Glucosidase inhibition enhances presentation of de-N-glycosylated hepatitis B virus epitopes by major histocompatibility complex class I in vitro and in woodchucks

Abstract

In this report, the possibility of pharmacologically altering the hepatitis B virus (HBV) epitopes presented by major histocompatibility complex class I on infected cells is demonstrated. The HBV middle envelope glycoprotein (MHBs) maturation appears to require calnexin-mediated folding. This interaction is dependent on glucosidases in the endoplasmic reticulum. Prevention of HBV envelope protein maturation in cultured cells through use of glucosidase inhibitors, such as 6-O-butanoyl castanospermine and N-nonyl deoxynorjirimycin, resulted in MHBs degradation by proteasomes. The de-N-glycosylation associated with polypeptide degradation was predicted to result in conversion of asparagine residues into aspartic acid residues. This prediction was confirmed by showing that peptides corresponding to the N-glycosylation sequons of MHBs, but with aspartic acid replacing asparagine, (1) can prime human cytotoxic T lymphocytes that recognize HBV-producing cells and (2) that the presentation of these envelope motifs by major histocompatibility complex class I is enhanced by incubation with glucosidase inhibitors. Moreover, although peripheral blood mononuclear cells isolated from woodchucks chronically infected with woodchuck hepatitis virus and vaccinated with woodchuck hepatitis virus surface antigen could be induced to recognize the natural MHBs asparagine-containing peptides, only cells isolated from animals treated with glucosidase inhibitor recognized the aspartic acid-containing peptides.

Conclusion: These data suggest that pharmacological intervention with glucosidase inhibitors can alter the MHBs epitopes presented. This editing of the amino acid sequence of the polypeptide results in a new epitope, or "editope", with possible medical significance.

Figure 1. Schematic representation of the consequences of endoplasmic reticulum associated degradation-linked de-N-glycosylation

The figure depicts interference of the interaction of MHBs with calnexin (CNX) in the ER by glucosidase inhibitor (GluI), with subsequent retrotranslocation to the cytoplasm. Both de-N-glycosylation by PNGase and degradation by the proteasome result in the production of a novel D-peptide in place of the original N-peptide. These peptides are now available for re-import into the ER and loading into empty MHC class I complexes. Inverted triangle, tri-glucosylated N-glycan chain.

Figure 2. CTLs raised against aspartic containing envelope protein epitopes recognize HBV producing cells

PBMCs isolated from healthy HLA-A2+ human donor blood were stimulated in vitro with peptides corresponding to the HLA-A2 restricted CTL epitope from HBs (KPSDGNCTC) or the ‘D’ substituted peptide (KPSDGDCTC). The ability of in vitro generated CTLs to recognize and secrete interferon-γ was evaluated by ELISpot assay. A. CTLs generated against ‘N’ containing peptide and the corresponding ‘D’ containing peptide were incubated with T2 cells pulsed with either ‘N’ or ‘D’ containing peptide to assess T cell cross-reactivity. B. HBV negative HepG2 cells or HBV positive HepG2.2.15 cells, either left untreated or treated with BuCas (1mg/ml) twice for three day intervals were used as targets. Target cells (5000 cells per well) were washed once before they were co-incubated with CTLs (100,000 cells/well) in an ELISpot plate. Error bars represent SEM of experimental replicates. P value was calculated from a Student’s t-Test analysis of experimental results.

Figure 3. Vaccination schedule for woodchucks, and proliferation of PBMCs in response to viral antigens

A, Scheduled treatment of woodchucks. Arrows indicate vaccination with complexes of alum and surface antigen for selected groups of animals. Circle, vaccination of animals with D-peptide. See text for details on animal groupings. B, BuCas; V, vaccine; L, L-FMAU; P, placebo. B, PBMCs were isolated at the indicated time points, and cultured as described in Materials and Methods. Peptide antigens are shown in Table 1; in addition, full length WHV core and HBs were used as antigens. Animals were scored as positive if cells proliferated above the cut-off value of ≥ 3.1. Treatment groups are designated as P, placebo; B, BuCas; V, vaccine; B+V, BuCa plus vaccine. Percentage of animals with a positive response to one or more WHsAg-related peptides is shown. C, As for B, with percentage of animals with a positive response to the entire WHsAg and/or WHcAg shown.

Figure 4. Proliferation of PBMCs induced by viral neo-antigen in response to drug treatment

A, Detailed responses of individual animals at a single time point to N-peptides versus D-peptides. Positive response is as defined in Fig. 4. Treatment groups are designated as Un, uninfected controls; P, placebo; B, BuCas; V, vaccine; B+V, BuCa plus vaccine. B, Summary of responses of groups to N-peptides and D-peptides over time.

Figure 5. Proliferation of PBMCs in response to neo-antigen vaccination

Detailed responses of individual animals either pre-inoculation (week 28) or 4 weeks post-inoculation with D-peptides. Treatment groups are designated as Un, uninfected controls; P, placebo; B, BuCas; V, vaccine; B+V, BuCa plus vaccine. Woodchuck 7092 died following week 20 of the study, and thus is unscored.