Two major histocompatibility complex class I-restricted epitopes of the Borna disease virus p10 protein identified by cytotoxic T lymphocytes induced by DNA-based immunization

Abstract

Borna disease virus (BDV) infection of Lewis rats is the most studied animal model of Borna disease, an often fatal encephalomyelitis. In this experimental model, BDV-specific CD8(+) cytotoxic T lymphocytes (CTLs) play a prominent role in the immunopathogenesis of infection by the noncytolytic, persistent BDV. Of the six open reading frames of BDV, CTLs to BDV X (p10) and the L-polymerase have never been studied. In this study, we used plasmid immunization to investigate the CTL response to BDV X and N. Plasmid-based immunization was a potent CTL inducer in Lewis rats. Anti-X CTLs were primed by a single injection of the p10 cDNA. Two codominant p10 epitopes, M(1)SSDLRLTLL(10) and T(8)LLELVRRL(16), associated with the RT1.A(l) major histocompatibility complex class I molecules of the Lewis rats, were identified. In addition, immunization with a BDV p40-expressing plasmid confirmed the previously reported RT1.A(l)-restricted A(230)SYAQMTTY(238) peptide as the CTL target for BDV N. In contrast to the CTL responses, plasmid vaccination was a poor inducer of an antibody response to p10. Three injections of a recombinant eukaryotic expression plasmid of BDV p10 were needed to generate a weak anti-p10 immunoglobulin M response. However, the antibody response could be optimized by a protein boost after priming with cDNA.

FIG. 1.

A single dose of priming with pcX-FLAG plasmid DNA is sufficient to induce an RT1.A^l-restricted, BDV-specific cytotoxic response. Lewis rats were immunized intramuscularly by one (○), two (▵), or three (□) doses of pcX-FLAG plasmid DNA (30 μg/dose) as indicated. Three weeks after the injections, splenic mononuclear cells were restimulated in vitro with recombinant His₆-p10 protein (75 μg/ml) for 5 days. Cytotoxicity was measured by use of the nonradiometric CytoTox 96 assay (Promega, Madison, Wis.), with Lewis F10 cells (A) and their BDV-infected counterpart, F10/BV cells (B), as targets. The anti-RT1.A MAb OX18 was used to treat the target cells (•) for 90 min at 37°C, prior to exposure to the effectors, to determine the class I MHC restriction of the antigen-specific cytotoxicity.

FIG. 2.

Only a small proportion of the in vitro restimulated cells are activated CD8⁺ cells. Lewis rats were immunized intramuscularly by three doses of pcX-FLAG plasmid DNA (30 μg/dose) at 3-week intervals. Three weeks after the last boost, splenic mononuclear cells were restimulated in vitro with recombinant His₆-p10 protein (75 μg/ml) for 5 days. The percentage of activated CD8⁺ cells was determined by two-color FACS after staining with the PE-conjugated OX8 (Serotec) and FITC-conjugated DB1 (Serotec) MAbs.

FIG. 3.

Two p10 epitopes are recognized by antigen-specific CTLs induced by plasmid immunization. Lewis rats were immunized intramuscularly with three doses of pcX-FLAG plasmid DNA (30 μg/dose) at 3-week intervals. Three weeks after the last boost, splenic mononuclear cells were restimulated in vitro with recombinant His₆-p10 protein (75 μg/ml) for 5 days. Cytotoxicity was measured by use of the nonradiometric CytoTox 96 assay (Promega), with Lewis F10 cells or F10 cells loaded with peptide 1 (M₁SSDLRLTTL₁₀) or 2 (T₈LLELVRRL₁₆) of p10 as targets. F10 cells loaded with the unrelated peptide 3 (A₂₃₀SYAQMTTY₂₃₈) of p40 served as control targets.