Research
Original Investigation
JC Virus in CD34+ and CD19+ Cells in Patients With Multiple Sclerosis Treated With Natalizumab Elliot M. Frohman, MD, PhD; Maria Chiara Monaco, PhD; Gina Remington, RN; Caroline Ryschkewitsch, MT; Peter N. Jensen, BS; Kory Johnson, PhD; Molly Perkins, PhD; Julia Liebner, BS; Benjamin Greenberg, MD; Nancy Monson, PhD; Teresa C. Frohman, PA-C; Daniel Douek, MD, PhD; Eugene O. Major, PhD
IMPORTANCE Infection with JC virus (JCV) may lead to development of demyelinating progressive multifocal leukoencephalopathy in patients with multiple sclerosis (MS) who are treated with natalizumab.
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OBJECTIVE To determine whether mononuclear cells in circulation from MS patients treated with natalizumab harbor JCV DNA. DESIGN, SETTING, AND PARTICIPANTS In this prospective investigation, we enrolled 49 MS patients from the Clinical Center for Multiple Sclerosis at The University of Texas Southwestern Medical Center and 18 healthy volunteers. We drew 120-mL blood samples from 26 MS patients at baseline and at approximately 3-month intervals to 10 months during the course of natalizumab infusions. One blood sample was drawn from 23 MS patients receiving natalizumab for more than 24 months and from 18 healthy volunteers. INTERVENTIONS Natalizumab treatment of MS. MAIN OUTCOMES AND MEASURES The blood samples were separated using flow cytometry into CD34+, CD19+, and CD3+ cell subsets; DNA templates were prepared using quantitative polymerase chain reaction for JCV DNA identification. Plasma samples were tested for anti-JCV antibodies by enzyme-linked immunosorbent assays performed at the Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological and Communicative Diseases and Stroke. RESULTS Thirteen of the 26 patients (50%) with baseline and follow-up blood samples had detectable viral DNA in at least 1 cell compartment at 1 or more points. Ten of the 23 patients (44%) receiving treatment for more than 24 months and 3 of the 18 healthy volunteers (17%) also had detectable viral DNA in 1 or more cell compartment. Fifteen of the 49 MS patients (31%) were confirmed to harbor JCV in CD34+ cells and 12 of 49 (24%) in CD19+ cells. Only 1 of 18 healthy volunteers were viremic in CD34+ cells and none in CD19+ cells. Nine patients and 1 healthy volunteer were viremic but had seronegative test results for JCV antibodies. CONCLUSIONS AND RELEVANCE JC virus DNA was detectable within cell compartments of natalizumab-treated MS patients after treatment inception and longer. JC virus DNA may harbor in CD34+ cells in bone marrow that mobilize into the peripheral circulation at high concentrations. Latently infected cells initiate differentiation to CD19+ cells that favors growth of JCV. These data link the mechanism of natalizumab treatment with progressive multifocal leukoencephalopathy.
JAMA Neurol. 2014;71(5):596-602. doi:10.1001/jamaneurol.2014.63 Published online March 24, 2014. 596
Author Affiliations: Author affiliations are listed at the end of this article. Corresponding Authors: Elliot M. Frohman, MD, PhD, Department of Neurology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390 (elliot.frohman@utsouthwestern .edu); Eugene O. Major, PhD, Laboratory of Molecular Medicine and Neuroscience, National Institutes of Health, 10 Center Dr, Room 3B14A, Bethesda, MD 20892 ([email protected]). jamaneurology.com
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JC Virus in CD34+ and CD19+ Cells in MS Patients
S
ince the reintroduction of natalizumab therapy in 2006 for relapsing remitting forms of multiple sclerosis (MS), more than 440 cases of progressive multifocal leukoencephalopathy (PML), the central nervous system virus– induced demyelinating disease, have been reported. The highest incidence of PML is approximately 1:80 in patients with a combination of risk factors, including receiving 24 or more natalizumab infusions, a history of immunosuppressive treatment, and seropositivity for antibodies to the JC virus (JCV). The overall incidence of PML in natalizumab-treated MS patients is 1:250, with approximately 11 new cases reported each month.1,2 Several hypotheses have focused on the direct association of natalizumab treatment and PML, particularly because few other opportunistic infections occur in this group.3 One putative mechanism centers on the lack of immune surveillance in the central nervous system because natalizumab blocks extravasation of T and B lymphocytes into the brain.4 This concept is predicated on the presence of JCV in the brain in a latent or persistent state of residency so that, at times of protracted immune suppression, a productive amplification of viral infection is established. Although this concept may be partly true, it does not explain why PML has not been reported in MS patients treated with a variety of other immunosuppressive agents (eg, azathioprine, mycophenolate, mitoxantrone hydrochloride, and rituximab). However, if protracted immune suppression was the primary mechanism of PML in such patients, we would anticipate PML occurring in MS patients whose immune systems are more severely compromised than seen in natalizumab-treated patients. Further, JCV infection in the brains of MS patients not treated with natalizumab has not been reported. The only reports of PML in MS patients are those treated with natalizumab.5 Another proposed mechanism to explain the unique link between natalizumab treatment and PML is the drug’s effect on the mobilization of mononuclear cells from the bone marrow,6,7 some of which may harbor latent JCV infection.8-10 These observations are not mutually exclusive, however, and may actually work in concert to provide the circumstances in which 2 very different demyelinating diseases can occur in the brain of a single patient. To investigate the link between natalizumab treatment and the presence of JCV in lymphoid cells, peripheral blood samples were collected from MS patients before the first dose of natalizumab, on the day treatment was initiated, and periodically during the course of the next 10 monthly infusions. Blood samples were also collected at a single point from MS patients who received at least 24 infusions and from healthy volunteers who were matched by age and sex to the natalizumabtreated MS patients.
Methods
fusion (baseline) and then at approximately 3-month intervals, depending on the patients’ return to clinic. The samples were immediately shipped overnight to the Laboratory of Molecular Medicine and Neuroscience (LMMN), National Institute of Neurological and Communicative Diseases and Stroke, for cell separation processing. Single 120-mL samples were collected from 23 MS patients undergoing more than 24 infusions at the Clinical Center for Multiple Sclerosis, and 150-mL samples of blood were collected from the healthy volunteers at the Transfusion Medicine Blood Bank of the National Institutes of Health. Signed informed consent was obtained in accordance with the respective institutional review boards.
Cell Isolation and Phenotypic Characterization Human CD34+ cells were enriched by positive selection using a CD34 purity selection kit (Stemcell Technologies Inc) or on lineage-depleted whole blood samples using a cocktail of monoclonal antibodies directed against CD2, CD3, CD14, CD16, CD19, CD24, CD36, CD56, and CD66b. Peripheral blood mononuclear cells and enriched CD34+ progenitor cells were washed in culture medium (RPMI-1640; Life Technologies) without fetal bovine serum (RO medium). The cells and RO medium were mixed with pretitered amounts of directly conjugated monoclonal antibodies using the following surface stains: for CD3 and CD8, fluorescein isothiocyanate conjugate; for CD19, peridinim-chlorophyll-cyanine 5.5 conjugated reagent or brilliant violet 421; for CD4, phycoerythrin; for CD34, phycoerythrin–cyanine 7; and for CD45, V500 (all reagents, BD Biosciences) in a total volume of 100 μL of RO medium. Cells were stained for 20 minutes at 4°C, washed and resuspended in 250 μL of phosphate-buffered solution, and then passed through a 40-μm strainer to ensure singlecell suspension. Flow cytometric analysis was performed immediately after this treatment.
Flow Cytometric Analysis Cells were analyzed using a flow cytometer (FACSAria II SORP; BD Biosciences) equipped for the detection of as many as 6 fluorescence variables. Events collected for each sample numbered from 5000 to 50 000. Electronic compensation was conducted with peripheral blood mononuclear cells stained separately with single monoclonal antibodies used in test samples. Electronic compensation was performed with antibody capture beads (BD Biosciences) for CD34+ cells only. The cells were then identified as mononuclear cells by sidescatter area and forward-scatter area and as singlet cells by forward-scatter height, forward-scatter width, side-scatter height, and side-scatter width. We defined and sorted CD34+, CD19+, and CD3+ cells by positive selection using the CD34+/CD45+, CD19+/CD45+, and CD3+/CD45+ gates, respectively.
Quantitative Polymerase Chain Reaction for JCV Genome
Patients Twenty-six MS patients starting natalizumab therapy (Tysabri; Biogen Idec) were recruited in the Clinical Center for Multiple Sclerosis at The University of Texas Southwestern Medical Center. To isolate sufficient CD34+ and CD19+ cells, 120-mL blood samples were collected immediately before the first injamaneurology.com
Original Investigation Research
Cell samples were examined using the validated, Clinical Laboratory Improvement Amendment–certified JCV quantitative polymerase chain reaction (PCR) method.11-13 Cellular DNA was extracted from a maximum of 5.0 × 106 cells per sample using a DNA blood and tissue kit (DNeasy, catalogue No. 69504; Qiagen) and eluted to a final volume of 25 μL. The primers and JAMA Neurology May 2014 Volume 71, Number 5
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Figure 1. Enrichment of CD34+ Cells and Gates Strategy Schematics A CD34+ Enrichment
B
CD2, CD3, CD14, CD16, CD19, CD24, CD36, CD56, and CD66b
All cells or CD34+-enriched cells
Gates Strategy
Label
P1 (mononuclear cells, FSC-A, and SSC-A) P2 (single cells, FSC-H, and FSC-W) P3 (single cells, SSC-H, and SSC-W) Incubate 20 min at room temperatue
CD34+/CD45+ (CD34+ cells) CD3+/CD45+ (T cells)
Layer over Ficoll-Paque
CD3–/CD45+ CD19+/CD45+ (B cells)
Ficoll-Paque
Spin
Plasma Enriched cells
Collect
Ficoll-Paque
Enriched cells (unrosetted)
Red cells (unwanted rosetted cells)
probes for the specific amplification and detection of the conserved N-terminal of the JCV T protein do not amplify BK virus or simian virus 40 DNA in this region. Extracted DNA templates were amplified using the PCR reagents (TaqMan Universal reagent, catalogue No. 4304437; Applied Biosystems) and a commercially available PCR system (7500 Real-Time System; Applied Biosystems). All samples were run in duplicate using 10 μL per reaction. The number of JCV genomes was calculated using the manufacturer’s system software and adjusted according to the dilution factors before reporting as copies per milliliter only when both duplicates detected viral DNA.
Enzyme-Linked Immunosorbent Assay for Determination of Anti-JCV Antibody The gene that codes for the major JCV structural protein, Vp-1, was amplified from SVG cells14 infected with the Mad-4 viral variant of persistently infected owl monkey 586 tumor cells.15 This amplified VP1 gene was ligated into the pBlueBacIII plasmid vector and cotransfected into Sf9 insect cells along with the baculovirus DNA expression vector system (BAc-N-Blue; Invitrogen) according to the manufacturer’s protocol. Subsequent proliferating baculovirus expressing the recombinant Vp-1 was used as the inoculum in Sf9 expansion cultures. Supernatant from the expansion cultures was ultracentrifuged through 40% (weight to volume ratio) sucrose cushions. The resulting recombinant Vp-1 pellets were subjected to deoxycholic acid treatment, low protein–binding polyethersulfone membrane filtration, and gradient banding on density gradient medium (OptiPrep; Nycomed) to remove cell lysate contaminants. This 598
A, Enrichment of CD34+ cells was achieved by positive selection using a cocktail of lineage-specific monoclonal antibodies. B, The enriched cells were further sort purified using a flow cytometer (FACSAria II SORP; BD Biosciences) gated on CD34+/CD45+. In parallel, peripheral blood mononuclear cells isolated by density gradient medium (Ficoll-Paque; Sigma-Aldrich) were identified using forward-scatter area (FSC-A) and side-scatter area (SSC-A) (gate P1). Subsequently, gate P2 forward-scatter height (FSC-H) and forward-scatter width (FSC-W) and P3 side-scatter height (SSC-H) and side-scatter width (SSC-W) were placed to include only single cells. Consequently, T and B cells were sorted by positive selection using the CD3+/CD45+ and CD19+/CD45+ gates, respectively.
highly purified recombinant Vp-1 was used as the antigen in establishing the enzyme-linked immunosorbent assay (ELISA) for determining the level of JCV antibody response present in the clinical samples. In the ELISA, 4× serial dilutions that generated an optical density reading of 0.05 greater than that of plate and well controls were labeled as positive at a dilution of 2560. The highest dilution of a sample registering positive findings was used to identify the titer of the sample.16 We compared the LMMN ELISA assay directly with a second-generation JCV antibody ELISA (STRATIFY JCV; Focus Diagnostics) supplied as a kit by the manufacturer using 17 plasma samples from these patients. Fifteen of the 17 samples showed identical serological results, whereas 2 other samples that the LMMN ELISA showed to have seronegative results had indeterminate results using the STRATIFY JCV ELISA after step 2 adsorption with viruslike particles as described below.
Results Natalizumab is known to promote mobilization of hematopoietic stem cells from the bone marrow, a putative site for JCV latency, into the peripheral circulation, resulting in higher-thannormal physiological levels of CD34+ cells for months to years after treatment. Therefore, we collected blood samples and separated them into CD34+, CD19+, and CD3+ cells using flow cytometry. The mononuclear cells were quantitated in relation to total number of peripheral blood mononuclear cells (Figure 1). We found an increased percentage of CD34+ cells from MS patients
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JC Virus in CD34+ and CD19+ Cells in MS Patients
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Figure 2. Comparison of Percentage of CD34+ Cells in Peripheral Blood Mononuclear Cells (PBMC) Between Healthy Volunteers and Patients With Multiple Sclerosis (MS) 7 6
CD34+Cells in PBMC, %
compared with healthy volunteers (Figure 2) as in previous observations.17 Percentages of CD34+ and CD19+ cells were persistently elevated in the blood samples from MS patients treated with natalizumab starting at 3 months and for extended periods. Although all blood samples were separated into cell compartments by flow cytometry, the data in Table 1 report only those samples confirmed to harbor detectable levels of JCV DNA. Patients described in Table 1 reflect the acquisition of the samples that were sent to the LMMN for analysis and not the total number of patients in either cohort. Blood samples were collected at baseline (before infusion) and at approximately 3-month intervals to 9 or 10 months, depending on patient status, for the 26 patients initiating treatment. A total of 92 samples from this cohort (4 samples of 120 mL each) underwent analysis. Table 1 shows the study participants with viremia, the specific compartment in which viremia was detected, the number of infusions, and the antibody titer of the plasma at the time of testing. Antibody titers of 2560 or greater are considered seropositive.16 Table 1 also shows that 3 of 18 healthy volunteers (17%) were viremic, although only in 2 cell compartments (CD19+ cells in 2 and CD34+ cells in 1) for a mean of 66 copies/mL of viral DNA. In the cohort just initiating treatment, 19 of 92 samples (21%) from 13 of 26 patients (50%) had detectable viral DNA. Three of these 13 patients (23%) had detectable viral DNA in CD34+ and CD19+ cells. In the cohort of 23 patients with more than 24 infusions from whom only 1 blood sample was collected, 10 (38%) had detectable viral DNA in cell compartments, including CD34+ and CD19+ cells in 1 of these patients. Regardless of the compartment in which viral DNA was detected across all 3 groups, the copy number of viral genomes was low, particularly when compared with the numbers usually seen in virus excreted into the urine, or viruria. None of the samples from any group had detectable viral DNA in the CD3+ T cells, the cell control from each patient irrespective of group designation. JC virus has not been shown to bind to or infect T cells.19 When both patient groups were combined (Table 2), 15 of 49 patients (31%) were viremic in CD34+ cells at different times, including 2 patients at baseline with a mean of 25 copies/mL in the initiating group and 212 copies/mL in the longer treatment group. Twelve of the 49 patients (24%) were viremic in CD19+ cells, with a mean of 96 copies/mL in the initiating group and 543 copies/mL in the longer treatment group. The plasma viremia in these patients has been reported previously.18 Seventeen patients (35%) had plasma viremia. Six patients were viremic only in the plasma (patients 3, 8, 10, 33, 36, and 48), including 4 at baseline with a mean of 86 copies/mL in the initiating group and 67 copies/mL in the longer treatment group. Three patients were found to be viremic in all 3 compartments (patients 2, 13, and 40). The high copy number in cell compartments in patient 40, who had 30 infusions, was much larger than those of the others. In general, however, the viral copy number was higher in the longer treatment group, with a mean of 230 copies/mL compared with 71 copies/mL in the initiating group. Analysis of viremia in the longer treatment group showed that viral DNA was primarily localized within CD34+ cells at a mean of 212 copies/mL compared with 25 copies/mL in the initiating group and within CD19+ cells at a mean of 543 copies/mL in the longer treatment group compared with 96 cop-
Original Investigation Research
P 24 natalizumab infusions
marrow lymphoid precursors compared with myeloid precursors, resulting in chronic increases in T- and B-cell populations in the peripheral circulation. Marginal zone–like memory B cells significantly increased expression of CD49d, which enables harboring in marginal zones in lymph nodes. Together these studies provide evidence that supports the role of these cell populations as possible disseminators of JCV because natalizumab triggers the mobilization of cells potentially infected with JCV into the circulation. The cell populations described in the present study showed the same pattern of responses with natalizumab treatment, a finding that persisted for several years (Figure 2). The observation of JCV latency within these characterized mononuclear cells makes a direct mechanistic link between natalizumab and the occurrence of PML. We used the highly sensitive, specific, and vali600
Only viremia data for 2 cell compartments are shown. Patients 4, 12, 19, 41, and 55 were viremic in the CD34+ cell compartment and in plasma. Patients 20, 26, and 52 were viremic in the CD19+ cell compartment and in plasma. Patients 2, 13, and 40 were viremic in all 3 compartments. Viremia in plasma only was reported previously18 (not shown) for patients 3, 8, 10, 33, 36, and 48.
dated quantitative PCR assay that provided the laboratory confirmation of PML diagnosis in nearly half of the MS cases in the present study to detect viral DNA in cell compartments. Detectable viral DNA in CD34+ cells was found in 15 of 49 patients (31%) and in CD19+ cells in 12 of 49 (24%) after initiation of treatment or after several years of continuous treatment. In contrast to our findings, other studies23,24 have not identified JCV DNA in CD34+ cells, suggesting that compartments such as bone marrow or lymphoid tissues may not be reservoirs for latent infections. However, the same studies failed to identify cytomegalovirus in CD34+ cells as a positive control in their study, despite evidence from multiple reports that confirm detection of cytomegalovirus DNA25,26 and JCV DNA9,10 in these lymphoid stem cells. The assays that did not detect JCV DNA in similar cell populations probably were not sensi-
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Table 2. Detectable JCV DNA by Quantitive Polymerase Chain Reaction MS Patients, No. (n = 49) Compartments With JCV DNA Detected
≤10 Infusions (n = 26)
>24 Infusions (n = 23)
Healthy Volunteers, No. (n = 18)
5
3
2
CD19 only
Viremia, No. (%)
MS patients
8/49 (16)
Healthy volunteers
2/18 (11)
CD34 only
5
6
11/49 (22)
Healthy volunteers
1/18 (6) 3
1
4/49 (8)
Healthy volunteers
0 13
10
23/49 (47)
Healthy volunteers
3/18 (17)
tive enough and probably were not used in sufficient numbers of cells to compare the outcomes of these investigations legitimately. In our study, more CD34+ cells were available from 120and 150-mL blood samples that were collected and processed within 24 hours, in contrast to the 18-mL samples used in the other reported study.25 In addition, the low viral copy number in our study is consistent with a latent or a persistent infection. In previous work,27,28 a cell culture model had shown that even a low level of persistence of JCV has significance because JCV can be transferred to other cells. In fact, JCV was first isolated by a similar blind passage in human fetal brain cells.29 Another important observation from our study is the identification of individuals with seronegative test findings but who were nevertheless viremic for JCV (Table 1). The ELISA used in the LMMN is validated using a 4-fold serial dilution of plasma with a cutoff titer of 640 or less, constituting seronegative status. Several samples had titers of 160, an unambiguous seronegative result. Of particular interest are patients 9 and 13, who are seronegative and viremic. In a separate analysis,30 both of these patients demonstrated T-cell responses to JCV proteins, indicating cell-mediated immune responses to JCV infection. These observations add to the complexity of finding a rigorous set of criteria for identifying patients at increased risk for PML when receiving natalizumab or similar therapies that modulate the immune system.
Accepted for Publication: January 27, 2014. Published Online: March 24, 2014. doi:10.1001/jamaneurol.2014.63. Author Affiliations: Department of Neurology, The University of Texas Southwestern Medical Center, Dallas (Elliot M. Frohman, Remington, Greenberg, Monson, T.C. Frohman); Department of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas (Elliot M. Frohman); Laboratory of Molecular Medicine and Neuroscience, National Institutes of Health (NIH), Bethesda, Maryland (Monaco, Ryschkewitsch, Jensen, Major); Bioinformatics Section, National
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.57 Abbreviations: JCV, JC virus; MS, multiple sclerosis.
3
MS patients
ARTICLE INFORMATION
.16
0
MS patients CD19 or CD34
.72
1
MS patients CD19 and CD34
P Valuea
a
.03
Calculated as 2-sided Fisher exact test using the method of summing small P values.
Although viral serology plays an important role in risk stratification algorithms,31 the use of serology alone may not be sufficient to identify all patients with prior exposure to JCV.32 Other measures of JCV infection may be of equal importance and should be considered, including T-cell responses, a rise in antibody titer indicating active infection, and the presence of JCV DNA variants in peripheral circulation, particularly in cell compartments. 33 These additional factors, previously suggested,18,34 could be helpful during periodic surveillance if included in stratification protocols.35 Continued studies seem warranted on the pathoetiologic factors that influence the development of PML in patients treated with natalizumab and other biological modulators of the human immune system.
Conclusions We detected JCV DNA within the cell compartments of natalizumab-treated MS patients after treatment inception and after 24 months. The JCV DNA may harbor in CD34+ cells in bone marrow that mobilize into the peripheral circulation at high concentrations. Cells with latent infection initiate differentiation to CD19+ cells that favors growth of JCV. Continued studies are needed to further investigate natalizumab treatment as the mechanism of PML.
Institute of Neurological and Communicative Diseases and Stroke, NIH, Bethesda, Maryland (Johnson); Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland (Perkins, Liebner, Douek). Author Contributions: Dr Frohman had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: E. M. Frohman, Monaco, Remington, Jensen, Monson, T. C. Frohman, Douek, Major. Acquisition, analysis, or interpretation of data: All authors.
Drafting of the manuscript: E. M. Frohman, Remington, T. C. Frohman. Critical revision of the manuscript for important intellectual content: All authors. Statistical analysis: E. M. Frohman, Johnson, Perkins. Obtained funding: E. M. Frohman, Remington, T. C. Frohman. Administrative, technical, or material support: E. M. Frohman, Monaco, Remington, Ryschkewitsch, Jensen, Monson, T. C. Frohman, Douek, Major. Study supervision: E. M. Frohman, Remington, Greenberg, T. C. Frohman, Douek, Major. Conflict of Interest Disclosures: Dr Frohman has received speaking and consulting fees from Biogen JAMA Neurology May 2014 Volume 71, Number 5
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Idec, TEVA Neuroscience, Acorda, Genzyme, and Novartis and has received consulting fees from Abbott Laboratories. Ms Remington serves on the speakers bureau for TEVA Neurosciences and Biogen Idec and has received honoraria from TEVA Neurosciences, Biogen Idec, Genzyme, Acorda Therapeutics, the Consortium for Multiple Sclerosis Centers, and the National Multiple Sclerosis Society. Dr Greenberg has received grant support from the Accelerated Cure Project, the Guthy Jackson Charitable Foundation for NMO (Neuromyelitis Optica), and Amplimmune, Inc; has received consulting fees from Biogen Idec, sanofi-aventis, DioGenix, The Greater Good Foundation, and Elan Pharmaceuticals; and owns equity in DioGenix, Inc. Ms Frohman has received speaker’s and consulting fees from Biogen Idec, Novartis, Genzyme, and Acorda. No other disclosures were reported. Funding/Support: This study was supported by Division of Intramural Research funds (National Institute of Neurological and Communicative Diseases [NINDS] and National Institute of Allergy and Infectious Disease [NIAID] laboratories), by contract HHSN 271200900268P (LMMN, The University of Texas Southwestern Medical Center LMMN, and NINDS), and by grant RG 3780A3/3 from the National Multiple Sclerosis Society (Dr Frohman). Role of the Sponsors: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. Additional Contributions: Rafaello Cimbor, PhD, from the NIAID, and Dragan Maric, PhD, from the NINDS, assisted with the flow cytometry and data analysis. Gloria von Geldern, MD, from the NINDS, provided critical review of the manuscript. No financial compensation was given.
8. Major EO. Reemergence of PML in natalizumab-treated patients: new cases, same concerns. N Engl J Med. 2009;361(11):1041-1043. 9. Houff SA, Major EO, Katz DA, et al. Involvement of JC virus–infected mononuclear cells from the bone marrow and spleen in the pathogenesis of progressive multifocal leukoencephalopathy. N Engl J Med. 1988;318(5):301-305. 10. Tan CS, Dezube BJ, Bhargava P, et al. Detection of JC virus DNA and proteins in the bone marrow of HIV-positive and HIV-negative patients: implications for viral latency and neurotropic transformation. J Infect Dis. 2009;199(6):881-888. 11. Ryschkewitsch C, Jensen P, Hou J, Fahle G, Fischer S, Major EO. Comparison of PCR–southern hybridization and quantitative real-time PCR for the detection of JC and BK viral nucleotide sequences in urine and cerebrospinal fluid. J Virol Methods. 2004;121(2):217-221. 12. Ryschkewitsch CF, Jensen PN, Monaco MC, Major EO. JC virus persistence following progressive multifocal leukoencephalopathy in multiple sclerosis patients treated with natalizumab. Ann Neurol. 2010;68(3):384-391. 13. Lindå H, von Heijne A, Major EO, et al. Progressive multifocal leukoencephalopathy after natalizumab monotherapy. N Engl J Med. 2009;361(11):1081-1087. 14. Major EO, Miller AE, Mourrain P, Traub RG, de Widt E, Sever J. Establishment of a line of human fetal glial cells that supports JC virus multiplication. Proc Natl Acad Sci U S A. 1985;82(4):1257-1261.
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antibody natalizumab mobilizes CD34+ hematopoietic progenitor cells in humans. Blood. 2008;111(7):3893-3895.
18. Major EO, Frohman EM, Douek D. JC viremia in natalizumab-treated patients with multiple sclerosis. N Engl J Med. 2013;368(23):2240-2241. 19. Wei G, Liu CK, Atwood WJ. JC virus binds to primary human glial cells, tonsillar stromal cells, and B-lymphocytes, but not to T lymphocytes. J Neurovirol. 2000;6(2):127-136. 20. Krumbholz M, Meinl I, Kümpfel T, Hohlfeld R, Meinl E. Natalizumab disproportionately increases circulating pre-B and B cells in multiple sclerosis. Neurology. 2008;71(17):1350-1354. 21. Skarica M, Eckstein C, Whartenby KA, Calabresi PA. Novel mechanism of immune modulation of natalizumab in multiple sclerosis. J Neuruoimmumology. 2011;235:70-76.
22. Planas R, Jelčić I, Schippling S, Martin R, Sospedra M. Natalizumab treatment perturbs memory- and marginal zone-like B-cell homing in secondary lymphoid organs in multiple sclerosis. Eur J Immunol. 2012;42(3):790-798. 23. Warnke C, Smolianov V, Dehmel T, et al. CD34+ progenitor cells mobilized by natalizumab are not a relevant reservoir for JC virus. Mult Scler. 2011;17(2):151-156. 24. Saure C, Warnke C, Zohren F, et al. Natalizumab and impedance of the homing of CD34+ hematopoietic progenitors. Arch Neurol. 2011;68(11):1428-1431. 25. Goodrum FD, Jordan CT, High K, Shenk T. Human cytomegalovirus gene expression during infection of primary hematopoietic progenitor cells: a model for latency. Proc Natl Acad Sci U S A. 2002;99(25):16255-16260. 26. Poole E, McGregor Dallas SR, Colston J, Joseph RS, Sinclair J. Virally induced changes in cellular microRNAs maintain latency of human cytomegalovirus in CD34+ progenitors. J Gen Virol. 2011;92(pt 7):1539-1549. 27. Atwood WJ, Amemiya K, Traub R, Harms J, Major EO. Interaction of the human polyomavirus, JCV, with human B-lymphocytes. Virology. 1992;190(2):716-723. 28. Monaco MC, Atwood WJ, Gravell M, Tornatore CS, Major EO. JC virus infection of hematopoietic progenitor cells, primary B lymphocytes, and tonsillar stromal cells: implications for viral latency. J Virol. 1996;70(10):7004-7012. 29. Padgett BL, Walker DL, ZuRhein GM, Eckroade RJ, Dessel BH. Cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy. Lancet. 1971;1(7712):12571260. 30. Perkins M, Ryschkewitsch CF, Liebner JC, et al. Changes in JC virus-specific T cell responses during natalizumab treatment and in natalizumabassociated progressive multifocal leukoencephalopathy. PLoS Pathog. 2012;8(11):e1003014. doi:10.1371/ journal.ppat.1003014. 31. Gorelik L, Lerner M, Bixler S, et al. Anti-JC virus antibodies: implications for PML risk. Ann Neurol. 2010;68(3):295-303. 32. Berger JR, Houff SA, Gurwell J, Vega N, Miller CS, Danaher RJ. JC virus antibody status underestimates infection rates. Ann Neurol. 2013;74(1):84-90. 33. Ryschkewitsch CF, Jensen PN, Major EO. Multiplex qPCR assay for ultra sensitive detection of JCV DNA with simultaneous identification of genotypes that discriminates non-virulent from virulent variants. J Clin Virol. 2013;57(3):243-248. 34. Major EO, Ryschkewitsch CF, Jensen PN, et al. Persistence of JCV in natalizumab treated MS patients who developed PML [reply]. Ann Neurol. 2011;69:430-431. 35. Sorenson PS, Bertolotto A, Edan G, et al. Risk stratification for progressive multifocal leukoencephalopathy in patients treated with natalizumab. Mult Scler. 2012;18(12):143-152.
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Original Investigation
JC Virus in CD34+ and CD19+ Cells in Patients With Multiple Sclerosis Treated With Natalizumab Elliot M. Frohman, MD, PhD; Maria Chiara Monaco, PhD; Gina Remington, RN; Caroline Ryschkewitsch, MT; Peter N. Jensen, BS; Kory Johnson, PhD; Molly Perkins, PhD; Julia Liebner, BS; Benjamin Greenberg, MD; Nancy Monson, PhD; Teresa C. Frohman, PA-C; Daniel Douek, MD, PhD; Eugene O. Major, PhD
IMPORTANCE Infection with JC virus (JCV) may lead to development of demyelinating progressive multifocal leukoencephalopathy in patients with multiple sclerosis (MS) who are treated with natalizumab.
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OBJECTIVE To determine whether mononuclear cells in circulation from MS patients treated with natalizumab harbor JCV DNA. DESIGN, SETTING, AND PARTICIPANTS In this prospective investigation, we enrolled 49 MS patients from the Clinical Center for Multiple Sclerosis at The University of Texas Southwestern Medical Center and 18 healthy volunteers. We drew 120-mL blood samples from 26 MS patients at baseline and at approximately 3-month intervals to 10 months during the course of natalizumab infusions. One blood sample was drawn from 23 MS patients receiving natalizumab for more than 24 months and from 18 healthy volunteers. INTERVENTIONS Natalizumab treatment of MS. MAIN OUTCOMES AND MEASURES The blood samples were separated using flow cytometry into CD34+, CD19+, and CD3+ cell subsets; DNA templates were prepared using quantitative polymerase chain reaction for JCV DNA identification. Plasma samples were tested for anti-JCV antibodies by enzyme-linked immunosorbent assays performed at the Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological and Communicative Diseases and Stroke. RESULTS Thirteen of the 26 patients (50%) with baseline and follow-up blood samples had detectable viral DNA in at least 1 cell compartment at 1 or more points. Ten of the 23 patients (44%) receiving treatment for more than 24 months and 3 of the 18 healthy volunteers (17%) also had detectable viral DNA in 1 or more cell compartment. Fifteen of the 49 MS patients (31%) were confirmed to harbor JCV in CD34+ cells and 12 of 49 (24%) in CD19+ cells. Only 1 of 18 healthy volunteers were viremic in CD34+ cells and none in CD19+ cells. Nine patients and 1 healthy volunteer were viremic but had seronegative test results for JCV antibodies. CONCLUSIONS AND RELEVANCE JC virus DNA was detectable within cell compartments of natalizumab-treated MS patients after treatment inception and longer. JC virus DNA may harbor in CD34+ cells in bone marrow that mobilize into the peripheral circulation at high concentrations. Latently infected cells initiate differentiation to CD19+ cells that favors growth of JCV. These data link the mechanism of natalizumab treatment with progressive multifocal leukoencephalopathy.
JAMA Neurol. 2014;71(5):596-602. doi:10.1001/jamaneurol.2014.63 Published online March 24, 2014. 596
Author Affiliations: Author affiliations are listed at the end of this article. Corresponding Authors: Elliot M. Frohman, MD, PhD, Department of Neurology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390 (elliot.frohman@utsouthwestern .edu); Eugene O. Major, PhD, Laboratory of Molecular Medicine and Neuroscience, National Institutes of Health, 10 Center Dr, Room 3B14A, Bethesda, MD 20892 ([email protected]). jamaneurology.com
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JC Virus in CD34+ and CD19+ Cells in MS Patients
S
ince the reintroduction of natalizumab therapy in 2006 for relapsing remitting forms of multiple sclerosis (MS), more than 440 cases of progressive multifocal leukoencephalopathy (PML), the central nervous system virus– induced demyelinating disease, have been reported. The highest incidence of PML is approximately 1:80 in patients with a combination of risk factors, including receiving 24 or more natalizumab infusions, a history of immunosuppressive treatment, and seropositivity for antibodies to the JC virus (JCV). The overall incidence of PML in natalizumab-treated MS patients is 1:250, with approximately 11 new cases reported each month.1,2 Several hypotheses have focused on the direct association of natalizumab treatment and PML, particularly because few other opportunistic infections occur in this group.3 One putative mechanism centers on the lack of immune surveillance in the central nervous system because natalizumab blocks extravasation of T and B lymphocytes into the brain.4 This concept is predicated on the presence of JCV in the brain in a latent or persistent state of residency so that, at times of protracted immune suppression, a productive amplification of viral infection is established. Although this concept may be partly true, it does not explain why PML has not been reported in MS patients treated with a variety of other immunosuppressive agents (eg, azathioprine, mycophenolate, mitoxantrone hydrochloride, and rituximab). However, if protracted immune suppression was the primary mechanism of PML in such patients, we would anticipate PML occurring in MS patients whose immune systems are more severely compromised than seen in natalizumab-treated patients. Further, JCV infection in the brains of MS patients not treated with natalizumab has not been reported. The only reports of PML in MS patients are those treated with natalizumab.5 Another proposed mechanism to explain the unique link between natalizumab treatment and PML is the drug’s effect on the mobilization of mononuclear cells from the bone marrow,6,7 some of which may harbor latent JCV infection.8-10 These observations are not mutually exclusive, however, and may actually work in concert to provide the circumstances in which 2 very different demyelinating diseases can occur in the brain of a single patient. To investigate the link between natalizumab treatment and the presence of JCV in lymphoid cells, peripheral blood samples were collected from MS patients before the first dose of natalizumab, on the day treatment was initiated, and periodically during the course of the next 10 monthly infusions. Blood samples were also collected at a single point from MS patients who received at least 24 infusions and from healthy volunteers who were matched by age and sex to the natalizumabtreated MS patients.
Methods
fusion (baseline) and then at approximately 3-month intervals, depending on the patients’ return to clinic. The samples were immediately shipped overnight to the Laboratory of Molecular Medicine and Neuroscience (LMMN), National Institute of Neurological and Communicative Diseases and Stroke, for cell separation processing. Single 120-mL samples were collected from 23 MS patients undergoing more than 24 infusions at the Clinical Center for Multiple Sclerosis, and 150-mL samples of blood were collected from the healthy volunteers at the Transfusion Medicine Blood Bank of the National Institutes of Health. Signed informed consent was obtained in accordance with the respective institutional review boards.
Cell Isolation and Phenotypic Characterization Human CD34+ cells were enriched by positive selection using a CD34 purity selection kit (Stemcell Technologies Inc) or on lineage-depleted whole blood samples using a cocktail of monoclonal antibodies directed against CD2, CD3, CD14, CD16, CD19, CD24, CD36, CD56, and CD66b. Peripheral blood mononuclear cells and enriched CD34+ progenitor cells were washed in culture medium (RPMI-1640; Life Technologies) without fetal bovine serum (RO medium). The cells and RO medium were mixed with pretitered amounts of directly conjugated monoclonal antibodies using the following surface stains: for CD3 and CD8, fluorescein isothiocyanate conjugate; for CD19, peridinim-chlorophyll-cyanine 5.5 conjugated reagent or brilliant violet 421; for CD4, phycoerythrin; for CD34, phycoerythrin–cyanine 7; and for CD45, V500 (all reagents, BD Biosciences) in a total volume of 100 μL of RO medium. Cells were stained for 20 minutes at 4°C, washed and resuspended in 250 μL of phosphate-buffered solution, and then passed through a 40-μm strainer to ensure singlecell suspension. Flow cytometric analysis was performed immediately after this treatment.
Flow Cytometric Analysis Cells were analyzed using a flow cytometer (FACSAria II SORP; BD Biosciences) equipped for the detection of as many as 6 fluorescence variables. Events collected for each sample numbered from 5000 to 50 000. Electronic compensation was conducted with peripheral blood mononuclear cells stained separately with single monoclonal antibodies used in test samples. Electronic compensation was performed with antibody capture beads (BD Biosciences) for CD34+ cells only. The cells were then identified as mononuclear cells by sidescatter area and forward-scatter area and as singlet cells by forward-scatter height, forward-scatter width, side-scatter height, and side-scatter width. We defined and sorted CD34+, CD19+, and CD3+ cells by positive selection using the CD34+/CD45+, CD19+/CD45+, and CD3+/CD45+ gates, respectively.
Quantitative Polymerase Chain Reaction for JCV Genome
Patients Twenty-six MS patients starting natalizumab therapy (Tysabri; Biogen Idec) were recruited in the Clinical Center for Multiple Sclerosis at The University of Texas Southwestern Medical Center. To isolate sufficient CD34+ and CD19+ cells, 120-mL blood samples were collected immediately before the first injamaneurology.com
Original Investigation Research
Cell samples were examined using the validated, Clinical Laboratory Improvement Amendment–certified JCV quantitative polymerase chain reaction (PCR) method.11-13 Cellular DNA was extracted from a maximum of 5.0 × 106 cells per sample using a DNA blood and tissue kit (DNeasy, catalogue No. 69504; Qiagen) and eluted to a final volume of 25 μL. The primers and JAMA Neurology May 2014 Volume 71, Number 5
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Figure 1. Enrichment of CD34+ Cells and Gates Strategy Schematics A CD34+ Enrichment
B
CD2, CD3, CD14, CD16, CD19, CD24, CD36, CD56, and CD66b
All cells or CD34+-enriched cells
Gates Strategy
Label
P1 (mononuclear cells, FSC-A, and SSC-A) P2 (single cells, FSC-H, and FSC-W) P3 (single cells, SSC-H, and SSC-W) Incubate 20 min at room temperatue
CD34+/CD45+ (CD34+ cells) CD3+/CD45+ (T cells)
Layer over Ficoll-Paque
CD3–/CD45+ CD19+/CD45+ (B cells)
Ficoll-Paque
Spin
Plasma Enriched cells
Collect
Ficoll-Paque
Enriched cells (unrosetted)
Red cells (unwanted rosetted cells)
probes for the specific amplification and detection of the conserved N-terminal of the JCV T protein do not amplify BK virus or simian virus 40 DNA in this region. Extracted DNA templates were amplified using the PCR reagents (TaqMan Universal reagent, catalogue No. 4304437; Applied Biosystems) and a commercially available PCR system (7500 Real-Time System; Applied Biosystems). All samples were run in duplicate using 10 μL per reaction. The number of JCV genomes was calculated using the manufacturer’s system software and adjusted according to the dilution factors before reporting as copies per milliliter only when both duplicates detected viral DNA.
Enzyme-Linked Immunosorbent Assay for Determination of Anti-JCV Antibody The gene that codes for the major JCV structural protein, Vp-1, was amplified from SVG cells14 infected with the Mad-4 viral variant of persistently infected owl monkey 586 tumor cells.15 This amplified VP1 gene was ligated into the pBlueBacIII plasmid vector and cotransfected into Sf9 insect cells along with the baculovirus DNA expression vector system (BAc-N-Blue; Invitrogen) according to the manufacturer’s protocol. Subsequent proliferating baculovirus expressing the recombinant Vp-1 was used as the inoculum in Sf9 expansion cultures. Supernatant from the expansion cultures was ultracentrifuged through 40% (weight to volume ratio) sucrose cushions. The resulting recombinant Vp-1 pellets were subjected to deoxycholic acid treatment, low protein–binding polyethersulfone membrane filtration, and gradient banding on density gradient medium (OptiPrep; Nycomed) to remove cell lysate contaminants. This 598
A, Enrichment of CD34+ cells was achieved by positive selection using a cocktail of lineage-specific monoclonal antibodies. B, The enriched cells were further sort purified using a flow cytometer (FACSAria II SORP; BD Biosciences) gated on CD34+/CD45+. In parallel, peripheral blood mononuclear cells isolated by density gradient medium (Ficoll-Paque; Sigma-Aldrich) were identified using forward-scatter area (FSC-A) and side-scatter area (SSC-A) (gate P1). Subsequently, gate P2 forward-scatter height (FSC-H) and forward-scatter width (FSC-W) and P3 side-scatter height (SSC-H) and side-scatter width (SSC-W) were placed to include only single cells. Consequently, T and B cells were sorted by positive selection using the CD3+/CD45+ and CD19+/CD45+ gates, respectively.
highly purified recombinant Vp-1 was used as the antigen in establishing the enzyme-linked immunosorbent assay (ELISA) for determining the level of JCV antibody response present in the clinical samples. In the ELISA, 4× serial dilutions that generated an optical density reading of 0.05 greater than that of plate and well controls were labeled as positive at a dilution of 2560. The highest dilution of a sample registering positive findings was used to identify the titer of the sample.16 We compared the LMMN ELISA assay directly with a second-generation JCV antibody ELISA (STRATIFY JCV; Focus Diagnostics) supplied as a kit by the manufacturer using 17 plasma samples from these patients. Fifteen of the 17 samples showed identical serological results, whereas 2 other samples that the LMMN ELISA showed to have seronegative results had indeterminate results using the STRATIFY JCV ELISA after step 2 adsorption with viruslike particles as described below.
Results Natalizumab is known to promote mobilization of hematopoietic stem cells from the bone marrow, a putative site for JCV latency, into the peripheral circulation, resulting in higher-thannormal physiological levels of CD34+ cells for months to years after treatment. Therefore, we collected blood samples and separated them into CD34+, CD19+, and CD3+ cells using flow cytometry. The mononuclear cells were quantitated in relation to total number of peripheral blood mononuclear cells (Figure 1). We found an increased percentage of CD34+ cells from MS patients
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JC Virus in CD34+ and CD19+ Cells in MS Patients
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Figure 2. Comparison of Percentage of CD34+ Cells in Peripheral Blood Mononuclear Cells (PBMC) Between Healthy Volunteers and Patients With Multiple Sclerosis (MS) 7 6
CD34+Cells in PBMC, %
compared with healthy volunteers (Figure 2) as in previous observations.17 Percentages of CD34+ and CD19+ cells were persistently elevated in the blood samples from MS patients treated with natalizumab starting at 3 months and for extended periods. Although all blood samples were separated into cell compartments by flow cytometry, the data in Table 1 report only those samples confirmed to harbor detectable levels of JCV DNA. Patients described in Table 1 reflect the acquisition of the samples that were sent to the LMMN for analysis and not the total number of patients in either cohort. Blood samples were collected at baseline (before infusion) and at approximately 3-month intervals to 9 or 10 months, depending on patient status, for the 26 patients initiating treatment. A total of 92 samples from this cohort (4 samples of 120 mL each) underwent analysis. Table 1 shows the study participants with viremia, the specific compartment in which viremia was detected, the number of infusions, and the antibody titer of the plasma at the time of testing. Antibody titers of 2560 or greater are considered seropositive.16 Table 1 also shows that 3 of 18 healthy volunteers (17%) were viremic, although only in 2 cell compartments (CD19+ cells in 2 and CD34+ cells in 1) for a mean of 66 copies/mL of viral DNA. In the cohort just initiating treatment, 19 of 92 samples (21%) from 13 of 26 patients (50%) had detectable viral DNA. Three of these 13 patients (23%) had detectable viral DNA in CD34+ and CD19+ cells. In the cohort of 23 patients with more than 24 infusions from whom only 1 blood sample was collected, 10 (38%) had detectable viral DNA in cell compartments, including CD34+ and CD19+ cells in 1 of these patients. Regardless of the compartment in which viral DNA was detected across all 3 groups, the copy number of viral genomes was low, particularly when compared with the numbers usually seen in virus excreted into the urine, or viruria. None of the samples from any group had detectable viral DNA in the CD3+ T cells, the cell control from each patient irrespective of group designation. JC virus has not been shown to bind to or infect T cells.19 When both patient groups were combined (Table 2), 15 of 49 patients (31%) were viremic in CD34+ cells at different times, including 2 patients at baseline with a mean of 25 copies/mL in the initiating group and 212 copies/mL in the longer treatment group. Twelve of the 49 patients (24%) were viremic in CD19+ cells, with a mean of 96 copies/mL in the initiating group and 543 copies/mL in the longer treatment group. The plasma viremia in these patients has been reported previously.18 Seventeen patients (35%) had plasma viremia. Six patients were viremic only in the plasma (patients 3, 8, 10, 33, 36, and 48), including 4 at baseline with a mean of 86 copies/mL in the initiating group and 67 copies/mL in the longer treatment group. Three patients were found to be viremic in all 3 compartments (patients 2, 13, and 40). The high copy number in cell compartments in patient 40, who had 30 infusions, was much larger than those of the others. In general, however, the viral copy number was higher in the longer treatment group, with a mean of 230 copies/mL compared with 71 copies/mL in the initiating group. Analysis of viremia in the longer treatment group showed that viral DNA was primarily localized within CD34+ cells at a mean of 212 copies/mL compared with 25 copies/mL in the initiating group and within CD19+ cells at a mean of 543 copies/mL in the longer treatment group compared with 96 cop-
Original Investigation Research
P 24 natalizumab infusions
marrow lymphoid precursors compared with myeloid precursors, resulting in chronic increases in T- and B-cell populations in the peripheral circulation. Marginal zone–like memory B cells significantly increased expression of CD49d, which enables harboring in marginal zones in lymph nodes. Together these studies provide evidence that supports the role of these cell populations as possible disseminators of JCV because natalizumab triggers the mobilization of cells potentially infected with JCV into the circulation. The cell populations described in the present study showed the same pattern of responses with natalizumab treatment, a finding that persisted for several years (Figure 2). The observation of JCV latency within these characterized mononuclear cells makes a direct mechanistic link between natalizumab and the occurrence of PML. We used the highly sensitive, specific, and vali600
Only viremia data for 2 cell compartments are shown. Patients 4, 12, 19, 41, and 55 were viremic in the CD34+ cell compartment and in plasma. Patients 20, 26, and 52 were viremic in the CD19+ cell compartment and in plasma. Patients 2, 13, and 40 were viremic in all 3 compartments. Viremia in plasma only was reported previously18 (not shown) for patients 3, 8, 10, 33, 36, and 48.
dated quantitative PCR assay that provided the laboratory confirmation of PML diagnosis in nearly half of the MS cases in the present study to detect viral DNA in cell compartments. Detectable viral DNA in CD34+ cells was found in 15 of 49 patients (31%) and in CD19+ cells in 12 of 49 (24%) after initiation of treatment or after several years of continuous treatment. In contrast to our findings, other studies23,24 have not identified JCV DNA in CD34+ cells, suggesting that compartments such as bone marrow or lymphoid tissues may not be reservoirs for latent infections. However, the same studies failed to identify cytomegalovirus in CD34+ cells as a positive control in their study, despite evidence from multiple reports that confirm detection of cytomegalovirus DNA25,26 and JCV DNA9,10 in these lymphoid stem cells. The assays that did not detect JCV DNA in similar cell populations probably were not sensi-
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Table 2. Detectable JCV DNA by Quantitive Polymerase Chain Reaction MS Patients, No. (n = 49) Compartments With JCV DNA Detected
≤10 Infusions (n = 26)
>24 Infusions (n = 23)
Healthy Volunteers, No. (n = 18)
5
3
2
CD19 only
Viremia, No. (%)
MS patients
8/49 (16)
Healthy volunteers
2/18 (11)
CD34 only
5
6
11/49 (22)
Healthy volunteers
1/18 (6) 3
1
4/49 (8)
Healthy volunteers
0 13
10
23/49 (47)
Healthy volunteers
3/18 (17)
tive enough and probably were not used in sufficient numbers of cells to compare the outcomes of these investigations legitimately. In our study, more CD34+ cells were available from 120and 150-mL blood samples that were collected and processed within 24 hours, in contrast to the 18-mL samples used in the other reported study.25 In addition, the low viral copy number in our study is consistent with a latent or a persistent infection. In previous work,27,28 a cell culture model had shown that even a low level of persistence of JCV has significance because JCV can be transferred to other cells. In fact, JCV was first isolated by a similar blind passage in human fetal brain cells.29 Another important observation from our study is the identification of individuals with seronegative test findings but who were nevertheless viremic for JCV (Table 1). The ELISA used in the LMMN is validated using a 4-fold serial dilution of plasma with a cutoff titer of 640 or less, constituting seronegative status. Several samples had titers of 160, an unambiguous seronegative result. Of particular interest are patients 9 and 13, who are seronegative and viremic. In a separate analysis,30 both of these patients demonstrated T-cell responses to JCV proteins, indicating cell-mediated immune responses to JCV infection. These observations add to the complexity of finding a rigorous set of criteria for identifying patients at increased risk for PML when receiving natalizumab or similar therapies that modulate the immune system.
Accepted for Publication: January 27, 2014. Published Online: March 24, 2014. doi:10.1001/jamaneurol.2014.63. Author Affiliations: Department of Neurology, The University of Texas Southwestern Medical Center, Dallas (Elliot M. Frohman, Remington, Greenberg, Monson, T.C. Frohman); Department of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas (Elliot M. Frohman); Laboratory of Molecular Medicine and Neuroscience, National Institutes of Health (NIH), Bethesda, Maryland (Monaco, Ryschkewitsch, Jensen, Major); Bioinformatics Section, National
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.57 Abbreviations: JCV, JC virus; MS, multiple sclerosis.
3
MS patients
ARTICLE INFORMATION
.16
0
MS patients CD19 or CD34
.72
1
MS patients CD19 and CD34
P Valuea
a
.03
Calculated as 2-sided Fisher exact test using the method of summing small P values.
Although viral serology plays an important role in risk stratification algorithms,31 the use of serology alone may not be sufficient to identify all patients with prior exposure to JCV.32 Other measures of JCV infection may be of equal importance and should be considered, including T-cell responses, a rise in antibody titer indicating active infection, and the presence of JCV DNA variants in peripheral circulation, particularly in cell compartments. 33 These additional factors, previously suggested,18,34 could be helpful during periodic surveillance if included in stratification protocols.35 Continued studies seem warranted on the pathoetiologic factors that influence the development of PML in patients treated with natalizumab and other biological modulators of the human immune system.
Conclusions We detected JCV DNA within the cell compartments of natalizumab-treated MS patients after treatment inception and after 24 months. The JCV DNA may harbor in CD34+ cells in bone marrow that mobilize into the peripheral circulation at high concentrations. Cells with latent infection initiate differentiation to CD19+ cells that favors growth of JCV. Continued studies are needed to further investigate natalizumab treatment as the mechanism of PML.
Institute of Neurological and Communicative Diseases and Stroke, NIH, Bethesda, Maryland (Johnson); Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland (Perkins, Liebner, Douek). Author Contributions: Dr Frohman had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: E. M. Frohman, Monaco, Remington, Jensen, Monson, T. C. Frohman, Douek, Major. Acquisition, analysis, or interpretation of data: All authors.
Drafting of the manuscript: E. M. Frohman, Remington, T. C. Frohman. Critical revision of the manuscript for important intellectual content: All authors. Statistical analysis: E. M. Frohman, Johnson, Perkins. Obtained funding: E. M. Frohman, Remington, T. C. Frohman. Administrative, technical, or material support: E. M. Frohman, Monaco, Remington, Ryschkewitsch, Jensen, Monson, T. C. Frohman, Douek, Major. Study supervision: E. M. Frohman, Remington, Greenberg, T. C. Frohman, Douek, Major. Conflict of Interest Disclosures: Dr Frohman has received speaking and consulting fees from Biogen JAMA Neurology May 2014 Volume 71, Number 5
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Research Original Investigation
Idec, TEVA Neuroscience, Acorda, Genzyme, and Novartis and has received consulting fees from Abbott Laboratories. Ms Remington serves on the speakers bureau for TEVA Neurosciences and Biogen Idec and has received honoraria from TEVA Neurosciences, Biogen Idec, Genzyme, Acorda Therapeutics, the Consortium for Multiple Sclerosis Centers, and the National Multiple Sclerosis Society. Dr Greenberg has received grant support from the Accelerated Cure Project, the Guthy Jackson Charitable Foundation for NMO (Neuromyelitis Optica), and Amplimmune, Inc; has received consulting fees from Biogen Idec, sanofi-aventis, DioGenix, The Greater Good Foundation, and Elan Pharmaceuticals; and owns equity in DioGenix, Inc. Ms Frohman has received speaker’s and consulting fees from Biogen Idec, Novartis, Genzyme, and Acorda. No other disclosures were reported. Funding/Support: This study was supported by Division of Intramural Research funds (National Institute of Neurological and Communicative Diseases [NINDS] and National Institute of Allergy and Infectious Disease [NIAID] laboratories), by contract HHSN 271200900268P (LMMN, The University of Texas Southwestern Medical Center LMMN, and NINDS), and by grant RG 3780A3/3 from the National Multiple Sclerosis Society (Dr Frohman). Role of the Sponsors: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. Additional Contributions: Rafaello Cimbor, PhD, from the NIAID, and Dragan Maric, PhD, from the NINDS, assisted with the flow cytometry and data analysis. Gloria von Geldern, MD, from the NINDS, provided critical review of the manuscript. No financial compensation was given.
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