1 Department of Virology, University of Freiburg, D-79104 Freiburg, Germany
2 Federal Research Center for Virus Diseases of Animals, Institute for Immunology, D-72076 Tuebingen, Germany
Correspondence
Peter Staeheli
peter.staeheli{at}uniklinik-freiburg.de
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ABSTRACT |
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Present address: Bavarian Nordic GmbH, Fraunhoferstraße 13, D-82152 Martinsried, Germany.
Present address: Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, UK.
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MAIN TEXT |
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Immunization of Lewis rats with a recombinant vaccinia virus expressing BDV-N prior to BDV challenge decreased viral burden at the cost of enhanced central nervous system (CNS) inflammation and aggravated disease (Lewis et al., 1999). By contrast, in a more recent study, immunization of Lewis rats with a recombinant parapoxvirus (orf virus) expressing BDV-N (D1701-VrVp40) induced a high degree of protection against intracerebral challenge with BDV (Henkel et al., 2005
). In persistently BDV-infected B10.BR mice, which are resistant to spontaneous development of Borna disease, post-exposure vaccination with vaccinia virus expressing BDV-N induced a lethal immune response (Hausmann et al., 1999
). Similarly, immunization with dendritic cells pulsed with the immunodominant peptide derived from BDV-N-induced neurological disease in persistently infected mice (Fassnacht et al., 2004
). However, if immunization of mice with dendritic cells was done before intracerebral challenge with BDV, partial protection from virus spread and neurological disease was achieved (Fassnacht et al., 2004
). It remains unclear whether vaccine-induced antiviral CD8+ T cells were mainly responsible for protection under these conditions and if so, whether perforin-mediated cytotoxic effects played a role.
We established a primeboost vaccination protocol in which 3-week-old mice first received an intramuscular injection of 107 p.f.u. of either parapoxvirus D1701-VrVp40 expressing BDV-N (Henkel et al., 2005) or parental D1701-VrV expressing bacterial
-galactosidase (
-gal) (Fischer et al., 2003
). Seven days later, boosting was done by intraperitoneal infection of the animals with 5x106 p.f.u. vaccinia virus expressing either BDV-N or
-gal (Schamel et al., 2001
). We chose this fast immunization protocol because susceptibility of MRL mice to BDV-induced neurological disease is most pronounced in young animals (Hallensleben et al., 1998
). A slower immunization protocol might have been more effective, but the immunized animals would also have acquired a higher degree of intrinsic resistance to BDV, which may have complicated the interpretation of subsequent virus-challenge experiments.
When spleen cells from immunized B10.BR mice were restimulated in vitro for 5 days with the BDV-specific peptide TELEISSI (Schamel et al., 2001), a high level of cytolytic activity was detected (Fig. 1
a, middle panel). If the immunization protocol was changed and parapoxvirus VrVp40 was used for immune priming as well as for boosting, no cytolytic activity was detected (Fig. 1a
, left panel). If the mice were subjected to a single round of immunization with vaccinia virus expressing BDV-N, BDV-specific lytic activity was only slightly above background level (Fig. 1a
, right panel), suggesting that the use of heterologous vectors for priming and boosting is of great advantage. Staining of T cells with tetrameric complexes of MHC class I molecules carrying TELEISSI peptides revealed that a mean of 76 % (range 5593 %) of the CD8+ T cells in spleen cultures from mice subjected to the heterologous primeboost immunization protocol were specific for BDV-N (Fig. 1b
). Thus, the newly established primeboost immunization protocol was far more efficient than a previous protocol that used peptide-loaded dendritic cells for immunization. Using the latter protocol, in vitro-restimulated spleen cultures contained a mean of only 5 % TELEISSI-specific CD8+ T cells after 7 days of restimulation (Fassnacht et al., 2004
).
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One way to determine whether CD8+ T cells are involved in vaccine-induced protection against BDV is to use MRL-2m0/0 mice that lack mature CD8+ T cells due to a defect in the gene encoding
2-microglobulin. Such animals are highly susceptible to BDV. However, because they lack CD8+ T cells, they do not experience immunopathological damage of the CNS and thus remain healthy in spite of a high virus load in the brain (Hallensleben et al., 1998
). None of the 10 MRL-
2m0/0 mice that were immunized with BDV-N by the same primeboost protocol was protected from BDV challenge. Upon analysis at 35 days post-challenge, BDV antigen was abundantly present throughout the brains of these animals (data not shown). This result demonstrated that immunization-induced protection against BDV was strongly dependent on CD8+ T cells.
However, is vaccine-induced protection of perforin-deficient mice also mediated by CD8+ T cells? We recently identified the BDV-N-derived peptide 129TELEISSI136 as the immunodominant CD8+ T-cell epitope of BDV in MRL and other H-2k mice (Schamel et al., 2001). If vaccine-induced BDV resistance in perforin-deficient mice is mediated by CD8+ T cells, we would expect that immunization with a mutant form of BDV-N that lacks the TELEISSI epitope would not be able to induce protective immunity. Since no appropriate parapoxvirus vector was available, we turned to a simplified immunization protocol in which animals were vaccinated by a single intraperitoneal infection with recombinant vaccinia virus expressing either wild-type BDV-N or mutant BDV-NE130K, I136T in which the two amino acids that anchor the peptide on the MHC I molecule were exchanged (Schamel et al., 2001
). Although less effective than the primeboost immunization protocol (Fig. 1a
), this simple protocol was previously shown to induce BDV-specific CD8+ T cells that could exhibit strong biological effects (Hausmann et al., 1999
). Eight of nine infected perforin-deficient mice (89 %) that were immunized with wild-type BDV-N remained healthy during the 5-week observation period, although most contained low to moderate numbers of infected neurons in the brain (Table 2
). By contrast, only three of the 12 perforin-deficient mice (25 %) that were immunized with BDV-NE130K, I136T remained healthy (P=0·006, MannWhitney U test), and the number of virus-infected cells in the brains of these mice was significantly higher than in mice vaccinated with BDV-N (P=0·036, MannWhitney U test). We concluded from these results that TELEISSI-specific CD8+ T cells also mediated protection against BDV in perforin-deficient mice.
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The results of our present study support the popular concept that virus-specific CD8+ T cells can either be pathogenic or protective, depending on the delicate balance between the speed of virus replication, the time of T-cell priming and the strength of the antiviral T-cell response. Our results further demonstrate that solid immunity against intracerebral challenge with a neurotropic virus can be induced if parapoxvirus and vaccinia virus vectors are combined in a primeboost immunization protocol.
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ACKNOWLEDGEMENTS |
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Received 31 August 2004;
accepted 9 November 2004.