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

Department of Virology, University of Freiburg, Hermann-Herder-Str. 11, D-79104 Freiburg, Germany

Correspondence
Jürgen Hausmann
juergen.hausmann{at}uniklinik-freiburg.de


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 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 (MRLxB10.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 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). Disease-inducing 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 (MRLxB10.BR) mice were infected intracerebrally under ether anaesthesia at 7 weeks of age with 10 µl 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 (MRLxB10.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), non-essential amino acids and 50 µM {beta}-mercaptoethanol. Cells were pooled and counted. Mean yields were 5–9x107 cells per mouse. Cells were plated at 5–7x106 cells per dish in 10 ml IMDM supplemented with non-essential amino acids, 50 µM {beta}-mercaptoethanol, 5 ng interleukin 4 (IL4) ml–1 (PromoCell) and 100 ng granulocyte–macrophage colony-stimulating factor (GM-CSF) ml–1, 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 concentration of 1 µg ml–1 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 10–4 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 µl 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 µg ml–1 at room temperature.

Flow cytometry.
For surface staining, suspensions of 2·5x105–106 DCs were incubated at 4 °C with anti-CD16/CD32 in 50–100 µl PBS supplemented with 1 % FCS and 0·1 % NaN3 to block non-specific 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-CD8{alpha} (1 µg ml–1, clone 53-6.7; Pharmingen) with or without PE-labelled Kk/TELEISSI tetramer (5 µg ml–1) 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 10–6 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 µM {beta}-mercaptoethanol. Cultures were used for tetramer staining and as effector cells in cytotoxicity assays.

In vitro cytotoxicity assay.
Cytolytic activity of in vitro-restimulated splenocytes from DC-vaccinated F1 (MRLxB10.BR) mice was determined by a standard 51Cr-release assay. Briefly, 5x106 L929 (H-2k) cells were labelled in suspension with 200 µCi Na251CrO4 (ICN Biomedicals) and 10–4 M peptide for 2 h at 37 °C. After three washes with IMDM, they were diluted to a final concentration of 4x104 cells ml–1, dispensed into 96-well round-bottom microtitre plates at 4x103 cells per well and coincubated with different dilutions of in vitro-restimulated lymphocyte cultures, as indicated, in a total volume of 200 µl for 6 h at 37 °C. The percentage of specific target cell lysis was calculated according to the following formula: 100x[(test release – spontaneous release)/(total release – spontaneous release)]. Target cells were pulsed with 10–4 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 protective efficacy of TELEISSI-specific CD8 T cells against a recombinant 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 °C 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 (MRLxB10.BR) animals were immunized twice at an interval of 1 week with 5x105 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 TELEISSI-specific lytic activity. Specific lysis of target cells was clearly detectable in restimulated splenocyte cultures of F1 (MRLxB10.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 (MRLxB10.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 TELEISSI-specific (Fig. 1c). Immunization with control peptide-loaded 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 % tetramer-positive CD8 T cells had moderate CTL activity (Fig. 1d). Cultures with intermediate numbers of tetramer-positive 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.



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Fig. 1. Induction of TELEISSI-specific CD8 T cells by DC immunization. (a) F1 (MRLxB10.BR) mice were immunized twice at a weekly interval with 5x105 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 (MRLxB10.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.

 
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 (MRLxB10.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).



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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 (MRLxB10.BR) mice persistently infected with BDV were immunized with a single, subcutaneous dose (7x106 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.

 

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Table 1. Breaking immunological ignorance in BDV-infected F1 (MRLxB10.BR) mice by immunization with peptide-loaded DCs

Persistently infected, symptomless F1 (MRLxB10.BR) mice were immunized at 8–10 weeks post-infection with peptide-loaded DCs in two independent experiments. Animals were observed for 14 (experiment 1) and 11 (experiment 2) days, respectively.

 
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 mononuclear infiltrates (Table 1). It is possible that non-specific 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, 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 (2x105 or 9x106 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 (MRLxB10.BR) mice were immunized with TELEISSI-loaded DCs from MRL 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.



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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 (2x105 cells; filled symbols) or high-dose (9x106 cells; open symbols) application of B10.BR-derived 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 (VV-N). 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 (MRLxB10.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 (MRLxB10.BR) mice were given two subcutaneous injections at a weekly interval of 3–4x106 F1 (MRLxB10.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.

 
To assess protective immunity against intracerebral challenge with BDV, F1 (MRLxB10.BR) mice were subcutaneously injected twice with DCs derived from F1 (MRLxB10.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 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 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.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 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 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 virus-specific 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, peptide-pulsed 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 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-{gamma}) 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 cell-derived IFN-{gamma} (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-{gamma} (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-{gamma}, although perforin or other effector mechanisms of CD8 T cells cannot be ruled out at present.


   ACKNOWLEDGEMENTS
 
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).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bartholdy, C., Christensen, J. P., Wodarz, D. & Thomsen, A. R. (2000). Persistent virus infection despite chronic cytotoxic T-lymphocyte activation in gamma interferon-deficient mice infected with lymphocytic choriomeningitis virus. J Virol 74, 10304–10311.[Abstract/Free Full Text]

Batra, A., Planz, O., Bilzer, T. & Stitz, L. (2003). Precursors of Borna disease virus-specific T cells in secondary lymphatic tissue of experimentally infected rats. J Neurovirol 9, 325–335.[Medline]

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Received 16 March 2004; accepted 3 May 2004.