Modified vaccinia virus Ankara (MVA) has a highly restricted host range in cell culture and is apathogenic in vivo. MVA was derived from the parental chorioallantois vaccinia virus Ankara (CVA) by more than 570 passages in chicken embryo fibroblast (CEF) cells. During CEF cell passaging, six major deletions comprising 24,668 nucleotides occurred in the CVA genome. We have cloned both the MVA and the parental CVA genome as bacterial artificial chromosomes (BACs) and have sequentially introduced the six major MVA deletions into the cloned CVA genome. Reconstituted mutant CVA viruses containing up to six major MVA deletions showed no detectable replication restriction in 12 of 14 mammalian cell lines tested; the exceptions were rabbit cell lines RK13 and SIRC. In mice, CVA mutants with up to three deletions showed slightly enhanced virulence, suggesting that gene deletion in replicating vaccinia virus (VACV) can result in gain of fitness in vivo. CVA mutants containing five or all six deletions were still pathogenic, with a moderate degree of attenuation. Deletion V was mainly responsible for the attenuated phenotype of these mutants. In conclusion, loss or truncation of all 31 open reading frames in the six major deletions is not sufficient to reproduce the specific MVA phenotype of strong attenuation and highly restricted host range. Mutations in viral genes outside or in association with the six major deletions appear to contribute significantly to this phenotype. Host range restriction and avirulence of MVA are most likely a cooperative effect of gene deletions and mutations involving the major deletions.

Efficient T-cell responses against recombinant antigens expressed by vaccinia virus vectors require expression of these antigens in the early phase of the virus replication cycle. The kinetics of recombinant gene expression in poxviruses are largely determined by the promoter chosen. We used the highly attenuated modified vaccinia virus Ankara (MVA) to determine the role of promoters in the induction of CD8 T-cell responses. We constructed MVA recombinants expressing either enhanced green fluorescent protein (EGFP) or chicken ovalbumin (OVA), each under the control of a hybrid early-late promoter (pHyb) containing five copies of a strong early element or the well-known early-late p7.5 or pS promoter for comparison. In primary or cultured cells, EGFP expression under the control of pHyb was detected within 30 min, as an immediateearly protein, and remained higher over the first 6 h of infection than p7.5-or pS-driven EGFP expression. Repeated immunizations of mice with recombinant MVA expressing OVA under the control of the pHyb promoter led to superior acute and memory CD8 T-cell responses compared to those to p7.5-and pS-driven OVA. Moreover, OVA expressed under the control of pHyb replaced the MVA-derived B8R protein as the immunodominant CD8 T-cell antigen after three or more immunizations. This is the first demonstration of an immediate-early neoantigen expressed by a poxviral vector resulting in superior induction of neoantigenspecific CD8 T-cell responses.

Borna disease virus (BDV) frequently causes meningoencephalitis and fatal neurological disease in young but not old mice of strain MRL. Disease does not result from the virus-induced destruction of infected neurons. Rather, it is mediated by H-2 k -restricted antiviral CD8 T cells that recognize a peptide derived from the BDV nucleoprotein N. Persistent BDV infection in mice is not spontaneously cleared. We report here that N-specific vaccination can protect wild-type MRL mice but not mutant MRL mice lacking gamma interferon (IFN-␥) from persistent infection with BDV. Furthermore, we observed a significant degree of resistance of old MRL mice to persistent BDV infection that depended on the presence of CD8 T cells. We found that virus initially infected hippocampal neurons around 2 weeks after intracerebral infection but was eventually cleared in most wild-type MRL mice. Unexpectedly, young as well as old IFN-␥-deficient MRL mice were completely susceptible to infection with BDV. Moreover, neurons in the CA1 region of the hippocampus were severely damaged in most diseased IFN-␥-deficient mice but not in wild-type mice. Furthermore, large numbers of eosinophils were present in the inflamed brains of IFN-␥-deficient mice but not in those of wild-type mice, presumably because of increased intracerebral synthesis of interleukin-13 and the chemokines CCL1 and CCL11, which can attract eosinophils. These results demonstrate that IFN-␥ plays a central role in host resistance against infection of the central nervous system with BDV and in clearance of BDV from neurons. They further indicate that IFN-␥ may function as a neuroprotective factor that can limit the loss of neurons in the course of antiviral immune responses in the brain.

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.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 noncytopathic 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-2 k 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...

Borna disease virus (BDV) infection of the central nervous system (CNS) leads to severe neurological symptoms in susceptible MRL mice. The disease is mainly mediated by CD8 + T cells specific for the immunodominant epitope TELEISSI in the BDV nucleoprotein. In this study, TELEISSI/MHC class I tetramers were used to directly visualize antigen-specific CD8 + T cells. We found that on average approximately 30% of the ex vivo analyzed CD8 + T cells in the CNS of diseased mice were specific for TELEISSI. Unexpectedly, the frequency of tetramer-reactive brain-derived CD8 + T cells doubled following overnight culture in the absence of antigen. The majority of CD8 + T cells showed enhanced tetramer binding without up-regulation of T cell receptor surface expression. The frequency of IFN-c-secreting CD8 + T cells after antigen-specific stimulation was higher in overnight cultures than in freshly isolated BDV-specific brain lymphocytes, and enhanced tetramer binding correlated with elevated sensitivity to lower levels of peptide antigen in cytotoxicity assays. These results indicate that the functional avidity of virus-specific CD8 + T cells was down-modulated in vivo. Thus, quantification of tissue-infiltrating CD8 + T cells by the tetramer technique must be interpreted with caution as it may underestimate the real frequency of antigen-specific CD8 + T cells.