Journal of General Virology (2004), 85, 2379–2387
DOI 10.1099/vir.0.80115-0
Immunization with dendritic cells can break
immunological ignorance toward a persisting virus
in the central nervous system and induce partial
protection against intracerebral viral challenge
Ulrike Fassnacht, Andreas Ackermann, Peter Staeheli
and Jürgen Hausmann
Correspondence
Jürgen Hausmann
Department of Virology, University of Freiburg, Hermann-Herder-Str. 11, D-79104 Freiburg,
Germany
juergen.hausmann@
uniklinik-freiburg.de
Received 16 March 2004
Accepted 3 May 2004
Dendritic cells (DCs) have been used successfully to induce CD8 T cells that control virus
infections and growth of tumours. The efficacy of DC-mediated immunization for the control of
neurotropic Borna disease virus (BDV) in mice was evaluated. Certain strains of mice only rarely
develop spontaneous neurological disease, despite massive BDV replication in the brain.
Resistance to disease is due to immunological ignorance toward BDV antigen in the central nervous
system. Ignorance in mice can be broken by immunization with DCs coated with TELEISSI, a
peptide derived from the N protein of BDV, which represents the immunodominant cytotoxic T
lymphocyte epitope in H-2k mice. Immunization with TELEISSI-coated DCs further induced solid
protective immunity against intravenous challenge with a recombinant vaccinia virus expressing
BDV-N. Interestingly, however, this immunization scheme induced only moderate protection against
intracerebral challenge with BDV, suggesting that immune memory raised against a shared
antigen may be sufficient to control a peripherally replicating virus, but not a highly neurotropic virus
that is able to avoid activation of T cells. This difference might be due to the lack of
BDV-specific CD4 T cells and/or inefficient reactivation of DC-primed, BDV-specific CD8 T cells
by the locally restricted BDV infection. Thus, a successful vaccine against persistent viruses
with strong neurotropism should probably induce antiviral CD8 (as well as CD4) T-cell responses
and should favour the accumulation of virus-specific memory T cells in cervical lymph nodes.
INTRODUCTION
Borna disease virus (BDV) is an enveloped virus with a
single-stranded RNA genome of negative polarity (Briese
et al., 1995; Schneemann et al., 1995). It is non-cytolytic
in vitro (Herzog & Rott, 1980; Ludwig et al., 1993), as well as
in vivo (Gosztonyi & Ludwig, 1995; Sauder et al., 2001), and
can readily establish a persistent infection of the central
nervous system (CNS) in a wide variety of vertebrate species.
Infection may lead to non-purulent meningoencephalitis in
susceptible hosts (Ludwig et al., 1988; Rott & Becht, 1995).
Induction of protective immunity against BDV is a
challenging task (Lewis et al., 1999; Richt & Rott, 2001).
As BDV-induced disease results from the cellular antiviral
immune response (Hallensleben et al., 1998; Stitz et al.,
2002), vaccination can have either a beneficial or a
detrimental outcome. In fact, attempts to induce antiviral
immunity by active immunization with a recombinant
vaccinia virus (VV) expressing BDV-N protein enhanced
immunopathology in Lewis rats (Lewis et al., 1999). Analysis
showed that virus replication after intracerebral challenge
0008-0115 G 2004 SGM
with BDV was reduced, at the cost of enhanced meningoencephalitis and clinical symptoms. Earlier attempts to
prime protective immune responses in horses by applying
an attenuated BDV strain showed limited success and
vaccination was eventually stopped, due to doubts about its
protective effect (Ludwig & Bode, 2000).
After intracerebral infection with BDV, mice develop either
severe meningoencephalitis or symptomless persistent infection, depending on the virus strain and age of the animal at
infection. MRL mice (H-2k) show high susceptibility to
virus-induced neurological disease, whereas B10.BR (H-2k)
or F1 (MRL6B10.BR) mice mostly remain healthy, despite
persistent virus infection of the CNS (Hallensleben et al.,
1998; Hausmann et al., 1999). It has been shown that B10.BR
mice ignore BDV infection of the CNS, as post-exposure
activation of BDV-specific T cells by peripheral immunization could induce disease, indicating that virus-specific
T cells were present, but failed to be activated (Hausmann
et al., 1999). A large body of experimental data indicates
that neurological disease and behavioural abnormalities in
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BDV-infected hosts result from immunopathological processes that are mediated by CD8 T cells, which require help
from CD4 T cells (Bilzer et al., 1995; Sobbe et al., 1997;
Hallensleben et al., 1998; Nöske et al., 1998). Diseaseinducing CD8 T cells in susceptible H-2k mice recognize the
immunodominant epitope TELEISSI, which is derived from
the viral N protein (Schamel et al., 2001).
Immunization with peptide-loaded dendritic cells (DCs)
was shown previously to elicit cytotoxic T lymphocyte
(CTL)-mediated protective immunity against systemic
infection with a non-cytolytic virus (Ludewig et al., 1998).
We therefore evaluated whether immunity against BDV
could similarly be induced by active immunization with
DCs. DC immunization efficiently terminated immunological ignorance of mice that were persistently infected with
BDV. It further induced solid protection against a
recombinant VV expressing BDV-N, but induced less
potent protection against intracerebral challenge with BDV.
METHODS
Mice. MRL/MpJ and B10.BR mice were originally purchased from
the Jackson Laboratory (Bar Harbor, ME, USA). Breeding colonies
were maintained in our local animal facility.
Viruses. The BDV stock that was used to infect mice was adapted
to the mouse by four consecutive passages of the rat-adapted BDV
strain 4p (Planz et al., 2003) through brains of newborn BALB/c
mice. After two more passages through adult MRL mouse brains,
stocks were amplified once in brains of 5-week-old rats and 10 %
(w/v) rat brain homogenates were prepared. Recombinant VVs
expressing BDV-N from strain He/80 and P450scc as irrelevant antigen have been described previously (Hausmann et al., 1999;
Ortmann et al., 2004) and were grown and titrated in CV-1 cells by
standard procedures (Mackett et al., 1984).
Animal infection. DC-immunized F1 (MRL6B10.BR) mice were
infected intracerebrally under ether anaesthesia at 7 weeks of age
with 10 ml samples of 10 % rat brain homogenates that contained
300 focus-forming units of mouse-adapted BDV. Naive B10.BR mice
were infected at 12–15 weeks of age with 300 focus-forming units of
the same stock of mouse-adapted BDV. For analysis of anti-VV
immunity after DC/TELEISSI vaccination, B10.BR and F1
(MRL6B10.BR) mice were infected intravenously with 107 p.f.u. of
the recombinant VVs indicated.
DC preparation and vaccination. DCs were prepared from
murine bone marrow essentially as described previously (Lutz et al.,
1999). Briefly, bone marrow was flushed out of fibiae and tibulae of
8–16-week-old mice with Iscove’s modified Dulbecco’s medium
(IMDM) supplemented with 10 % fetal calf serum (FCS), nonessential amino acids and 50 mM b-mercaptoethanol. Cells were
pooled and counted. Mean yields were 5–96107 cells per mouse.
Cells were plated at 5–76106 cells per dish in 10 ml IMDM supplemented with non-essential amino acids, 50 mM b-mercaptoethanol,
5 ng interleukin 4 (IL4) ml21 (PromoCell) and 100 ng granulocyte–
macrophage colony-stimulating factor (GM-CSF) ml21, which
was obtained from the culture supernatant of Ag8653 mouse
myeloma cells. On days 3 and 5 of culture, 80 % of the medium was
replaced by fresh medium supplemented with growth factors. DCs
were usually harvested and used for immunization without further
in vitro maturation, except where indicated. If maximal maturation was required, lipopolysaccharide (LPS) was added to a final
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concentration of 1 mg ml21 on day 7 and the cells were incubated
overnight. Cells were harvested either on day 7 or after LPS maturation on day 8 by pooling collected cells from the supernatant with
trypsinized adherent cells. DCs were washed with IMDM/10 % FCS,
counted and pulsed with 1024 M of the indicated peptides for
2–3 h. After three washes with PBS, cells were transferred into animals in the quantities indicated. Vaccination was performed either
by injecting 200 ml DC suspension into the lateral tail veins or by
subcutaneous injection at the left and right flanks of the animals.
Peptides and major histocompatibility complex (MHC) class
I tetramer. Peptides TELEISSI (Schamel et al., 2001) and
FEANGNLI (Gould et al., 1991) were purchased from Neosystem at
a purity of >65 % (immunograde) for in vitro assays. TELEISSI–
H-2Kk tetrameric complexes labelled with phycoerythrin (PE) were
kindly provided by the NIAID tetramer facility, Atlanta, GA, USA.
Tetramers were tested, together with anti-CD8 antibodies, over a
range of doses and temperatures for optimal binding to specific
brain lymphocytes by flow cytometry and were used at a concentration of 5 mg ml21 at room temperature.
Flow cytometry. For surface staining, suspensions of 2?56105–
106 DCs were incubated at 4 uC with anti-CD16/CD32 in 50–100 ml
PBS supplemented with 1 % FCS and 0?1 % NaN3 to block nonspecific IgG binding, followed by incubation with properly diluted
mAbs anti-CD11c–PE, anti-I-Ek–FITC (fluorescein isothiocyanate)
and/or anti-CD86–biotin. Streptavidin–FITC (Caltag) was used as
the secondary reagent. For detection of TELEISSI-specific CD8
T cells, in vitro-restimulated splenocytes were incubated with allophycocyanin (APC)-conjugated anti-CD8a (1 mg ml21, clone 53-6.7;
Pharmingen) with or without PE-labelled Kk/TELEISSI tetramer
(5 mg ml21) for 30 min at room temperature. Analysis of cells was
performed on a FACSort flow cytometer (BD).
Isolation
and
in
vitro
restimulation
of
splenocytes.
Splenocytes were obtained by gently pressing the spleen through a
metal grid (60 mesh; Sigma) in PBS. The tissue suspension was pelleted and resuspended in IMDM/10 % FCS. After sedimentation of
large debris, the supernatant was collected and the lymphocytes were
used for in vitro restimulation as effector cells. Naive splenocytes
were treated with mitomycin C and pulsed with TELEISSI peptide at
a concentration of 1026 M for 60 min to serve as restimulator cells.
After washing, the peptide-pulsed splenocytes were mixed with
splenocytes from immunized animals at an effector/stimulator ratio
of 10 : 1 for 9–14 days in IMDM supplemented with 10 % FCS and
50 mM b-mercaptoethanol. Cultures were used for tetramer staining
and as effector cells in cytotoxicity assays.
In vitro cytotoxicity assay. Cytolytic activity of in vitro-restimu-
lated splenocytes from DC-vaccinated F1 (MRL6B10.BR) mice was
determined by a standard 51Cr-release assay. Briefly, 56106 L929
(H-2k) cells were labelled in suspension with 200 mCi Na251CrO4
(ICN Biomedicals) and 1024 M peptide for 2 h at 37 uC. After three
washes with IMDM, they were diluted to a final concentration of
46104 cells ml21, dispensed into 96-well round-bottom microtitre
plates at 46103 cells per well and coincubated with different dilutions of in vitro-restimulated lymphocyte cultures, as indicated, in a
total volume of 200 ml for 6 h at 37 uC. The percentage of specific
target cell lysis was calculated according to the following formula:
1006[(test release 2 spontaneous release)/(total release 2 spontaneous release)]. Target cells were pulsed with 1024 M TELEISSI for
the determination of specific lysis or, as control, with the irrelevant,
H-2Kk-binding peptide FEANGNLI that is derived from the HA
protein of influenza virus A/PR/8/34 (H1N1) (Gould et al., 1991).
Spontaneous release did not exceed 28 % on average.
Determination of VV titres in ovaries. To measure the protec-
tive efficacy of TELEISSI-specific CD8 T cells against a recombinant
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VV expressing this epitope, vaccinated mice were challenged with
107 p.f.u. VV-N and sacrificed 7 days later. Ovaries were removed,
weighed and homogenized by using glass douncers. To completely
release virus particles, cell lysates were subjected to three cycles of
freeze–thawing followed by 10 min sonication. Cellular debris was
pelleted by centrifuging for 5 min at 1000 g and VV in the supernatants was titrated on BSC-40 cells by using standard protocols
(Mackett et al., 1984).
Histology and immunohistochemical analysis. Brains from
sacrificed animals were divided along the midline on removal and
the hemispheres were immersed in Zamboni’s fixative (4 % paraformaldehyde and 15 % picric acid in 0?25 M sodium phosphate,
pH 7?5) for at least 24 h. Fixed brain hemispheres were embedded
in paraffin. Immunostaining of brain sections was performed overnight at 4 uC by using a mouse mAb against BDV-N [Bo18 (Haas
et al., 1986), kindly provided by J. Richt, Giessen, Germany].
Blocking and antibody dilutions were done in PBS that contained
5 % normal goat serum. After extensive washing, bound antibody
was detected by using a peroxidase-based Vectastain elite ABC kit
(Vector Laboratories). Diaminobenzidine was used as the substrate,
according to the manufacturer’s instructions. Counterstaining was
done with haematoxylin.
RESULTS
DCs were prepared from bone marrow of mice by standard procedures (Lutz et al., 1999) by using GM-CSF
and IL4 as growth and differentiation factors. After
7 days culture with media replacement every other day,
bone marrow-derived cell cultures usually contained
between 40 and 60 % CD11c+/MHC II+ cells, representing
DCs. To induce TELEISSI-specific CD8 T-cell responses, F1
(MRL6B10.BR) animals were immunized twice at an
interval of 1 week with 56105 peptide-loaded DCs from
MRL mice by the intravenous route. One day before peptide
loading, DC maturation was induced by addition of LPS to
the culture medium. Eight days after the second immunization, splenocytes from immunized mice were restimulated
in vitro for 10 days and subsequently tested for TELEISSIspecific lytic activity. Specific lysis of target cells was clearly
detectable in restimulated splenocyte cultures of F1
(MRL6B10.BR) mice (Fig. 1a), albeit at rather low levels,
and was similarly detectable in splenocyte cultures of MRL
mice that were immunized in parallel (data not shown). To
improve detection of TELEISSI-specific CD8 T cells after DC
immunization, we performed an additional booster immunization with peptide-pulsed DCs in F1 (MRL6B10.BR)
mice. Following this vaccination scheme, we obtained high
levels of lytic activity after secondary in vitro restimulation of
splenocytes from immunized mice (Fig. 1b). Analysis of
restimulated cultures by staining with H-2Kk/TELEISSI
tetramers showed that, depending on the immunization
schedule, 2–14 % (mean, 5?5 %) of all restimulated CD8
T cells were tetramer-positive and therefore TELEISSIspecific (Fig. 1c). Immunization with control peptideloaded DCs induced no TELEISSI-specific T cells and
staining remained at background level (~1 %) (Fig. 1c).
Cultures that contained >10 % tetramer-positive CD8
T cells displayed high TELEISSI-specific lytic activity,
whereas cultures that contained only around 1–2 %
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tetramer-positive CD8 T cells had moderate CTL activity
(Fig. 1d). Cultures with intermediate numbers of tetramerpositive CD8 T cells showed intermediate lytic activity
(Fig. 1d). Thus, the number of tetramer-positive CD8
T cells in restimulated cultures correlated well with the
TELEISSI-specific lytic activity of these splenocytes, again
demonstrating that the observed lytic activity was specific
for the immunogen. In conclusion, significant numbers
of TELEISSI-specific CD8 T cells could be induced by
immunization with peptide-loaded DCs.
We next determined whether DC immunization would be
efficient enough to induce TELEISSI-specific CD8 T cells
that can break immunological ignorance toward BDV in
persistently infected mice. We demonstrated previously that
this was possible if recombinant VV expressing the BDV-N
was used for immunization (Hausmann et al., 1999). For
these experiments, we employed F1 (MRL6B10.BR) mice
that were infected with BDV at about 2 weeks of age. Like
parental B10.BR mice, F1 animals rarely developed
spontaneous disease after infection with BDV (data not
shown); instead, BDV established symptomless, persistent
infection in the CNS of these animals. Before immunization
at 10 weeks post-infection, all recipient animals were
healthy. They were immunized by the subcutaneous route
with MRL-derived DCs that were loaded with either
TELEISSI or control peptide. At 5–8 days after DC
application, all five TELEISSI-immunized mice developed
severe neurological symptoms. In three animals, disease was
so strong that they had to be sacrificed prematurely
(Fig. 2a). In two of these mice, onset of symptoms occurred
early and disease progressed very rapidly without significant
weight loss. In the remaining animals, neurological
symptoms were accompanied by massive weight loss
(Fig. 2a). All three control-vaccinated mice showed no
signs of disease and no significant weight loss (Fig. 2a).
Similar results were obtained in a second experiment in
which four animals received TELEISSI-loaded DCs and
three animals were immunized with control peptide-loaded
DCs (results of both experiments are summarized in
Table 1). Analysis of virus spread by immunohistochemical
analysis of brain sections confirmed that BDV had
established persistent infection in the brains of all animals
(Fig. 2h, i; Table 1), which excluded variability of CNS
infection as the cause of the observed differences. Infection
even affected a number of CA1 region neurons, which are
typically only rarely infected in mice (Sauder et al., 2001;
Rauer et al., 2004), except after prolonged times of BDV
persistence. As BDV had persisted in brains of mice used in
this experiment for >10 weeks, the infection had spread to
a number of CA1 neurons (Fig. 2h, i).
The hippocampus (Fig. 2b), cortex (Fig. 2d) and cerebellum (Fig. 2f) of TELEISSI-immunized mice were massively
infiltrated by mononuclear cells. In contrast, only very
few inflammatory infiltrates were detectable in the various
brain regions of control-immunized mice (Fig. 2c, e, g),
with the exception of two animals that showed stronger
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Fig. 1. Induction of TELEISSI-specific CD8 T cells by DC immunization. (a) F1 (MRL6B10.BR) mice were immunized twice
at a weekly interval with 56105 DCs that were derived from the bone marrow of MRL mice. Splenocytes of recipient mice
were prepared 8 days after the last immunization. Restimulation in vitro was for 10 days using TELEISSI-loaded, mitomycin
C-treated splenocytes from syngenic mice as antigen-presenting cells. Lytic activity of cultures in serial threefold dilutions was
determined by a standard 6 h 51Cr-release assay using RDM-4 cells that were pulsed with either 10”4 M TELEISSI or the
H-2Kk-binding control peptide FEANGNLI. (b, d) F1 (MRL6B10.BR) mice were immunized three times at weeks 0, 1 and 6
with DCs that were derived from the bone marrow of MRL mice loaded with TELEISSI. Splenocytes were harvested 11 days
after the last immunization. Restimulation in vitro was done for 6–7 days by using TELEISSI-pulsed, mitomycin C-treated
splenocytes from syngenic mice. Cultures were either tested for lytic activity in serial threefold dilutions or stained with
Kk/TELEISSI tetramer. Lytic activity was assessed by a standard 6 h 51Cr-release assay after 6 days culture using L929 cells
pulsed with either 10”4 M TELEISSI or 10”4 M of the H-2Kk-binding control peptide FEANGNLI. Results are shown as mean
values ±SEM (b) or individual curves from cultures of three immunized mice that contained the indicated percentages of
Kk/TELEISSI tetramer-positive CD8 T cells (d). (c) Restimulated splenocytes were stained with anti-CD8-APC and
Kk/TELEISSI-PE tetramer after 7 days culture and analysed by flow cytometry. Representative flow cytometry profiles are
shown for one animal that was immunized with TELEISSI and one animal that was immunized with the control peptide
FEANGNLI. Numbers in the upper right quadrant indicate percentages of tetramer-positive CD8 T cells.
mononuclear infiltrates (Table 1). It is possible that nonspecific activation of immune cells by control DCs was
particularly strong in these animals, leading to an influx of
such activated, non-specific cells into the infected brain
without causing disease. In conclusion, immunization with
TELEISSI-loaded DCs could break ignorance toward BDV
in the CNS. Efficiency appeared to be similar to that for
immunization with a recombinant VV expressing BDV-N
(Hausmann et al., 1999).
To measure protective immunity induced by immunization with TELEISSI-loaded DCs, in a first experiment,
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vaccinated B10.BR mice were challenged with VV expressing
BDV-N. VV replication was assessed in the ovaries, where it
replicates best (Karupiah & Blanden, 1990; Binder &
Kundig, 1991). Immunization was done by a single
application of either a low or a high dose (26105 or
96106 cells) of peptide-loaded, syngenic DCs. Animals that
were immunized with either dose of TELEISSI-loaded
DCs showed solid protection against challenge with VV-N.
In contrast, the virus grew well in the ovaries of control mice
that had been immunized with control peptide-loaded DCs
(Fig. 3a). In a second experiment, F1 (MRL6B10.BR) mice
were immunized with TELEISSI-loaded DCs from MRL
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Cell vaccination against neurotropic BDV infection
Table 1. Breaking immunological ignorance in BDV-infected F1 (MRL6B10.BR) mice by
immunization with peptide-loaded DCs
Persistently infected, symptomless F1 (MRL6B10.BR) mice were immunized at 8–10 weeks postinfection with peptide-loaded DCs in two independent experiments. Animals were observed for 14
(experiment 1) and 11 (experiment 2) days, respectively.
Peptide
Neurological disease*
MeningoencephalitisD
CNS virus loadd
+++
++
++
+++
++
+++
+
+++
++
+++
+++
+++
+++
++
+++
+++
+++
+++
+++
+++
++
++
+++
+++
+++
+++
++
2
2
2
2
2
2
+
+
2
++
++
+
+++
++
++
+++
+++
+++
TELEISSI:
Control peptide:
*Scores: 2, no disease; +, mild disease; ++, severe, but not life-threatening disease; +++, lifethreatening disease.
DScores: 2, no infiltrates; +, one or two perivascular infiltrates per brain section with one or two layers of
cells, some mononuclear cells in meninges; ++, three to five perivascular infiltrates per brain section,
mostly with multilayer appearance and incidental spread into parenchyma, intermediate meningitis; +++,
more than five perivascular infiltrates per brain section with multiple layers of cells and strong infiltration of
parenchyma at multiple sites, strong meningitis.
dVirus load was assessed by immunohistochemical staining of sagittal brain sections with mAb Bo18
(recognizing BDV-N). Scoring was according to an arbitrary scale: 2, no virus antigen-positive cells detected
in at least three brain sections; +, <100 antigen-positive cells per brain section; ++, large number of
antigen-positive cells in certain brain regions only; +++, large number of antigen-positive cells in most
brain regions.
mice and challenged with two different VV recombinants,
one expressing BDV-N (VV-N) and one expressing an
irrelevant control protein (VVscc). Vaccination induced
solid protection against VV-N, but not against VVscc
(Fig. 3b), demonstrating the expected specificity of the
induced antiviral immune response.
To assess protective immunity against intracerebral
challenge with BDV, F1 (MRL6B10.BR) mice were
subcutaneously injected twice with DCs derived from
F1 (MRL6B10.BR) mice that were loaded with either
TELEISSI or control peptide. Seven days after the second
DC injection, mice were infected intracerebrally with
BDV. In contrast to the post-exposure immunization
protocol described above, immunized animals did not
show exacerbation of disease and all mice remained healthy
until 4 weeks post-infection, when the experiment was
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terminated. Immunohistological analysis of virus spread
showed that four out of six TELEISSI-immunized animals
had no virus antigen in the CNS, one presented with a
low-level infection and only one presented with disseminated BDV infection (Fig. 3c). In contrast, only one of six
control-vaccinated animals contained no BDV antigen in
the CNS, two presented with low-level infection and three
animals showed disseminated BDV infection (Fig. 3c).
Thus, DC immunization provided partial, but not complete,
protection against intracerebral BDV challenge (P=0?12;
Fisher’s exact test). BDV challenge of naive adult B10.BR
mice showed that a proportion of such animals (25 %) also
had low-level or even undetectable infection in the brain
(Fig. 3d). In contrast, in naive B10.BR mice that were
infected as suckling mice with the same or even lower doses
of virus, BDV always spread efficiently through the CNS
(Hausmann et al., 1999; data not shown), suggesting that
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Fig. 2. Immunization with TELEISSI-loaded
DCs can break immunological ignorance
in healthy mice persistently infected with
BDV. (a) Healthy F1 (MRL6B10.BR) mice
persistently infected with BDV were
immunized with a single, subcutaneous dose
(76106 cells) of MRL-derived DCs that
were loaded either with 10”4 M TELEISSI
(filled symbols) or with the same concentration of the control peptide FEANGNLI (open
symbols). Mice were observed for clinical
symptoms for up to 14 days post-immunization. They were then sacrificed for histological analysis of brain sections. Crosses
indicate animals that were sacrificed prematurely, due to severe neurological symptoms.
(b–i) Histological analysis of brains from two
representative animals immunized with either
TELEISSI-loaded DCs (b, d, f, h) or
FEANGNLI-loaded DCs (c, e, g, i). Brain
sections were stained with haematoxylin/
eosin (b–g) or immunostained with the
BDV-specific mAb Bo18 (brown stain) and
counterstained with haematoxylin (h, i). In
(b), note the higher number of nuclei in the
hilus region and the molecular layer of the
dentate gyrus, which represent mononuclear
cells infiltrating the CNS parenchyma.
older B10.BR mice are somewhat less susceptible than
suckling mice to intracerebral BDV challenge. In conclusion,
DC immunization showed a moderately protective effect
against intracerebral BDV challenge without exacerbating
disease.
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DISCUSSION
Interestingly, immune priming with one particular immunogen showed different protective efficacy in the two
infection models tested. Whereas protection against intracerebral BDV challenge by DC/TELEISSI vaccination was
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Cell vaccination against neurotropic BDV infection
Fig. 3. Immunization with TELEISSI-loaded DCs induces resistance to VV expressing BDV-N and inhibits replication of BDV
in the CNS. (a) B10.BR mice were immunized with a single,
subcutaneous, low-dose (26105 cells; filled symbols) or highdose (96106 cells; open symbols) application of B10.BRderived DCs that were loaded with either TELEISSI or control
peptide. Seven days post-immunization, animals were challenged with 107 p.f.u. recombinant VV expressing BDV-N (VVN). Seven days later, virus titres in ovaries were determined by
plaque assay using CV-1 cells. Horizontal lines indicate mean
values of titres from one group of mice. Dashed line indicates
detection limit. (b) F1 (MRL6B10.BR) mice were immunized by
a single, subcutaneous injection of 106 MRL-derived DCs that
were loaded with TELEISSI peptide. Immunity of vaccinated
animals was tested by using either VV-N (expressing BDV-N)
or VVscc (expressing the control protein P450scc). Horizontal
lines indicate mean values of titres from each group of mice.
Dashed line indicates detection limit. (c) F1 (MRL6B10.BR)
mice were given two subcutaneous injections at a weekly interval of 3–46106 F1 (MRL6B10.BR)-derived DCs that were
loaded with either TELEISSI or control peptide. One week after
the last immunization, mice were infected intracerebrally with
BDV. They were observed for clinical symptoms until sacrifice
at 4 weeks post-infection. Virus spread in the brain was
assessed by immunohistochemical staining with mAb Bo18.
(d) Naive B10.BR mice were infected intracerebrally with BDV
at age 12–15 weeks and brains of infected animals were
analysed 5 weeks post-infection for spread of BDV by immunohistochemical staining of paraffin sections with the N-specific
mAb Bo18.
not complete, replication of VV in ovaries was prevented
completely by this vaccination. One possible explanation is
that the two challenge viruses might reactivate primed
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memory CD8 T cells with different efficacy. Reactivation
presumably occurs at the site of immune priming, which is
still not conclusively defined for BDV. Available evidence
suggests that BDV-specific CD8 T cells are primed in
cervical lymph nodes (Batra et al., 2003). Priming of virusspecific CD8 T cells by other neurotropic viruses, such as
Lymphocytic choriomeningitis virus (LCMV) or a neurotropic variant of mouse hepatitis virus, also occurs in deep
cervical lymph nodes (Doherty et al., 1990; Marten et al.,
2003). If the encounter of BDV antigens and the immune
system in fact occurs at this site, the number of DC/
TELEISSI-induced CD8 memory T cells that is available
in the deep cervical lymph nodes is certainly of critical
importance. It is conceivable that the migration of specific
CD8 T cells primed by subcutaneous DC immunization into
these lymph nodes is limited. Thus, the BDV-specific CD8
T-cell response might not be reactivated efficiently enough
to protect completely against BDV infection. This view is
supported by the finding that intracerebral BDV infection
of newborn B10.BR mice did not detectably activate the
T-cell arm of the immune system, although such T cells
were not deleted or anergic, which indicated immunological ignorance at the T-cell level (Hausmann et al., 1999).
In contrast, intravenous VV inoculation leads to inflammatory infection of several organs, including secondary
lymphoid tissues (Moss, 1996), which could favour reactivation of DC-primed CD8 T cells and induce maturation
into fully differentiated effector CTLs. Interestingly, it was
shown that protection of mice by DC vaccination against
fatal neurological disease resulting from intracerebral
challenge with LCMV had already waned by 16 days after
immunization. In contrast, the same immunization protocol provided long-lasting protection against systemic
infection with LCMV (Ludewig et al., 1999). This result was
attributed to the reduced ability of locally produced LCMV
to reactivate CD8 T cells that had been primed by DC
immunization (Ludewig et al., 1999). In particular, peptidepulsed DCs may have limitations in the induction of fully
differentiated effector and memory CD8 T cells, as peptide
is removed rapidly from DCs (Ludewig et al., 2001), which
might lead to special requirements for reactivation of these
T cells.
A second reason for incomplete protection against BDV
infection might be the lack of BDV-specific CD4 T-cell
priming after immunization with the MHC class I-restricted
peptide TELEISSI. Induction of TELEISSI-specific CD8
T cells by peptide-pulsed DCs was performed in the absence
of BDV-specific CD4 T cells. In our experiments, T-cell help
was presumably provided by CD4 T cells that were activated
by DCs presenting helper epitopes that were derived from
bovine serum components. We assume that after BDV
challenge, virus-specific CD4 T cells might be necessary for
reactivation of specific CD8 memory T cells. Alternatively,
CD4 T cells might be necessary for maintenance of CTL
effector function in the CNS, as has been shown in a
neurotropic mouse hepatitis virus infection model
(Stohlman et al., 1998). We demonstrated previously that
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U. Fassnacht and others
CD4 T cells are required for antiviral immune responses that
lead to BDV-induced immunopathology (Hausmann et al.,
1999). Vaccine-induced T-cell responses that are restricted
to CD8 T cells might therefore be insufficient to completely
control infection of the CNS by BDV.
The effector mechanisms of CD8 T cells to control BDV
infection have not yet been determined conclusively. Data
from protection experiments in rats that used adoptive
immune-cell transfer suggested that perforin might be
involved (Nöske et al., 1998), whereas in vitro experiments
with BDV-infected murine organotypic slice cultures
showed that gamma interferon (IFN-c) might play an
important role (Friedl et al., 2004). Moreover, several
persistent virus infections of the CNS and especially of
neurons have been shown to be controlled by CD8 T cellderived IFN-c (Tishon et al., 1995; Bartholdy et al., 2000;
Binder & Griffin, 2001; Rodriguez et al., 2003). As immune
control of VV has been shown to be dependent on IFN-c
(Huang et al., 1993; Muller et al., 1994), TELEISSI-specific
CD8 T cells were apparently able to secrete this cytokine
in sufficient amounts to achieve protection against
infection of the ovaries. Thus, protective effects against
intracerebral BDV challenge might be mediated by IFN-c,
although perforin or other effector mechanisms of CD8
T cells cannot be ruled out at present.
Doherty, P. C., Allan, J. E., Lynch, F. & Ceredig, R. (1990). Dissection
of an inflammatory process induced by CD8+ T cells. Immunol
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& Pagenstecher, A. (2004). Borna disease virus multiplication in
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ACKNOWLEDGEMENTS
Karupiah, G. & Blanden, R. V. (1990). Anti-asialo-GM1 inhibits
We thank Rosita Frank for expert technical assistance in histology and
the MHC Tetramer Core Facility of the National Institute of Allergy
and Infectious Diseases, Emory University Vaccine Center, Atlanta, GA
30329, USA, and the NIH AIDS Research and Reference Reagent
Program for providing the MHC class I tetramer. This work was
supported by the Deutsche Forschungsgemeinschaft (SFB 620).
vaccinia virus infection of murine ovaries: asialo-GM1 as an
additional virus receptor? Immunol Cell Biol 68, 343–346.
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