Epstein–Barr virus infections: prospects for treatment

Abstract

Epstein–Barr virus (EBV) causes infectious mononucleosis and oral hairy leucoplakia, and is associated with a number of malignancies. There are, however, no regulatory agency-approved treatments for EBV-related diseases. Several antiviral drugs inhibit replication of EBV in cell culture including acyclic nucleoside and nucleotide analogues and pyrophosphate analogues, all of which inhibit the EBV DNA polymerase. Despite their potency in vitro, these drugs have limited use in vivo for treatment of acute primary EBV infection as well as EBV-associated malignancies for several reasons. Here we discuss novel anti-EBV compounds, including maribavir, potentially useful for the treatment of acute EBV infections. A number of experimental approaches for treatment of EBV-related malignancies that are not susceptible to conventional antiviral drug treatment are also discussed.

EBV, infectious mononucleosis, antiviral drugs

Why is it that there is no effective treatment for infectious mononucleosis (IM)? At least 90% of cases of IM are caused by primary Epstein–Barr virus (EBV) infection. Although most persons are infected by age 30, only some have the typical syndrome of IM, usually those who are infected in the late teens or early twenties. The rest have subclinical infection or non-specific symptoms, especially when infected in childhood.¹ Despite the availability of many antiviral drugs starting with aciclovir^2–6 that are potent inhibitors of EBV replication in cell culture, clinical trials of aciclovir for the treatment of patients with IM have failed to detect benefit.^7,8 There are several possible explanations for these outcomes.

First, the symptoms of IM are insidious in onset, which, coupled with a long incubation period (4–6 weeks), make for late diagnosis—in contrast, for example, to herpes labialis or chickenpox. Second, EBV is shed in the saliva. The levels of aciclovir achieved in the oropharynx, particularly after oral administration of the drug, may be inadequate as judged in part by failure to suppress virus titres in the saliva,^7,8 in contrast to the reduction in titres that are produced by aciclovir given intravenously.⁹ Presumably, the orally administered aciclovir prodrug, valaciclovir,¹⁰ would do almost as well as intravenously administered aciclovir, since the prodrug produces high blood levels of aciclovir. Third, and probably the most important for the failure to respond to antiviral therapy, is the strong evidence that most of the symptoms and signs of IM are due not directly to viral cytopathology in infected tissues, but to immunopathic responses to EBV-infected cells, particularly EBV-infected B-lymphocytes that circulate in the blood and infiltrate tissues of affected organs. The virus is usually restricted to infection of epithelium in the oropharynx and the cervix and of B-lymphocytes, initially in the tonsillar region. In immunocompetent hosts, the virus does not usually infect liver cells, neural cells or haematological cells other than lymphocytes, although liver, neural and bone marrow tissues and cells may be affected indirectly by immunopathic responses in complications of IM. Indeed, the atypical lymphocytosis (up to 40% or more of total circulating white blood cells) characteristic of IM consists of T cells, not EBV-infected B cells, and signifies the massive cell-mediated immune response mounted in the course of infection to the proliferating infected B cells.¹ Ideally then it seems apparent that antiviral therapy coupled with an immunomodulatory drug might be effective. Corticosteroids are used empirically by physicians in treating IM, especially if there is severe pharyngitis, tonsillar swelling or airway obstruction,¹¹ and in fact a controlled trial of prednisolone given with aciclovir for treatment of IM was carried out, but without apparent benefit.¹² However, the design of this trial may not have been optimal, as indicated by the fact that prednisolone alone did not produce the expected results.¹³ Also, use of aciclovir may not have produced adequate levels of the drug, although there was suppression of shedding of virus in the oropharynx.

From the target perspective, the drugs that might be candidates for treatment of EBV infection fall into two groups. The first group includes acyclic nucleoside analogues (aciclovir, ganciclovir, penciclovir, as well as their prodrugs valaciclovir,¹⁰ valganciclovir¹⁴ and famciclovir,¹⁵ respectively); acyclic nucleotide analogues (cidofovir¹⁶ and adefovir¹⁷); pyrophosphate analogues [phosphonoformic (foscarnet)¹⁸ and phosphonoacetic¹⁹ acids]; and possibly 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792).^20,21 All these compounds target viral DNA polymerase (detailed below). The second group contains compounds of mixed nature that have a distinct structure such as maribavir,²² β-l-5-iododioxolane uracil²³ and indolocarbazole NIGC-I.^24–27 Mechanisms of action of these drugs are still under study, but they do not involve inhibition of the viral DNA polymerase. All listed compounds are potent inhibitors of EBV replication, but are not equal candidates clinically because they differ greatly in toxicity—a crucial consideration, especially in the otherwise healthy young persons and children in whom IM occurs.

Antiviral drugs that have been used thus far to inhibit EBV replication belong to the first group and target viral DNA polymerase. Foscarnet and phosphonoacetic acid interact directly with the pyrophosphate-binding site of the enzyme, whereas others act at two levels: as competitive alternative substrates, competing with GTP on the substrate-binding site, and as DNA chain terminators, by incorporating into the growing DNA chain and blocking its elongation due to their acyclic structure (reviewed in ref 28). Acyclic nucleoside and nucleotide analogues require phosphorylation to their triphosphorylated forms in order to become active. However, in the case of the nucleoside analogues, the antiviral specificity is based in part on the fact that viral kinases are more efficient in catalysing the first step in the drug intracellular metabolism (monophosphorylation), and so these compounds are more specific. EBV encodes two kinases: a thymidine kinase (BXLF1 gene product) and a protein kinase (BGLF4 gene product). However, in contrast to all the other herpesviruses, it is unclear which of these enzymes is responsible for the monophosphorylation and activation of nucleoside analogues.^29,30 Specificity is also imparted by the ultimate target of these drugs, viral DNA polymerase, which explains why the phosphonated nucleoside analogues like cidofovir are still specific. It is unclear if herpesvirus DNA polymerases differ in sensitivity to these drugs since rigorous comparative determinations of K_m's and K_i's of the different herpesvirus DNA polymerases for any given drug have not been attempted, in part because of issues of enzyme purification.³¹ While usable, all these compounds suffer from a number of drawbacks (i.e. toxic side effects, poor oral bioavailability and risk for emergence of drug-resistant virus strains). These effects have prompted the search for novel compounds that are more specific in their antiviral action and devoid of the shortcomings of the currently used drugs. Of note, however, since the diseases related to the EBV lytic cycle occur less frequently compared with other herpesviruses, this search has been primarily directed toward herpes simplex virus type 1 (HSV-1), HSV-2, varicella-zoster virus (VZV) and human cytomegalovirus (HCMV), and only prominent candidates have been tested for possible efficacy in treatment of EBV infections. This approach somewhat reduces the total number of the ‘EBV-tested’ antiviral compounds, but narrows the selection to the potentially more effective ones.

This search has yielded a number of new compounds, which demonstrate unique modes of action. Previous studies with the benzimidazole d-ribonucleosides 2,5,6-trichloro-1-β-d-ribofuranosylbenzimidazole (TCRB) and 2-bromo-5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (BDCRB) demonstrated their effectiveness in inhibition of HCMV replication;³² however, neither inhibited EBV replication.³³ These compounds prevent the processing and maturation of concatameric viral DNA to monomeric genomes.^34,35 The short plasma half-lives of these compounds led to the design of additional analogues, including maribavir,²² which in contrast to BDCRB is an l-ribonucleoside. Surprisingly, this novel compound has a mechanism of action that is different from that of BDCRB and in fact not yet fully understood. Maribavir is active against both HCMV^36–40 and EBV,^27,33,40,41 and its effects on these viruses seem to involve direct or indirect inhibition of viral protein kinases^{27,36,41–43} as well as possible interference with nuclear egress of virions during viral maturation.⁴⁴ Maribavir resistance in HCMV has been mapped to UL97³⁶ and UL27^45,46 genes. In contrast, the EBV BGLF4 gene product, a protein kinase that is the homologue of HCMV UL97, was not inhibited directly by the drug,^27,41 and EBV does not encode a homologue of UL27. However, phosphorylation of the EBV DNA-processivity factor, EA-D, by the EBV protein kinase is inhibited by maribavir in infected cells.⁴¹ Maribavir is the only drug in this group for which Phase I clinical trials have been completed, viz., for HCMV infection,^47,48 and Phase II trials were initiated in July 2004.⁴⁹

Another class of compounds, namely, indolocarbazoles, has emerged recently as potential inhibitors of HCMV²⁴ and EBV²⁷ replication. The mechanism of action of these compounds seems to involve the inhibition of HCMV UL97 protein kinase;^25,26 however, the same compounds (except for one, K252a) failed to directly inhibit EBV BGLF4 protein kinase.²⁷ These observations suggest that although some compounds may affect different groups of viruses, the mechanism of their action might be different for each of these groups.

Finally, a number of 4-oxo-dihydroquinolines (4-oxo-DHQs) (as represented by PNU-182171 and PNU-183792)^20,21 showed a broad spectrum of anti-herpesvirus activity. Although non-nucleosides, the compounds seem to affect viral DNA polymerase and inhibit HSV-1 and -2, VZV, HCMV, kaposi's sarcoma herpesvirus (KSHV)⁵⁰ and EBV⁵¹ in cell culture. The mechanism of action of these drugs has not been reported, but it is clearly different from that of aciclovir and related drugs. Resistance to 4-oxo-DHQs has been mapped to V823 of the viral DNA polymerase, a residue that is conserved in the DNA polymerases of six (HSV-1 and -2, VZV, HCMV, EBV and KSHV) of the eight human herpesviruses, which implies that the compounds specifically inhibit these enzymes.⁵⁰

In addition to IM, EBV causes hairy leucoplakia (HLP) of the tongue, another acute infection, but one that occurs almost exclusively in immunodeficient patients. The virus is associated with, or may contribute aetiologically to, a variety of neoplasms, such as: EBV lymphoproliferative syndromes in immunocompromised persons, especially patients with AIDS, recipients of organ transplants, and in persons with rare genetically determined immunological dysfunctions; nasopharyngeal carcinoma; Burkitt's lymphomas; Hodgkin's lymphomas; and a subset of EBV-positive gastric carcinomas.^52,53 Except for HLP, which is benign and represents a purely lytic infection with large amounts of EBV replicating freely in the lesions, all the other EBV diseases are malignancies characterized by latent infection that relies on cellular enzymes for EBV episomal DNA synthesis. Therefore, it is not expected that antiviral drugs directed at viral synthetic processes would affect EBV in the latent phase. In latent infection, the linear double-stranded genomes characteristic of productive lytic infection and encapsidated in virus particles are not made. Rather infection persists via controlled replication of viral episomes that are found only in the nucleus in a nucleosomal form. These circular supercoiled genomes are replicated once during cell division and perpetuated in progeny cells indefinitely. Replication is mediated by host DNA polymerase (and other enzymes of the cellular replicative machinery). Episomal copy number is tightly regulated and remains constant while expression of viral genes is greatly limited to several latency genes.^52,53 None of these processes is affected by, or sensitive to, antiviral drugs to any degree, as shown by their lack of effect on latently EBV-infected cell lines or tumours.³¹ A possible exception might be the initially polyclonal EBV lymphoproliferative disorders, in which occasional cells do exhibit lytic rather than latent infection; in such cases, theoretically antiviral therapy might prevent secondary infection of a new population of B cells. There is only anecdotal evidence for such an effect clinically, and it is scant. However, Chodosh et al. have reported experiments in which treatment of latently EBV-infected cells with hydroxyurea led to loss of episomes,⁵⁴ and in a subsequent case report detailed apparently successful use of hydroxyurea in a patient with an EBV-related CNS lymphoma.⁵⁵

It should be noted though, that a number of groups are intensively studying means to induce a switch from latent to cytolytic infection as a therapeutic approach for the treatment of EBV-positive neoplasms, the rationale being that viral replication causes cytolysis of the infected cells and could do so in tumours.⁵⁶ The approaches reported so far include the chemotherapeutic agents gemcitabine and doxorubicin,^57,58 arginine butyrate,^59–62 as well as radiation therapy,⁶³ all of which alone or in combination induce viral reactivation. Combinations of such inducers with ganciclovir, which is activated by EBV PK (or TK) and thus toxic for infected and some neighbouring cells, has led to some progress in treatment of EBV-positive malignancies in animal models^57,58,63 and in Phase I/II clinical trials.⁶⁰

In quite another experimental approach to treatment of EBV-associated malignancies, phosphonated nucleoside analogues have been used to cause apoptosis of human EBV-positive nasopharyngeal carcinomas (NPCs) grown in athymic mice. Since NPCs are latently infected, again the antitumour effect must be independent of the antiviral effects of these drugs.^64–66 Interestingly, cidofovir has been reported to produce complete responses in vivo in another virus-associated neoplasm, laryngeal papillomatosis, which is caused by human papillomavirus.^67,68 Abdulkarim et al.⁶⁹ reported that such effects of cidofovir might be linked to decreased expression of EBV latent membrane protein 1 (LMP1) and consequent aberrations in apoptotic mechanisms, but there is evidence that weighs against these conclusions,⁶⁴ and so how these nucleotide analogues affect neoplastic growths is far from clear and remains an important topic for continued study.⁷⁰

In conclusion, although a number of the licensed antiviral drugs can inhibit EBV replication, none of them has been licensed for the treatment of EBV infection in the clinic. Regimens that have been used empirically and in clinical trials have been largely ineffective, and as such have prompted the search for more effective approaches. For latent infection and EBV-related malignancies, induction of the viral lytic cycle in order to destroy infected cells or to convert drugs into cytotoxic forms, as well as development of novel antiviral drugs that target non-replicative viral proteins, are among the more promising approaches that might lead to effective treatment of EBV-related diseases.