Wolfgang Zauner

Cross-genotype-reactivity of the immunodominant HCV CD8 T-cell epitope NS3-1073

The HCV-specific HLA-A2-restricted NS3(1073) epitope is one of the most frequently recognized epitopes in hepatitis C. NS3(1073)-specific T-cell responses are associated with clearance of acute HCV-infection. Therefore this epitope is an interesting candidate for a HCV-peptide vaccine. However, heterogeneity between genotypes and mutations in the epitope has to be considered as an obstacle. We here identified 34 naturally occurring NS3(1073)-variants, as compared with the wild type genotype-1 variants (CVNGVCWTV/CINGVCWTV) by sequencing sera of 251 Greek and German patients and searching for published HCV-genomes. The frequency of variants among genotype-1 patients was 10%. Importantly, HLA-A2 binding was reduced only in 3 genotype 1 mutants while all non-genotype 1 variants showed strong HLA-A2-binding. By screening 28 variants in ELISPOT assays from T cell lines we could demonstrate that HCV-NS3(1073)-wild-type-specific T-cells displayed cross-genotype-reactivity, in particular against genotypes 4-6 variants. However, single aa changes within the TCR-binding domain completely abolished recognition even in case of conservative aa exchanges within genotype-1. NS3(1073)-specific T-cell lines from recovered, chronically infected, and HCV-negative individuals showed no major difference in the pattern of cross-recognition although the proliferation of NS3(1073)-specific T-cells differed significantly between the groups. Importantly, the recognition pattern against the 28 variants was also identical directly ex vivo in a patient with acute HCV infection and a healthy volunteer vaccinated with the peptide vaccine IC41 containing the NS3(1073)-wild-type peptide. Thus, partial cross-genotype recognition of HCV NS3(1073)-specific CD8 T cells is possible; however, even single aa exchanges can significantly limit the potential efficacy of vaccines containing the NS3(1073)-wild-type peptide.

Immunodominant T-cell epitopes of hnRNP-A2 associated with disease activity in patients with rheumatoid arthritis.

The heterogeneous nuclear ribonucleoprotein A2 (hnRNP‐A2) has been described as an important autoantigen in rheumatoid arthritis (RA) since it is targeted by autoantibodies, autoreactive T cells, and is aberrantly expressed in synovial cells in patients. To identify hnRNP‐A2‐specific T‐cell epitopes possibly associated with pathogenicity, we used an innovative approach. We first scanned 280 overlapping hnRNP‐A2 peptides for binding to the RA‐associated class II molecules HLA‐DR4 and HLA‐DR1, leading to a comprehensive selection of binders. The selected peptides were tested in IFN‐γ‐specific ELISPOT assay: PBMC from 18% of RA patients showed a significant IFN‐γ response to hnRNP‐A2 peptides, 15% to the overlapping sequences 117–133 and/or 120–133, whereas PBMC from healthy individuals tested negative. We measured proliferative responses to these two peptides in another cohort of patients with RA or osteoarthritis: positive responses were found in 28% of RA, but also in 11% of osteoarthritis patients and these responses could be blocked by anti‐MHC class II Ab. Remarkably, the presence of 117/120–133‐specific T cells was significantly associated with active disease in RA patients, and bone erosion appeared to be more common in T‐cell positive patients. These data suggest involvement of hnRNP‐A2 specific cellular autoimmune responses in RA pathogenesis.

Unique membrane-interacting properties of the immunostimulatory cationic peptide KLKL5KLK (KLK)

We have monitored the effects of KLKL5KLK (KLK), a derivative of a natural cationic antimicrobial peptide (CAP) on isolated membrane vesicles, and investigated the partition of the peptide within these structures. KLK readily interacted with fluorescent dyes entrapped in the vesicles without apparent pore formation. Fractionation of vesicles revealed KLK predominantly in the membrane. Peptide‐treated vesicles appeared with generally disorganized bilayers. While KLK showed no effect on osmotic resistance of human erythrocytes, dramatic decrease in core and surface membrane fluidity was observed in peptide‐treated erythrocyte ghosts as measured by fluorescence anisotropy. Finally, CD spectroscopy revealed lipid‐induced random coil to β‐sheet and β‐sheet to α‐helix conformational transitions of KLK. Together with the oligonucleotide oligo‐d(IC)13 [ODN1a], KLK functions as a novel adjuvant, termed IC31™. Among other immunological effects, KLK appears to facilitate the uptake and delivery of ODN1a into cellular compartments, but the nature of KLKs interaction with the cell surface and other membrane‐bordered compartments remains unknown. Our results suggest a profound membrane interacting property of KLK that might contribute to the immunostimulatory activities of IC31™.

Hepatitis C virus-specific T cell responses against conserved regions in recovered patients

For the design of peptide-based vaccines against the hepatitis C virus it is essential to acquire more information on frequently recognized epitopes in patients with successful immune control of HCV in the context of different HLA types. A matrix approach using 393 15mer peptides from conserved HCV regions overlapping by 13 amino acids was applied in 52 HCV-recovered individuals. Candidate peptides were further tested in two independent laboratories. 38 peptides induced IFN-gamma responses in ELISPOT assays including 15 previously unknown epitopes. There was no particular immune dominance as only five peptides were recognized by more than three individuals. Seven out of 14 peptides tested in more detail could be confirmed to be immunogenic using ELISPOT and cytotoxicity assays. While only 33% of HCV-recovered individuals recognized recombinant HCV proteins, 81% of individuals tested positive in the matrix approach (p<0.001). The strength, frequency and breadth of HCV-specific T cell responses were similar in spontaneously recovered patients than in interferon-recovered patients. In conclusion (i) we identified novel HCV epitopes in conserved regions, (ii) confirmed the inter-individual diversity of HCV-specific T cell responses and (iii) found no significant differences in HCV-specific T cell responses between spontaneously recovered and IFN-recovered patients.