Borna Disease (BD) is a mostly fatal disease of horses and sheep endemic in central Europe. Antibodies to Borna disease virus (BDV) have been described in sheep and other species living in BD non-endemic areas. Meaningful clinical BDV serology is hampered by difficulties in defining serological cut-offs, which require the investigation of populations from endemic areas. Here we studied BD serology in sheep from endemic and non-endemic areas of similar geography in Switzerland. Antibodies to BDV antigens were detected by ELISA and indirect immunofluorescence analysis (IFA) only in sera from 3 of 6 sheep with autopsy confirmed BD. One serum was positive by IFA but not by ELISA, while 2 sera were negative in both assays, indicating that not all diseased animals develop BDV specific antibodies. Six % of clinically healthy animals (6/106) from an endemic area and 2% from a non-endemic area (4/192) had serum antibody to either BDV p40 or p24 as detected by ELISA. None of the animals showed a cellular immune response to BDV p40. In some healthy sheep from the endemic area, serum antibody titers to BDV p24 antigen remained elevated over several months without onset of disease symptoms. Infections with either BDV or related viruses may thus occur at low frequency in sheep from non-endemic areas leading to the production of antibodies to BDV antigens. We further propose viral strain differences or environmental factor(s) may determine the clinical outcome.

Cells with dendritic morphology obtained from several organs of mice lacking both type I and II IFN receptors were immortalized by a retrovirus and analysed for their phenotype and for their function to induce cognate immune responses in vitro and in vivo. Two cell lines called AG101 (skin) and AG116 (brain) were cloned and analysed in more detail. They constitutively expressed the cell surface markers CD45, CD11b, MHC class II, F4/80, N418, B7-2 and ICAM1 but were CD8- and B220-negative. Cells from both lines were capable of taking up ovalbumin (OVA). The processed protein was presented to the OVA-specific T cell hybridoma BO97.105 which responded specifically with the production of IL-2. AG101 and AG116 cells were able to induce a mixed lymphocyte reaction as shown by a 50-fold increase of IL-2 production over background. Naive T cells were stimulated by antigen-primed AG101 and AG116, resulting in a T cell proliferation which was 20-30 times over background, and in IL-2 production it was 10 times the background. The capacity of AG101 or AG116 cells to prime naive T cells was directly compared with freshly isolated and cultured cutaneous dendritic cells (DC) from 129 Sv/Ev mice (wtDC). After cognate T cell interaction, IL-6 (20-100-fold) and IL-12 p40 (100-1000-fold) were similarly upregulated in either AG101, AG116 or mature wtDC. To analyse the capacity of the immortalized DC to induce antibodies in vivo, cell line AG116 was permanently infected with Borna disease virus (BDV) which is unable to replicate in adult mice. One hundred and twenty-nine Sv/Ev mice injected with different cell numbers of AG116 carrying BDV (but not control cells) produced antibodies against the viral BDVp40 and BDVp24 protein. Therefore, the cell lines AG101 and AG116 appear to unify some functions of immature and mature DC. They are able to pick up antigen and process it. In the absence of externally added cytokines, the antigen presented on AG101 or AG116 cells drives T cells with an efficiency similar to mature DC. The cloned cell lines may prove to be useful to study both immune response and replication of infectious agents in the absence of functional interferon receptors.

ABSTRACTThe importance of each of the two interferon (IFN) systems in impeding herpesvirus replication and in stimulating virus-specific lymphocytes to control an acute systemic infection is not completely understood. To further our knowledge, pseudorabies virus, attenuated by deletion of the glycoprotein E gene to impair its neurovirulence and by deletion of the thymidine kinase gene (gE−TK−PRV), was used to infect wild-type 129Sv/Ev and congenic mice with immune system-associated genetic deficiencies. Mice with mature B and T lymphocytes but lacking either one or both functional receptors for members of each of the two IFN families were infected with gE−TK−PRV. At 3 and 7 but not 14 days after infection, replicating gE−TK−PRV could be isolated only from livers or spleens of mice lacking the receptors for both IFN families, and these mice survived the infection. Therefore, functional IFN receptors were not required to induce a protective immune response against an acute infection with gE−TK−PRV. Furthermore, PRV-specific antibodies of all immunoglobulin G isotypes were produced in these mice. Mice without mature B and T lymphocytes and lacking either one or both functional receptors for members of each of the two IFN families were also infected with gE−TK−PRV. Three days after infection, replicating virus could be isolated only from mice lacking both mature B and T lymphocytes and functional IFN receptors, and these mice were not able to clear the virus. We present evidence that mice with an intact gamma IFN system but without mature B and T cells were able to prevent systemic dissemination of gE−TK−PRV.

This study aimed to exploit bacterial artificial chromosomes (BAC) as large antigen-capacity DNA vaccines (BAC-VAC) against complex pathogens, such as herpes simplex virus 1 (HSV-1). The 152-kbp HSV-1 genome recently has been cloned as an F-plasmid-based BAC in Escherichia coli (fHSV), which can efficiently produce infectious virus progeny upon transfection into mammalian cells. A safe modification of fHSV, fHSVDeltapac, does not give rise to progeny virus because the signals necessary to package DNA into virions have been excluded. However, in mammalian cells fHSVDeltapac DNA can still replicate, express the HSV-1 genes, cause cytotoxic effects, and produce virus-like particles. Because these functions mimic the lytic cycle of the HSV-1 infection, fHSVDeltapac was expected to stimulate the immune system as efficiently as a modified live virus vaccine. To test this hypothesis, mice were immunized with fHSVDeltapac DNA applied intradermally by gold-particle bombardment, and the immune responses were compared with those induced by infection with disabled infectious single cycle HSV-1. Immunization with either fHSVDeltapac or disabled infectious single cycle HSV-1 induced the priming of HSV-1-specific cytotoxic T cells and the production of virus-specific antibodies and conferred protection against intracerebral injection of wild-type HSV-1 at a dose of 200 LD(50). Protection probably was cell-mediated, as transfer of serum from immunized mice did not protect naive animals. We conclude that BAC-VACs per se, or in combination with genetic elements that support replicative amplification of the DNA in the cell nucleus, represent a useful new generation of DNA-based vaccination strategies for many viral and nonviral antigens.

Background: To perform an ELISA or for panning phage particles which display recombinant proteins, one of the reactants is immobilized on solid phase. Immobilization in ELISA is generally performed by passive adsorption of ligands to plastic. However, protein is denatured during the adsorption process. This may result in low efficiency interaction between ligands and receptors which depend on native structures. In contrast, indirect immobilization has been shown to prevent protein denaturation. Objectives: The aim was to develop a system that allows efficient and stable indirect immobilization of a variety of recombinant multimeric proteins to solid phase. Results: A new vector was constructed which allows the expression of up to three proteins linked by the Jun/Fos leucine zipper. Purification of the resulting protein was achieved by Ni+ affinity chromatography utilizing the 6xHis-ABP (albumin binding protein) protein fused to the N-terminus of the Jun polypeptide. The high binding affinity of ABP to rat serum albumin (RSA) was exploited for indirect immobilization of recombinant proteins to solid phase. In an enzyme linked assay, the binding of ABP to immobilized RSA was shown to be 10-1000 times more efficient than other immobilization systems. Using the ZZ IgG binding domain of staphylococcal protein A as bait, the RSA-ABP immobilization system was successfully used to screen and enrich IgG Fc encoding DNA fragments from a cDNA phage library. Conclusion: The newly designed vector termed pJuFoexpress allows production and purification of mullimeric protein complexes linked by the Jun/Fos leucine zipper. Without chemical modifications, the recombinant proteins can be immobilized indirectly to solid phase. The immobilization results in the stable display of native, biologically active proteins which can be used in ELISA and phage display systems. (C) 1998 Elsevier Science B.V. All rights reserved.

Immobilization of proteins to a solid phase leads to denaturation of the adsorbed molecules which may subsequently affect biological interactions. However, for many applications maintenance of the native structure is desired. Therefore, an indirect immobilization system was developed, based on binding of the albumin binding domain (ABP) of streptococcal protein G to rat serum albumin (RSA) precoated on a solid phase (RSA-microtiter plates). Escherichia coli vectors were adapted for production of recombinant protein fused to ABP and the 6 X His-tag. The expressed ABP tag was found to form homodimers. Plasmon resonance was used to study the interaction between an ABP fusion protein and immobilized RSA. Apparent on- and off-rates were calculated using a model for a bivalent analyte (k(a1) = 3.37 x 10(4) M(-1) s(-1), k(d1) = 1.23 x 10(-4) s(-1)). Thus, the stability of the ABP-RSA interaction can be explained by a slow off-rate. This was confirmed by chase experiments in an ELISA format. The ABP-RSA interaction remained stable after addition of different albumins. This immobilization system was used for the development of an ELISA to detect antibodies against Borna disease virus protein p40. The use of RSA-microtiter plates for indirect immobilization of ABP fusion protein was shown to be superior to direct adsorption on plastic. To obtain maximal antibody binding ten times less antigen was needed for indirect immobilization compared to direct adsorption. The binding capacity of the RSA-microtiter plates was determined to be about 0.8 pmol of monomeric ABP protein.