimantadine is metabolized differently from amantadine, and it causes fewer central nervous system side effects such as insomnia and difficulty concentrating (110, 514). It is also a more potent in vitro inhibitor of influenza A viruses (431), although both drugs have been equally effective in treating patients. Amantadine became available in 1966; rimantadine is being considered for approval by the U.S. Food and Drug Administration. It has been known for some time that amantadine inhibits an early phase of viral replication (207, 232), more specifically, virus uncoating (48, 381). Recent studies of avian influenza viruses have also demonstrated a block at a later stage, virus maturation and assembly (184, 461). Susceptibility to amantadine is determined principally by the M2 protein (184, 281), a virus-specified matrix protein present on the surface of infected cells and in the virion in small amounts (530). It has been proposed (28) that this protein forms ion channels through which protons pass across the membranes of intracellular endocytic and exocytic vesicles. During virus uncoating, protons are transferred from the endocytic vesicle to the virion, allowing the fall in ph that releases free viral nucleoprotein into the cell cytoplasm. During virus assembly, protons are transferred out of the exocytic vesicle, thus maintaining the ph above the level at which the viral hemagglutinin, a major surface glycoprotein, would lose its structural integrity and fail to be incorporated into the viral envelope. Amantadine appears to block this M2-mediated transfer of protons and thus inhibits viral

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6 VOL. 5, 1992 ANTIVIRAL THERAPY 151 5H ACYCLOVIR GUANOSINE RIBAVIRIN H2N' OH N3 GANCICLOVIR ZIDOVUDINE DIDEOXYINOSINE DI DEOXYCYT I DINE FIG. 5. The purine nucleoside guanosine and some nucleoside analogs currently in use for antiviral therapy. uncoating or viral maturation or both, depending on the strain of virus (185). At in vitro concentrations much higher than those at which these effects occur, adamantanes inhibit other RNA viruses, including influenza B virus, and paramyxoviruses (114). With current drug formulations, these high concentrations cannot be achieved in patients, and thus the adamantanes remain useful only for the management of influenza A virus infections (Table 1). Both drugs are very effective in preventing illness due to influenza A virus. When given prophylactically during a community outbreak, either compound reduces the risk of acquiring influenzal illness by 50 to 90% ( 68, 110, 161, 317, 331, 500). Rimantadine has fewer side effects when used in NH2 HCI NH2 *HCI H-C-CH3 this manner, although lowering the dose of amantadine also reduces adverse effects (378, 419). The efficacy of low-dose amantadine has not been proven in this situation, however, and the higher dose remains the prophylactic regimen of choice until rimantadine becomes available. Both drugs are more effective in preventing illness than in preventing infection with influenza A virus (110, 317). This may be beneficial, however, in that it allows the patient to produce protective antibodies without developing frank illness. Currently, the Immunization Practices Advisory Committee recommends using amantadine prophylactically to protect those at risk of influenza complications who cannot or have not been vaccinated (60). The drug can also be given at the same time as [ 0-0-p-cl gi Na AMANTADINE RIMANTADINE FOSCARNET FIG. 6. Nonnucleoside antiviral agents.

7 152 BEAN CLIN. MICROBIOL. REV. TABLE 1. Antiviral agents currently available and their uses Agent Route of administration Use Adult dosage Acyclovir Oral Initial genital herpes 200 mg 5 times daily for 10 days Recurrent genital herpes 200 mg 5 times daily for 5 days Suppression, genital herpes 400 mg twice daily for up to 1 yr Suppression, mucocutaneous herpes 200 mg 3 to 5 times daily in immunocompromised host Treatment, mucocutaneous herpes in mg 5 times daily until healed immunocompromised host Zoster in immunocompetent host 800 mg 5 times daily for 7-10 days Ganciclovir Zidovudine Vidarabine Amantadine Ribavirin Interferon Foscarnet Didanosine Intravenous Topical Intravenous Oral Intravenous Ophthalmic ointment Oral Aerosol Intravenous Subcutaneous Intralesional Intravenous Oral Herpes encephalitis Neonatal herpesa Severe genital herpes Treatment, mucocutaneous herpes, immunocompromised host Suppression, mucocutaneous herpes in immunocompromised hosta Zoster or varicella in immunocompromised host Initial genital herpes Cutaneous herpes in immunocompromised host CMV retinitis in AIDS HIV infection, CD4 cells <500/mm3, or symptomatic Herpes encephalitis Neonatal herpes Zoster in immunocompromised host Herpes keratitis Influenza A: treatment Influenza A: prophylaxis Severe RSV, infants and children Lassa fever' Hepatitis B-chronic active liver disease' Hepatitis C-chronic liver disease Condyloma acuminata (warts) CMV retinitis Acyclovir-resistant HSV or VZVa Zidovudine intolerance or treatment failure in HIV-infected host 10 mg/kg 3 times daily for days 500 mg/mth 3 times daily for 10 days 5 mg/kg 3 times daily for 5 days 5 mg/kg 3 times daily for 7 days 5 mg/kg 3 times daily Adult: 10 mg/kg 3 times daily for 7 days Child: 500 mg/mrn 3 times daily for 7 days Apply every 3 h up to 6 times daily for 7 days Apply every 3 h up to 6 times daily for 7 days Induction: 5 mg/kg twice daily for days Maintenance: 5 mg/kg daily 100 mg 5 times daily 15 mg/kg daily over h for 10 days 15 mg/kg daily over h for 10 days 15 mg/kg daily over h for 5 days 0.5 in. [1.27 cm] to eye 5 times daily for 7-21 days 200 mg daily for 5-7 days 200 mg daily h daily for 3-7 days (concn, 20 mg/ml) 2-g load, then g 3-4 times daily for 10 days 5 x 106 U daily for 4 mo 2 x x 106 U 3 times weekly for 6 mo 1 x 106 U per wart in up to 5 warts 3 times weekly Induction: 60 mg/kg 3 times daily for 2-3 wk Maintenance: mg/kg daily mg/kg 3 times daily Tablet: mg (as 2 tablets) 2 times daily Powder: mg 2 times daily Trifluridine Ophthalmic solution Herpes keratitis 1 drop every 2 h up to 9 drops daily until healed Idoxuridine Ophthalmic solution a The package insert is not approved for this indication. b Square meters of body surface area. Herpes keratitis 1 drop/h during day and every 2 h at night until healed

8 VOL. 5, 1992 influenza virus vaccine to protect the patient until an antibody response to the vaccine occurs. Both amantadine and rimantadine have been used for the treatment of influenza. They are most effective when given within the first 48 h of illness and may allow the patient to return to routine daily activities 1 to 2 days earlier than no treatment would (191, 475, 483, 514). Amantadine also appears superior to aspirin in this regard. In one comparative study, patients taking 3.25 g of aspirin daily became afebrile sooner but had more side effects (insomnia, nausea, ringing in the ears) than amantadine recipients, who experienced an earlier reduction in symptoms and fewer adverse reactions (528). The peripheral pulmonary airway dysfunction accompanying influenza is also improved by amantadine (279), and this may be important for earlier resolution in normal hosts and for minimizing disease in those with underlying pulmonary or cardiac conditions. The effects of therapy on the complications of influenza, such as pneumonia and myocarditis, are unknown. Although drug resistance does not occur naturally among influenza viruses (27), it can be induced by therapy, and these resistant viruses can cause influenzal illness. When patients ill with influenza were treated with rimantadine, they improved symptomatically, but 50% of them began shedding rimantadine-resistant viruses within 4 to 6 days (177, 188). In one study, these resistant viruses were apparently transmitted to family members, who also became ill with influenza (188). Resistance was mapped to the M2 protein (29), and subsequent studies of similarly resistant avian viruses have shown that these resistant strains are genetically stable and equal in virulence to wild-type viruses (25). These findings raise the possibility that widespread use of adamantanes for treatment of influenza might result in antiviral pressure and selection of predominantly resistant virus populations. To prevent the spread of resistant virus, it has been suggested that patients be isolated or have limited contact with others while they are being treated for influenza (186). The effects of this and other methods to control transmission are unknown, however. In view of these problems and of the unknown effect of therapy in preventing or relieving complications, no recommendation for therapy of influenza is currently made by the Centers for Disease Control (60), although amantadine is approved for this purpose (package insert). Ribavirin Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) was first synthesized in the early 1970s as part of an intensive effort to identify new antiviral agents (517). At first glance, it appears to be a nucleoside analog with an open pyrimidine ring (Fig. 5). Structurally (362) and functionally (458), however, it most closely resembles guanosine. Ribavirin is active in vitro against a wide variety of RNA and DNA viruses, including adenoviruses, herpesviruses, influenza A and B viruses (429, 517), respiratory syncytial virus (RSV) (212), bunyaviruses, arenaviruses (214), reoviruses (367), and HIV (292). The mode of action has not been clearly defined. Three mechanisms have been proposed, and all may operate to some extent in cells infected by different viruses. This may also account for the wide spectrum of activity. Ribavirin readily diffuses into eukaryotic cells, where it is converted by cellular enzymes to the mono-, di-, and triphosphate forms (437, 458). The monophosphate is a potent competitive inhibitor of IMP dehydrogenase, an enzyme essential for ANTIVIRAL THERAPY 153 the synthesis of GTP. This inhibition results in a decrease in the cellular pools of guanine nucleotide necessary for both cellular and viral replication (458, 459). Ribavirin inhibits capping of viral mrna (170, 493), a critical step in the replication of most viruses. Its most important effect, however, appears to be on the early events of viral replication. Ribavirin directly inhibits viral RNA-dependent RNA polymerase of influenza viruses (129), and it can retard both initiation (54, 338, 367, 521) and elongation (129, 521) of mrna transcripts. It is not incorporated into the growing chain of viral nucleic acid and does not cause chain termination (129, 477). Only one strain of virus, a mutant of fowl plague virus, is known to be resistant to ribavirin (215). In addition to its antiviral properties, ribavirin has immunoregulatory effects. In cell culture and in animals, it inhibits macromolecular synthesis and cell division (270), lymphocyte proliferation, and nucleic acid synthesis (341). It also suppresses B lymphocytes and subsequent antibody production (358) and tumor growth (357). To date, however, none of these effects has been shown to be of consequence in patients treated with ribavirin. The major toxicities of ribavirin are anemia and embryotoxicity. Anemia occurs when ribavirin diffuses into erythrocytes and accumulates, because erythrocytes lack phosphatases and are unable to hydrolyze (dephosphorylate) the drug. The erythrocytes then become damaged as they age and are removed prematurely from the circulation (52, 334). At very high doses of ribavirin in animals, bone marrow suppression of erythroid precursors also occurs (52). All of these effects are reversible upon removal of the drug, but the half-life of ribavirin in human erythrocytes is 40 days, so the effect is prolonged. In pregnant rodents treated with ribavirin, skeletal defects in the developing embryo as well as fetal resorption have been observed (242). Because of this, the drug is contraindicated during pregnancy, and safety measures must be taken by pregnant health care workers who administer ribavirin to patients (see below). Ribavirin became commercially available for use as an aerosol in Both the oral and intravenous forms have been studied, but the drug was found to be most effective when given to animals by pulmonary aerosol, so it was developed for that purpose. The drug is diluted in a reservoir, nebulized by a small-particle aerosol generator, and delivered to the patient via face mask, ventilator, or infant oxygen hood. Aerosolized drug can leak from the system during administration and may be inhaled by health care workers. This may represent a hazard to pregnant workers, although it is not known whether the small amounts absorbed into the bloodstream will damage the fetus (57). In college students with uncomplicated influenza, aerosol ribavirin reduced the duration of fever and symptoms and decreased viral shedding (239, 291, 513). This has not been true of all trials and for all influenza variants, however (33, 167). Because of this inconsistency and because aerosol therapy is an expensive and cumbersome mode of therapy for a self-limited illness, ribavirin is not often used in this setting. In infants with bronchiolitis due to RSV, ribavirin has shown some effect in decreasing viral shedding, improving oxygenation, and reducing the duration of illness (178, 179, 388, 469), although questions have been raised about the methods used in treatment trials (494). In a recent trial, ribavirin decreased the duration of oxygen therapy, mechanical ventilation, and hospital stay in otherwise healthy infants with severe RSV illness (439). Nevertheless, important questions remain unanswered, including duration of therapy,

9 154 BEAN response of children with underlying cardiac and pulmonary disease, and efficacy of the drug when administered late in the course of illness (368). Ribavirin is extremely expensive ($400 per day in drug costs alone), and all children with self-limited RSV cannot be hospitalized and treated. Along with the possible toxicity for health care workers, these unanswered questions indicate the great need for additional studies to define the optimum use of this drug for respiratory viral disease. Because of its in vitro activity against bunyaviruses and arenaviruses, intravenous ribavirin has been investigated for treatment of viral hemorrhagic fevers (214). Most notable has been the success with Lassa fever. When patients at high risk of death from Lassa fever were treated intravenously within 6 days of fever onset, the death rate of 55 to 76% in placebo-treated patients decreased to 5 to 9% in ribavirin recipients (293). Oral ribavirin is also beneficial against Lassa fever and is given as prophylaxis to high-risk contacts of Lassa fever patients (58). Recent small-scale studies have shown that ribavirin crosses the blood-brain barrier well, giving drug levels in the cerebrospinal fluid that are 50 to 100% of those in serum (82, 330). For this reason, it may also be of use in the treatment of common viral encephalitides caused by bunyaviruses, such as La Crosse encephalitis (54, 214). Ribavirin has recently been investigated for possible use in HIV-infected patients. Although the drug given orally in high doses appeared to delay progression to AIDS (384), many questions have been raised about the study methodology (39). In addition, no effects on surrogate markers of HIV progression (CD4 counts, p24 antigenemia, viremia, or immune function) were found, and it is not clear that clinically achievable drug levels are inhibitory for viral replication (292, ). In view of these problems, licensure of ribavirin for treatment of HIV is no longer being pursued in the United States, although the drug is available in other countries. The place of ribavirin in our armamentarium of antiviral agents is not yet clear. Though useful in aerosol form, it is cumbersome, expensive, and perhaps toxic. Intravenous ribavirin is very useful for life-threatening illness, such as Lassa fever, but toxicity limits the use of this form in less severe diseases. Liposome-encapsulated ribavirin has been used successfully in animal models for treatment of Rift Valley fever and influenza (163, 236), and it may represent a way to achieve improved drug delivery to target organs while minimizing toxicity. Analogs of ribavirin have been synthesized and tested for antiviral activity. One compound, 5-ethynyl-1-beta-ribofuranosylimidazole-4-carboxamide (EICAR), was originally studied as an antileukemic agent but has now been shown to be 10 to 30 times as active as ribavirin against RSV and influenza viruses (98). Whether it will be clinically useful remains to be seen. CLIN. MICROBIOL. REV. Vidarabine Vidarabine (9-beta-D-arabinofuranosyladenine) was the first antiviral agent licensed for intravenous use in the United States, in It is an analog of the nucleoside adenosine (Fig. 1) and was originally synthesized and studied in the early 1960s as an antitumor agent. Subsequently, it was found to be active in vitro against HSV, varicella-zoster virus (VZV), cytomegalovirus (CMV), vaccinia virus, and some RNA tumor viruses. In animal models, it was active against HSV and vaccinia virus (409). The mechanism of action has not been completely delineated, but it involves phosphorylation of the nucleoside by cellular enzymes (44, 355) and incorporation into the growing chains of both cellular and viral DNAs, with resultant slowing of DNA synthesis (343). Vidarabine triphosphate is also a competitive inhibitor of DNA polymerase, both cellular and viral. It is more potent, however, against viral enzymes, and this potency may account for its selective inhibition of viral DNA synthesis (117, 417). Vidarabine inhibits other steps in nucleic acid synthesis, such as RNA polyadenylation (393) and terminal deoxynucleotidyl transferase (108), but whether or not these contribute to its antiviral activity is unknown. It also inhibits S-adenosylhomocysteine hydrolase (398), an enzyme necessary for methylation of trna and mrna and for other cellular transmethylation reactions. This inhibition results in cytotoxicity for mononuclear cells and in the possible accumulation of biogenic amines (117), both of which may contribute to the toxicity of this compound. Vidarabine became available as an eye ointment in 1977 and remains useful for the treatment of herpes keratitis (Table 1). Although it is also useful for the treatment of HSV and VZV in immunocompromised patients (502, 504, 507, 510) and for herpes encephalitis (508, 509) and neonatal herpes (506, 511), it has been almost entirely replaced by acyclovir for these diseases. Vidarabine is difficult to use. It is poorly soluble in water and rapidly deaminated in vivo to arahypoxanthine, a much less active compound. It must therefore be administered continuously by the intravenous route in large volumes of fluid. Toxicities, including nausea and vomiting, weakness, weight loss, megaloblastosis of bone marrow erythroid precursors, tremors, and myoclonus have been seen in a high proportion of treated patients (287, 394). Acyclovir is easier to administer, less toxic, and more active against herpesviruses. Although vidarabine does not require a virus-coded thymidine kinase for activation, and thymidine kinase-deficient HSV isolates are susceptible to the drug in vitro, vidarabine has not proven useful for treatment of acyclovir-resistant HSV in HIV-infected patients, and foscarnet is preferred (402). Acyclovir Acyclovir [9-(2-hydroxyethoxymethyl)guanine] has the best therapeutic index of available antiviral agents, and it is widely used for the treatment of herpesvirus infections. It was "discovered" in the mid-1970s by Elion and colleagues while they were screening for antiviral activity of nucleoside analogs (410). The compound is an analog of guanosine (Fig. 5), but instead of a complete ribose ring attached to the purine base, it has an acyclic side chain. Acyclovir diffuses freely into cells but is activated and accumulates only in cells infected with herpesviruses because the first step in activation, phosphorylation at the 5' position of the side chain, is catalyzed only by a virus-specified thymidine kinase and not to any appreciable extent by cellular kinases (Fig. 4) (122, 159). Further phosphorylations to the diphosphate and the active triphosphate forms are carried out by cellular enzymes (122, 312). During DNA replication, acyclovir triphosphate competes with the natural substrate, dgtp, for the viral DNA polymerase. It has a higher affinity for the enzyme, however, and is preferentially incorporated into the growing chain of viral DNA (122, 453). Because the 3' hydroxyl is missing from the attached acyclovir molecule, the next nucleotide cannot be attached, and DNA replication is terminated (103, 156, 294). Furthermore, when acyclovir is bound to the DNA template in the presence of the next

10 VOL. 5, 1992 nucleotide, a tight irreversible complex is formed and the polymerase is completely inactivated, further slowing viral DNA synthesis (103, 158, 369). Taken together, these properties make acyclovir highly selective in inhibiting viral replication while having little effect on host cell function. There is, for instance, a 3,000-fold difference between the concentration of acyclovir needed to inhibit HSV type 1 (HSV-1) and that needed to inhibit the cells in which it is grown (122). The herpesviruses vary in their susceptibilities to acyclovir. HSV-1 and HSV-2 are exquisitely susceptible, requiring mean in vitro concentrations of 0.04 and 0.4,ug/ml, respectively, for 50% inhibition of viral replication (86, 329). The thymidine kinase of VZV does not phosphorylate acyclovir as effectively, and the virus is 8 to 10 times less susceptible to the drug than HSV (35, 86, 329). Although its DNA polymerase is very susceptible to acyclovir (452), CMV does not encode a thymidine kinase (133), and high drug levels (5 to 25,ug/ml) are required for inhibition (86, 353). It is not clear that Epstein-Barr virus (EBV) encodes a thymidine kinase capable of activating acyclovir. Its DNA polymerase is, however, very susceptible to the drug (91), and the 50% inhibitory concentrations are low (73, 278). Acyclovir has no effect on the latent phase of any of the herpesviruses. Acyclovir is available in three formulations: an ointment, pills or capsules, and an intravenous suspension. Systemic absorption and other side effects of the ointment are minimal, but it is not very effective (see below) and has largely been replaced by oral formulations. Levels achieved in serum after intravenous administration are well above the inhibitory concentrations of HSV, VZV, EBV, and some strains of CMV. With oral dosing, however, only 15 to 20% of the medication is absorbed, and levels in serum are not consistently above the inhibitory concentrations for any of the herpesviruses except HSV (100). Acyclovir is remarkably free of serious toxicity. When given intravenously in high doses, it can cause a transient rise in serum creatinine that is apparently due to obstructed renal tubules (22, 407). A reversible neurologic syndrome consisting of confusion, lethargy, and occasionally hallucinations and coma has also been described. This syndrome is usually seen in very ill, immunocompromised patients receiving high-dose intravenous therapy (137, 253, 491). With oral therapy, nausea, diarrhea, and headache have occasionally been reported. Acyclovir is not carcinogenic and is mitogenic or teratogenic only at very high levels in animals (479). It does cross the placenta, however, and therefore must be used with caution in pregnant patients (45, 172). Acyclovir is very effective for the treatment of HSV in otherwise healthy patients (Table 1). When given orally or intravenously, it shortens the course of initial genital herpes by one-third to one-half (46, 79, 314, 323). Recurrent genital herpes does not respond as dramatically, even when patients initiate therapy themselves at the first sign of disease (323, 374). This may be because recurrences normally last only a few days, and it is difficult to demonstrate a shortening of the course with drug therapy. Acyclovir is, however, very useful in preventing recurrent disease. When given daily in low doses to patients with frequent eruptions, it suppresses them almost completely and causes no side effects even with prolonged use (112, 230, 306, 313). It does not prevent asymptomatic viral shedding (457), and transmission has occurred when patients were free of genital lesions (392). Topical acyclovir, a 5% ointment, is useful, though not as effective as oral therapy, for treatment of initial genital herpes (79, 80, 472). In recurrent disease it offers no benefit ANTIVIRAL THERAPY 155 (282, 373). Topical therapy is occasionally used in pregnant patients who have mild genital herpes and should not receive systemic acyclovir. Oral treatment of herpes labialis (the common cold sore) shortens the course of illness in those patients who are prone to more severe eruptions and are able to initiate therapy themselves at the first sign of an eruption (451). When given prophylactically to patients exposed to the sun or to artificial UV light, oral acyclovir decreased the frequency of herpetic lesions appearing within the next 2 or 7 days, respectively (448, 449) but did not affect the lesions which appeared immediately (within 48 h) after the exposure (448). Topical therapy is of little benefit in otherwise healthy patients with cold sores (448, 520). In the treatment of herpes encephalitis, acyclovir has proven both more effective and less toxic than vidarabine (434, 503). It reduces morbidity to 14% (vidarabine reduces it to 38%), and it is now the treatment of choice for that disease. A recent trial of acyclovir versus vidarabine for the treatment of neonatal herpes showed neither drug to be superior (501). Given the sample size, however, as much as a 25% difference in morbidity and mortality could have been present and gone undetected. Acyclovir has also been very useful for management of mucocutaneous HSV in immunocompromised patients. Administered orally or intravenously, it decreases pain and viral shedding and accelerates healing (310, 425). Though not as effective, topical therapy can also be useful in this regard (505). When given suppressively to HSV-seropositive patients, acyclovir greatly reduces the chances of a recurrence during subsequent chemotherapy (405) or transplantation (406, 418, 423, 492). VZV infection responds better to intravenous than to oral acyclovir, probably because levels in serum after oral administration are often below the 50% inhibitory dose for the virus. In immunocompromised patients with chickenpox or zoster, intravenous acyclovir has been very effective in decreasing dissemination and other complications (15, 326, 361, 424). It can also be given orally in the post-bone marrow transplantation period and appears to decrease the frequency of zoster during that time (346, 421). In otherwise healthy patients with zoster, intravenous acyclovir decreases acute pain and accelerates lesion healing (23, 350), but oral therapy has had only a modest effect against this disease (213, 296). Neither form of therapy reduces postherpetic neuralgia. Of importance, oral acyclovir decreases the frequency of eye complications in patients with ophthalmic zoster (69, 70). In one study of otherwise healthy children with chickenpox, oral acyclovir reduced the duration of disease by 1 day but did not influence the complication rate, days off from school, or transmission rate within the family (18). In otherwise healthy adults with chickenpox pneumonia, intravenous acyclovir appears to hasten recovery (175). Despite the reduced susceptibility of CMV to acyclovir, the drug may yet prove useful in the management of CMV disease in immunocompromised patients. Two studies, one of bone marrow recipients (309) and one of renal allograft recipients (17), have suggested that high-dose intravenous acyclovir can reduce the frequency of CMV infection and disease when given suppressively in the peritransplantation period. The reasons for this are not clear, but may relate to the fact that only low numbers of viruses are present early after reactivation, and the small amount of active acyclovir present may be sufficient to limit viral replication enough to prevent clinical disease. Acyclovir therapy of established CMV infections in immunocompromised patients has not been successful (16, 489).

11 156 BEAN Treatment of diseases due to EBV has also been disappointing, despite the apparent susceptibility of the virus to acyclovir. The course and symptoms of mononucleosis are not affected by treatment, although viral shedding from the oropharynx is decreased (3, 481). On the other hand, acyclovir does appear to be effective for treating hairy leukoplakia, an EBV-associated condition characterized by painful tongue lesions in patients infected with HIV (377). Improvement is transient, however, and the lesions recur when the drug is stopped. Resistance of HSV to acyclovir has been extensively studied. It results from changes in the virus-specified thymidine kinase and/or the DNA polymerase and occurs by one of three mechanisms: a deletion or mutation conferring inability or reduced ability to express thymidine kinase, resulting in lack of acyclovir phosphorylation (a TK- or thymidine kinase-deficient mutant) (72, 413); expression of a DNA polymerase with reduced affinity for acyclovir triphosphate (72, 265, 413); or expression of a thymidine kinase with altered substrate binding properties for the drug (90, 154). Both of the last two mechanisms result in lack of recognition of acyclovir as a substrate for the enzyme. Among patient isolates, thymidine kinase-deficient mutants are most common, but viruses expressing the other mechanisms have also been found (49, 76, 123, 399, 426, 490). In nature, HSV populations consist of a mixture of TKand normal (TK+) virus variants (337). They are predominantly TK+, however, because these viruses have a selective advantage in being more capable of establishing latent, reactivatable infections in host sensory ganglia. They are also more neurovirulent (121, 142, 143, 471). Under pressure of antiviral drug therapy, a population can become predominantly TK-, as the TK+ variants are unable to replicate. When the drug is withdrawn, the reverse occurs. In practice, resistance develops when patients are treated for prolonged periods with acyclovir in doses that do not suppress viral replication completely but allow "breakthrough" replication to occur. To date, this has been seen almost entirely in immunocompromised patients, (19, 24, 131, 220, 335). Treatment consists of withdrawing acyclovir and, when necessary, administering a drug with a different mode of action, such as foscarnet (see below). The importance of resistance for the future of acyclovir therapy is not yet clear. Thymidine kinase-deficient mutants are generally not as hardy as their TK+ counterparts. This not true, however, of most thymidine kinase-altered and DNA polymerase-altered viruses (90, 141, 142, 265). Whether resistant virus populations will be able to establish themselves and compete with wild-type viruses remains to be seen. Because of acyclovir's poor oral availability and lack of effect against some herpesviruses, other drugs are being sought for treatment of herpesvirus infections. Famciclovir is the diacetyl ester of penciclovir, an acyclic nucleoside analog with a long intracellular half-life (484) and good activity against HSV and VZV but poor oral absorption (485). Famciclovir is well absorbed and rapidly converted to penciclovir (485) and is very promising for treatment of herpesvirus infections. 1-Beta-D-arabinofurasonyl-E-5-(2- bromovinyl)uracil, or BV-ara-U, is a thymidine analog which is 1,000 times more active against VZV than acyclovir (283). Cellular toxicity is seen at concentrations 1 millionfold higher than those needed to inhibit viral replication, making this drug highly selective and very promising for treatment of VZV infections. It is currently in patient trials. Ganciclovir CLIN. MICROBIOL. REV. Ganciclovir, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, is a nucleoside analog similar to acyclovir except that it contains a carbon with attached hydroxyl group at the 3' position of the ribose ring (Fig. 5). It is active against all the human herpesviruses (139) and is up to 100 times more active than acyclovir against human CMV (436, 474). It is also more toxic than acyclovir and for this reason has been used only for treatment of serious CMV disease in immunocompromised patients. It is not clear why ganciclovir is so much more effective than acyclovir against CMV. In HSV-infected cells, ganciclovir, like acyclovir, is activated by a virus-specified thymidine kinase (37, 139, 436). In CMV-infected cells, in which no viral thymidine kinase is induced, ganciclovir is also phosphorylated and accumulates to much higher levels than acyclovir (37, 151). It is removed from these cells very slowly, persisting in stable form for several days (37). The molecular mechanisms of activation, accumulation, and degradation are not known but probably account to some extent for the difference in activity, as ganciclovir is actually a less potent inhibitor of viral DNA polymerase than acyclovir is (151, 286). Ganciclovir triphosphate is incorporated into the growing viral DNA chain and markedly slows DNA synthesis (285). Because the 3' hydroxyl is present on the molecule, the next incoming nucleotide can be bound to the nascent DNA chain, and ganciclovir is thus not a chain terminator (65, 285). Despite the fact that ganciclovir, like acyclovir, is a more potent inhibitor of viral than cellular polymerases (151, 436), it is more toxic than acyclovir. In vitro inhibition of the myeloid elements of bone marrow occurs at levels in serum seen with intravenous administration of ganciclovir (443, 444). The drug is also mitogenic in mammalian cells and carcinogenic and embryotoxic in animals (467). In humans, ganciclovir causes a reversible bone marrow toxicity in approximately 25% of patients, a factor that frequently limits its use (62, 208, 422). There have been no placebo-controlled trials to evaluate the efficacy of ganciclovir. Historically controlled trials have shown the drug to be generally useful in the management of CMV disease in immunocompromised patients, although efficacy varies among different patient groups. AIDS patients with CMV retinitis respond well to intravenous therapy, with 80% or more achieving stabilization or improvement in their vision (47, 74, 138, 197, 208, 217, 271). Almost all relapse, however, unless maintenance therapy is instituted, and even then many patients experience breakthrough disease or cannot tolerate the medication. These problems and the difficulties of long-term intravenous therapy have prompted the investigation of other modes of treatment. Intravitreal administration of ganciclovir avoids myelosuppression and has stabilized vision in some patients (195, 198). Oral therapy is also being investigated. While the oral bioavailability of ganciclovir is very poor (6 to 8% [96]), levels in serum may be high enough to inhibit some strains of CMV (222). AIDS patients with CMV pneumonia may have a response rate as high as 60 to 80% when given intravenous ganciclovir (47, 271), although strict criteria for diagnosis were not applied in published studies. On the other hand, only 10 to 40% of bone marrow transplant patients respond, and their mortality remains high despite suppression of viral replication and shedding by the drug (47, 83, 422). One hypothesis for this difference is that, in bone marrow recipients, CMV pneumonia is an immune-mediated disease resulting from T-cell cytotoxic responses to viral antigens

12 VOL. 5, 1992 expressed on the surface of infected lung cells, whereas in AIDS patients, whose cellular cytotoxic responses are poor, the disease is due to direct damage to the lungs (174). To neutralize viral antigens and prevent recognition by immune effector cells, high-titered CMV immunoglobulin has been administered with ganciclovir in bone marrow transplant patients. Such combined antiviral and immunomodulator therapy has increased the survival rate to 50 to 70% (125, 371). In addition, it may permit a decrease in ganciclovir dosage, with an attendant decrease in bone marrow toxicity. In one recent trial, suppressive ganciclovir therapy was very successful in preventing clinical disease in bone marrow recipients who had CMV in their respiratory secretions but had not yet developed pneumonia (411). An additional placebo-controlled trial has confirmed and extended these findings; early ganciclovir therapy in bone marrow recipients excreting CMV but not yet ill reduced the frequency of CMV disease by 93% (from 15 of 35 to 1 of 37 patients) and significantly improved patient survival at 6 months posttransplantation (163). In uncontrolled trials of AIDS patients with CMV gastrointestinal disease, ganciclovir appeared to reduce diarrhea and the pain and dysphagia of esophagitis (62, 74, 109). A recent, well-controlled study of bone marrow recipients, however, indicated no benefit in this patient group (372). In patients who have undergone solid-organ transplantation and developed CMV disease, there is some evidence that ganciclovir therapy improves the recovery rate (119, 180, 193) and may improve graft survival (119). Ganciclovir resistance has been reported in three immunocompromised patients being treated for CMV disease (127) and was due to lack of phosphorylation of the drug by the CMV-infected cells (452). This finding is consistent with phosphorylation of ganciclovir by a virus-induced cellular enzyme or by an enzyme that is virus encoded. In addition, a CMV strain produced in the laboratory has been found to possess two mutations which confer ganciclovir-resistance: one in the DNA polymerase gene giving rise to an altered DNA polymerase and one at an unknown site giving rise to an inability to phosphorylate the drug (463). An HSV strain resistant to ganciclovir because of an altered DNA polymerase has been identified (84). A recent prospective survey estimated that 8% of patients receiving ganciclovir for more than 3 months developed resistant CMV (118), suggesting that prolonged therapy may facilitate the emergence of resistant strains. Although the importance of ganciclovir resistance is presently unknown, the three reported patients all had progressive CMV disease, and this suggests that resistance can be associated with therapeutic failure. Foscarnet Foscarnet (trisodium phosphonoformate) is a pyrophosphate analog unlike any other antiviral agent currently in use (Fig. 6). It was developed in the late 1970s as a less toxic, more effective alternative to phosphonoacetic acid, another pyrophosphate analog with antiviral properties (196). The Food and Drug Administration recently granted an indication for treatment of CMV retinitis in AIDS patients. In clinical trials, foscarnet has also proven useful for treatment of acyclovir-resistant herpesvirus infections. Unlike nucleoside analogs, foscarnet does not need to be activated by cellular or viral kinases. Instead, the molecule binds directly to the pyrophosphate-binding sites of RNA and DNA polymerases, inhibiting them in a noncompetitive fashion with respect to nucleotide substrates (328). While the mechanism of inhibition is not well understood, one hypoth- ANTIVIRAL THERAPY 157 esis is that polymerase-bound foscamet forms an unstable intermediate with nucleoside monophosphates, resulting in degradation of the nucleic acids (275, 328). Both cellular and viral polymerases are affected by foscarnet, but some viral enzymes are inhibited by concentrations 1/10 to 1/2 those needed for cellular enzyme inhibition. These include the polymerases of some strains of influenza A virus, the human herpesviruses, hepatitis B virus, and the reverse transcriptase of HIV (269, 328, 404, 488, 493). Foscarnet is difficult to use. Oral bioavailability is poor (433), although efforts are being made to increase absorption and prolong the half-life by conjugation to fatty alcohols (322). Currently, the drug is available only in intravenous formulations, and it must be given frequently by means of an infusion pump, as the half-life in plasma is very short (9, 433). At physiologic ph, foscarnet is highly ionized and has limited cell penetration (328, 433). Levels in spinal fluid are usually about 40% of those in plasma but may vary by a wide margin (7). Up to 30% of the drug may be deposited in bone, with a subsequent half-life of several months (432). The importance of such deposition, especially for bone development in children, is not known. The major adverse effects of foscarnet are renal impairment (present in most patients and dose limiting in 10 to 23% of them), imbalances of electrolytes such as calcium, phosphate, potassium, and magnesium (101, 134, 221, 224, 382, 495), and seizures (7). It appears that foscamet chelates divalent metal ions such as calcium, resulting in a doserelated but reversible drop in serum calcium that may be severe or even fatal (223). Penile ulcers have also been seen with systemic therapy and may result from the localized irritant effect of high levels of ionized foscamet in the urine (136, 168, 276, 319, 482). In AIDS patients with CMV retinitis, foscarnet and ganciclovir appear to be equally effective in preventing progression of disease, as assessed in clinical trials with historical controls. Vision stabilized or improved in 80 to 100% of patients, but maintenance therapy was required, and even so, 70% or more relapsed (134, 224, 274, 336, 495). The potential advantage of foscarnet in this setting is that it has less bone marrow toxicity than ganciclovir, and it may be possible to use it in conjunction with zidovudine. The additive bone marrow toxicities of ganciclovir and zidovudine preclude their simultaneous administration. A controlled trial directly comparing ganciclovir and foscamet for CMV retinitis was recently completed. Preliminary analysis confirmed that they are equally effective in halting progression and suggested that foscarnet may prolong survival by a few months in some patients. Other uncontrolled trials have indicated that some bone marrow and renal transplant patients with severe CMV pneumonia or with fever and leukopenia may improve with foscarnet therapy. Many died, however, and in the absence of a control group, it is difficult to assess the value of therapy in these patients (4, 238, 382). In AIDS patients who develop acyclovir-resistant HSV or VZV infections after prolonged courses of therapy, foscarnet has proven very useful (Table 1). About 80% of patients respond with ulcer healing and symptom resolution (63, 130, 400, 401). In this setting, foscarnet is also superior to vidarabine; the rate of lesion healing is higher and there is less toxicity (402). Maintenance therapy with foscarnet is needed, as with other antiherpesvirus agents, and is successful in about 70% of patients. Apparent resistance to foscarnet has developed, however, after several courses of therapy in HIV-infected patients (34) and in one bone marrow allograft recipient treated for CMV pneumonia (241). The mechanisms of resistance have not yet been determined for

13 158 BEAN these viruses but probably involve altered DNA polymerases, as seen when viruses are exposed to foscarnet in vitro (102). When patients are infected with thymidine kinasedeficient, acyclovir-resistant HSV or VZV mutants, as is most commonly the case, foscarnet is useful for therapy, as it does not require activation by thymidine kinases. When acyclovir resistance is due to changes in the viral DNA polymerase, however, it may be accompanied by resistance to foscarnet, and in these cases the drug may not be useful. Foscarnet is effective against HIV at levels achieved in patients' serum (404, 488). In preliminary studies, it decreased viral (p24) antigenemia and raised helper lymphocyte (CD4) counts (30, 221, 328), but the effect was transient, and patients relapsed when the drug was stopped. The need for intravenous administration has made it difficult to study foscarnet for long-term maintenance therapy in HIV infection and may preclude its use in favor of more easily administered drugs. Zidovudine Zidovudine (3'-azido-3'-deoxythymidine; formerly known as azidothymidine, or AZT), is a synthetic dideoxynucleoside, one of a family of nucleoside analogs in which the 2' and 3' hydroxyls of the ribose ring have been replaced by hydrogens or other moieties. It was first studied as an antitumor compound and then as a drug for feline leukemia virus, and in 1985, it was found to inhibit HIV (316). The drug became commercially available in 1987 and is now used for management of HIV infections. Zidovudine is an analog of deoxythymidine in which the 3' hydroxyl has been replaced by an azido (N3) group (Fig. 5). The drug is activated to its mono- di-, and triphosphate forms by cellular enzymes in both HIV-infected and uninfected cells (155). The triphosphate is a competitive inhibitor of HIV reverse transcriptase, the RNA-dependent DNA polymerase needed for transcription of viral genomic RNA into double-stranded DNA that can be incorporated into host cell DNA (Fig. 2). In fact, zidovudine binds to reverse transcriptase much better than does its natural competitor dttp and is thus the preferred substrate (66, 155, 454). It also inhibits reverse transcriptase at about 1/100 the concentration needed for inhibition of cellular DNA polymerase alpha (66, 155, 316). Zidovudine triphosphate is incorporated into the growing chain of DNA, and because the azido group occupies the 3' hydroxyl site, additional nucleotides cannot attach and the chain is terminated (155, 454). Zidovudine is active in vitro against mammalian and avian retroviruses at low concentrations (153) and against EBV at concentrations 10 to 100 times those needed to inhibit HIV (277). It inhibits some bacteria of the Enterobacteriaceae family when they are actively dividing (124, 234). Zidovudine is available in both oral and intravenous forms. It is absorbed rapidly and completely from the gastrointestinal tract and so is well suited to oral use (38). It also penetrates well into the cerebrospinal fluid and is useful for treating central nervous system manifestations of AIDS. The drug is metabolized primarily by hepatic glucuronidation, and thus its metabolism may be inhibited by other drugs that share this pathway, such as sulfa-containing compounds, nonsteroidal anti-inflammatory agents, and narcotic analgesics. Probenecid, another compound that inhibits glucuronidation, can also be used to prolong the half-life of zidovudine (247). One of the major drawbacks to the successful use of zidovudine has been its rapid elimination and short plasma half-life, necessitating frequent administration. CLIN. MICROBIOL. REV. However, its intracellular half-life is considerably longer (8, 155), and more convenient dosing schedules have recently been proven effective. Zidovudine also crosses the placenta (497) and has recently been associated with fetal resorption when administered to mice prior to and early in pregnancy (476). The importance of this for patients remains to be seen. Despite the fact that mammalian cells do not have reverse transcriptases to be inhibited by zidovudine, it is nonetheless a moderately toxic drug. This may be explained by recent observations. Zidovudine monophosphate competitively inhibits thymidylate kinase, a cellular enzyme that catalyzes the conversion of nucleoside monophosphates to diphosphates (155). As a consequence, when cells are exposed to the drug, intracellular pools of nucleoside di- and triphosphates decrease, and cellular DNA synthesis is slowed accordingly (8). In addition, when zidovudine is incorporated into a growing chain of cellular DNA, it causes chain termination (8). In patients, zidovudine can cause anemia severe enough to require transfusion or discontinuation of therapy. Neutropenia is less frequent, but other reactions such as nausea and headache are common, especially at higher doses and in patients with more advanced AIDS (166, 379). A myopathy characterized clinically by muscle weakness and anatomically by abnormal mitochondria in muscle fibers has also been described (88). Patients on zidovudine appear to have a high risk of non-hodgkin lymphoma (318, 354), although whether this susceptibility is due to the drug or simply to prolonged survival in the face of profound immunosuppression is not yet clear. The bone marrowsuppressive effects of zidovudine are at least additive (206) and may be synergistic with those of ganciclovir (359). Few patients can tolerate concurrent use of these drugs. Zidovudine is useful for treatment of all stages of HIV infection (Table 1). In patients with AIDS or AIDS-related complex, high-dose zidovudine (1,200 to 1,500 mg daily) prolongs survival, reduces the frequency of opportunistic infections, and improves functional capacity, weight gain, and immunologic function (116, 146). Viremia or HIV (p24) antigenemia also decreases during therapy (218, 446). Although the survival benefit remains after 1 to 2 years of therapy (81, 145), the other benefits tend to disappear, with a return of antigenemia and opportunistic infections and a deterioration of immunologic function. This is partly because drug toxicity requires dose reductions or discontinuation of therapy in many patients. A recent study has shown that a lower daily dose, 600 mg, is as beneficial as the higher dose and much less toxic, at least in nonadvanced AIDS (144). It is now the recommended dosage. As little as 300 mg daily may be beneficial in patients with AIDS-related complex (75), although it is not recommended unless patients are unable to tolerate higher doses. High-dose zidovudine is still used for treatment of HIV neurologic disease (412, 523, 527). The bone marrow-suppressive effects of zidovudine spare the megakaryocytes and platelets, and the drug can be used to treat HIV-induced thrombocytopenia (332, 466). Although zidovudine reaches high concentrations in semen (199), it does not appear to decrease shedding from this site (252), and this lack may have important implications for sexual transmission of disease. In asymptomatic or mildly symptomatic HIV patients with CD4 (helper) T-lymphocyte counts of less than 500/mm3, both high- and low-dose zidovudine delayed disease progression and transiently improved immunologic function and antigenemia (147, 487). Adverse effects were also less common in these patients than in those with more advanced disease. These patients have not been followed long enough

14 VOL. 5, 1992 to determine whether zidovudine prolongs survival in early HIV infection. A recent cost effectiveness analysis, however, indicates that zidovudine therapy in early HIV infection compares favorably with other well-accepted medical interventions. The cost per year of life saved, estimated at $6,553 (1989 dollars), is very similar to that of counseling patients to quit smoking (415). Resistance of HIV to zidovudine has been seen in patients receiving therapy for prolonged periods (258, 267, 391). Resistance appears to develop more rapidly and to higher levels in patients with more advanced disease and greater viral burdens (380), a situation somewhat analogous to the development of isoniazid resistance in patients with cavitary tuberculosis and high burdens of mycobacteria. The clinical importance of resistance is not known; thus far, there has been no correlation with individual patient responses to therapy. At the molecular level, resistance is due to specific changes in three or four amino acid residues of the reverse transcriptase molecule (267). In patients, high-level resistance is associated with accumulation of multiple mutations, and it appears to develop in a stepwise fashion over time. The only cross-resistance observed has been to other 3'- azido-containing nucleosides, suggesting decreased recognition of this moiety by the altered reverse transcriptase (264). Despite rapid and remarkable advances in the development and use of zidovudine, many questions remain unanswered. The minimum effective dose is not yet known, nor are the effects of resistance and cumulative toxicity. Whether the drug prolongs survival when given early in disease remains to be seen, and whether it will prevent HIV infection if given prophylactically after exposure is unknown, though anecdotal experience suggests that this is not the case (263). Didanosine Like zidovudine, didanosine (2',3'-dideoxyinosine [ddi]; Fig. 5) is a dideoxynucleoside. It became available for management of HIV infections in late 1991, before definitive patient trials were completed. It is the first compound to follow a "fast track" to Food and Drug Administration approval and be made available prior to completion of safety and efficacy studies in an attempt to make promising new drugs quickly available to desperately ill patients. Didanosine is an analog of deoxyadenosine. It is converted by cellular enzymes to ddl monophosphate, then to dideoxyadenosine (dda) monophosphate, to dda diphosphate, and finally to dda triphosphate (ddatp), the active form (1, 78, 227). Like the triphosphates of other dideoxynucleosides, ddatp competitively inhibits HIV reverse transcriptase and acts as a chain terminator for proviral DNA (524). Human DNA polymerase alpha is relatively resistant to ddatp, but polymerases beta and gamma are more sensitive to it (496), perhaps accounting for some of the toxicity seen in patients. In vitro, ddl is less potent against HIV than dideoxycytidine (ddc) (315), but intracellularly ddatp is as active as zidovudine (344), and it has an intracellular half-life of 8 to 12 h (compared with about 3 h for zidovudine [1, 227]). Didanosine is also active against zidovudine-resistant HIV isolates (264). Didanosine is available in oral form but is somewhat difficult for patients to take correctly. At acid ph, hydrolysis occurs at the glycosidic bond between the sugar and the base moieties of the nucleoside, inactivating the compound. The drug is thus supplied as both a buffered powder to be made into solution and as buffered chewable tablets which must be ANTIVIRAL THERAPY 159 taken at least two at a time to provide adequate buffering capacity in the stomach. The major side effects have been a painful peripheral neuropathy in 16 to 34% of patients treated to date; pancreatitis, seen in 8 to 9% of patients and occasionally fatal (77, 257, 525); and diarrhea in 18 to 34% of patients, possibly related to buffering agents used in the powder (182). Didanosine is avoided in patients at high risk for pancreatitis, such as those with a history of heavy alcohol consumption or previous pancreatitis, and it increases the risk of pancreatitis when given simultaneously with intravenous pentamidine or ganciclovir. Development of pancreatitis or peripheral neuropathy requires discontinuing the drug. In phase I uncontrolled dose escalation studies of patients previously treated with zidovudine, didanosine therapy resulted in significant rises in CD4 cell counts and decreases in p24 antigenemia (50, 77, 257, 525). Phase II controlled trials are under way to determine whether ddi has any effect on patient survival, time to progression of disease, or frequency of opportunistic infections. In view of the desperate need for additional anti-hiv therapies and because rising CD4 cell counts are a surrogate marker for disease improvement, didanosine was approved for use in both adult and pediatric HIV-infected patients who are unable to tolerate zidovudine (usually because of bone marrow toxicity) or who experience clinical or immunologic deterioration while receiving it. Zidovudine is still regarded as first-line HIV therapy. The place of ddl, both by itself and in combination with other agents for HIV therapy, will need to be determined in future trials. Investigational Antiretroviral Agents Many compounds are under investigation as candidate drugs for the management of HIV infection. It is not possible to discuss them all, and this section will concentrate on those which are especially promising or represent a new approach to therapy and for which there is adequate description in the scientific literature. The in vitro antiretroviral activity of dideoxynucleosides other than zidovudine was first described in 1986 (315). ddc (Fig. 5) protected cells against the cytopathic effect of HIV at 1/20 to 1/50 the concentrations needed for dda, ddl, and dideoxyguanosine, and it was the first of these to begin clinical trials. ddc is well absorbed orally, has a short half-life, and penetrates the central nervous system, although not as well as zidovudine (526). Its phosphorylation pathway is different from that of zidovudine, and, unlike zidovudine, it is excreted by the kidneys (526). Its toxicity profile is also substantially different from that of zidovudine. In early trials, patients were given high doses and many of them developed a painful peripheral neuropathy, necessitating discontinuance of the drug. Decreases in p24 antigenemia and rises in CD4 counts were also seen, however, and no bone marrow toxicity was observed (304, 526). Currently, trials are in progress to determine the efficacy of lower doses and of dosage schedules that employ zidovudine and ddc together, given either simultaneously or in an alternating fashion (43, 351, 435). Approval of ddc by the Food and Drug Administration is expected in the near future. Two other dideoxynucleosides are currently in clinical trials. Dideoxythymidinine, also known as D4T, has good activity against HIV but is phosphorylated differently from zidovudine and has less bone marrow toxicity (205, 284). Azidouridine is slightly less active against HIV than zidovudine, but is also less toxic to bone marrow (67, 531), and it

15 160 BEAN H CH3 TIBO R HO 4H 0 OH oaoh K No CH2OH I CH2CH2CH" CH3 N-BUTYL-DNJ o"t. PROTEASE INHIBITOR XVII FIG. 7. Investigational antiretroviral agents. Ph, phenyl; tbu, tertiary butyl; N-butyl-DNJ, N-butyl-deoxynojirimycin. inhibits the virus very well in peripheral blood mononuclear cells. It is currently in phase I trials. Several nonnucleoside compounds have shown excellent activity in vitro against HIV reverse transcriptase and are candidates for patient trials. Among the most potent and specific are the tetrahydroimidazobenzodiazepinone (TIBO) compounds, benzodiazepine derivatives that inhibit HIV type 1 (HIV-1) but not HIV-2 or other retroviruses. In vitro, the selectivity index of TIBO R82150 (Fig. 7) is greater than 31,000, compared with 6,200 for zidovudine and 191 for didanosine in the same cell system (339). These agents do not require phosphorylation and inhibit HIV-1 reverse transcriptase by an as yet unidentified mechanism. B1-RG-587 (nevirapine) is a dipyridodiazepinone with properties similar to those of the TIBO compounds (249, 305). It is beginning phase I trials in the near future. A group of six-substituted acylouridine derivatives, the HEPT [1-(2-hydroxyethoxymethyl)-6-phenylthiothymine] congeners, are also highly potent and specific for HIV-1, do not require phosphorylation, and appear to interfere with reverse transcriptase by a mechanism different from that of zidovudine (10, 12). One compound, HEPT-S, also has pharmacokinetic properties suitable for oral administration. Reverse transcriptase inhibitors can prevent new DNA from being made in newly infected cells but they cannot prevent the reactivation of HIV from previously infected cells, as reverse transcriptase is not involved in this process (Fig. 2). Identification of agents capable of interfering with HIV at later points in the replication cycle, and perhaps preventing reactivation, would represent a major advance in the management of HIV infection. One such group of agents is the HIV protease inhibitors. HIV reverse transcriptase and other gag and gag-pol gene products are synthesized as precursor polypeptides and must be cleaved and processed before they can be packaged into virions. HIV protease, an aspartic proteinase encoded by the virus, is responsible for this processing and is essential for the proper assembly and maturation of fully infectious HIV-1 virions. It can also be CLIN. MICROBIOL. REV. inhibited by protease inhibitors, small peptide molecules that mimic the natural substrate for the reaction (97) (Fig. 7). Several such compounds have been identified and are being developed for clinical use (128, 248, 383). They are highly potent and selective inhibitors of HIV in vitro, and at least one has been shown to have antiviral activity when added to cell cultures late (18 to 20 h) after HIV infection and in chronically infected cells (162). Another promising compound, which acts later in the HIV replication cycle, is N-butyldeoxynojirimycin (Fig. 7). It reduces infectious virus in vitro in acutely infected cells and eliminates HIV entirely from chronically infected cells (231). The compound works by inhibiting glycosylation of the envelope glycoprotein gp120, thereby reducing its ability to bind to CD4 receptors and initiate new viral infection. Although glycosylation of viral glycoproteins is nonspecific and can be carried out by cellular enzymes, N-butyl-deoxynojirimycin is highly specific for HIV-infected cells in vitro. Phase I trials are under way to determine its clinical utility. Another approach is interference with the viral mrna itself. This was first proposed for Rous sarcoma virus in 1978 (529) and has been vigorously pursued for the treatment of many diverse diseases in addition to viral infections. It consists of constructing small oligonucleotides that are complementary to specific genes or mrna sequences (antisense oligonucleotides). These bind to the nucleic acid, ultimately preventing gene expression and protein synthesis. A molecule antisense to the mrna of rev, a critical regulatory protein of HIV, has been synthesized, and it inhibits protein synthesis in vitro in chronically infected cells (289). While this approach is extremely exciting, several problems with oligonucleotides, including degradation by RNases, cellular permeability, specificity, and large-scale production, must be solved before they can be used as therapeutic agents. All of these problems are under active investigation (397). It is also possible to interfere with HIV infection at its first step, the binding of HIV to CD4 lymphocytes (Fig. 2). Recombinant, soluble CD4 (rscd4) is the synthetic gpl20 binding portion of the complete CD4 molecule (the transmembrane and cytoplasmic portions are missing). In vitro, rscd4 binds to gpl20 on the virion surface and prevents attachment of the virion to CD4 lymphocytes (438). In patients, no toxicities were seen when the compound was administered in phase I trials, but the half-life in serum was very short, and sustained changes in p24 antigenemia and CD4 cell counts were not seen (229, 414). Currently, efforts are being directed toward the construction of rscd4-immunoglobulin G (IgG) and rscd4-igm hybrid molecules that have longer half-lives (53), may cross the placenta (53), have greater activity (478), and may recruit antibody-dependent (51) or complement-mediated (478) immune functions to destroy infected cells. Other efforts have resulted in the coupling of rscd4 to toxins such as Pseudomonas exotoxin A (64) and the plant toxin ricin (473). When bound to the HIV-infected lymphocyte and internalized, these compounds result in cell death. The use of Pseudomonas exotoxin has been especially effective in vitro and "cures" cell cultures of HIV infection when used in conjunction with reverse transcriptase inhibitors (6). IMMUNOGLOBULINS Immunoglobulins are available in three different formulations: an intramuscular preparation termed immune globulin or IG; an intravenous form termed IVIG; and several different intravenous preparations with high titers of antibodies to

16 VOL. 5, 1992 individual viruses. All are made from pooled human plasma and contain predominantly IgG, though small amounts of other globulins are also present. High-titered (hyperimmune) globulins are derived from pooled units of plasma selected for high antibody titers to specific viruses. IG has been available for many years, but it cannot be administered intravenously because of the tendency for IgG to form aggregates and fix complement in vivo. Recent advances in the cold fractionation procedure used, however, have made it possible to prevent this aggregation, and newer preparations can be given intravenously. Large volumes of fluid cannot be administered intramuscularly, so the amount of immunoglobulin that can be given by this route has also been limited. It is possible to give much larger amounts by the intravenous route, although these preparations are extremely costly. Regardless of route of administration, immunoglobulins are quite safe and are associated with very few side effects. The mechanism of action is unknown, but in all likelihood it involves the antibodies present. As will be discussed below, immunoglobulins are more effective when used prophylactically than therapeutically. For viruses, this is probably because the administered antibodies are present during the extracellular phase of infection, when it is possible to neutralize or opsonize virions before they enter cells and are protected from humoral immunity. Currently, IG is used primarily for prevention of hepatitis A. It is administered to patients within a few days of exposure to contaminated food or to an infected person and prevents infection or chemical illness in 80 to 95% of recipients (176, 255). It is also given at regular intervals to travelers who will be spending prolonged periods in conditions of poor or uncertain sanitation (61, 518). Although IG has not been unequivocally shown to decrease infection with non-a, non-b hepatitis, it may be "reasonable" to administer it to persons who sustain percutaneous exposures to infected blood (61). IG has not been studied for prevention of hepatitis C. For prevention of measles, IG should be given to persons who have not had the disease or been vaccinated against it and who are household contacts of measles patients, to those who have been exposed while pregnant, and to those who are immunocompromised when exposed (59). IG does not replace vaccine for control of measles outbreaks. Hyperimmune globulins are used for postexposure prevention of hepatitis B, chickenpox, and rabies. Hepatitis B IG prevents disease in 75% of persons with needlestick exposures (420). It also prevents hepatitis B surface antigen carriage in 85 to 95% of babies born to e-antigen-positive mothers (26, 61). Along with hepatitis B vaccine, hepatitis B IG is given within a few hours of birth to babies of hepatitis B surface antigen-positive mothers. Varicella-zoster IG modifies the severity of chickenpox but does not prevent infection when given to exposed susceptible persons. In immunocompromised children of less than 15 years of age (many of whom have not already had chickenpox and thus are not immune), it is administered as soon as possible after exposure to chickenpox. It is also given to babies who have no maternal antibody, including newborns whose mothers develop chickenpox within 5 days before or 2 days after delivery, premature babies whose mothers are seronegative, and all premature babies of less than 28 weeks of gestation. Varicella-zoster IG is very expensive, the supply is limited, and its use in adults, including exposed seronegative pregnant women, is limited to those situations in which the physician seeks to reduce disease severity. It has not been ANTIVIRAL THERAPY 161 shown to decrease the frequency of congenital or neonatal varicella when administered to exposed pregnant women and, in fact, may do the opposite by making the disease less severe in the mother while allowing transmission to the fetus (56). Human rabies IG became available in 1975 and is made from the plasma of hyperimmunized human donors. It is used in conjunction with human rabies vaccine to decrease transmission and death after exposure to rabid animals (55). CMV IG was recently approved for management of CMV infections in renal transplant patients. When given prophylactically, it reduced the frequency of serious CMV disease in seronegative recipients of seropositive kidneys, although it did not decrease transmission of the virus in this setting (442). In bone marrow transplant patients the use of prophylactic CMV IG is controversial. In two studies it decreased infection rates in seronegative recipients (41, 308), in one it did not (42), and in one it decreased CMV disease severity but not infection rates (516). These discrepancies may be due to differences in antibody titers of the immunoglobulins or to the fact that small numbers of patients were studied. Intravenous immunoglobulins were originally developed for the treatment of primary immune deficiency states. They have, however, been studied and used for many other purposes, including treatment and prophylaxis of viral infections. Preparations are available from several different manufacturers, and while they are frequently considered equivalent, both manufacturing processes and donor populations differ considerably and may result in substantially different end products. Moreover, manufacturers are not permitted to publish information about antibody titers of their products, so it is difficult to know whether preparations have equal efficacy unless they are directly compared in patient trials. As discussed earlier, IVIG improves survival when used in conjunction with ganciclovir for treatment of CMV pneumonia in bone marrow recipients (125, 371). In a recent trial, IVIG also decreased the frequency of graft-versus-host disease and interstitial pneumonia when given alone to seropositive bone marrow recipients (462). Although CMV was not confirmed as the cause of pneumonia in this study, it is the most common cause of interstitial pneumonia following bone marrow transplantation and it is associated with graft-versus-host disease. This suggests that IVIG may be beneficial in this setting. Another important use of IVIG is in the treatment of chronic enteroviral meningoencephalitis in children with agammaglobulinemia. Given intravenously, immunoglobulin in one study did not reach very high levels in the cerebrospinal fluid, and children almost always relapsed, though they had responded initially (297). When immunoglobulin was given through an intraventricular catheter, however, 50% of the children responded, and a few appear to have been cured of their infection (120, 297). Further studies need to be done, but intraventricular immunoglobulin appears to be a major advance in the treatment of this devastating illness. There has been a great deal of interest in the use of IVIG for the management of HIV infection and its complications. IVIG itself does not contain antibodies to HIV, but it may provide functioning antibodies to other infectious agents in patients who are functionally hypogammaglobulinemic despite having elevated serum globulin levels (261). Children, unlike adults, do not have protective antibodies from previous illnesses, and they may benefit from exogenous immunoglobulins. One large multicenter trial of IVIG therapy recently demonstrated that symptomatic HIV-infected children with CD4 counts of greater than 200 per mm3 had

17 162 BEAN significantly fewer serious bacterial infections and fewer hospital days than those treated with placebo (320). A similar benefit was not seen among those with fewer CD4 cells, but this may have been because of the small number of children studied. A second trial is under way to further investigate these findings. No preparation of hyperimmune HIV globulin is available. A recent trial using a high-titered preparation made from the plasma of two donors with high anti-p24 antibody titers showed that AIDS patients given the plasma had immediate clearing of p24 antigenemia, fewer symptoms, transient rises in T lymphocytes, fewer opportunistic infections, and lower rates of HIV isolation from blood (219). Such results are intriguing but have not been confirmed. IMMUNOMODULATORS In the strict sense of the word, immunomodulators are substances that modify the response of immune competent cells through signaling mechanisms (519). Many discussions, however, including this one, include immune-based therapies with a direct effect on cells such as the use of specific antibodies, exogenous immune effector cells, and procedures such as bone marrow transplantation. Immunomodulators are administered in the hope of augmenting or restoring host immune responses to infectious agents or malignancies. A few older agents, for instance, levamisole, isoprinosine, and transfer factor, have been used for several years in patients with viral infections (455). Their effects have never been convincingly demonstrated, however, and they remain largely investigational. The AIDS epidemic has rekindled interest in immunomodulators, and with the use of recombinant DNA technology it has become possible to manufacture these compounds in large quantities and to study their effects in patients, animal models, and in vitro systems. Immunomodulation in HIV infection (and other viral infections) can be a two-edged sword, however. Potent immunomodulators such as cytokines have the potential for increasing viral replication and perhaps triggering the transition from latent to active infection by stimulating target cells and making them more capable of supporting viral infection. They may also induce new targets by stimulating cells otherwise nonpermissive for viral infection. Last but not least, they may worsen or cause opportunistic infections by stimulating the target cells for these organisms. If some of the features of HIV disease are autoimmune in nature, it is possible that immunomodulators will also worsen them (519). Tumor necrosis factor alpha, interleukin 6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been shown in vitro to induce expression of HIV from chronically infected cells. Interleukins 1, 2, 3, and 4 and gamma interferon do not, while alpha interferon and transforming growth factor beta actually down-regulate HIV expression (135). Only two immunomodulators, alpha interferon and GM-CSF, are commercially available, and this discussion will concentrate on them. Many other agents are in the early phases of investigation for treatment of HIV infection. Three of these, inosine pranobex (isoprinosine), ditiocarb sodium (diethyldithiocarbamate), and IMREG-1, have been administered to patients in controlled trials and have shown some possible short-term effects in decreasing progression of the disease (171, 200, 342). More and better studies are needed, however, to establish their utility in treatment of HIV infection. Other agents such as thymic hormones (21, 430), enkephalins (333), interleukin 2 (416), CLIN. MICROBIOL. REV. and activated natural killer cells (301) and bone marrow transplantation (262) are in earlier stages of investigation. One other intriguing approach is that proposed by Salk: the use of inactivated HIV vaccine or its components in HIVinfected asymptomatic individuals to stimulate cytotoxic responses to infected cells and eliminate them, preventing progression of disease and development of full-blown AIDS (403). An early, phase I trial of this approach has shown it to be safe and immunogenic (370). Patients were followed for less than 1 year, however, and further trials will be necessary to determine the effect of postinfection immunization on the course of HIV disease. Interferons are natural products, small proteins, or glycoproteins that are elaborated by eukaryotic cells in response to viral infection. In turn, they induce in other noninfected cells biochemical changes that render these cells resistant to subsequent viral infection. The "antiviral state" induced by interferons is not specific with respect to infecting virus but is relatively specific with respect to species of host cell or animal. Interferons were first described by Isaacs and Lindemann in 1957 (216) and have subsequently been found to have potent antiproliferative and immunomodulating effects in addition to their antiviral properties (349). Their primary function is, in fact, related to the regulation of cell growth, differentiation, and immune function (228). Interferons are currently being used and actively investigated for the treatment of malignancies as well as viral diseases. In nature there are three types, alpha, beta, and gamma, classified by cell of origin, primary function, and other chemical and physical properties. Alpha and beta interferons are secreted in response to viral infection and are involved in the response to it, while gamma interferon is produced by activated T lymphocytes and is involved in the response to antigens and mitogens. While gamma interferon is the primary immunomodulator, all interferons affect the immune system to some extent. The only products available commercially are alpha interferons, and this review will concentrate on them. Interferons are now known to be cytokines, hormonelike polypeptides that play an important regulatory role in the early nonspecific phases of the host response to microbial invasion. They are elaborated by almost all nucleated cells after exposure to a variety of stimuli, including viruses, viral antigens, and double-stranded RNA. They are then secreted from the infected cells and attach to receptors on other, uninfected cells. After internalization, they trigger the formation of enzymes that, upon exposure to double-stranded RNA, degrade viral mrna and inhibit protein synthesis, resulting in an aborted viral infection. The best studied of these enzymes are 2,5-oligo(A), a family of oligonucleotides that synthesize an activator of RNase L, an enzyme that cleaves RNA; and protein kinase, which phosphorylates and thereby inactivates eif-2, a protein initiation factor (228). These are not the only mechanisms by which virus infections are halted, however. The multiplication of HIV, for instance, is directly inhibited by interferons, apparently at a late stage of replication involving release of virus particles from the cell membrane (356). While these mechanisms are clear-cut in vitro, the in vivo actions of interferons are undoubtedly more complex because of the effects of interferons on immune functions, including stimulation of natural killer cells and of B-lymphocyte proliferation, inhibition of lymphocyte blastogenesis, and enhanced expression of major histocompatibility class I receptors on cell surfaces (240). Interferons are responsible for the fever, chills, arthralgias,

18 VOL. 5, 1992 and myalgias frequently seen with viral infections, and they may actually cause immune-mediated tissue damage. Most interferons in use today are made by using recombinant DNA technology. They are not absorbed from the gastrointestinal tract when given orally and thus are administered by intramuscular or subcutaneous injection (512). The serum half-life is short, but the antiviral state induced in cells can last several days (228). The most common early adverse effect is an influenzalike illness characterized by fever, headache, myalgias, and arthralgias. These symptoms can usually be controlled with medication, and tolerance soon develops, so that therapy can be continued. Later, however, fatigue, anorexia, and weight loss can develop, and these often limit the use of the drug. Other adverse effects have been described, though they are much less common: bone marrow toxicity, psychological abnormalities (376), and cardiac toxicities (445). Interferon neutralizing antibodies are produced in 2 to 3% of patients receiving systemic therapy (447). While the significance of these antibodies is not completely understood, their presence has not yet been associated with a diminished response in patients being treated for viral infections. Interferons were first used clinically for the management of herpesvirus infections in immunocompromised patients. Although they were useful in controlling the complications of chickenpox and zoster (5, 303, 515) and in preventing the reactivation of CMV after renal transplantation (203), they had no effect on prevention of CMV after bone marrow transplantation (307) and were not as effective as acyclovir for treatment of genital herpes (256, 340). In addition, the frequency of adverse effects was high, and they were soon replaced by acyclovir and ganciclovir. Because the common cold is caused by a variety of different viruses, a nonspecific agent such as interferon would seem an ideal mode of treatment and/or prophylaxis. Leukocyte interferon was first administered intranasally for this purpose in 1973 (302). Unfortunately, it produced a very high frequency of local side effects such as nasal stuffiness, dryness, and bleeding and actually prolonged cold symptoms rather than shortening them (190). When given prophylactically to exposed family members, intranasal recombinant interferon decreased the frequency of rhinovirus colds, but it has little effect on colds due to other viruses (115, 187), and it was again associated with very bothersome side effects. It has now been abandoned for this purpose. Anogenital warts (condyloma acuminata) lend themselves to local, intralesional therapy, and interferons may be useful for this purpose. Compared with placebo, they promote faster healing and fewer recurrences both in untreated patients and in those with refractory warts (132, 152, 375). Side effects were less common and less severe than in patients receiving systemic therapy, although intralesional interferon is absorbed systemically. Interferons used in conjunction with other local therapies such as podophyllin (113) and laser vaporization (480) also appear superior to these agents alone. The role of intralesional interferon therapy in genital warts is not yet clear. Such therapy is very expensive, and 20 to 30% of patients still suffer recurrences. Intralesional injections are painful, limiting the number of warts than can be treated. Cervical warts are very difficult to inject and may not be treatable by this mode. In adults with chronic hepatitis B, a lack of endogenous interferon may play a role in failure to clear the acute viral infection and in progression to chronic disease (94). Treatment with exogenous interferons has been under investigation for several years. Earlier trials suggested that the best ANTIVIRAL THERAPY 163 results were achieved by pretreating patients with prednisone, stopping this treatment, and then initiating interferon therapy during the rebound from corticosteroid therapy (347). A recent well-controlled trial, however, shows that this approach is effective in only a small subset of hepatitis patients with minimal liver dysfunction (348). Another larger subset of sicker patients responded to long-term (4-month) treatment with interferon alone, showing improvements in liver function and in abnormal histology and disappearance of hepatitis B e antigen, surface antigen, and DNA from the serum (348). A recent long-term follow-up study of treated patients indicated that many of these remissions were sustained for 3 to 7 years and may, in fact, represent cures of the disease (209, 245). In total, however, only 30 to 40% of chronic hepatitis B patients respond to interferon, and when lower doses are given, the response rate is lower (280). Further studies are needed to better determine which patients will benefit from interferon therapy and whether it will have any effect on long-term progression of disease, on survival, and on the frequency of subsequent hepatocellular carcinoma. In recent studies of interferon for chronic hepatitis C, about 50% of treated patients responded with decreases in liver function abnormalities, hepatic inflammation, and viral RNA in serum (93, 107, 427). Six months later, 50% of those responding had relapsed, resulting in a sustained response rate of only 20 to 25%. Although alpha interferon 2b has been approved for treatment of chronic hepatitis C (Table 1), many important questions remain about optimum dose and duration of treatment and the need for maintenance therapy (106). Better ways to identify patients likely to respond are also needed. In view of the cost and side effects of interferon, it should not be routinely used in hepatitis C until these problems are solved. Interferons have been known for some time to inhibit animal retroviruses, and they were shown in 1985 to inhibit HIV (204). In latently infected cells, interferons appear to act at a late point in virus replication to inhibit virion assembly and/or release (356). In acute infection, they prevent new infection of cells (204) and may act at an earlier point in replication. In mature macrophages, interferons appear to limit the formation of proviral DNA (246). When administered to AIDS patients for treatment of Kaposi sarcoma, interferon resulted in decreased p24 antigenemia and increased CD4 cells, although there was a "rebound" increase in p24 in some patients when the drug was stopped (105, 260). A subsequent placebo-controlled trial in asymptomatic HIV patients confirmed the ability of interferon to decrease viremia and perhaps opportunistic infections in this population (259). It also appears that interferons work best when given in early infection, while the immune system is still reasonably intact. The role of interferons, including those produced endogenously in HIV infection, is complex, however, and not completely understood. It may involve mechanisms by which the virus is able to avoid the effects of the drug, such as down-regulation of alpha interferon receptors on cell surfaces after prolonged exposure to these agents (272). As noted earlier, interferons also fail to cross the blood-brain barrier and can be associated with intolerable side effects, especially in those asymptomatic patients who stand to benefit the most from them (259). For these reasons, use of interferons alone for HIV infection may be problematic, and they are probably best used in combination with other agents such as zidovudine and other dideoxynucleosides that inhibit viral replication at a different stage. In vitro, alpha interferon and zidovudine are synergistic in

19 164 BEAN inhibiting HIV (183). In early, dose-finding patient trials, it has been possible to administer both agents together in doses that appear to have an antiviral effect, although bone marrow toxicity has been dose limiting in many cases (31, 250, 254). Much work remains to be done in this area, including investigation of gamma interferons for therapy of AIDS and use of additional agents, such as CSFs to alleviate the dose-limiting toxicities of such combinations as zidovudine and interferon. CSFs are naturally occurring cytokines that stimulate the reproduction and differentiation of granulocytes (G-CSF) or granulocytes, monocytes, and macrophages (GM-CSF) from bone marrow precursor cells. They also enhance the effectiveness of mature cells by, for instance, preventing neutrophil migration, potentiating antigen processing by macrophages, and stimulating phagocytosis of yeast cells and bacteria (499). Both G-CSF and GM-CSF are available commercially and are produced in Escherichia coli or in yeast cells by using recombinant DNA techniques. They were originally developed for prevention and treatment of neutropenias induced by cancer chemotherapy and bone marrow transplantation. A recent study indicates that they are extremely useful for this purpose, even reducing the number of hospital days required following autologous marrow transplantation (321). A great deal of interest has also developed in using them to control the complications of HIV infection, especially the neutropenias resulting from antiretroviral therapy. CSFs have no direct effect on HIV and do not affect HIV-infected lymphocytes. In monocytes, however, GM-CSF stimulates virus replication in vitro (251, 345), an effect consistent with the cell stimulatory properties of cytokines discussed earlier. If zidovudine is added to these in vitro systems, its potency is enhanced, and much lower concentrations are required to inhibit HIV than in the absence of GM-CSF (345). This effect is probably due to enhanced drug phosphorylation by the stimulated cells, and while it may be useful in the treatment of AIDS patients, it also raises the possibility of increased zidovudine-induced bone marrow toxicity when both drugs are administered simultaneously. These issues have not been resolved, but they have not prevented substantial progress in the use of CSFs. Groopman and colleagues first showed that GM-CSF was biologically active in raising granulocyte counts in leukopenic AIDS patients (173) and went on to demonstrate that the induced neutrophils functioned normally in killing Staphylococcus aureus. The same investigators also identified two patients with defective bacterial killing mechanisms (one in phagocytosis and one in intracellular killing), but these, too, appeared to be corrected by GM-CSF (14). Subsequent studies have shown both G-CSF and GM-CSF to be very effective in correcting the neutropenias seen with zidovudine therapy alone (311) or combined with interferon, and the two CSFs have allowed treatment to continue in patients otherwise unable to tolerate it because of bone marrow toxicity (92, 408). This has been true of patients with advanced (408) as well as early (92) HIV disease. There is also preliminary evidence that GM-CSF may ameliorate the neutropenia associated with ganciclovir therapy (181). The doses of CSFs needed in AIDS patients are usually much lower than those used in cancer patients, and the side effects are more closely related to the use of zidovudine and interferon. CSFs are, however, prohibitively expensive. In the dosages used for cancer patients, the cost is approximately $200 per day (300). Whether or not it will be lower in AIDS patients remains to be seen. In the face of this, it is important for future studies CLIN. MICROBIOL. REV. to identify the patients most likely to benefit from these expensive agents. PROBLEMS OF ANTWIRAL THERAPY Resistance Unlike bacteria, viruses do not have a wide variety of mechanisms for developing resistance to chemotherapeutic agents. They are genetically simple and metabolically inert, and changes like those seen in bacteria, such as decreased uptake of antibiotics, acquisition of resistance plasmids, or induction of inactivating enzymes, have not been seen. Viruses do, however, have two properties that allow them to evade potential inhibitors: they have the ability to replicate to high titers in host cells, and they can mutate very rapidly. As a consequence, most viral resistance arises from genetic mutation, giving rise to changes in either enzymes or structural components of the virion. The success of this mechanism can be seen in cell culture, where viruses readily develop resistance after only a few passes in the presence of an inhibitory agent (for a review, see reference 140). In contrast, very few resistant viruses have been isolated from immunocompetent patients being treated for viral diseases (19, 126). Resistant herpesviruses are isolated from patients immunocompromised by cancer chemotherapy or transplantation, although this is relatively infrequent. In addition, most of these resistant viruses appear to have reduced virulence and to cause predominantly indolent, non-lifethreatening infections (19, 85, 428). There are however, increasing reports of AIDS patients with life-threatening or very serious illnesses due to resistant herpesviruses (127, 131, 164, 288), suggesting that severely immunocompromised patients may provide a milieu in which mutant viruses are not at a selective disadvantage and can prosper. In addition, a larger viral load creates a larger gene pool from which mutations can emerge. Under these circumstances, selective pressure from antiviral agents could result in the propagation and perhaps the transmission of resistant viruses, eventually replacing wild-type susceptible populations with more resistant ones. Despite what is known about viral resistance in vitro, a great deal remains to be learned about its importance and about the management of patients infected with resistant viruses. A summary of unanswered questions follows. Under what circumstances does resistance arise? In general, it seems that the greatest risk is in severely immunocompromised patients receiving prolonged courses of antiviral therapy, but the details of the relationship among resistance, severity of underlying disease, and treatment are unknown. Although the herpesviruses do not become resistant easily in immunocompetent patients (325), influenza viruses do (188). The reasons for this difference are not known. What are the properties of the resistant viruses? Virulence may be reduced, but this appears to depend on the host, at least among herpesviruses. Rimantadine-resistant influenza A virus strains, on the other hand, are as virulent for ferrets (464) and for people (188) as their wild-type counterparts. Whether resistant viruses can establish latency is another important but unanswered question. This ability may also depend on the host and the mechanism of resistance. Likewise, little is known about transmissibility of resistant viruses. To date, transmission of resistant herpesviruses has not been documented, but it may only be a matter of time, unless resistance is accompanied by reduced transmissibil-

20 VOL. 5, 1992 ity. Resistant influenza viruses, on the other hand, appear to be readily transmitted, at least in a household setting (188). Another very important issue is cross-resistance among antiviral drugs. A great deal is known about in vitro crossresistance, and a few clinically useful patterns have emerged, such as cross-resistance of zidovudine-resistant HIV to other azido-containing nucleosides and susceptibility of thymidine kinase-deficient HSV to drugs not needing phosphorylation or not requiring a virus-induced enzyme for phosphorylation. A great deal remains to be learned, however. At least 20 different DNA polymerase mutants have been found in vitro among HSV variants, for instance, and most of them possess altered drug-binding properties. Resistance to one compound is usually accompanied by decreased susceptibility to other drugs of the same class, but predictable cross-resistance among different classes of drugs has also been found and is thought to indicate overlap of drug-binding sites (71). Exceptions to known patterns also exist, further confirming the complexity of resistance due to DNA polymerase changes. Consequently, predicting crossresistance in DNA polymerase mutants may not be possible unless the viruses are carefully characterized. What is the result of the development of drug resistance in the patient? As discussed above, the appearance of zidovudine-resistant HIV has not yet been associated with clinical deterioration in individual patients, although it may play a role in the reduced effectiveness of zidovudine after many months of therapy. Failure of herpes lesions to heal and even serious illness are frequently due to acyclovir-resistant herpesviruses in AIDS patients, but this is not true in other immunocompromised patients in whom failure to heal may or may not be due to acyclovir resistance and in whom lesions due to resistant virus may heal despite the lack of effective therapy (470). Rimantadine prophylaxis against influenza A was ineffective in persons with household exposures to rimantadine-treated patients, but the influenzal illness itself (due to rimantadine-resistant influenza) was typical and resolved in the usual self-limited fashion (188). Thus, viral resistance is clinically important in some, but not all, situations and much remains to be learned. What are the most reliable ways of identifying resistance in the laboratory? Traditional research methods that involve propagation of viruses in cell culture, exposure to inhibitory agents, and measurement of subsequent changes in host cells or in amount of virus produced are time-consuming and expensive. They may also be unreliable in that they can select for virus strains that grow well in cell culture but are not necessarily representative of the virus population present in the patient. It is increasingly appreciated that viruses isolated from patients are not clonal derivatives homogeneous in their properties but instead are heterogeneous populations of viruses with differing drug susceptibilities and other properties, including the ability to grow in different cell lines (123, 149). As a result, new methods of measuring resistance are being developed for the research laboratory, including use of the polymerase chain reaction for detection of resistance genes, cell lines which allow growth of a broader spectrum of viral strains, and improved methods of nucleic acid hybridization (226, 264, 266). It is hoped that these can be adapted to the clinical laboratory so that patients can benefit directly from the improved methods. What can be done to minimize problems due to drug resistance? As with tuberculosis, it is hoped that combination therapy in HIV disease will forestall or even prevent clinical manifestations of drug resistance (see below). Other ANTIVIRAL THERAPY 165 measures that have been successful in herpesvirus-infected patients include avoiding therapy with prolonged courses of drugs at "subinhibitory" levels and discontinuation of therapy whenever possible. Latency With the possible exception of a new agent that may "cure'" cell cultures of HIV infection (see N-butyl-deoxynojirimycin, above), antiviral agents are virustatic rather than virucidal. In vitro, such agents are most active against actively proliferating viruses, and when these agents are removed from cell cultures, virus replication resumes. In patients, a course of treatment with acyclovir does not prevent future recurrences of HSV, nor does zidovudine prevent progression of AIDS, indicating that these drugs do not eliminate these viruses when they are in the latent state. Furthermore, the presence of latent virus in treated patients could afford the opportunity for prolonged contact between virus and drug, with attendant opportunities for development of drug resistance (140). The successful treatment of latent viral infections will depend, as it does in acute viral infections, on the identification of specific viral processes that can be specifically attacked by antiviral agents. Recently, a great deal of progress has been made in defining the molecular events associated with latent HSV infections. A group of RNAs, termed latency-associated transcript, has been described and possibly linked to reactivation of latent HSV (201); for a review, see reference 456. Advances such as these should eventually make it possible to identify agents capable of interfering with viruses in the latent state. Immunosuppression by Antiviral Agents Because antiviral agents are virustatic but not virucidal, host immune responses remain critical to the recovery of patients from viral infections. As discussed above, many antiviral agents have antiproliferative activity against rapidly dividing cells, and inhibition of host immune responses has always been a concern in antiviral therapy. A recent direct comparison of the effects of several antiviral agents on the proliferative responses of peripheral blood mononuclear cells from healthy individuals indicated that zidovudine, ganciclovir, and ribavirin decreased mitogenesis, while acyclovir and didanosine had no effect (192). Another study showed that zidovudine, didanosine, and ddc had no inhibitory effect on the bactericidal activity of polymorphonuclear leukocytes and possibly enhanced it (389). Further studies are needed, but these trials demonstrate the need for identifying the immunosuppressive properties of individual antiviral agents, establishing their importance in patients, and using this information to individualize patient therapy. PROSPECTS FOR THE FUTURE Liposomes Liposomes are synthetic phospholipid vesicles that can be used as carriers of many biologic molecules, including antitumor and antimicrobial agents, immunomodulators, toxins, and enzymes. When administered to animals or humans, they are naturally distributed to the reticuloendothelial organs and cleared by phagocytic cells, especially blood mononuclear cells and tissue macrophages. Liposomes can also be targeted to specific cells by incorporating the appropriate antibodies into them. For these reasons,

21 166 BEAN they are ideal agents for delivery of drugs to intracellular organisms such as viruses. Although no patient trials of antiviral therapy have yet been reported, animal and in vitro studies are intriguing and encouraging. Liposomes have been used to increase the cellular permeability of highly ionized drugs such as foscarnet (468) and to lengthen the half-life of ddc in the central nervous system of rats (237). HSV-infected cells can be selectively destroyed by liposomes encapsulated with acyclovir and tagged with anti- HSV antibodies (324) or by mononuclear phagocytes activated by liposomes encapsulated with immunomodulators such as gamma interferon or macrophage-activating factor (243). Of importance, CD4-bearing liposomes interact with HIV-infected lymphocytes and deliver their contents to the interior of these cells (87). This raises the possibility of loading these liposomes with antiviral agents such as reverse transcriptase inhibitors and using them to deliver the drugs to the interior of HIV-infected cells. HIV-1 also fuses directly with negatively charged liposomes, and its infectivity is thereby decreased (244), another promising approach to treatment of HIV. Combination Therapy Prompted by the success of combination chemotherapy for cancer and stimulated by the need for effective treatment of HIV infection, combination therapy has come to the forefront of antiviral treatment. The purpose is threefold: to enhance the efficacy of single-agent therapy, to minimize toxicity by reducing individual drug dosages, and to prevent or forestall the development of drug resistance. There are many reports of drug combinations synergistic in vitro against HIV and other viruses. Many patient trials are also under way, but few have been completed and most data are preliminary. The most promising combinations consist of a nonnucleoside and a nucleoside inhibitor of HIV reverse transcriptase or of two drugs active at different sites of viral replication. Different toxicities are also very important. In AIDS patients with Kaposi sarcoma, for instance, zidovudine and alpha interferon together showed early evidence of suppressing antigenemia more effectively than either drug alone and, possibly, of delaying drug resistance (31, 148). ddc is a reverse transcriptase inhibitor like zidovudine, but it is phosphorylated by a different mechanism and has different toxicities. When ddc is administered with zidovudine in an alternating regimen, less toxicity is seen than with either agent alone at full doses (43, 351, 435). Very exciting results have been obtained by giving GM-CSF to patients who develop neutropenia on zidovudine-interferon regimens. GM-CSF rapidly alleviated the neutropenia and allowed patients to remain on this very effective regimen when they would otherwise have been unable to tolerate it (92, 408). One recent trial demonstrated what appears to be an additive effect of zidovudine and foscarnet in suppressing p24 antigenemia (225). Although it has no activity against retroviruses, acyclovir can be added to zidovudine to control concomitant HSV infections. If herpesviruses, especially CMV, can transactivate HIV or induce cellular receptors for it, as has been shown in vitro (95, 295; for a review, see reference 273), control of herpesvirus infections may become an important part of HIV management. Despite the enthusiasm for combination therapy, adverse interactions must be closely monitored, and prospective combinations of agents must be tested in vitro and in animals before they are given to patients. Ribavirin, for instance, is phosphorylated by the same enzymes as zidovudine and in vitro inhibits its CLIN. MICROBIOL. REV. effectiveness against HIV (11, 486). In vitro, when didanosine was added to zidovudine, the combination synergistically inhibited HIV, but at high concentrations it also additively inhibited bone marrow progenitor cells (111), indicating that careful dosage adjustment will be needed in treating patients with this combination. Computer-Aided Drug Design Traditionally, the search for antiviral agents has consisted of randomly screening compounds for viral inhibitory activity in cell culture, identifying a few, making structural modifications in them, and retesting for changes in activity. Computer-aided drug design uses X-ray crystallography to determine the three-dimensional structures of viral macromolecules and then employs computer simulation and sophisticated thermodynamic computations to study atomic interactions with other molecules and to delineate the structures of those with favorable binding characteristics (290). In this way, the effect of structural modifications can be determined and promising compounds can be identified before an investment is made in synthesizing and testing them. The first human virus to be studied in this fashion was the human rhinovirus. Its three-dimensional atomic structure was described by Rossmann and colleagues in 1985 (396). In 1986, the interaction of the virus with a known picornavirus inhibitor, WIN 51711, was described: the drug was bound within a deep depression on the virion surface and induced conformational changes that increased stability and prevented uncoating of the virus (150, 441). A series of compounds with similar bridging properties were subsequently synthesized and studied (13), and some of them are currently being tested in patients for the treatment of rhinovirus colds. The structure of the influenza virus hemagglutinin and its interaction with its cellular receptor, sialic acid, have also been described and suggest that agents that would interfere with attachment of the influenza virus to cells could be designed (498). It has been proposed that HIV has a surface similar to that of rhinoviruses and thus might also be inhibited at the uncoating step (395). DesJarlais and colleagues (104) have recently used the structure of HIV-1 protease and a computer-assisted searching program to identify and study potential inhibitors of this enzyme. In addition, the structure of HIV reverse transcriptase itself is being studied by X-ray crystallography in the hope of identifying specific inhibitory compounds (268). Computerassisted drug design is still in its infancy, and no antiviral agents have yet been designed de novo by this technique. Nevertheless, it is advancing rapidly and holds great promise for the efficient, selective identification of new drugs. Role of the Clinical Microbiology Laboratory Most antiviral agents in use have a narrow spectrum of activity, and many of them cause substantial toxicity at the usual dosages. Consequently, empiric patient therapy with antiviral agents is seldom possible, although it is a common practice with antibacterial agents. Identification of a specific viral pathogen is usually needed, and although antiviral susceptibility testing is currently only necessary in referral centers, the rising number of HIV-infected patients and the increasing use of antiviral agents will eventually demand that it be available in general and community hospitals as well. Recent advances in diagnostic methodology have made it possible to identify many common viral pathogens from patient specimens within a few hours to a few days (440).

22 VOL. 5, 1992 Standard cell culture is still necessary for identification of all possible pathogens if no specific virus is suspected of causing a given illness. When only a single or a small number of viruses is implicated, however, immunofluorescent or enzyme immunoassays can frequently be performed directly on patient specimens and can detect the presence of viral antigens within a few hours. RSV is commonly identified in this way. In other cases, shell vials are used to allow a period of viral amplification before identification is attempted. The specimen is centrifuged onto a monolayer of cells grown on a coverslip in the bottom of a 1-dram (3.7-ml; shell) vial. Nutrient medium is added and the vial is incubated, usually for 18 to 72 h. The coverslip is then removed, the cells are fixed, and, most commonly, a fluorescein-conjugated antibody is added to detect the presence of viral antigen in the cells. HSV, VZV, CMV, and influenza viruses, as well as some others, can be identified in 1 to 3 days by using this method. Almost no convenient, practical methods for antiviral susceptibility testing are available to the clinical laboratory. The plaque reduction assay, a method commonly used by the research laboratory to measure antiviral activity (86, 189), is time-consuming, cumbersome, and difficult to reproduce. In addition, it depends on the ability of the virus to grow in a given cell line and thus may select virus populations capable of replicating well in vitro but not necessarily representative of the population infecting the patient. Many other methods have been used to quantitate viral replication in the presence of inhibitory agents including nucleic acid hybridization (160), shell vials with immunofluorescent staining (470), immunoperoxidase staining (211), enzyme immunoassay (366), dye uptake assay (299), and yield reduction assay (360). None of these methods has been standardized among laboratories, however. One method of nucleic acid hybridization for HSV susceptibility has been made commercially available in kit form. It compares well with the plaque reduction assay and involves wicking of lysed, virusinfected cells onto membranes followed by hybridization with a radioiodinated DNA probe (465). Although this method is more adaptable to clinical laboratories than many others and is in use in some, 3 to 4 days are required for susceptibility testing after the virus is isolated in cell culture and radioisotopes, frequently a disposal problem for microbiology laboratories, are still employed. As the demand for antiviral susceptibility testing grows, it is hoped that practical diagnostic techniques will also become available. CONCLUSIONS Although early progress in antiviral therapy was slow, rapid advances have been made in the last decade. As our understanding of viral replication increases, more and more viral processes are being identified as possible targets for inhibition by antiviral compounds. Immunomodulators are being used increasingly to augment the immune response in the treatment of viral diseases. Resistance to antiviral drugs has been noted among diverse groups of viruses but thus far has caused treatment failure primarily in highly immunosuppressed patients, such as those with AIDS. It is hoped that combination antiviral therapy, in addition to improving the effectiveness of individual drugs, will limit the impact of resistance on patient care. Other new approaches, such as the use of liposomes to deliver antiviral drugs intracellularly and the use of computers to identify potential new agents rapidly and inexpensively, should result in increasingly safe ANTIVIRAL THERAPY 167 and effective antiviral drugs. 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