Department of Virology, University of Freiburg, Hermann-Herder-Str. 11, D-79104 Freiburg, Germany
Correspondence
Jürgen Hausmann
juergen.hausmann{at}uniklinik-freiburg.de
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ABSTRACT |
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INTRODUCTION |
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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.
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METHODS |
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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 1215 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 816-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
-mercaptoethanol. Cells were pooled and counted. Mean yields were 59x107 cells per mouse. Cells were plated at 57x106 cells per dish in 10 ml IMDM supplemented with non-essential amino acids, 50 µM
-mercaptoethanol, 5 ng interleukin 4 (IL4) ml1 (PromoCell) and 100 ng granulocytemacrophage colony-stimulating factor (GM-CSF) ml1, 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 ml1 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 104 M of the indicated peptides for 23 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. TELEISSIH-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 ml1 at room temperature.
Flow cytometry.
For surface staining, suspensions of 2·5x105106 DCs were incubated at 4 °C with anti-CD16/CD32 in 50100 µl PBS supplemented with 1 % FCS and 0·1 % NaN3 to block non-specific IgG binding, followed by incubation with properly diluted mAbs anti-CD11cPE, anti-I-EkFITC (fluorescein isothiocyanate) and/or anti-CD86biotin. StreptavidinFITC (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 (1 µg ml1, clone 53-6.7; Pharmingen) with or without PE-labelled Kk/TELEISSI tetramer (5 µg ml1) 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 106 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 914 days in IMDM supplemented with 10 % FCS and 50 µM -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 104 M peptide for 2 h at 37 °C. After three washes with IMDM, they were diluted to a final concentration of 4x104 cells ml1, 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 104 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 freezethawing 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.
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RESULTS |
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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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DISCUSSION |
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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-
) 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-
(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-
(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-
, although perforin or other effector mechanisms of CD8 T cells cannot be ruled out at present.
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ACKNOWLEDGEMENTS |
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Received 16 March 2004;
accepted 3 May 2004.