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Virus-specific CD8 T cell response seems to play a significant role in the outcome of hepatitis delta virus (HDV) infection. However, the HDV-specific T cell epitope repertoire and mechanisms of CD8 T cell failure in HDV infection have been poorly characterized. We therefore aimed to characterize HDV-specific CD8 T cell epitopes and the impacts of viral mutations on immune escape. In this study, we predicted peptide epitopes binding the most frequent human leukocyte antigen (HLA) types and assessed their HLA binding capacities. These epitopes were characterized in HDV-infected patients by intracellular gamma interferon (IFN-γ) staining. Sequence analysis of large hepatitis delta antigen (L-HDAg) and HLA typing were performed in 104 patients. The impacts of substitutions within epitopes on the CD8 T cell response were evaluated experimentally and by in silico studies. We identified two HLA-B*27-restricted CD8 T cell epitopes within L-HDAg. These novel epitopes are located in a relatively conserved region of L-HDAg. However, we detected molecular footprints within the epitopes in HLA-B*27-positive patients with chronic HDV infections. The variant peptides were not cross-recognized in HLA-B*27-positive patients with resolved HDV infections, indicating that the substitutions represent viral escape mutations. Molecular modeling of HLA-B*27 complexes with the L-HDAg epitope and its potential viral escape mutations indicated that the structural and electrostatic properties of the bound peptides differ considerably at the T cell receptor interface, which provides a possible molecular explanation for the escape mechanism. This viral escape from the HLA-B*27-restricted CD8 T cell response correlates with a chronic outcome of hepatitis D infection. T cell failure resulting from immune escape may contribute to the high chronicity rate in HDV infection. IMPORTANCE Hepatitis delta virus (HDV) causes severe chronic hepatitis, which affects 20 million people worldwide. Only a small number of patients are able to clear the virus, possibly mediated by a virus-specific T cell response. Here, we performed a systematic screen to define CD8 epitopes and investigated the role of CD8 T cells in the outcome of hepatitis delta and how they fail to eliminate HDV. Overall the number of epitopes identified was very low compared to other hepatotropic viruses. We identified, two HLA-B*27-restricted epitopes in patients with resolved infections. In HLA-B*27-positive patients with chronic HDV infections, however, we detected escape mutations within these identified epitopes that could lead to viral evasion of immune responses. These findings support evidence showing that HLA-B*27 is important for virus-specific CD8 T cell responses, similar to other viral infections. These results have implications for the clinical prognosis of HDV infection and for vaccine development.

Hepatitis delta antigen (HDAg) consists of two species, small HDAg (SHDAg) and large HDAg (LHDAg), which are identical in sequence with the exception that the large form contains an additional 19 amino acids at the C-terminus. Both HDAgs are nuclear phosphoproteins. However, LHDAg is hyperphosphorylated, i.e. it is at least 10 times more phosphorylated than SHDAg. To determine the phosphorylation site(s) of the LHDAg, we mutated all the conserved serine residues and expressed these mutant proteins using a recombinant baculovirus expression system. By labeling insect cells in vivo with 32P-orthophosphate and immunoprecipitation, we showed that LHDAg is phosphorylated at multiple serine residues. Although LHDAg contains two additional serines at its 19-amino acid extension, mutations of these two amino acids did not affect the overall phosphorylation level. Most importantly, the phosphorylation level of middle domain-deleted LHDAg (M75del) was significantly higher than that of wild-type LHDAg. We conclude that phosphorylation of the LHDAg occurs at multiple sites and that hyperphosphorylation is associated with alteration of protein conformation.

326 PRO-205 OF LARGE HEPATITIS DELTA ANTIGEN AND PRO-62 OF MAJOR HEPATITIS B SURFACE ANTIGEN INFLUENCE THE ASSEMBLY OF HEPATITIS D VIRUS OF DIFFERENT GENOTYPES H.H. Shih, C. Shih, H.-W. Wang, I.-J. Sheen, C.-W. Su, J.-C. Wu. Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Institute of BioMedical Sciences, Academia Sinica, Institute of Microbiology and Immunology, National Yang-Ming University, Institute of Clinical Medicine, National Yang-Ming University, Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, Department of Medical Research and Education, Taipei Veterans General Hospital; Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan R.O.C. E-mail: jcwu@vghtpe.gov.tw