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.

-The aim of this study was to determine the immunomodulatory effects of IL-12, IL-18 and CD154 (CD40 ligand, CD40L) in DNA-vaccination against the classical swine fever virus. Four recombinant plasmids were constructed including the CSFV coding region for the glycoprotein gp55/E2 alone or together with porcine IL-12, IL-18 or CD154 genes. Five groups of four pigs each were immunized intramuscularly (i.m.) three times with the respective constructs. The control group was inoculated with empty plasmid DNA. Eighteen days after the final immunization, the pigs were challenged with a lethal dose of CSFV strain Eystrup and monitored for a further 16 days. This study showed that co-delivery of IL-18 and CD154 induced an earlier appearance of serum antibodies, reduced B-cell deficiency after infection and protected pigs against a lethal CSFV infection. In contrast, co-delivery of IL-12 led to a reduced titer of neutralizing antibodies and protection against a lethal CSFV challenge in comparison to the other pigs and to pigs that were immunized with a gp55/E2 plasmid alone.

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Background The tubes used for collection and storage and their fill volumes have been identified as critical elements for standardized CSF collection as they have proven to significantly influence recovery of CSF biomarker levels and β‐amyloid1‐42 (Aβ1‐42) in particular. The aim of the current study was to compare different collection protocols in terms of CSF biomarker recovery to assess the potential impact on studies establishing cut‐off values. Method Two pre‐analytical protocols, used in two experienced CSF biomarker testing sites in Europe, were compared by exchanging the collection and storage tubes. In addition, two different fill volumes were tested for the largest storage tube. In total, six protocols were compared of which protocol 1 and 2 represent the routine practice in the respective sites (Table 1). Twelve consecutive patients were sampled according to all six protocols and the order of the protocols was sequentially assigned per patient. CSF biomarker analyses were done using the LUMIPULSE G platform and related assays. A linear mixed effects model was applied using R. Result Protocols 1 and 3, both of which used storage tube 1 with a 0.5 mL fill volume, resulted in significantly lower CSF Aβ1‐42 and CSF Aβ1‐42/Aβ1‐40 ratio compared to all other protocols (p<0.001). Compared to protocol 6, which gave the highest recovery for the Aβ markers, the relative difference in Aβ1‐42 was ‐13.9% (95% CI: ‐16.1% to ‐11.6%) for protocol 1 and ‐14.8% (95% CI: ‐17.1% to ‐12.6%) for protocol 3. For the Aβ1‐42/Aβ1‐40 ratio, this resulted in a difference of ‐6.7% (95%CI: ‐8.8% to ‐4.5%) and ‐6.3% (95%CI: ‐8.5% to ‐4.1%) for protocol 1 and 3, respectively. No significant differences were observed for total Tau and hyperphosphorylated Tau. Conclusion In the current study, and with the tubes studied in these protocols, CSF biomarker recovery did not depend on the tubes in which the samples were collected or stored. However, fill volume did have a significant impact on the Aβ1‐42 levels and the Aβ1‐42/Aβ1‐40 ratio, implying that the combination of tube plus fill volume is a factor that needs to be taken into account when comparing studies calculating cut‐off values.