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. Author manuscript; available in PMC: 2010 Feb 5.
Published in final edited form as: Virology. 2008 Dec 16;384(1):12–15. doi: 10.1016/j.virol.2008.11.027

Inhibition of cellular alpha-glucosidases results in increased presentation of hepatitis B virus glycoprotein-derived peptides by MHC class I

Ender Simsek a, Gomathinayagam Sinnathamby b, Timothy M Block a,b, Yuanjie Liu a, Ramila Philip b, Anand S Mehta a, Pamela A Norton a,*
PMCID: PMC2765373  NIHMSID: NIHMS145420  PMID: 19091367

Abstract

Inhibitors of alpha glucosidases prevent the trimming of oligosaccharides on certain nascent glycoproteins, including the hepatitis B virus MHBs envelope glycoprotein. MHBs proteins with untrimmed oligosaccharides do not interact with calnexin, increasing protein misfolding and subsequent degradation by proteasomes. As peptides loaded onto newly synthesized MHC class I complexes are predominantly derived from proteasomes, the possibility that glucosidase inhibition could increase presentation by MHC class I was determined. Using either a model epitope, or a natural MHBs epitope, it was demonstrated that glucosidase inhibitors enhanced presentation by MHC class I and promoted activation of antigen-specific CTLs, suggesting a pharmacologic approach to immune modulation.

Keywords: HBV envelope glycoprotein, Cytotoxic T lymphocytes, Glucosidase inhibitor

Introduction

Major histocompatibility complex class I (MHC class I) molecules present peptides generated by the proteasome for recognition by T lymphocytes (Cresswell, 2001). Recognition of a peptide-MHC class complex by CD8+ cytolytic T cells (CTLs) results in T cell activation and is critical to mounting a sustained cellular immune response. Presentation of foreign intracellular antigenic peptides (such as from viral proteins) by the MHC class I complex contributes to a successful CTL-mediated immune response that eliminates infected cells. Conversely, it is thought that failure to mount an adequate T cell response is a key weakness that permits viruses such as hepatitis B virus (HBV) to establish chronic infections (Chisari and Ferrari, 1995). It is thus hypothesized that pharmacologic agents that selectively cause HBV envelope proteins to misfold and become degraded would enhance presentation of peptide epitopes by MHC class I. This hypothesis was tested using a transfection assay that exploits an unusual dependence of HBV on a host cell function. HBV specifies three envelope proteins called LHBs, MHBs and HBs (Bruss, 2004), all derived from a single open reading frame with a common C terminal region. Folding and secretion of MHBs are much more dependent upon the endoplasmic reticulum (ER) chaperone calnexin than HBs (Werr and Prange, 1998), and secretion of MHBs specifically requires glycan added at pre-S2 (Mehta et al., 1997). Calnexin recognizes monoglucosylated glycans present on nascent polypeptides as a consequence of normal processing by ER glucosidases I and II (Helenius and Aebi, 2001). Pharmacologic inhibition of ER glucosidases prevents trimming to monoglucose, and thus prevents interaction with calnexin. The calnexin-dependent biogenesis of MHBs renders the protein extremely sensitive to glucosidase inhibitors, a property shared by a number of viral glycoproteins (Block and Jordan, 2002). We asked what impact such inhibitors will have on the presentation of HBV epitopes to CTLs.

Results

The three HBV envelope proteins have differing amino termini, but share a common C-terminus (Bruss, 2004). MHBs contain an additional 55 amino acids N-terminal to HBs, referred to as the pre-S2 region (Fig. 1A); LHBs contain pre-S2 and an additional ca. 110 amino acid N-terminal extension, termed pre-S1 region (not shown). All three proteins can undergo N-linked glycosylation at amino acid 146 within the shared HBs domain. However, MHBs contain an additional N-glycosylation site at amino acid 4 within pre-S2 (Fig. 1A). Previously, it was established that genetic destabilization of MHBs resulted in enhanced presentation by MHC class I to CTLs (Liu et al., 2007a). We hypothesized that pharmacologic inhibition of glucosidases might have a similar effect, as improper processing of glycan structure is likely to destabilize MHBs. To determine whether pharmacological inhibition of glucosidases could result in enhanced presentation by class I to CTLs, a well-characterized T-cell epitope tag presented by the mouse MHC class I Kb (SIINFEKL) that is derived from ovalbumin was introduced into MHBs (W/T-M-Ova, (Liu et al., 2007a)). Briefly, oligodeoxynucleotides encoding amino acids 257–264 (SIIN-FEKL, Ova257–264) along with five flanking amino acids on each side were inserted into the unique EcoRI site within the pre-S2 sequence of MHBs expression construct pM(S–)X (Fig. 1A). Plasmid pM(S–)X expresses MHBs but not HBs, due to mutation of the initiation codon for the latter (Norton et al., 2005); the plasmid also contains the HBxAg coding region, although expression of HBxAg has not been established. The glycosylation site normally present at pre-S2 amino acid 4 and required for secretion (Mehta et al., 1997) is now present downstream of Ova257–264 in plasmid W/T-M-Ova, in a sequence context that should still become glycosylated.

Fig. 1.

Fig. 1

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Expression of ovalbumin-tagged HBV middle glycoprotein MHBs in L-Kb cells. (A) Diagram of the construct for expression of MHBs with the embedded Ova257–264 tag. Complementary oligonucleotides were designed with cohesive ends to insert the underlined sequences in frame with Pre-S2 at the EcoRI site (inverted triangle). Diamonds indicate the position of sites of N-glycan addition, indicating that the N-glycan site normally present at amino acid 4 is now at amino acid 23 (NST). The asparagine at amino acid 4 is no longer within the context of an N-glycosylation sequon (N-X-S/T) and thus will not be modified. (B) Immunoblot analysis of lysates prepared from L-Kb cells transfected with a plasmid W/T-M-Ova and treated as indicated or left untreated for 3 days post-transfection. Blot was probed with antibody directed against the pre-S2 region; the three possible glycoforms of MHBs are indicated.

The mouse cell line L-Kb stably expresses the murine MHC class Kb protein (Golovina et al., 2005). Transfection of mouse L-Kb cells with W/T-M-Ova resulted in production of MHBs, which migrated as two species, representing protein either singly glycosylated at amino acid 4 or doubly glycosylated at both sites (Fig. 1B). The second site is glycosylated 50% of the time (Bruss, 2004), accounting for the doublet appearance of the protein. Addition of the competitive glucosidase inhibitor 6-O-butanoyl castanospermine (BuCas) at 1.0 mg/ml resulted in a 2-to 3-fold reduction of the steady state level of intracellular MHBs relative to untreated controls (Fig. 1B, lanes 1 and 2). Drug treatment resulted in reduced oligosaccharide trimming, with both glycosylated species exhibiting reduced mobility relative to untreated cells, indicating a failure to trim terminal glucoses, whereas the mobility of unglycosylated p30 was unaffected. This indicates that the Ova-tagged version of MHBs is sensitive to glucosidase inhibition in L-Kb cells, as we have previously found for wild type MHBs in cells of hepatocyte origin (Norton et al., 2005; Simsek et al., 2005).

Next, BuCas treated and untreated cultures were incubated with B3.Z CTL hybridoma cells, which specifically recognize Ova257–264 presented in the context of H-2Kb. These cells contain a lacZ reporter under the control of the NF-AT element from the interleukin-2 promoter (Karttunen and Shastri, 1991). Thus, occupancy of the T cell receptor by the peptide/MHC complex triggers the expression of the lacZ gene product, β-galactosidase, in the co-cultured B3.Z cells. In the absence of BuCas treatment, baseline β-galactosidase levels were slightly above the background levels observed B3.Z cells transfected with L-Kb cells expressing non-Ova tagged MHBs (Ova-) (Fig. 2A). Since glucosidase inhibition resulted in a proteasome-dependent degradation of MHBs (Simsek et al., 2005), and proteasomes are the major source of the peptides that are loaded into MHC class complexes, we determined whether CTL recognition of L-Kb target cells would increase following their treatment with BuCas. As shown in Fig. 2A, treatment of MHBs-Ova transfected cells with BuCas resulted in a dose-dependent increase in CTL activation relative to untreated cells, indicating that drug treatment enhanced presentation of Ova257–264 by MHC class I.

Fig. 2.

Fig. 2

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Glucosidase inhibition increases epitope presentation by MHC class I. (A) Relative levels of CTL activation following incubation with L-Kb target cells in the presence of increasing amounts of drug. L-Kb cells were transfected with W/T-M-Ova then treated as indicated for 3 days prior to incubation with B3.Z CD8+ hybridoma cells, which specifically recognize the Ova epitope when presented in the context of H-2Kb. Following incubation, cells were lysed and assayed for β-galactosidase activity. Values shown indicate light output in counts per second (cps); each bar is the average of duplicate determinations from a single experiment which varied by <10%. (B) Cell treatments were as in A, with the addition of epoximicin (Epox) to some wells, but levels shown are normalized to enzyme activity produced by incubation with untreated transfected cells (set to unity). Error bars represent SEM of replicates (n=4). b-gal, β-galactosidase.

Wild type MHBs apparently undergoes little proteasomal degradation under normal conditions (Liu et al., 2007b; Simsek et al., 2005). Although the nature of the resistance to degradation is not fully understood, one possibility is that assembly of envelope glycoproteins into larger lipoprotein particles shields them from the endoplasmic reticulum-associated degradation (Block et al., 2006). However, alpha-glucosidase inhibitors render the MHBs and LHBs glycoproteins much more sensitive to proteasomal degradation (Simsek et al., 2005). Since proteasomes are the predominant source of MHC-I presented peptides and, in cells in which glucosidase function is inhibited, proteasomal degradation of HBV MHBs is increased (Simsek et al., 2005), we reasoned that BuCas-induced increased antigen processing and presentation would be blocked by proteasome inhibitors. As expected, inclusion of epoximicin (EPOX), a specific proteasome inhibitor that does not inhibit non-proteasomal proteases (Schwarz et al., 2000), blocked the BuCas-mediated increase in antigen-dependent CTL activation (Fig. 2B).

It was of interest to establish whether the increased antigen presentation obtained with glucosidase inhibition in this model system would also be observed with naturally processed HBV antigenic peptides. HepG2.2.15 cells were derived from the human hepatoma line HepG2 and contain an integrated HBV genome that produces all HBV nucleic acids and proteins and releases infectious progeny (Sells et al., 1987). These cells are known to express HLA-A2 (Ito et al., 1993), so these cells were chosen as target cells for CTLs that had been primed with previously described HBV-derived HLA-A2 peptides presented by human MHC class I HLA-A2.

PBMCs were isolated from a HLA-A2+ healthy human donor, and incubated in the presence of a known HLA-A2-specific envelope peptide derived from the common S domain, 183-FLLTRILTI (Chisari and Ferrari, 1995), to induce antigen-specific cells. After three rounds of stimulation of cells with peptide-pulsed autologous antigen presenting cells, activated CTLs were incubated with drug treated or untreated HepG2.2.15 target cells (incubation of effector and target cells was performed overnight in the absence of drug). Co-cultures were assayed for antigen-specific activated CTLs using the ELISpot assay for IFN-gamma secretion as a function of CTL response, at a T cell:target ratio of 20:1 (Fig. 3). Antigen presenting T2 cells lacking any HBV proteins served as a negative control; pulsing these cells with peptide resulted in an increase in T cell response, showing antigen specificity (Fig. 3, upper right). HepG2.2.15 cells presented the 183-FLLTRILTI epitope at a basal level, presumably processed from viral envelope proteins. However, BuCas-treated HepG2.2.15 cells stimulated twice as much IFN-gamma production as untreated cells, suggesting increased presentation of the 183-FLLTRILTI epitope by the drug treated target cells (Fig. 3, lower right). CTL activation was completely blocked by inclusion of epoximicin, indicating proteasome dependence.

Fig. 3.

Fig. 3

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Glucosidase inhibition increases envelope peptide-specific CTL response. In vitro generated peptide specific CTLs were co-cultured with various target cells at an effector to target ratio of 20:1 overnight and IFN-gamma production was measured using human IFN-gamma ELISpot reagents. Targets were unpulsed or peptide (Pep) pulsed T2 cells or HepG2 or HepG2.2.15 (HBV expressing cells) that were either left untreated or treated with the indicated drugs (BuCas or Epoximicin). Results are presented as number of IFN-gamma producing cells per 106 CD8+ T cells. Error bars represent SEM of experimental replicates (n=3).

In contrast to the effect of BuCas treatment on 183-FLLTRILTI presentation, no effect was observed when CTLs were primed with another HLA-A2-restricted HBV peptide 18-FLPSDFFPSV derived from the viral core antigen (Chisari and Ferrari, 1995), that is not known to be glycosylated (Fig. 3, lower left). Thus, glucosidase inhibition resulted in enhanced presentation of a viral epitope due to specific destabilization of one or more of the envelope glycoproteins.

Discussion

These data demonstrate that pharmacologic inhibition of cellular glucosidases results in enhanced proteasomal degradation of the viral glycoprotein MHBs, with increased MHC class I-mediated presentation of an embedded T cell epitope tag. This is presumably due to reduction of viral glycoprotein secretion as a consequence of interfering with calnexin-mediated protein folding. Moreover, the increased recognition of HBV-infected cells by antigen-primed CTLs suggests that by rendering MHBs more susceptible to the proteasome, the glucosidase inhibitor has thus increased the possibility that viral antigens will be processed and be available for loading into MHC class I. However, as the accumulation of intracellular MHBs (Fig. 1B) is more modest than might be expected based on the CTL assay (Fig. 2), there might be other factors promoting antigen presentation. The relationship between protein degradation rate and presentation efficiency is incompletely understood (Eisenlohr et al., 2007), with multiple steps that must occur for glycoproteins that undergo endoplasmic reticulum-associated degradation.

Although the nature of the resistance of HBV MHBs to proteasomal degradation that we have reported previously is not clear (Liu et al., 2007b; Simsek et al., 2005), the consequences are of potential importance in the context of chronic viral infection. If viral glycoproteins are unable to be degraded, peptides will not be available for presentation by MHC class I, which may result in deficient CTL responses to virally infected cells. As summarized in a recent review, there are likely to be several reasons for the inadequate CTL response observed in chronic HBV infection, including deficiencies in antigen presentation (Guidotti and Chisari, 2006). HBV and the related woodchuck hepatitis virus have also been reported to reduce expression of MHC class I (Chen et al., 2006; Wang and Michalak, 2006), suggesting a more active role in immune evasion. In the case of the woodchuck virus, WHV, the pre-S2 region of the MHBs protein (see Fig. 1) has been implicated in a modest but significant reduction in surface MHC class I levels on hepatocytes that had been transfected with WHV MHBs (Wang and Michalak, 2006), although a similar role for pre-S2 as encoded by the human virus is not known. Thus, presentation of HBV antigens may be affected by several factors.

Imino sugars that inhibit cellular glucosidases, such as BuCas, have been approved for use in lysosomal glycosphingolipidoses (Butters et al., 2005). BuCas has been advanced to Phase II clinical trials against hepatitis C virus, with some promise when used in combination with other (Durantel et al., 2007). The immunomodulatory activity of glucosidase inhibitors was previously unrecognized, and is somewhat unexpected, as it has been reported that glucosidase inhibition can destabilize unassembled MHC class I molecules (Moore and Spiro, 1993). The results presented in the present report suggest a possible new therapeutic role for using this class of pharmacologic agents against other chronic viral infections in which a CD8+ CTL response is deficient, and the viral envelope glycoprotein might be sensitive to glucosidase inhibition, such as HBV.

Materials and methods

The mouse cell lines L-Kb and B3.Z CTL cells were a kind gift of by Dr. L. Eisenlohr, Thomas Jefferson University, Philadelphia, PA. T2 cells, obtained from American Type Culture Collection (ATCC; Manassas, VA), are HLA-A2 expressing human T cell–B cell somatic hybrid cells that are defective in transporting of MHC class I presented antigenic peptides across ER membrane. Consequently, T2 cells express ‘empty’ (peptide receptive) HLA-A2 molecules on the cell surface. Human HepG2 (ATCC) cells were derived from a well-differentiated hepato-cellular carcinoma. HepG2.2.15 cells were derived from HepG2 cells by stable transfection of HBV genome and were a gift of Dr. G Acs. 6-O-butanoylcastanospermine (BuCas, also called Celgosivir) was a gift of Migenix, Inc., Vancouver, BC. The construction of plasmid W/T-M-Ova, transfection of cells and immunoblotting with an antibody that recognizes the pre-S2 region (Research Diagnostics, Inc., Somerset, NJ) was as described previously (Liu et al., 2007a).

Target L-Kb cells were treated with BuCas for a total of 3 days. B3.Z CTL cells were added on day two and the co-cultures incubated overnight. No cell toxicity was observed at any dose tested (data not shown). In cases when epoximicinwas included, it was added only for the final overnight incubation. The lacZ gene product, β-galactosidase, was measured in cell lysates using a chemiluminescence-based assay (Galactolight Plus, Applied Biosystems, Bedford, MA).

In vitro generation of peptide specific human CTLs was achieved according to standard methods. Briefly, heparinized blood from a healthy HLA-A2+ human donor (Research Blood Components, Brighton, MA). Peripheral blood mononuclear cells were purified using lymphocyte separation medium (Mediatech, Manassas, VA) using differential centrifugation according to standard methods. 20×106 cells were plated per well in 2 ml RPMI 1640 medium supplemented with 10% fetal bovine serum, l-glutamine (300 µg/ml), non-essential amino acids, sodium pyruvate, penicillin and streptomycin (complete medium) in 6-well tissue culture plates (BD, Franklin Lakes, NJ) overnight. Non-adherent cells were removed and saved. Plastic adherent cells were pulsed with 50 µg/ml synthetic peptide and 1.5 µg/ml human β2-microglobulin (Sigma-Aldrich, St. Louis, MO) in complete medium for 2 h. Medium was removed and non-adherent cells were added in 5 ml complete medium supplemented with IL-7 at 5 ng/ml, Keyhole Limpet Hemocyanin (KLH; Sigma) at 5 µg/ml, Granulocyte Monocyte Colony Stimulating Factor (GM-CSF) at 25 ng/ml and IL-4 at 50 ng/ml (all cytokines and growth factors were from Peprotech, Rocky Hill, NJ). Plates were incubated at 37 °C in a humidified incubator with 5% CO2 for 12 days. 2.5 ml medium was removed from each well and replaced with fresh complete medium supplemented with 10 U/ml IL-2 for 2 days. T cells were restimulated with CD4/CD8 T cell depleted autologous monocytes pulsed with synthetic peptide at 10 µg/ml and 1.5 µg/ml human β2-microglobulin in complete medium containing 5 ng/ml IL-7 and 5 µg/ml KLH for 5 days. IL-2 treatment and in vitro restimulation were repeated thrice at weekly intervals prior to use of in vitro expanded T cells in ELISpot assays. In vitro expanded T cells were used as effectors in ELISpot assays to assess antigen stimulated interferon-γ release using human interferon-γ assay kit (BD-Pharmingen, San Jose, CA) according to the manufacturer's instructions.

Acknowledgments

This work was supported by grants from NIAID, the Hepatitis B Foundation and an appropriation from the Commonwealth of Pennsylvania.

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