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Cellular and humoral immune response in progressive multifocal leukoencephalopathy.

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Cellular and Humoral Immune Response in
Progressive Multifocal Leukoencephalopathy
Frank Weber, MD,1 Claudia Goldmann,2 Marcus Krämer,2 Franz Josef Kaup, MD,3 Marcus Pickhardt, PhD,4
Peter Young, MD,5 Harald Petry, PhD,2 Thomas Weber, MD,4 and Wolfgang Lüke, PhD2
Progressive multifocal leukoencephalopathy (PML) is a fatal, demyelinating disease caused by JC virus (JCV) in patients
with severe immunosuppression. We studied the JCV-specific cellular and humoral immune response in 7 healthy donors
(HD), 6 human immunodeficiency virus-1 (HIV-1)-infected patients without PML (HIV), 4 HIV-1-negative patients
with PML (PML), and 8 HIV-1-positive patients with PML (HIV/PML). As antigens, recombinant virus-like particles of
the major structural protein VP1 (VP1-VLP) of JCV, tetanus toxoid (TT), or the mitogen phytohemagglutinin (PHA)
were used. Proliferation of peripheral blood mononuclear cells (PBMC) after stimulation with the VP1-VLP was significantly suppressed in PML and HIV/PML patients compared to HD. After antigen stimulation the production of
interferon-␥ (IFN-␥) was reduced in PML, in HIV/PML, and in HIV patients. The production of interleukin-10 (IL-10),
however, was elevated in HIV/PML patients. Neither proliferation nor cytokine production correlated with the presence
of JCV DNA in PBMC. The immunoglobulin G serum antibody titer to the VP1-VLP was slightly elevated in HIV,
elevated in PML, and highly elevated in HIV/PML patients compared to HD. The development of PML appears to
coincide with a general impairment of the Th1-type T-helper cell function of cell-mediated immunity.
Ann Neurol 2001;49:636 – 642
Progressive multifocal leukoencephalopathy (PML) was
first described in 1958.1 PML is a demyelinating disease of immunocompromised patients caused by the
human polyomavirus JC virus (JCV). Once PML was a
rare disorder, but the incidence has risen dramatically
as a result of the worldwide AIDS pandemic. The acquired immune deficiency syndrome (AIDS) is the underlying immunosuppression in about 85% of PML
patients and about 4 – 6% of all patients with AIDS
may develop PML.2,3 The disease is also observed in
patients with neoplastic diseases and in immunocompromised patients after transplantation or treatment
with immunosuppressants.3,4 Very rarely, PML is seen
in patients without any signs of immunodeficiency.5
PML results from a lytic infection of oligodendrocytes
by JCV, which causes death of oligodendrocytes and
demyelination. Clinical symptoms of PML are nonspecific and include motor weakness, cognitive changes,
visual deficits, and cranial nerve palsies.6 By magnetic
resonance imaging (MRI), multifocal demyelinating lesions are most often found at the gray matter–white
matter junction. Detection of JCV DNA in the cerebrospinal fluid (CSF)7 or of an intrathecal JCV-specific antibody synthesis8 is used to confirm diagnosis of PML.
From the 1Department of Neurology, University of Göttingen; Departments of 2Virology and Immunology and 3Experimental Pathology, German Primate Center, Göttingen; 4Department of Neurology,
Marienkrankenhaus, Hamburg; and 5Department of Neurology, University of Münster, Germany.
Received Dec 15, 1999, and in revised form Aug 11. Accepted for
publication Nov 27, 2000.
636
© 2001 Wiley-Liss, Inc.
Whereas many studies address the molecular biology
of JCV, studies on the cellular and humoral immune
response are rare.9,10 This is mainly due to the limited
availability of viral antigen based on the difficulties of
propagating JCV in cell culture. We describe a systematic analysis of the humoral and cellular immune response to JCV. To obtain sufficient amounts of JCVrelated antigen, we expressed the major structural
protein VP1 of JCV as virus-like particles (VP1VLP).11 These VP1-VLP were used to investigate the
proliferation of peripheral blood mononuclear cells
(PBMC) as well as the cytokine production in seven
healthy donors (HD), six HIV-1-infected patients
without PML (HIV), eight HIV-1-infected patients
with PML (HIV/PML), and four HIV-1-negative patients with PML (PML). In addition, JCV-specific serum immunoglobulin G antibodies were assessed using
VP1-VLP as antigen.8,12
Patients and Methods
Patients and Controls
Up to 40 ml EDTA blood was obtained from 7 HD, 6 HIV,
4 PML, and 8 HIV/PML patients after informed consent
was obtained. There was no statistically significant difference
Published online 2 April 2001.
Address correspondence to Dr Weber, Section of Neurology, MPI
of Psychiatry, Kraepelinstrasse 2–10, 80804 Munich, Germany. Email: fweber@mpipsykl.mpg.de
with respect to CD4⫹/CD8⫹ ratio and HIV-1 virus load
between HIV and HIV/PML patients (Table 1). Among the
4 PML patients, 2 suffered from chronic lymphatic leukemia
and 1 from immunosuppression after organ transplantation.
In the fourth patient, no underlying immunosuppressive illness could be detected. PML was clinically suspected based
on the occurrence of neurological deficits and typical lesions
on magnetic resonance imaging (MRI).3,13 Diagnosis was
proved by the detection of JCV DNA in cerebrospinal fluid7
or by the demonstration of a JCV-specific intrathecal antibody response.8,12 In 2 HIV-1-negative PML patients, the
diagnosis was proved by brain biopsy. PBMC of HD were
included in the study, to investigate the proliferative capacity
and the cytokine production in healthy volunteers. This
study was approved by the ethics committee of the GeorgAugust-University Gättingen and the institutional review
board of the Ärztekammer, Hamburg, Germany.
Template Preparation
From patients who donated 40 ml EDTA blood or more,
sufficient amounts of PMBC could be isolated to analyze the
presence of JCV DNA in PBMC by polymerase chain reaction (PCR). These were 4 HIV, 3 PML, and 5 HIV/PML
patients. In addition, 3 HD with and 3 HD without JCVspecific antibodies were investigated. DNA was isolated from
106 PBMC. Cells were lysed by 1% sodium dodecyl sulfate
(SDS) and incubated in the presence of proteinase K (300
␮g/ml) overnight at 56°C. Thereafter, an equal volume of
buffer-saturated phenol was added and mixed, and DNA was
extracted by phenol-chloroform extraction. Finally, the DNA
was precipitated by the addition of 0.1 volumes of a saturated NaCl solution followed by an equal volume of isopro-
panol for 1 hr at –20°C. DNA was pelleted at 20,000g for
15 minutes; DNA pellets were washed repeatedly with 70%
ethanol, dried, and finally suspended in 100 ␮l H2O.
PCR Amplification
Nested PCR was carried out using 1 ␮g of purified DNA as
previously described,7 with some modifications. The first
round of PCR consisted of 10 cycles with the primer pair
JC4284/JC4961 flanking a 656-base-pair fragment of the
large T gene. The sequence of primer JC4284 in 5⬘ to 3⬘
orientation was from position 4,284 to position 4,527 TTT
TTA GGT GGG GTA GAG TGT TG and primer JC4961
from position 4,982 to 4,961 TGG AGC TTA TGG ATT
TAT TAG G. The second round of PCR was carried out
with 4 ␮l of the first round for 30 cycles using the primer
pair JC36/JC39 spanning a 389-base-pair fragment located
in the large T gene region.6 In 5⬘ to 3⬘ orientation the
primer JC36 has the sequence AGA TCC CTG TAG GGG
GGT GTC TCC from position 4,505 to position 4,527 and
primer JC 39 from position 4,894 to position 4,874 the sequence AAT GCA AAG AAC TCC ACC CTG ATA AAG
GTG G. Each amplification round was initiated at 95°C for
2 minutes 30 seconds. Cycles of first-round PCR consisted
of 95°C for 30 seconds, 55°C for 1 minute, and 72°C for 1
minute. Second-round PCR consisted of 95°C for 30 seconds, 60°C for 1 minute, and 72°C for 1 minute. Both
rounds were concluded with an extension cycle of 72°C for 5
minutes. By this PCR, at least 10 copies of target JCV DNA
molecules were detected in a background of a JCV-negative
cell extract from 106 cells of a permanent human T cell line,
which was spiked with 106 to 0 JCV DNA molecules.
Table 1. Characteristics of HIV-1-Infected Patients
Patients
CD4
(Cells/␮l)
CD4/CD8
(Ratio)
HIV
RO
HW
LK
660
220
160
0.50
0.60
0.32
5,500
1,600
0
KR
300
nd
0
22
204
0.02
0.38
560,000
24,000
443
373
346
0.50
0.40
0.14
158,000
nd
14,500
BT
60
0.19
600
HA
250
0.24
nd
UM
BK
nd
nd
nd
nd
nd
nd
OR
77
0.07
3,700
MM
GE
HIV/PML
MF
VP
WM
HIV-1 Virus Load
(RNA Copies/ml)
Antiretroviral Therapy
None
Azidothymidin, lamivudin
Azidothymidin, lamivudin,
saquinavir
Azidothymidin, lamivudin,
saquinavir
None
None
None
IL-2, lamivudin, stavudin
Dideoxyinosin, saquinavir,
stavudin
Azidothymidin, lamivudin,
saquinavir
Azidothymidin, indinavir,
lamivudin
Fivefold combination
Azidothymidin, indinavir,
lamivudin
Fivefold combination
HIV ⫽ HIV-1-infected patients without PML; HIV/PML ⫽ HIV-1-infected PML patients. There was no difference between HIV-1-infected
patients with or without PML with respect to CD4/CD8 ratio and HIV-1 virus load ( p ⫽ 0.428 and p ⫽ 0.699, respectively).
Weber et al: Immune Response in PML
637
Analysis of PCR Products
Second-round PCR products were purified by a PCR purification kit according to the manufacturers procedure (Qiagen, Hilden, Germany). For proving JCV specificity, the purified DNA was digested with the restriction endonuclease
Rsa I according to the instructions of the manufacturer (Amersham Pharmacia Biotech, Freiburg, Germany) and separated on a 2% agarose gel. In case of JCV specificity, two
fragments of about 100 and 300 base pairs will arise after
restriction enzyme digestion.
Antigens
The major structural protein VP1 of JCV was cloned and
expressed in insect cells as previously described.11 Recombinant VP1 spontaneously assembled to virus-like particles resembling empty JCV capsids.11 Tetanus toxoid (TT) was a
kind gift of Dr Blackkolb (Behringwerke, Marburg, Germany), and phytohemagglutinin (PHA) was purchased from
Sigma (Deisenhofen, Germany).
Preparation of PBMC and Proliferation Assay
Peripheral venous blood was separated within 24 hr by
Ficoll-Hypaque gradient centrifugation. PBMC were washed
twice with phosphate-buffered saline (PBS) and seeded into
round-bottomed 96 well culture plates (Nunc, Roskilde,
Denmark) at 2 ⫻ 105 cells per well in a final volume of 100
␮l CG medium (Vitromax, Selters, Germany) supplemented
with 2 mM L-glutamine, 103 U/ml penicillin and 103 U/ml
streptomycin (Gibco, Eggenstein, Germany). VP1-VLP were
added at a final concentration of 10 ␮g/ml. Parallel sets of
wells containing TT (10 ␮g/ml), PHA (5 ␮g/ml), or medium alone were used as controls. Cells were maintained at
37°C in a humidified atmosphere (5% CO2).
Four days after antigen stimulation, PBMC were labeled
with 0.1 ␮Ci [3H]thymidine (Amersham, Braunschweig,
Germany) and harvested 18 hr later. [3H]thymidine incorporation was measured with a scintillation counter (Tri-Carb
4000 Series; Packard, Frankfurt, Germany). All determinations were set up at least in triplicate. The stimulation index
(SI) was calculated as follows: counts per minute (cpm) of
antigen-stimulated culture/cpm of culture with medium.
Determination of Cytokines
PBMC cultures were set up as described above for the proliferation assay. After 2, 3, and 4 days the content of 3 wells
was pooled and pelleted (2,200g, 5minutes). The supernatant
was stored immediately at – 80°C. The Th1-associated cytokine interferon-␥ (IFN-␥) and the Th2-associated cytokine
interleukin-10 (IL-10) were determined by a sandwich
enzyme-linked immunosorbent assay (ELISA). Antibodies
and standard were purchased from Biosource (Ratingen,
Germany). The ELISA was performed according to the instructions of the manufacturer, with minor modifications.
All samples were determined in duplicate. The detection limits of IFN-␥ and IL-10 were determined at 3.4 pg/ml and
2.4 pg/ml, respectively.
VP1-Specific Antibody ELISA
The titer of VP1-specific immunoglobulin G antibodies in
sera of patients and HD was determined as described previ-
638
Annals of Neurology
Vol 49
No 5
May 2001
ously.8 In brief, ELISA plates (Nunc) were coated with 100
␮l VP1-VLP (1 ␮g/ml) and incubated with serial dilutions
of sera. Human IgG was detected by a POD-conjugated goat
antihuman IgG (Dianova, Hamburg, Germany). Results
were expressed as arbitrary units, which were standardized
using a human serum pool.
Statistical Analysis
The Mann-Whitney test was used to calculate significance of
results. A p value ⬍ 0.05 was regarded as significant.
Results
Optimization of PBMC Proliferation
In pilot experiments, the dose response of PBMC to
increasing amounts of VP1-VLP was investigated in 4
members of the HD group. In the presence of 0.1–10
␮g/ml VP1-VLP, a dose-dependent proliferation was
demonstrated in all HD. A representative result is
shown in Figure 1. A similar dose response was obtained with TT as the stimulating antigen (data not
shown). In the following experiments, VP1-VLP and
TT were used at a concentration of 10 ␮g/ml. Further
analysis showed that not only CD4⫹ but also CD8⫹
cells were able to proliferate in response to the VP1VLP (data not shown).
Proliferation of PBMC Obtained from HD and
HIV, PML, or HIV/PML Patients
Figure 2A shows that proliferation after stimulation
with the VP1-VLP was highest in the HD group.
Compared to the case in the HD group, the proliferation was significantly suppressed in both the PML
( p ⫽ 0.004; Fig 2A) and the HIV/PML groups ( p ⫽
0.0002; Fig 2A). In the HIV/PML group, the SI values
were lower than in the HIV group. However, this difference was not statistically significant ( p ⫽ 0.21; Fig
Fig 1. The proliferation of peripheral blood mononuclear cells
against VP1-VLP is dose-dependent. A representative result of
a healthy donor (HD) is shown. Values represent mean ⫾
SD. All assays were performed in quadruplicate.
PHA, the PBMC proliferation in the HD group was
higher than in all other groups (Fig 2C), but differences between the groups were not statistically significant (PML and HD: p ⫽ 0.57; HIV and HIV/PML:
p ⫽ 0.6).
Cytokine Production of PBMC from HD and HIV,
PML, or HIV/PML Patients
IFN-␥ secretion after stimulation with VP1-VLP, TT,
and PHA was highest in the HD group (Fig 3A–C). A
marginal IFN-␥ production was found in all other
groups after stimulation with VP1-VLP or TT (Fig
3A,B). After stimulation with PHA, the IFN-␥ production was much lower in the HIV, PML, and HIV/
PML groups compared to the HD group (Fig 3C). Secretion of IL-10 by PBMC obtained from HIV/PML
patients in response to VP1-VLP or TT was clearly elevated compared to all other groups (Fig 3D,E). After
stimulation with PHA, however, there was a slight difference in favor of the HD and PML groups in comparison to both HIV-1-infected groups (Fig 3F).
Fig 3. Peripheral blood mononuclear cells (PBMC) of healthy
donors (HD), progressive multifocal leukoencephalopathy
(PML) patients without HIV-1 infection (PML), HIV-1infected patients without clinical signs of PML (HIV), and
HIV-1-infected PML patients (HIV/PML) were stimulated
with VP1-VLP (10 ␮g/ml; A,D), TT (10 ␮g/ml; B,E), and
PHA (5 ␮g/ml; C,F). Production of IFN-␥ and IL-10 was
determined by ELISA on days 2, 3, and 4 after stimulation.
Values are given as mean over 3 days for each patient. In
addition, the median of each group is indicated.
Fig 2. Proliferation of peripheral blood mononuclear cells
(PBMC) of healthy donors (HD), progressive multifocal leukoencephalopathy (PML) patients without HIV-1 infection
(PML), HIV-1-infected patients without clinical signs of PML
(HIV), and HIV-1-infected PML patients (HIV/PML) in the
presence of VP1-VLP (10 ␮g/ml; A), TT (10 ␮g/ml; B), and
PHA (5 ␮g/ml; C) is shown. The difference in proliferation
to VP1-VLP (A) and TT (B) between HD and PML is statistically significant ( p ⫽ 0.004 and p ⫽ 0.012, respectively).
The difference in proliferation between HIV and HIV/PML is
statistically not significant ( p ⫽ 0.21 and p ⫽ 0.19, respectively). Proliferation was determined by [3H]thymidine incorporation. Values are given as stimulation index and median.
2A). Stimulation of PBMC with TT yielded similar
results. The HD group showed the highest proliferation (Fig 2B). Whereas the response to TT in the PML
group was significantly lower than in the HD group
( p ⫽ 0.012), the difference between the HIV group
and the HIV/PML group was small and not statistically significant ( p ⫽ 0.19). After stimulation with
Weber et al: Immune Response in PML
639
JCV DNA Detection in PBMC and JCV-Specific
Humoral Immune Response
PBMC JCV DNA was detected in 50% of HD, in
75% of HIV patients, in 66% of PML patients, and in
60% of HIV/PML patients (Table 2). All PCR products were demonstrated to be JCV-specific by restriction endonuclease digestion (data not shown). In the
HD group, the presence of JCV DNA in PBMC correlated with the occurrence of JCV-specific antibodies.
No correlation, however, was found in any other group
(Table 2). The highest VP1-specific antibody titers
were found for HIV/PML patients, who showed a very
low proliferation in response to VP1-VLP. No correlation of the SI values after PBMC stimulation with
VP1-VLP, TT, or PHA and the presence of JCV DNA
in the PBMC was found in any of the groups. The
high VP1-specific antibody titer in the HIV/PML
group is in agreement with the IL-10 production
found in this group. However, there was no significant
difference between the antibody titers of the PML and
the HD groups or between the HIV and the HIV/
PML groups ( p ⫽ 0.11 and p ⫽ 0.06, respectively).
response to purified natural JCV particles in a PML
patient compared to HD.16 This observation is in line
with the findings reported here for VP1-VLP, which
appeared to be a very useful antigen for the assessment
of the JCV-specific cellular and humoral immune response.
The present study demonstrates that a pronounced
JCV-specific proliferative response in HD is obviously
one of the important mechanisms to control JCV. A
significantly lower JCV-specific proliferation of PBMC
compared to HD was found in PML and HIV/PML
patients. Furthermore, the proliferative response of
PBMC to TT and PHA was also impaired in PML
patients compared to HD. Our observation of a reduced lymphocyte proliferation in response to PHA
stimulation is in line with earlier reports10,16 and the
previous description of the absence of a delayed-type
hypersensitivity to various antigens in PML patients.10,15
In parallel with the suppressed proliferation, the production of the Th1-type cytokine IFN-␥, which is associated with an antiviral cellular immune response,
was strongly reduced in PML, HIV/PML, and HIV
patients after stimulation with VP1-VLP, TT, and
PHA. In contrast, the production of the Th2-type cytokine IL-10, which is associated with an antiviral humoral immune response, was elevated in response to
VP1-VLP and TT in HIV/PML. Neither the proliferative capacity nor the cytokine production was correlated to the JCV DNA detected in the PBMC. The
frequency of JCV DNA was in line with several studies.17 The relatively high percentage of JCV DNA detected in the PBMC of patients in the HIV group was
similarly correlated with the low CD4⫹ T cell count,
as previously reported.18
The elevated titer of VP1-specific IgG antibodies in
PML and HIV/PML patients compared to HD and
HIV patients provides further evidence for an antiviral
humoral immune response. These findings support the
Discussion
Investigations of the immunopathogenesis of PML are
limited to humans, because no animal model for PML
is known.14 Only a few studies have addressed the cellular and humoral immune response to JCV in
PML.9,10,15 We analyzed the JCV-specific cellular and
humoral immune response in HIV-1-infected patients
without or with PML and in PML patients without
HIV-1 infection. HD were included in the study to
analyze the normal situation at baseline. The present
investigation became feasible with the availability of
sufficient amounts of the recombinant major structural
protein VP1 of JCV.11 The recombinant VP1 formed
VP1-VLP, which exhibits the same structural and functional properties as natural JCV capsids.11 In an earlier
study, we demonstrated a highly reduced proliferative
Table 2. JCV-Specific Humoral Immune Response and Detection of JCV DNA in PBMC by PCR
HD
Median:
PML
HIV
HIV/PML
Donor
Titers
PCR
Donor
Titers
PCR
Donor
Titers
PCR
Donor
WL
MK
SZ
SP
EM
CG
KL
4,800
1,664
0
0
4,500
0
17,500
nd
⫹
⫺
⫺
⫹
⫺
⫹
ED
GH
PW
0
60,000
3,500
⫹
⫺
⫹
RO
HW
LK
KR
MM
GE
6,800
0
44,000
43,200
0
0
⫹
⫺
nd
⫹
nd
⫹
MF
VP
WM
BT
HA
UM
BK
0
OR
1,664
33,550
3,400
Titers
8,400
316,000
7,584,000
353,600
2,480
87,000
150,000
118,700
PCR
nd
nd
⫺
⫹
⫺
⫹
nd
⫹
HD ⫽ healthy donors; PML ⫽ PML patients without HIV-1 infection; HIV ⫽ HIV-1-infected patients without PML; HIV/PML ⫽ HIV1-infected PML patients; nd ⫽ not done; PCR ⫽ polymerase chain reaction; ⫹ ⫽ ⬎10 JCV molecules; ⫺ ⫽ ⬍10 JCV molecules; PBMC ⫽
peripheral blood mononuclear cells. Antibody titers were expressed as arbitrary units.
640
Annals of Neurology
Vol 49
No 5
May 2001
hypothesis of an impaired cellular immune response
leading to the reactivation of JCV and the development of PML. Although Willoughby et al.10 described
a specific deficiency in the cellular immune response to
JCV in PML patients, our more detailed study does
not support a JCV-specific immunodeficiency. This is
corroborated by the observation of a reduced PBMC
proliferation and suppressed production of IFN-␥ after
stimulation with TT, PHA, and VP1-VLP. Our results
indicate a general loss of Th1-type T-helper functions
in PML patients as evidenced by the reduction of proliferation and IFN-␥ production in the presence of all
three stimulants. However, to support this observation,
more Th1-type cytokines (interleukin-2, tumor-necrosisfactor-␣, and lymphotoxin) should be investigated in
further studies. This hypothesis is in agreement with several reports emphasizing the importance of IFN-␥ in the
control of a number of virus infections.19 In addition,
the effect of highly active antiretroviral therapy
(HAART) argues against a selective JCV-specific immunodeficiency. It has recently been shown that HAART
significantly prolongs the survival of HIV-1-infected
PML patients.12,20 –27 The positive effect of HAART
appears to be due to the reconstitution of the immune
system and correlates with an increase of the number of
CD4⫹ T cells.23–25
In contrast to the diminished proliferation of
PBMC, JCV-specific serum antibodies are elevated in
most PML patients, in particular in the HIV/PML patients. Obviously, the B cell response against JCV is
not impaired in most PML patients and may be T cellindependent. Such a T cell-independent humoral immune response occurs after infection with the mouse
polyomavirus28 and provides resistance to acute mortality.29 The finding of a high antibody titer in HIV/
PML patients together with an elevated IL-10 production after stimulation with VP1-VLP supports a
functional Th2-type T-helper response. It may reflect a
shift from a Th1-type to a Th2-type immune response
in these patients. Recent evidence also suggests an association of prolonged survival of PML patients receiving HAART with the development of a VP1-specific
intrathecal humoral immune response.12 The demonstration of a JCV-neutralizing capacity of serum or intrathecal VP1-specific antibodies would elucidate the
role of the humoral immune response in the development of PML. Because the VP1-VLP carry authentic
neutralizing epitopes of JCV,11 they represent an ideal
antigen to perform such a detailed study for JCVspecific neutralizing antibodies.
Furthermore, recent findings of a comparable incidence of PML in patients receiving HAART and historical controls indicate the need for a further detailed
and systematic study of the cellular and humoral immune response in these patients.19,20 This study should
include the analysis of more Th1- and Th2-type cyto-
kines to elucidate further the importance of the Th1and Th2-type response in controlling JCV. Such a
study with a higher number of patients appears now to
be feasible, with a median survival of more than 46
weeks in PML patients receiving HAART19,20 and the
availability of a useful JCV-authentic antigen in sufficient amounts.11
The study was supported by the Deutsche Forschungsgemeinschaft
(We1297/3–1 and Lu397/5–1) and by a grant from the Werner
Otto Stiftung to T.W. The work of M. Pickhardt is supported by
the Werner Otto Stiftung, Hamburg, Germany.
We thank Drs A. Argyrakis and C. Trebst for providing blood samples and case histories of patients with PML. W.L. and T.W. contributed equally to this work.
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