Vaccine-induced protection against Borna disease in wild-type and perforin-deficient mice

Jürgen Hausmann1,{dagger}, Karen Baur1, Karin R. Engelhardt1,{ddagger}, Timo Fischer2, Hanns-Joachim Rziha2 and Peter Staeheli1

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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Borna disease virus (BDV) can persistently infect the central nervous system and induce CD8+ T-cell-mediated neurological disease in MRL mice. To determine whether specific immune priming would prevent disease, a prime–boost immunization protocol was established in which intramuscular injection of a recombinant parapoxvirus expressing BDV nucleoprotein (BDV-N) was followed by intraperitoneal infection with vaccinia virus expressing BDV-N. Immunized wild-type and perforin-deficient mice remained healthy after intracerebral infection with BDV and contained almost no virus in the brain at 5 weeks post-challenge. Immunization failed to induce resistance against BDV in mice lacking mature CD8+ T cells. Immunization of perforin-deficient mice with a poxvirus vector expressing mutant BDV-N lacking the known CD8+ T-cell epitope did not efficiently block multiplication of BDV in the brain and did not prevent neurological disease, indicating that vaccine-induced immunity to BDV in wild-type and perforin-deficient mice resulted from the action of CD8+ T cells.

{dagger}Present address: Bavarian Nordic GmbH, Fraunhoferstraße 13, D-82152 Martinsried, Germany.

{ddagger}Present address: Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, UK.


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Borna disease virus (BDV) is an enveloped virus with a single-stranded RNA genome of negative polarity that transcribes and replicates in the nucleus of infected cells (Briese et al., 1994; Cubitt et al., 1994). BDV is non-cytopathic and readily establishes persistent infections in the central nervous system of animals (Gonzalez-Dunia et al., 1997). Studies in rats and disease-susceptible MRL mice have demonstrated that BDV-induced pathology is caused by CD8+ T cells, which require help from the CD4+ T-cell subset (Bilzer et al., 1995; Bilzer & Stitz, 1994; Hallensleben et al., 1998; Hausmann et al., 1999; Sobbe et al., 1997). From immunohistological studies, it was concluded that these T-cell subsets probably play similar roles in natural Borna disease of horses (Bilzer et al., 1995). In Lewis rats and mice of the H-2k haplotype, epitopes in the BDV nucleoprotein (BDV-N) are the main targets of cytotoxic T cells (Planz et al., 2001; Schamel et al., 2001).

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 prime–boost 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 {beta}-galactosidase ({beta}-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 {beta}-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. 1a, 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 55–93 %) of the CD8+ T cells in spleen cultures from mice subjected to the heterologous prime–boost immunization protocol were specific for BDV-N (Fig. 1b). Thus, the newly established prime–boost 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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Fig. 1. Heterologous prime–boost immunization induces a strong BDV-N-specific cytolytic CD8+ T-cell response. Groups of B10.BR mice (H-2k) were subjected to various immunization protocols. Left panel: priming and boosting (on day 7 post-priming) with parapoxvirus D1701-VrVp40 expressing BDV-N (PPV+PPV). Middle panel: priming with parapoxvirus D1701-VrVp40 expressing BDV-N followed by boosting 7 days later with vaccinia virus expressing BDV-N (PPV+VV). Right panel: vaccination by infection with recombinant vaccinia virus expressing BDV-N with no subsequent boosting (VV 1x). Two weeks after the last immunization, splenocytes were prepared. Restimulation in vitro was done for 5 days using naïve splenocytes pulsed with the peptide TELEISSI as described previously (Fassnacht et al., 2004). Specific lytic activity of restimulated cultures was tested on TELEISSI-pulsed L929 target cells (H-2k) in a standard 51Cr release assay incubated for 6 h at 37 °C (filled symbols). L929 target cells pulsed with the influenza A virus haemagglutinin-derived H-2k-binding epitope FEANGNLI served as a control (open symbols). Data represent mean values of groups of two to three animals. (b) High numbers of BDV-specific CD8+ T cells in spleen cell cultures from mice immunized by the heterologous prime–boost protocol. In vitro restimulated spleen cells from immunized B10.BR mice were stained with anti-CD8+ conjugated to allophycocyanin and Kk/TELEISSI conjugated to phycoerythrin tetramer after 7 days in culture as described previously (Fassnacht et al., 2004). Results of flow cytometric analyses of two individual mice are shown. The numbers in the upper right quadrants indicate the frequency of TELEISSI-specific CD8+ T cells from the total number of CD8+ T cells.

 
To determine whether the new vaccination protocol would efficiently prevent BDV-induced neurological disease in susceptible MRL mice, immunized animals were challenged by intracerebral infection with a mouse-adapted strain of BDV (Freude et al., 2002) at day 10 after the final booster immunization. All 13 wild-type MRL mice that were immunized with BDV-N remained healthy after challenge during the complete observation period of 5 weeks (Table 1). No virus-infected cells were detected upon post-mortem immunohistochemical (IHC) analysis of paraffin-embedded brain sections from these mice with the BDV-N-specific monoclonal antibody Bo18. However, mild encephalitis was observed in most cases (data not shown), indicating that the incoming virus was successfully cleared by the antiviral immune response. Six of the 12 BDV-challenged mice that were immunized in the same way by control vectors expressing {beta}-gal showed strong neurological disease, with a mean onset of symptoms around day 30 post-challenge (Table 1). Post-mortem IHC analyses revealed that all six diseased animals had high levels of BDV antigen in the brain. The brains of three control-immunized animals that remained healthy until sacrifice at day 35 post-infection also contained large numbers of BDV-infected cells, while the brains of the three remaining animals lacked evidence of successful infection (Table 1). Control infections of 6-week-old, naïve MRL mice showed that a similar percentage of animals (5/21) was resistant to infection with BDV (data not shown). With the exception of one healthy animal that had strong encephalitis, the degree of CNS inflammation correlated directly with the severity of disease (data not shown). These results demonstrated that the prime–boost vaccination protocol worked extremely well.


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Table 1. Effect of prime–boost immunization on susceptibility to BDV infection and virus-induced neurological disease in MRL mice

 
We next determined whether the newly established immunization protocol would also protect against BDV challenge in mice lacking perforin. Perforin is an essential component of cytotoxic granules in CD8+ T cells (Kagi & Hengartner, 1996). The antiviral immune defence against lymphocytic choriomeningitis virus and Theiler's virus infecting the CNS is dependent on perforin (Kagi & Hengartner, 1996; Pena-Rossi et al., 1998). By contrast, the host defence against the neurotropic JHM strain of mouse hepatitis virus is not dependent on perforin, although perforin contributes to the clearance of the virus from the brain (Lin et al., 1997). Perforin-dependent lytic activity of CD8+ T cells has been implicated in the limited spread of BDV in rats after adoptive transfer of a BDV-specific CD4+ T-cell line (Nöske et al., 1998). In our experiments, none of the 11 perforin-deficient MRL mice (Hausmann et al., 2001) immunized with BDV-N became ill after challenge, and only a few BDV-infected neurons in the CA3 region of the hippocampus were found in the brain of one animal (Table 1). In the remaining 10 animals, we observed no evidence for productive BDV infection by IHC analysis. Seven of the 11 BDV-challenged perforin-deficient mice that were immunized with control vectors expressing {beta}-gal showed strong neurological disease (Table 1) with a mean onset at about 28 days post-infection. Again, pronounced meningoencephalitis was noted in the brains of all {beta}-gal-immunized animals with strong neurological disease (data not shown). Analysis of brain sections by IHC revealed that all seven diseased animals contained high numbers of BDV-infected cells. The brains of the remaining four healthy animals were also infected (Table 1), although far fewer cells carried BDV antigen (data not shown). Thus, vaccine-induced protection in the absence of perforin was surprisingly effective, raising the question of whether protection was mediated by CD8+ T cells in these animals.

One way to determine whether CD8+ T cells are involved in vaccine-induced protection against BDV is to use MRL-{beta}2m0/0 mice that lack mature CD8+ T cells due to a defect in the gene encoding {beta}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-{beta}2m0/0 mice that were immunized with BDV-N by the same prime–boost 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 prime–boost 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, Mann–Whitney 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, Mann–Whitney 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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Table 2. Immunization-induced protection of perforin-deficient MRL mice depends on the presence of the immunodominant CD8+ T-cell epitope in BDV-N

 
Our results demonstrated that pre-exposure vaccination of susceptible MRL mice enables the immune system to control BDV replication in the CNS efficiently without enhancing clinical symptoms. Similar conclusions were drawn from a recent study with Lewis rats immunized with parapoxvirus D1701-VrVp40 (Henkel et al., 2005) and from experiments with MRL mice immunized with peptide-loaded dendritic cells (Fassnacht et al., 2004). Our experiments with {beta}2m0/0 and perforin-deficient mice provide important clues regarding the nature of the protective immune response. Since the vaccine was completely ineffective in {beta}2m0/0 mice, it is clear that CD8+ T cells are the major players. Since perforin-deficient mice were able to mount vaccination-induced immunity towards BDV, it appears that CD8+ T-cell-dependent, non-cytolytic antiviral mechanisms are important for the control of this neurotropic virus. These results are in good agreement with a recent study showing that interferon (IFN)-{gamma} produced by infiltrating immune cells was responsible for Sindbis virus elimination from certain types of neurons (Binder & Griffin, 2001), suggesting that cytokine secretion by CD8+ T cells might also control BDV replication in the mouse brain. Work with CD8+ T cells specific for hepatitis B virus antigens has further indicated that the in vivo antiviral effect of these cells is mediated by the cytokines TNF-{alpha} and IFN-{gamma} (reviewed by Guidotti, 2002). By using brain slice cultures, we recently demonstrated that IFN-{gamma} has antiviral activity against BDV in cells derived from the mouse cerebellum (Friedl et al., 2004).

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 prime–boost immunization protocol.


   ACKNOWLEDGEMENTS
 
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 620). We thank Rosita Frank for excellent technical assistance.


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Received 31 August 2004; accepted 9 November 2004.