Identification of T-cell epitopes in the structural and non-structural proteins of classical swine fever virus

Abstract

To identify new T-cell epitopes of classical swine fever virus (CSFV), 573 overlapping, synthetic pentadecapeptides spanning 82 % of the CSFV (strain Glentorf) genome sequence were synthesized and screened. In proliferation assays, 26 peptides distributed throughout the CSFV viral protein sequences were able to induce specific T-cell responses in PBMCs from a CSFVGlentorf-infected d/d haplotype pig. Of these 26 peptides, 18 were also recognized by PBMCs from a CSFV-Alfort/187-infected d/d haplotype pig. In further experiments, it could be shown that peptide 290 (KHKVRNEVMVHWFDD), which corresponds to amino acid residues 1446-1460 of the CSFV non-structural protein NS2-3 could induce interferon-γ secretion after secondary in vitro restimulation. The major histocompatibility complex (MHC) restriction for stimulation of T-cells by this pentadecapeptide was identified as being mainly MHC class II and partially MHC class I. In cytolytic assays, CSFV-specific cytotoxic T-lymphocytes (CTLs) were able to lyse peptide 290-loaded target cells. These findings indicate the existence of a CSFV-specific helper T-cell epitope and a CTL epitope in this peptide.

Immunological properties of recombinant classical swine fever virus NS3 protein in vitro and in vivo

“…Overall, induction of the T cell responses by NS3, especially the stimulated effector CTL, was comparable to the whole virus, indicating that NS3 contains immunodominant CTL epitope(s). This demonstrates that viral NS3 contains CTL epitopes in addition to the NS2 [1] and NS4 proteins [25].…”

“…Nevertheless, with viruses such as CSFV which produce a non-cytopathic infection in cells, and can thus persist in the host, one must consider both cytotoxic (CTL) and humoral (B lymphocyte) responses in the induction of protective immunity. T cell epitopes relating to the induction of virusspecific cytotoxic T lymphocytes have been identified on non-structural proteins of CSFV [1,25]. Consequently, the present work analysed for the first time porcine in vitro and in vivo immune responses against the recombinant non-structural NS3 protein of CSFV.…”

“…ELISPOT assays for the detection of IFN-γ secreting T cells were performed as described elsewhere [1] with minor modifications [2]. The threshold for identification of CSFV-specific IFN-γ spots was set at 10 spots/10 6 PBMC, based on tests performed with PBMC from naïve animals (n = 15), which were repeatedly bleeded.…”

“…Thus, the induction of potent CD8 + cytotoxic T cell responses has the potential to improve CSF vaccines. Consequently, the present study analysed the immunological properties of recombinant NS3, due to the observations that CSFV non-structural proteins carry T cell epitopes [1,25]. This was analysed in vitro and in vivo in terms of its contribution to protective immune responses induced in combination with an E2 subunit vaccine.…”

Generation of Th1 T cell responses directed to a HLA Class II restricted epitope from theAspergillusf16 allergen

“…The effectiveness of determining T cell epitopes and CD8 + /CD4 + T cell frequencies against viral antigens by use of protein-spanning pools of overlapping pentadecapeptides, with each 15 amino acids (aa) peptide overlapping the previous peptide by 11-aa, has been described recently [36][37][38]. Based on these results, we prepared a series of 104 overlapping pentadecapeptides spanning the entire 427-aa coding region of Asp f16 and used a complete pentadecapeptide pool (Asp f16-PPC) pulsed onto dendritic cells (DC) to prime autologous proliferative and cytotoxic T lymphocyte (CTL) responses.…”