Progressive multifocal leukoencephalopathy: current treatment options and future perspectives

Introduction

Progressive multifocal leukoencephalopathy (PML) was first described in the late 1950s [Astrom et al. 1958]. The causative virus was first isolated in 1971 [Padgett et al. 1971] and named JC virus (JCV) after the patient from whose brain it had been isolated. JCV is a polyomavirus, genetically related to BK virus (BKV) and simian virus 40 (SV40), composed of a 5–13 kb double-stranded circular DNA enclosed in a capsid without a lipoprotein envelope. The virus is widespread among human populations throughout the world. The routes of transmission are not well established, but oral/fecal route seems most plausible [Brew et al. 2010; Bofill-Mas et al. 2001]. Studies have shown that probably half of all infections occur at home in a parent-to-child transmission [Kitamura et al. 1994; Kunitake et al. 1995]. Most individuals are infected by age 30–40 [Knowles, 2006; Matos et al. 2010]. However, it is important to note that although JCV is commonly spread in the community, the JCV archetype is not pathogenic.

The JCV archetype can infect various cell types, including brain cells [Tominaga et al. 1992; O’Neill et al. 2003; Newman and Frisque, 1997]. It resides in epithelial cells of the kidney, and is frequently shed in urine [Kitamura et al. 1997; Lagatie et al. 2014], but it does not cause disease. The JCV variant that causes PML differs from the archetype in the noncoding regulatory region of the viral DNA. The JCV archetype regulatory region has one 98-bp element with a 23-bp and a 66-bp insert, whereas the regulatory regions of pathogenic PML prototypes usually consist of tandem repeats of a 98-bp element (various Mad strains) [Tan and Koralnik, 2010]. The regulatory region rearrangement is unique to each affected individual, suggesting the pathogenic prototype is not acquired by transmission, but rather emerges within its host from the commonly acquired JCV archetype [Agostini et al. 1997].

The pathological transformation of JCV is still poorly understood. However, there is strong evidence for an increased number of JCV prototypes in immunosuppressed individuals without PML [Tan et al. 2010; Bayliss et al. 2013], as well as correlation between duration of immunosuppression and PML incidence [Bloomgreen et al. 2012; ClinicSpeak, 2015]. It may take years before this transformation is accomplished, and the transformed virus induces clinically manifested brain lesions. The hallmark of PML is demyelination of axons caused by the lysis of infected oligodentrocytes by JCV. Clinical presentation depends on the extent of demyelination and brain structures involved.

Medical literature describes PML and PML–immune reconstitution inflammatory syndrome (PML-IRIS). PML-IRIS is the same disease as PML, but with one important difference, which is seemingly a paradox: worsening of PML symptoms or new onset PML following discontinuation of immunosuppression [Tan et al. 2011]. Whereas in PML, demyelination of axons is caused by the lysis of infected oligodentrocytes by JCV, in PML-IRIS this demyelination can be initiated or further enhanced by excessive destruction of brain tissue by the host’s immune system. For that reason PML-IRIS is usually treated with high doses of corticosteroids [Dahlhaus et al. 2013; Tan et al. 2009], a treatment that would be counterintuitive in PML. It is important to note that frequency of PML-IRIS in a given population correlates with more favorable outcomes (Table 1).

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Table 1.

Characteristics of three major PML populations.

PML population	Population size	PML-IRIS	Survival
HIV+	~80%	Observed	70% up to 1 year
		Unknown frequency	40-50% up to 2 years
			(pre-cART 9% up to 1 year)
Hematological malignancies	~10%	Rare	10% beyond 2 months
RRMS patients treated with natalizumab	<5%*	Frequent (70%)	77% up to 3 years

*

Estimate based on number of reported PML cases associated with natalizumab in the period 2005-2014 and assumption that contribution of PML cases by HIV+ and hematological malignancies populations remained stable since 2005.

PML, progressive multifocal leukoencephalopathy; IRIS, immune reconstitution inflammatory syndrome; cART, combined antiretroviral therapy; RRMS, relapsing–remitting multiple sclerosis

There are several distinct PML risk populations. The largest is the human immunodeficiency virus positive (HIV+) PML population. Although combined antiretroviral therapy (cART) significantly decreased the incidence of PML, in absolute terms this population is still the most significant contributor of PML cases. The incidence rates in the HIV+ population declined from levels of 2–10 per 1000 person-years in the pre-cART era, to around 1 per 1000 person-years in the post-cART era [Sacktor et al. 2001; D’Arminio Monforte et al. 2004; Khanna et al. 2009; Engsig et al. 2009]. In a period between 1998 and 2005, thus during the cART era, about 80% of 9675 PML cases in the United States were attributed to HIV [Molloy and Calabrese, 2009]. Survival of HIV+ PML patients up to 1 year from diagnosis was 9% in the pre-cART era [Berger et al. 1998]. Survival rates increased significantly with cART; however, the mortality rate remains high: up to ~30% after 1 year and ~50–60% after 2 years [Khanna et al. 2009; Engsig et al. 2009]. A subpopulation of HIV+ patients with low CD4+ T-cell count appears to be at particularly high risk of developing PML [Engsig et al. 2009]. PML-IRIS occurs in the HIV+ population [Cinque et al. 2001], but the frequency has not been defined.

In the same period (1998–2005), the second-largest risk population was patients with various forms of hematological malignancies [Molloy and Calabrese, 2009]. They constituted about 10% of all PML cases in the United States. This number is probably an underestimate, because the rapidly progressing complications secondary to the underlying malignancy and to chemotherapy that are common in this population are likely to hinder diagnosis of PML in many cases. Overall prognosis in this population is poor, with about 90% of subjects dying within 2 months of diagnosis, and PML-IRIS is rarely observed [Carson et al. 2014; von Geldern et al. 2012].

Today, the third-largest risk population is relapsing–remitting multiple sclerosis (RRMS) patients treated with natalizumab. First natalizumab related PML cases were reported in 2005. Current overall PML risk incidence in this population based on 540 cases is reported to be 3.96 per 1000 patients (see http://www.tysabri.com/ms-facts/). Risk stratification based on data from March 2013 shows that the risk affects mainly anti-JCV antibody positive patients. In such patients without prior immunosuppression, the risk is relatively low in the first 2 years of treatment (0.7 per 1000 patients years), but increases to 5.3 per 1000 patients years after 2–4 years of treatment and 6.1 per 1000 after 4–6 years without prior immunosuppression. Prior immunosuppression increases the incidence to 1.8 and 11.2 per 1000 patient years [Biogen-Idec, 2014] for 0–2 and 2–4 years of natalizumab treatment, respectively. Anti-JCV antibody negative patients show a much lower risk, estimated at less than 0.1 per 1000 patients [ClinicSpeak, 2015]. PML-IRIS is observed in the majority (70%) of RRMS PML patients within days or weeks following discontinuation and removal of natalizumab [Biogen-Idec, 2011, 2014]. Survival rate is 77% based on 517 PML patients with average follow up of almost 3 years after PML diagnosis [ClinicSpeak, 2015]. However, 40% of survivors had severe disability, 47% had moderate disability, and 13% had mild disability [Kappos, 2011].

PML is also associated with other conditions such as organ transplantation, solid malignancies, sarcoidosis, autoimmune disorders (e.g. lupus, rheumatoid arthritis), and congenital immune deficiencies; however, these populations individually contribute a relatively small number of cases and together account for less than 10% of all reported PML cases [Neff et al. 2008; Molloy and Calabrese, 2009; Clifford et al. 2011].

Although the epidemiological data presented in Table 1 summarizes two different periods spanning 1998–2005 for patients with HIV and hematological malignancies and 2005–2014 for natalizumab-treated patients, it is still estimated that these three primary PML populations contribute >90% of all PML cases. Taking into account that even in the population with the most favorable outcome, 40% of survivors are left with major disabilities, this means only a small proportion of all PML patients (estimated ~20%) will have a relatively favorable outcome, i.e. survival beyond 2 years with mild to moderate disability.

Current approach to PML treatment and prophylaxis

Currently there is neither a specific prophylaxis for PML, nor an effective anti-JCV treatment in the setting of PML. In the HIV+ population, timely treatment with highly effective cART is an effective way of preventing the onset of acquired immune deficiency syndrome (AIDS) and with it PML, which is a constitutive part of this syndrome in ~5% of AIDS patients [Berger et al. 1998]. The incidence of AIDS and PML has declined significantly since cART was introduced [Engsig et al. 2009; Khanna et al. 2009].

In the absence of an effective anti-JCV treatment, PML outcomes depend entirely on an individual’s capacity to recover immune system function and respond to JCV. In some cases this can happen after reversal of immunosuppression caused by a disease (e.g. cART in HIV+) or removal of immunosuppressive/immunomodulating treatments (e.g. natalizumab) that had enabled the development of PML. Most natalizumab-treated patients with PML are able to recover immune surveillance to JCV within the central nervous system (CNS) once the treatment limiting cellular immune response in the brain has cleared; this also happens in some HIV+ patients on cART. In fact, in these patients the response to JCV can be so vigorous (PML-IRIS) that it is necessary to inhibit the immune system with corticosteroids to prevent excessive damage to the infected tissue. Currently the most practical approach to treating PML is optimal immune reconstitution to control JCV without causing brain-damaging IRIS [Clifford, 2014]. In RRMS patients who developed PML while treated with natalizumab, immune reconstitution is achieved by plasma exchange (PLEX) in order to remove natalizumab from the peripheral circulation. Following this procedure, IRIS will develop in the majority of these patients within a few days to several weeks. This effect of immune reconstitution is balanced by corticosteroid therapy. Due to lack of tools that could predict the onset and severity of IRIS and because of concerns that a premature and overly aggressive corticosteroid treatment may be counter effective [Antoniol et al. 2012], most clinicians withhold corticosteroids until a well-demonstrated IRIS response is identified, and then conservatively titrate the inflammatory response [Clifford, 2014].

The approach to treatment of PML and IRIS in other PML groups is similar. This includes immune reconstitution by cART in case of HIV+ or discontinuation of immunosuppression in other PML populations, and aggressive corticosteroid treatment in case of IRIS. Unfortunately, in some PML populations immune reconstitution cannot be achieved for various reasons. For example, many of the patients with hematological malignancies who develop PML have depressed bone marrow or have been treated with drugs that cause long term immune cell depletion, even after treatment withdrawal. Immune reconstitution is possible in allograft organ recipients, but this could lead to fatal allograft rejection, thus preventing use of this treatment approach. The only effective treatment option for these PML patients would be a direct anti-JCV therapy, which is currently unavailable.

Potential candidates for PML treatment and prophylaxis

Potential candidates for treatment or prophylaxis of PML can be broadly categorized into three groups: antiviral agents, immune response modulators, and immunization strategies (Table 2).

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Table 2.

Drug candidates for PML treatment.

Class	Drug name	Mechanism of action	Preclinical evidence	Clinical experience
Antiviral agents
JCV cell entry inhibitors	Chlorpromazine	Block serotonin receptors	Inhibits JCV infection and replication in glial tissue culture [Pho et al. 2000; Atwood, 2001]	None
	Citalopram		All shown to block entry of JCV into serotonin 5HT2AR receptor transfected cell line [Elphick et al. 2004] Risperidone failed to prevent JCV infection of glial cells in vitro [Chapagain et al. 2008]	None
	Mirtazapine			Four case reports claiming benefit [Epperla and Yale, 2013; Park et al. 2011; Verma et al. 2007; Cettomai and McArthur, 2009].
	Risperidone			One case report claiming benefit [Kast et al. 2007].
	Ziprasidone			One case report claiming lack of effect [Kharfan-Dabaja et al. 2007].
Retrograde transport Inhibitors	Retro-2cycl	Inhibits retrograde transport of polyomaviruses to the endoplasmic reticulum.	Inhibits both initial virus infection [Maginnis et al. 2014; Nelson et al. 2013] and infectious spread in in cultured cells [Maginnis et al. 2014]	None
	Brefeldin A	Arf1 GTPase inhibitor. Inhibits transport to the ER and virus disassembly.	Treatment of SVG-A cells with Brefeldin A reduced JCV infectivity by 50% [Nelson et al. 2012]	None
Inhibitors of DNA replication	Cidofovir	Inhibits viral replication by incorporating itself into viral DNA, and by inhibiting viral DNA polymerases	None for JCV. Inhibits BK polyomavirus infection of renal tubular epithelial cells [Bernhoff et al. 2008]	Clinical study with PML subjects [Marra et al. 2002] and clinical data meta-analysis [De Luca et al. 2008] showed no benefit
	Brincidofovir [CMX001]	Lipid-ester derivative of cidofovir	Inhibits JCV replication in human brain derived cells [Hou and Major, 1998; Gosert et al. 2011; Jiang et al. 2010]	One case report claiming benefit [Patel et al. 2010].
				Clinical study in subjects with BK polyomavirus infection showed reduction in incidence of BKV associated hematuria and serum creatinine [Mommeja-Marin et al. 2012]
	Cytarabine	Inhibits both DNA and RNA polymerases and nucleotide reductase enzymes.	Effective in decreasing JCV replication in vitro [Hou and Major, 1998]	Clinical study with PML subjects showed no benefit [Hall et al. 1998].
	Ganciclovir	Inhibits viral DNA polymerases.	Concomitant inhibition of cytomegalovirus and JC virus replication in human fibroblasts [Heilbronn et al. 1993].	One case report claiming benefit [Demir et al. 2005]
	Leflunomide	Inhibits DNA and RNA synthesis by inhibiting the mitochondrial enzyme dihydroorotate dehydrogenase involved in de novo pyrimidine synthesis.	Suppression of BK virus replication in kidney epithelial cells [Bernhoff et al. 2010], in vitro suppression of BK and JCV [Josephson et al. 2006]	One case report claiming benefit [Epker et al. 2009]
				None on JCV. BK virus study completed and result published [Williams et al. 2005]
	Topotecan	Inhibits DNA replication by inducing double-stranded DNA breaks during DNA replication.	Suppressed replication of JCV DNA in human glioblastoma cells [Kerr et al. 1993]	Clinical study with PML subjects was inconclusive [Royal et al. 2003].
Anti-malarials	Mefloquine	Unknown	Inhibit JCV infection and replication in cultured cells [Brickelmaier et al. 2009]	Clinical study terminated prematurely; interim analysis showed no difference between the treatment groups [Clifford et al. 2013]
Poly ADP-ribose polymerase 1 (PARP-1) inhibitors	3-AB (3-aminobenzamide)	Inhibits DNA replication by inhibiting single strand DNA breaks repairs	In vitro suppression of JCV replication and propagation in neuroblastoma cell line [Nukuzuma et al. 2013]	None
Tyrosine kinase inhibitors	Inhibikase IkT-001Pro (imatinib)	Unknown mechanism for JCV infection	None	None
Silencing RNA (siRNA)	Ag122	siRNA against JCV agnoprotein	Ag122 siRNA treatment effectively inhibits JCV infection in vitro in SVG-A cells. Ag122 injected into the brains of nude mice 4 days after injecting JCV-positive JCI cells significantly reduced the percentage of JCV-infected cells compared with the control treatments [Orba et al. 2004].	None
Immune response modulators
Cytokines	IFN-alpha	Stimulates innate and adaptive cell-mediated immune responses against viral infection (e.g. hepatitis B & C).	None	Retrospective study suggested increased survival compared to historical controls. [Huang et al. 1998].
				Prospective pilot clinical study with PML subjects showed no benefit [Berger et al. 1992].
	IL-2	Stimulates growth of T-cell lymphocytes and provides other biochemical signaling to the immune system	None	Benefit claimed in individual case reports [Przepiorka et al. 1997; Re et al. 1999; Buckanovich et al. 2002; Kunschner and Scott, 2005]
	IL-7 (CYT107)	Stimulates proliferation of all cells in the lymphoid lineage and their development, survival and homeostasis.	None	Benefit claimed in individual case reports [Patel et al. 2010; Gasnault et al. 2014; Alstadhaug et al. 2014]
Inflammation inhibitors	Maraviroc	Blocks CCR5 mediated tissue inflammation	None	Benefit claimed in one case report [Giacomini et al. 2014]
	Glucocorticoids	General immune system suppression	None	Benefit claimed in individual case reports [Dahlhaus et al. 2013; Tan et al. 2009]
Immunization
Passive immunization	Recombinant human anti-JCV VP-1 monoclonal antibodies	Neutralization of JCV	Neutralize wild type JCV Mad-4 strain and prevent infection of SVG-A cells. [Grimm et al. 2014]	none
	JCV-specific cytotoxic T lymphocyte (CTL) therapy	Lysis and clearance of JCV-infected cells	None	Benefit claimed in one case report [Balduzzi et al. 2011]
Active immunization	IL-7 + JCV VP1 Vaccine	JCV capsid protein with added recombinant IL-7 to boost JCV-specific T cell responses.	None	Benefit claimed in two case reports [Sospedra et al. 2014]
	JCV oral vaccine	JCV peptide antigens adapted to trigger a JCV-specific immune response in the human intestinal tract.	None	None

Antiviral agents can be further subcategorized into three major groups: JCV cell entry inhibitors, JCV cellular retrograde transport inhibitors, and inhibitors of DNA replication. JCV entry into permissive cells is mediated by attachment to host cell sialic acids and facilitated by serotonin receptors [Elphick et al. 2004; Maginnis et al. 2014; Nelson et al. 2012; Atwood, 2001]. Several compounds targeting the serotonin receptor have shown antiviral activity in vitro: chlorpromazine, citalopram, risperidone, ziprasidone, and mirtazapine [Atwood, 2001; Elphick et al. 2004]. The inhibitory effect of risperidone was later challenged by negative findings in glial cells [Chapagain et al. 2008]. One case report claimed treatment with risperidone was beneficial [Kast et al. 2007]. Several case reports published in the literature claimed beneficial effects of mitrazapine for the treatment of PML [Epperla and Yale, 2013; Park et al. 2011; Verma et al. 2007; Cettomai and McArthur, 2009]. One case report was published for ziprasidone, claiming lack of effect [Kharfan-Dabaja et al. 2007]. Since chlorpromazine has also been shown to inhibit clathrin-dependent endocytosis, inhibition of JCV infection of glial cells may occur at two points during the infection process: attachment of JCV to host cells, and internalization by clathrin-dependent endocytosis [Pho et al. 2000; Atwood, 2001].

Two compounds, retro-2cycl and brefeldin A, are inhibitors of retrograde transport of JCV to the endoplasmic reticulum, a step necessary for disassembly of virions in the cell and productive infection [Querbes et al. 2006; Nelson et al. 2012, 2013]. Both retro-2cycl and brefeldin A were shown to inhibit JCV infection in vitro [Maginnis et al. 2014; Nelson et al. 2012, 2013], and retro-2cycl was also shown to inhibit infectious spread in cultured cells [Maginnis et al. 2014].

Inhibitors of DNA replication are a broad spectrum of small-molecule chemotherapeutics that interrupt DNA synthesis. JCV is a DNA virus, and thus these drugs are expected to have an inhibitory effect on JCV replication. Nucleoside analog cytarabine, and nucleotide analogs cidofovir and brincidofovir (CMX001) are chemical entities that block DNA polymerase by competing with nucleotides to be incorporated into growing DNA strands. Cytarabine and brincidofovir inhibit JCV replication in human brain derived cell lines [Hou and Major, 1998; Gosert et al. 2011; Jiang et al. 2010]. Cidofovir is able to limit replication of another polyomavirus closely related to JCV, the BKV, in renal tubular epithelial cells in vitro [Berhoff et al. 2008]. However, their actions are not specific to viral DNA and can also affect host cell DNA, resulting in toxicity at higher doses. Cytarabine and cidofovir were studied in PML treatment clinical studies. Both drugs failed to show any benefit and they are discussed in more detail below. One individual PML case report claimed benefit after treatment with brincidofovir, following unsuccessful treatment with risperidone, IV cidofovir and mefloquine [Patel et al. 2010]. In a phase IIb clinical study in subjects with pre-existing BKV infection, brincidofovir appeared to reduce the incidence of BKV-associated hematuria and serum creatinine [Mommeja-Marin et al. 2012].

Ganciclovir, topotecan, and leflunomide are small molecules that act at different stages of RNA and DNA replication, such as DNA and RNA polymerases and nucleotide reductase enzymes, causing DNA damage, or inhibiting chain elongation and duplication during DNA synthesis, or cell cycle, respectively. These compounds are effective at decreasing JCV or BKV replication in vitro [Heilbronn et al. 1993; Bernhoff et al. 2010; Josephson et al. 2006; Kerr et al. 1993]. Relatively successful treatment of PML was claimed with ganciclovir and leflunomide in individual case reports [Demir et al. 2005; Epker et al. 2009]. A clinical study of topotecan did not demonstrate any benefit, and is discussed below. In a leflunomide study in subjects with biopsy-proven BKV nephropathy, all subjects with blood levels of leflunomide that remained above a level consistent with concentrations required for blockade of BKV replication in vitro, had either clearance of the virus or progressive reductions in the viral load in blood and urine [Williams et al. 2005].

Other potential anti-JCV drug candidates are mefloquine, poly-ADP ribose polymerase (PARP) inhibitors, IkT-001Pro, and Ag122. Mefloquine, an antimalarial quinolone, was shown to inhibit JCV DNA replication in human glial cell line SVG-A, primary human fetal glial cells, and primary human astrocytes, in concentrations generally achieved in the brains of patients given mefloquine for malaria [Brickelmaier et al. 2009]. A clinical study testing mefloquine in subjects with PML was conducted; however, it was terminated prematurely because it failed to achieve the primary endpoint of reduction of JCV DNA in cerebrospinal fluid (CSF) [Friedman, 2011; Clifford et al. 2013]. This study is discussed in more detail below. A PARP-1 inhibitor, 3-aminobenzamide (3-AB), was tested in vitro and showed significant suppression of JCV replication and propagation in neuroblastoma cell lines [Nukuzuma et al. 2013]. IkT-001Pro is an engineered medication whose active ingredient, imatinib, is a host-directed protein kinase inhibitor shown to disrupt JCV replication. Inhibikase, the company that is developing this compound, claims that IkT-001Pro can prevent replication of JCV. The company received Orphan Drug Designation for imatinib to treat PML from the U.S. Food and Drug Administration (Newman M,. 2014). Ag122, a siRNA targeting JCV agnoprotein, was shown to reduce the percentage of JCV infected SVG-A cells in vitro [Orba et al. 2004] and in vivo in brains of mice when it was injected 4 days after instillation of engineered JCV-carrier cell line JCI [Matoba et al. 2008].

Immune modulation treatment is focused on the restoration of protective anti-JCV responses in PML or inhibition of exaggerated immune responses in PML-IRIS. The first group is represented by cytokines and antiretroviral drugs used to treat HIV+ patients. Type 1 interferons (IFNs) are able to stimulate direct antiviral mechanisms in cells, and IFNα has been developed for treatment or prevention of viral infections, such as hepatitis B and C virus and oral human papillomavirus. One retrospective study claimed survival prolongation in HIV+ PML patients treated with IFNα [Huang et al. 1998], but recombinant α2a interferon was shown to be ineffective in a pilot study for the treatment of AIDS-related PML as reported at the American Academy of Neurology meeting in 1992 [Berger et al. 1992]. Interest for type I IFNs as a drug candidate for treatment of PML further diminished following an outbreak of PML cases in patients simultaneously treated with natalizumab and IFNβ, suggesting that type I IFNs are unlikely to be an effective treatment option. In addition, IFNβ has been demonstrated to have no effect on absolute copy numbers or frequency of JCV expression in the urine of MS patients, further suggesting its lack of efficacy in suppressing JC viral replication [Miller et al. 2012].

Attempts to restore depleted T-cell responses in JCV-PML have employed interleukin-2 (IL-2) and interleukin-7 (IL-7). IL-2 stimulates the growth of T cells and other lymphocytes and provides other biochemical signaling to the immune system [Gaffena and Liub, 2004]. IL-2 treatments administered to PML patients with lymphopenia showed positive results in case reports in which patients displayed steady improvement in neurological and cognitive functioning, and in some cases near-complete resolution of symptoms and magnetic resonance imaging (MRI) abnormalities, coupled with recovery of CD4 counts [Przepiorka et al. 1997; Re et al. 1999; Buckanovich et al. 2002; Kunschner and Scott, 2005].

IL-7 plays a key role in the development, proliferation, survival, and homeostasis of all lymphocytes [Mackall et al. 2011]. Some promising results have been reported with IL-7 treatment of PML patients given as a single agent, together with or following treatment with antivirals such as mirtazapine or cidofivir, or as a component of a JCV vaccine discussed below [Alstadhaug et al. 2014; Patel et al. 2010; Gasnault et al. 2014].

The second group of immune modulators consists of drugs that are used to reduce an over-reactive immune response to JCV-infected brain tissue. Corticosteroids are currently used as standard of care for PML-IRIS [Dahlhaus et al. 2013; Tan et al. 2009]; however, no controlled studies have been conducted to prove its effectiveness.

The newcomer in this category is maraviroc, a C–C chemokine receptor type 5 (CCR5) antagonist that blocks CCR5-mediated inflammation. Maraviroc is a drug approved for the treatment of CCR5 tropic HIV infection, but it was also claimed to reduce graft-versus-host disease in patients treated with allogeneic bone marrow transplantation [Reshef et al. 2012], and CCR5 receptor was found to be implicated in IRIS pathophysiology in natalizumab associated PML [Schwab et al. 2012]. Successful treatment with maraviroc was reported in a case of natalizumab associated PML [Giacomini et al. 2014]. While on maraviroc, the patient had stable disease with no signs of IRIS, and CSF showed a selective decrease in CCR5-positive immune cells. After accidental omission of maraviroc treatment for a period of 5 days, the patient presented with cognitive and behavioral changes and an MRI consistent with IRIS. Treatment with maraviroc was reintroduced and IRIS subsided. The dose of maraviroc was reduced after imaging evidence of IRIS regression, and the dose was eventually tapered off once the lesions on MRI were stable and no longer showed gadolinium enhancement. In this patient, IRIS was controlled without use of corticosteroids.

Immunization strategies are novel concepts for prophylaxis or treatment of PML. A form of passive immunization is being developed by Neurimmune Holding Ag, which is developing recombinant JCV VP1-specific monoclonal antibodies (mAbs) generated from memory B-cell pools from human donors, including donors recovered from PML and PML-IRIS [Grimm et al. 2014]. Antibodies generated from donor pools were either JCV specific or also cross-reactive to VP1 protein from BKV. In addition to binding to wild-type JCV VP1, these mAbs are also able to bind to the most common PML-associated VP1 mutants. A lead mAb has been selected and is being further characterized.

Another form of passive immunization was reported in a recent case describing a JCV-specific cytotoxic T lymphocyte (CTL) treatment of a patient with PML following hematopoietic stem cell transplantation and immunosuppression for graft-versus-host disease [Balduzzi et al. 2011]. JCV antigen-specific CTLs were generated from stem cell donor peripheral blood mononuclear cells by in vitro stimulation with overlapping peptide pools spanning JCV VP1 and large T (LT) antigen. The CTLs were then transferred to the PML patient upon cessation of imatinib and budesonide treatment. The patient also received citalopram and cidofovir following immune suppressive treatment discontinuation, and overlapping with the first of two CTL infusions. CTL activity against JCV VP1 and LT that had been absent pre-infusion was detectable post-infusion. The virus was cleared from the CSF, and the patient showed clinical and MRI improvement.

Active immunization with JCV vaccine was proposed as prophylaxis for PML (reviewed by Katona [2009]). Potential JCV vaccines are in various stages of drug development. These include an oral JCV vaccine containing one or more JCV antigens in a carrier adapted to trigger a JCV-specific immune response in the human intestinal tract [Boland, 2012], a vaccine consisting of recombinant JCV major capsid protein VP1 CD4 T-cell epitope formulated with an adjuvant and recombinant human IL-7 to boost JCV-specific T-cell responses [Martin et al. 2011], and vaccines consisting of IL-7 and JCV virus-like particles (VLPs) consisting of JCV VP1 protein in the form of a conformational spherical particle resembling virus [Jelcic et al. 2013]. Positive result of immunization with IL-7 and JCV VP1 vaccine was claimed in a case report showing that vaccination of 2 PML patients resulted in an increase in neutralizing titer of antibodies against both wild-type (archetype) and PML mutant JCV. This correlated with resolution of PML progression as determined by brain MRI, slight contrast enhancement around the PML lesions as determined by brain MRI, induction of robust JCV VP-1 specific CD4 T cell proliferation, substantial reductions in JCV DNA viral load in CSF, and clinical improvement with slight delay after developing a JCV-specific immune response [Sospedra et al. 2014].

PML treatment clinical studies and their results

Over the past 20 years, only six prospective interventional clinical studies that were specifically designed to test potential treatment for PML have been conducted. These studies were either completed or prematurely terminated, and the results of five of them were published. Key features, including the study design, inclusion criteria for subjects, intervention, and measured outcomes of the five published studies are summarized in Table 3.

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Table 3.

Published clinical studies.

Study	Year	Status	Intervention	Number of subjects	Study design	Measured outcome	Population	Result
A Phase II Multicenter Study Comparing Antiretroviral Therapy Alone to Antiretroviral Therapy Plus Cytosine Arabinoside (Cytarabine; Ara-C) for the Treatment of Progressive Multifocal Leukoencephalopathy (PML) in Human Immunodeficiency Virus (HIV)-Infected Subjects	1994–1997	Completed	Cytarabine	Planned 90,Randomized 57	Phase II, open label, randomizedThree groups: HAART alone, HAART + intravenous cytarabine, HAART + intrathecal cytarabine	Rate of survival	HIV+	Only seven patients completed 24 weeks of treatment; 14 patients in each group died; no differences in survival among the three groups [Hall et al. 1998].
						PML ⩽2 monthsLife expectancy ⩾3 months
An Open, Comparative Phase II Study of Immediate Versus Delayed Treatment With Topotecan HCl Given as a Continuous 21-Day Infusion Every 28 Days to Patients With AIDS-Related Progressive Multifocal Leukoencephalopathy	1997–1999	Completed	Topotecan	Planned 54,Randomized 12	Phase II, open label, randomized, immediate versus 8 weeks delayed treatment	Safety and tolerability; Kurtzke score assessment	HIV+	11 evaluable subjects, 10 on immediate, one delayed treatment3/11 treatment responders, one partial response [Royal et al. 2003]
A Pilot Study of the Effect of Cidofovir for the Treatment of Progressive Multifocal Leukoencephalopathy (PML) in Subjects With Acquired Immunodeficiency Syndrome (AIDS)	1999–2001	Completed	Cidofovir	24	Phase I, open label	Primary: Safety and tolerability and change in neurological examination scores between baseline and week 8Secondary: Change in the concentration of JCV DNA in CSF and PBMC, and change in MRI abnormalities	HIV+	12 deaths within 24 weeks, no improvements of neurological examination scores
							PML ⩽3 months	No response to HAART [Marra et al. 2002].
							Life expectancy ⩾6 months
Early Intensification of Combination Antiretroviral Therapy Including FUZEON® in the Treatment of Progressive Multifocal Leucoencephalopathy During HIV-1 Infection	2005–2007	Completed	cART	Planned 30,	Phase II, open label	Primary: Rate of survival at 12 months	HIV+	Early use of five-drug cART after PML diagnosis appears to improve survival. This is associated with recovery of anti-JCV T-cell responses and JCV clearance from CSF [Gasnault et al. 2011].
				enrolled 28		Secondary: Functional score at M12, JC V CSF load and JCV CSF clearance at M3 and M6, CD4 & CD8 T sub-populations and JCV specific T-cell responses at 12 months, and concentration of enfuvirtide in the CSF	PML ⩽3 months
A Randomized, Rater-Blinded Study to Explore the Effect of Mefloquine in Subjects With Progressive Multifocal Leukoencephalopathy(PML)	2008–2010	Terminated	Mefloquine	37	Phase I/II, randomized to standard of care (SOC) or SOC+mefloquine;	Primary: Change in JCV DNA levels in CSF in 4 weeks and 8 weeks from baselineSecondary: Change in KPSI, EDSS, SDMT, VAS, changes in lesions at week 8 from baseline, and assessment of mortality at 6 months	HIV+ and HIV-PML ⩽ 6 monthsLife expectancy ⩾6 months	Only 12 patients completed treatment. Study terminated prematurely, interim analysis showed no difference [Clifford et al. 2013].

The first study for the treatment of PML, a phase II study comparing antiretroviral therapy alone to antiretroviral therapy plus cytosine arabinoside (Cytarabine; Ara-C), was conducted between 1994 and 1997. Cytosine arabinose is a nucleoside analogue that interferes with DNA synthesis and is used for the treatment of hematological cancers. In the 1970s, this drug was also considered as a potential treatment for herpes simplex encephalitis and generalized herpes simplex. Prior to this study, there were anecdotal reports of PML improvement with cytarabine [Moreno et al. 1996]. The study enrolled 57 of the initially planned 90 HIV+ subjects with biopsy-confirmed PML [Hall et al. 1998]. They were randomly assigned to receive antiretroviral therapy alone, antiretroviral therapy plus intravenous cytarabine, or antiretroviral therapy plus intrathecal cytarabine. Active treatment was to be given for 24 weeks, but only seven subjects completed the 24 weeks of treatment. The primary outcome of this study was overall rate of survival. There were no significant differences in survival among the three groups, with median survival of 11, 8, and 15 weeks, respectively. The conclusion of this study was that neither intravenously nor intrathecal administration of cytarabine improved the prognosis of HIV+ subjects with PML who were treated with the antiretroviral agents.

The second study, conducted between 1997 and 1999, was a phase II study of immediate versus 8-week delayed treatment with topotecan in HIV+ subjects with PML [Royal et al. 2003]. Topotecan is an anticancer drug that causes double-stranded DNA breaks during DNA replication, which eventually leads to death of the most actively replicating cells. Because the mechanism of action is not cancer-specific, use of this drug is associated with significant toxicities, and in particular bone marrow depression. Prior to this study, the only evidence that topotecan may be effective in treatment of PML came from an in vitro experiment showing that camptothecin, of which topotecan is an analogue, inhibits replication of JCV DNA expressed in a chimeric plasmid [Kerr et al. 1993]. The study enrolled only 12 of the initially planned 54 subjects. One subject died due to accidental overdose of topotecan, and five subjects had dose delays, all due to hematologic adverse events. The primary outcomes of this study were safety, tolerability, and response to treatment measured through Kurtzke score assessment. Clinical response was defined as an improvement in the Kurtzke score of ⩾0.5 and a radiological response of ⩾10%. Of 11 evaluable subjects, only one received delayed treatment. Three subjects who responded to treatment had higher pretreatment Karnofsky and lower Kurtzke expanded disability status scale scores than non-responders. Because of the small number of enrolled subjects, this study was inconclusive.

In 1999, two pilot studies of the potential treatment of PML in subjects with HIV infection were initiated: a study of the effect of cidofovir, and a study of efficacy of recombinant alpha interferon (IFN-A2b) and zidovudine. Both studies were completed, but only the results of the cidofovir study were published [Marra et al. 2002]. Cidofovir is an antiviral agent approved for treatment of cytomegalovirus retinitis. Prior to this study, evidence that cidofovir may be beneficial for treatment of PML was anecdotal [Taoufik et al. 1998; Brosgart et al. 1997]. Primary endpoints of the cidofovir study were assessment of safety, tolerability, and efficacy. Secondary endpoints were to assess JCV DNA in CSF and peripheral blood mononuclear cells and to assess changes in PML lesions by MRI. A total of 24 HIV+ PML subjects (with clinically manifested disease for 90 days or less) received cidofovir intravenously at baseline and 1 week, followed by infusions every 2 weeks. A total of 17 subjects were also receiving antiretroviral agents. Subjects were followed for 24 weeks. Survival at 12 weeks was 54%. Five subjects discontinued treatment because of toxicity. Two subjects experienced a ⩾25% improvement in neurological examination scores at week 8, but overall, MRI abnormalities and neurological examination scores worsened. The authors concluded that although cidofovir did not improve neurological examination scores at week 8, these scores were significantly better in subjects who entered with suppressed plasma HIV-1-RNA levels, which could be the result of control of HIV-1 infection itself or cidofovir. Subsequent to this study, an analysis of raw data from one prospective study and five cohort studies concluded that cidofovir in combination with antiretroviral therapy did not influence mortality or residual disability in HIV+ PML subjects [De Luca et al. 2008].

A study of the efficacy of IFN-A2b and zidovudine was presented as an abstract at the American Association of Neurologist (AAN) meeting in 1992 [De Luca et al. 2008], and showed negative results. The rational for use of IFNα as a potential treatment for PML was the known antiviral effect of IFNs and the results of a retrospective study that claimed survival prolongation in HIV+ PML subjects treated with IFNα [Huang et al. 1998]. It is important to note that IFNα and IFNβ are no longer considered as potential treatments for PML, particularly after association of PML with natalizumab in subjects who were concomitantly treated with IFNβ.

One of the more recent studies, conducted between 2005 and 2007, investigated cART with three or more drugs in the treatment of PML [Gasnault et al. 2011]. This cART was designed to accelerate HIV replication decay and JCV-specific immune recovery. A total of 28 subjects received cART with three or more drugs for 12 months, plus the fusion HIV inhibitor enfuvirtide during the first 6 months. The primary endpoint was the 1-year survival rate. Secondary endpoints included functional score at 12 months, JC viral load in the CSF, percentage of patients with JCV clearance of the CSF after 3 and 6 months, CD4 and CD8 T cells subpopulations and anti-JC specific T-cell responses at 12 months, and concentration of enfuvirtide in the CSF. Seven patients died, all within first 4 months of treatment. The 1-year survival estimate was 0.75. The conclusion of the study was that early use of five-drug cART in HIV+ patients after PML diagnosis appears to improve survival, and that this is associated with recovery of anti-JCV T-cell responses and JCV clearance from CSF.

The latest study, conducted between 2008 and 2010, evaluated the effect of mefloquine on PML and factors that may predict PML outcomes [Clifford et al. 2013]. Mefloquine, which is an antimalaria drug, was selected as a potential candidate for treatment of PML based on demonstrated in vitro activity against JCV infection of human astrocytes and the expectation that it would achieve efficacious concentrations in the brain due to its ability to penetrate the CNS [Brickelmaier et al. 2009]. This randomized, open-label study compared subjects with PML who received standard of care (SOC) with those who received SOC plus mefloquine. Also, pati-ents who were randomized to SOC were allowed to add mefloquine treatment at week 4. The primary endpoint was the change from baseline to weeks 4 and 8 in JCV DNA load in CSF. The study was open to all PML patients, but a majority of enrolled subjects were HIV+. The study was terminated prematurely because preplanned interim data analyses suggested that the study was unlikely to demonstrate a significant difference between groups. There was no clinical evidence of anti-JCV activity by mefloquine.

No clinical studies aimed specifically at prophylaxis of PML have been conducted. One phase III study evaluated the effect of IFNγ on incidence of opportunistic infections in subjects with advanced HIV infection [Riddell et al. 2001]. Although this study failed to show a significant decrease in overall incidence of opportunistic infections and increased survival, it is noteworthy than none of the subjects in the IFNγ cohort developed PML as opposed to four subjects in the placebo cohort. This is a peculiar finding because treatment with IFNγ did not induce significant changes in lymphocytes, and the incidence of most of the other opportunistic infections was similar between the two cohorts. This raises the possibility that IFNγ may have a direct effect on JCV replication. A recently published finding that IFNγ is involved in the regulation of JCV gene expression via downregulation of the major viral regulatory protein, T-antigen, supports this hypothesis [De-Simone et al. 2015].

Lessons learned from PML treatment clinical studies

One of the most significant challenges in the development of therapeutics for PML is the lack of good animal PML models. Because of this limitation, it has been difficult to screen compound libraries or conduct proof-of-concept animal studies to identify promising therapeutic candidates. Therapeutic candidates tested in the previously described studies were selected based on theoretical consideration, observations in sporadic case reports, or on the compound’s demonstrated ability to suppress JCV replication in JCV-permissive cell lines in vitro. Thus the first challenge facing PML treatment studies was the relatively modest evidence of their candidate drug’s potential efficacy. The second and no less important problem with these drug candidates was dose selection. Lack of animal PML models meant that there were no means to define the potentially effective dose range in humans, and the small number of study subjects and urgency to treat did not favor dose finding. The third problem was the potential drug candidate’s ability to cross the blood–brain barrier (BBB) and reach a concentration that was sufficient to suppress JCV replication in the brain. Only one of the drugs tested in PML treatment studies, mefloquine, was known to efficiently cross BBB. Intrathecal administration of cytosine arabinose was provided to a subset of study subjects to compensate for the BBB obstacle. However, even a drug that efficiently crosses BBB may not be able to significantly suppress JCV replication. Drug candidates used in PML treatment studies affect DNA synthesis in a nonspecific way. This means that in addition to inhibition of JCV replication, they will also inhibit DNA synthesis in the host cells and in particular those that are rapidly dividing. In severe PML, JCV replication may be so highly active that even the highest tolerated dose would not be sufficient to significantly suppress JCV replication.

One of the PML treatment studies investigated use of cART to treat PML. cART does not have any direct impact on JCV, but induces rapid reconstitution of immune responses in HIV+ patients. cART is effective prophylaxis for AIDS and PML when used in a timely manner, but the benefit of cART in subjects with already active PML is more modest.

This raises the question as to whether any of the studied drugs was actually a viable PML treatment option. These drugs might have been better suited for prevention of PML in high-risk patients rather than treatment of active disease. Also, a strategy that combines more treatment options such as DNA synthesis inhibition, viral entry inhibition, and immune system response boosting may be more effective.

Recruitment of subjects for rare disease studies is always a challenge, and this was true for PML treatment studies. Of the five studies assessed, the largest sought to enroll 90 patients and ultimately enrolled 57; another enrolled 12 out of 54 planned. All other studies ranged from 24–30 patients. One recently initiated study was terminated early due to lack of enrollment. The reason for difficulties in study subject enrollment is not just the rarity of PML, but also the difficulties in diagnosing this disease. Clinical presentation is not PML-specific, and unfortunately PML occurs in populations that may have an underlying CNS disorder or are prone to develop other CNS disorders with similar clinical manifestations. Therefore enrollment of study subjects based on simplified criteria such as clinical presentation is not possible. Confirmation of PML requires either direct evidence from brain biopsies or characteristic brain imaging findings with demonstrated presence of JCV in CSF. These invasive diagnostic techniques are justifiable only if other causes of clinical CNS manifestations could be excluded based on differential diagnoses examination. This can substantially delay the diagnosis, which is likely to adversely impact the outcome. There is evidence that early diagnosis of PML, even prior to clinical manifestation, is associated with better clinical outcome [Phan-Ba et al. 2012]. It is conceivable that even an otherwise effective treatment could fail if implementation of treatment is delayed. Depending on the brain structures involved with demyelination, the damage may be irreversible by the time a PML diagnosis has been confirmed, thus rendering futile any therapeutic treatment of PML.

Survival was an appropriate primary outcome for earlier PML treatment studies, in which few study subjects were expected to survive beyond 3 months. Since the introduction of highly effective cART, most PML patients that are HIV+ are expected to survive well beyond 6 months. For that reason, more recent studies are no longer looking at death rates as a study outcome, but are instead focused on changes in neurological examination scores, MRI lesions, and changes in JCV DNA levels in CSF. Theoretically these changes could show evidence of efficacy, even if the final outcome is not different. For example, an effective treatment could extend life, limit neurological deficits, or improve quality of life while still having no impact on the final outcome: death rates. This approach was not successful in HIV+ PML study subjects treated with mefloquine. This does not exclude lack of effect as a possible explanation, but in case of HIV+ patients and natalizumab-associated PML, one must notice that improvements in neurological scores, MRI lesions, or JCV viremia resulting from immune reconstitution under cART/PLEX-accelerated natalizumab concentration reduction may mask the effect of any additional treatment. Because of the lethal consequences of PML, it is unethical to delay or restrict access to other therapies that increase survival and improve outcomes; however, this presents a challenge when developing new therapies.

Conclusions

PML remains a significant concern for individuals with compromised immune systems regardless of their medical history. Advances in HIV infection treatments considerably reduced incidence of PML in this population. However, HIV+ patients with PML still face poor outcomes, with ~50% of them dying within 2 years from disease onset. PML incidence in other high-risk populations is slowly but steadily increasing, mostly due to the introduction of new therapies that significantly extend the life expectancy of patients with cancer and autoimmune disorders, but also increase the risk for PML. Survival rates are better in multiple sclerosis patients who develop PML while being treated with natalizumab, but only a minority of these patients recover with relatively favorable outcomes (i.e. mild to moderate disability). Outcomes in other PML populations are poor. Therefore, there is still high unmet need for an effective prophylaxis and treatment of PML.

Completed and published PML treatment clinical studies reveal the difficulty of studying potential therapies. Identification and selection of appropriate PML treatment candidates is significantly restrained by the lack of an adequate animal PML model. Recruitment of study subjects is negatively impacted by the rarity of the disease, time required to confirm diagnosis of PML, and rapid progression of the disease. Highly active JCV replication, relatively short life expectancy in a proportion of study subjects, and/or concomitant treatment with cART likely compromises the detection of the investigational treatment’s impact.

Because all of the factors that have negatively impacted previous PML treatment studies are still present, there is no reason to expect that new treatment studies would be more successful in delivering positive results. Studies aimed at prevention of PML may have a better chance at delivering positive outcomes. A prophylactic treatment trial most likely to deliver positive outcome, measured as delayed onset, reduced severity or reduction in incidence of PML, should be studied in a population at high risk for developing PML with a drug that crosses BBB and has demonstrated inhibitory effect on polyomavirus in vitro and/or in clinical setting.

The same antiviral drugs that have already been evaluated in PML treatment studies might prove effective as PML prophylaxis. For example, cART has been shown to be an effective prophylaxis for AIDS and PML; however, it is less beneficial in treating active PML. There are several factors that favor PML prophylaxis studies. Smaller, well-tolerated dosage formulations of anti-JCV drugs might still be effective in controlling JCV replication, and might prevent or delay the emergence of pathologic JCV prototypes. A PML prophylaxis study in high-risk subjects would also be easier to conduct. Screening and recruitment of study subjects would be simplified and the primary outcome of confirmed PML would be easier to measure.

Some progress has been made in identifying potential treatments for PML, despite the constraints on development. Currently identified potential drug candidates can be divided into three categories based on their mechanism of action: anti-JCV therapies, immune modulators, and immunization strategies. A successful anti-JCV drug candidate should demonstrate in vitro activity against JCV, be able to cross the BBB, and have an acceptable toxicity profile. The toxicity profile may be the key limiting factor for many of the potential anti-JCV drugs, because even at the highest tolerated dose, they may still have insufficient impact on widely spread and highly active replicating JCV in the brain. Immune response modulators such as IL-2, IL-7, and IFNγ are not JCV specific, rather they boost immune system responses to viral infection in general. These drug candidates are promising both as prophylaxis and as treatment options; however, their use excludes some of the PML populations in which immune system response cannot be boosted due to chronic bone marrow depression, concerns for worsening autoimmune disease, or risk of allograft rejection in organ transplant recipients. For these PML populations, passive immunization would be an attractive alternative.