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Journal of Virology, June 2002, p. 5305-5314, Vol. 76, No. 11
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.11.5305-5314.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Immunization with Surface Antigen Vaccine Alone and after Treatment with 1-(2-Fluoro-5-Methyl-ß-L-Arabinofuranosyl)-Uracil (L-FMAU) Breaks Humoral and Cell-Mediated Immune Tolerance in Chronic Woodchuck Hepatitis Virus Infection

Stephan Menne,1* Carol A. Roneker,1 Brent E. Korba,2 John L. Gerin,2 Bud C. Tennant,1 and Paul J. Cote2

Gastrointestinal Unit, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York,1 Division of Molecular Virology and Immunology, Georgetown University Medical Center, Rockville, Maryland2

Received 18 December 2001/ Accepted 26 February 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) were treated with the antiviral drug 1-(2-fluoro-5-methyl-ß-L-arabinofuranosyl)-uracil (L-FMAU) or placebo for 32 weeks. Half the woodchucks in each group then received four injections of surface antigen vaccine during the next 16 weeks. Vaccination alone elicited a low-level antibody response to surface antigen in most carriers but did not affect serum WHV DNA and surface antigen. Carriers treated first with L-FMAU to reduce serum WHV DNA and surface antigen and then vaccinated had a similar low-level antibody response to surface antigen. Following vaccinations, cell-mediated immunity to surface antigen was demonstrated in both groups, independent of serum viral and antigen load, but was significantly enhanced in woodchucks treated with L-FMAU and was broadened to include other viral antigens (core, e, and x antigens and selected core peptides). Cell-mediated immunity and antibody responses to surface antigen were observed after drug discontinuation in half of the carriers that received L-FMAU alone. Surface antigen vaccine alone or in combination with drug broke humoral and cell-mediated immune tolerance in chronic WHV infection, but the combination with drug was more effective. This suggested that a high viral and antigen load in carriers is important in maintaining immunologic tolerance during chronicity. The humoral and cellular immunity associated with the combination of L-FMAU and vaccine resembled that observed in self-limited WHV infection. Such combination therapy represents a potentially useful approach to the control of chronic hepatitis B virus infection in humans.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
More than 350 million people worldwide are chronically infected with hepatitis B virus (HBV) (19) and are at high risk of developing chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. Such individuals could benefit immensely from effective therapy, and a combination of antiviral drugs and immunomodulation is a strategy currently under consideration. Successful immunotherapeutic eradication of established HBV infection might be possible if there was better understanding of the underlying mechanisms of chronicity (1, 2, 11, 18, 22, 24, 30, 31, 39; reviewed in references 3 and 26). While approaches using antiviral drugs in combination with alpha interferon (IFN-{alpha}) or HBV surface antigen (HBsAg) vaccines have shown promise as therapies for established chronic HBV infection (9, 10, 12, 16, 20, 23, 35), such treatments have not been optimized based on a full understanding of the mechanisms of chronicity.

Chronic HBV infection is associated with defects in immunity (reviewed in references 3 and 26) that hinder the development of successful immunotherapy. In contrast to self-limited HBV infection, the HBV carrier appears to be immunologically tolerant. The T helper (Th) cell responses to viral antigens are usually deficient, and antibodies to HBsAg (anti-HBs) are rarely detected. Whether additional B-cell tolerance is involved here is not clear. Antibodies to the HBV core antigen (HBcAg) (anti-HBc antibodies) are detected in carriers with hyporesponsive Th cells to core gene products (HBcAg and HBV e antigen [HBeAg]) because anti-HBc can be elicited via Th-independent mechanisms (32). Th cell responses to HBcAg and HBeAg can be favorable for the carrier when viral replication is diminished during seroconversion to antibodies to HBeAg (anti-HBe) (11, 18, 22, 24, 39). However, T-cell responses in the HBV carrier are generally dysfunctional overall and contribute more to disease progression than to viral clearance (11, 24, 39; reviewed in reference 3).

The Eastern woodchuck (Marmota monax) chronically infected with the woodchuck hepatitis virus (WHV) has been used as an animal model to investigate the basic pathogenesis of chronic HBV infection and in the preclinical development of drugs for therapy of HBV (reviewed in references 6, 26, 36, and 38). This animal model mimics many of the immune response features observed in human HBV infections (4, 5, 7, 14, 15, 25, 27-29, 33; reviewed in references 6, 26, 36, and 38) and can predict human responses to antiviral agents as well (21).

Experimental studies in chronic WHV carrier woodchucks have been performed using the new and potent antiviral drug 1-(2-fluoro-5-methyl-ß-L-arabinofuranosyl)-uracil (L-FMAU) (34), which may represent a promising therapeutic approach for chronic HBV infection. L-FMAU significantly reduces the concentration in serum of both WHV DNA and WHV surface antigen (WHsAg) and also the levels of covalently closed circular viral DNA in the liver (34). A controlled study has now been performed in woodchucks involving treatment with a combination of L-FMAU followed by WHsAg vaccination. The purpose of this report is to show how it is possible using combination therapy to increasingly break immunologic tolerance in chronic WHV carrier woodchucks and to modulate the humoral and cellular immune response profiles toward that observed in self-limited WHV infections.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Woodchucks. Experimental woodchucks were born in the laboratory animal facilities of the College of Veterinary Medicine, Cornell University, Ithaca, N.Y. The use of woodchucks for these studies was reviewed and approved by the Cornell University Institutional Animal Care and Use Committee. Thirty-two chronic WHV carrier woodchucks, 1 to 2 years of age, stratified into four experimental groups, were used to investigate the influence of antiviral drug treatment on the immune response to therapeutic vaccination. The WHV carriers were infected as neonates with WHV7P1 as described previously (4). The carrier status of these woodchucks was verified by detection of persistent WHV viremia and WHs antigenemia from 3 months of age. On entry into the study, the carriers had minimal chronic hepatitis based on histologic examination of liver biopsies and serum enzyme profiles. All were considered to be free of hepatocellular carcinoma based on hepatic ultrasound examinations and on the {gamma}-glutamyl transferase activity of serum.

Drug and vaccine. The antiviral drug L-FMAU (clevudine) was provided as a dry powder by Triangle Pharmaceuticals, Inc. (Research Triangle Park, N.C.). L-FMAU was weighed, suspended in a saline vehicle, and administered to woodchucks orally using a dose syringe as described (34). The subunit vaccine consisted of serum-derived, 22-nm WHsAg particles purified by zonal ultracentrifugation (13), which were formalin inactivated and adsorbed to alum. The purified 22-nm WHsAg particles were not treated with enzymes that remove pre-S sequences. The WHsAg was isolated from the serum of WHV7P1-infected chronic carriers and was therefore homologous with the WHsAg circulating in the serum of the WHV carrier woodchucks in the present study. Prior to the study, the safety and immunogenicity of the WHsAg vaccine were demonstrated in WHV-susceptible woodchucks negative for serologic markers of WHV infection (data not shown).

Study design. For the drug treatment phase of the study, 16 chronic WHV carrier woodchucks received L-FMAU (L+ carriers; 10 mg/kg/day, once per day orally for 32 weeks) and 16 received placebo (L- carriers). Two of the woodchucks (one in each group) died before the start of the vaccine phase of the study. For the vaccine phase of the study, eight L+ carriers received 50 µg of the WHsAg vaccine at week 32 (L+V+ carriers; referred to as combination therapy) and seven L+ carriers received saline as an injection control (L+V- carriers). Eight L- carriers received 50 µg of the WHsAg vaccine at week 32 (L-V+ carriers), and seven L- carriers received saline as an injection control (L-V- carriers; referred to as carrier controls). All of the initially vaccinated carriers (L+V+ and L-V+ groups) received three additional 50-µg doses of the WHsAg vaccine at weeks 36, 40, and 48. All of the vaccine controls (L+V- and L-V- carriers) received additional saline injections at these times.

The immune responses in each experimental group were compared during the drug treatment phase (weeks 0 to 32) and the vaccine phase (weeks 32 to 60). During the study, four WHV-susceptible, unvaccinated woodchucks provided concurrent negative-control materials for the various assays. Serum samples were obtained throughout the study for measurement of viral markers (WHV DNA and WHsAg) and antibody (anti-WHs) to WHsAg. Cell-mediated immunity (CMI; referred to as vCMI when denoting virus-specific CMI) was monitored by an in vitro proliferation assay using peripheral blood mononuclear cells (PBMC) isolated from whole blood. Most of the woodchucks in each group (63% to 86%) were monitored at 2- to 4-week intervals. The remainder were tested at intervals of 8 to 12 weeks.

Assays of viral and antibody markers. Serum WHV DNA was measured by dot blot hybridization (4, 21, 34) either directly or following PCR amplification (limit of sensitivity, 30 virus genomic equivalents [vge]/ml). Serum WHsAg was measured by enzyme-linked immunosorbent assay (ELISA) (8) using a 1:10 or greater dilution of serum (lower sensitivity, ca. 30 ng/ml of serum). Anti-WHs antibody was quantified by ELISA (8) in reciprocal standard dilution units.

Briefly, values above the assay cutoff were compared against a standard dilution curve for a hyperimmune rabbit anti-WHs serum that contained 8,400 U/ml by endpoint titration. The cutoff of the assay was defined as 1 U. Thus, the minimal positive detection for sera assayed at a dilution of 1:100 was 101 U/ml. This assay uses a biotinylated protein G probe (8), which binds to the Fc region of immunoglobulin G. Theoretically, this enables detection of WHsAg/anti-WHs immune complexes bound to the assay catch antigen even under the conditions of antigen excess that usually exist in the WHV carrier. Detection may also result if there is exchange between the catch antigen and the anti-WHs of immune complexes in serum. Free anti-WHs also could become available in WHV carriers after reduction in serum surface antigen load.

Polyclonal activators, viral antigens, and synthetic peptides. In vitro stimulators were generated and used at concentrations optimal for woodchuck PBMC cultures as described previously (7, 17, 25, 27-29, 37). Concanavalin A (ConA; 8 µg/ml) was purchased from Sigma (St. Louis, Mo.). Human recombinant interleukin-2 (rIL-2; 100 IU/ml) was obtained from Cetus (Emeryville, Calif.). Viral antigen stimulators consisted of (i) native 22-nm WHsAg (0.5 and 2 µg/ml), the same purified antigen used to prepare the experimental vaccine and for the assay of anti-WHs, (ii) recombinant WHV core antigen (rWHcAg; 0.5 and 1 µg/ml), (iii) recombinant WHV e antigen (rWHeAg; 0.5 and 1 µg/ml), consisting of rWHcAg truncated C-terminally by 39 amino acids and without precore residues, and (iv) recombinant WHV x antigen (rWHxAg), 0.5 and 1 µg/ml. Three synthetic peptides of WHcAg (20 amino acids in length; 10 µg/ml) corresponded to sequences 1 to 20, 100 to 119, and 112 to 131 of WHcAg (C1-20, C100-119, and C112-131, respectively) (25, 27, 28).

In vitro PBMC proliferation assay. The in vitro proliferation assays using woodchuck PBMC were comparable to those for human studies (11, 18, 22, 24) except that dividing cells were labeled with [2-3H]adenine (25, 28) (37 kBq/well; specific activity, 703 GBq/mM; Amersham Pharmacia Biotech, Inc., Arlington Heights, Ill.). Woodchuck PBMC were isolated from whole blood and stimulated in vitro as described (7, 25, 27-29). Counts per minute (cpm) of triplicate cultures were averaged and expressed as a stimulation index (SI) by dividing the average sample cpm in the presence of stimulator by that in the absence of stimulator (seven replicates). An SI value of >=3.1 represents a positive, specific cell-mediated immune response. This positive cutoff is conservative, given that the range of maximal antigen-specific SI values for positive stimulation by antigens and peptides in our experience with this assay is usually in the range of 7 to 12. The 3.1 cutoff level represents 25 to 45% of the maximal SI values associated with positive stimulation induced by antigens or peptides. At the 3.1 SI cutoff, the cpm for positive samples were always greater than 2 standard deviations (SDs) above the mean cpm of unstimulated control cultures from the same woodchuck and were usually more than 2 SDs above the cpm for antigen- and peptide-stimulated PBMC from uninfected control woodchucks.

Parameters of humoral and cellular immune responses. The frequency of responding woodchucks was defined as the percentage of woodchucks in each group that developed positive responses at one or more time points. This was expressed as a cumulative frequency for a given study interval or incidentally as a frequency for a given time point. The frequency of positive samples was defined as the percentage of samples testing positive above the assay cutoff during a given study interval.

Statistical analysis. The frequency of responding woodchucks and frequency of positive samples from treatment and control groups were compared by Fisher's test for proportions. One-tailed test criteria were applied. P values of <0.05 were considered statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Use of WHsAg as a therapeutic vaccine in chronic WHV carriers with high versus low viral and antigen load. Typical virologic and immunologic responses for one representative woodchuck from each of the four experimental groups from the double-placebo-controlled trial are shown in Fig. 1 through week 60 of the study. Administration of L-FMAU to WHV carriers for 32 weeks resulted in marked reductions both in serum WHV DNA and in WHsAg compared with placebo controls (Fig. 1; L+V+ and L+V- versus L-V+ and L-V-). After drug treatment was discontinued at week 32, viral markers remained at the low levels in most of the drug-treated carriers for at least 2 months. This feature afforded the opportunity to compare the responses of carriers with either low (L+V+) or high (L-V+) viral and antigen load to vaccination with WHsAg. Anti-WHs and vCMI responses were elicited concurrent with the vaccinations in the L+V+ and L-V+ carriers (Fig. 1). In the L-V+ carriers, serum viral and antigen load were not significantly affected as a result of vaccination alone compared to L-V- control carriers (Fig. 1). There were no hepatic flare reactions associated with vaccination based on the assay of standard serum biochemical markers (i.e., liver enzymes) (data not shown). No other adverse effects were associated with vaccination in either the high or low viral load settings.



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  		FIG. 1. Kinetics of virologic and immunologic responses for one representative chronic WHV carrier woodchuck from each of the four experimental groups during the drug treatment and vaccine phases of the study. L+V+, L-FMAU/vaccine group, woodchuck 5496; L-V+, placebo/vaccine group, woodchuck 5371; L+V-, L-FMAU/injection control group, woodchuck 5342; L-V-, placebo/injection control group, woodchuck 5343. {circ}, serum WHV DNA; {triangleup}, serum WHsAg; {diamondsuit}, serum anti-WHs antibody response. - and +, undetectable (SI < 3.1) and detectable (SI >= 3.1) vCMI to WHV antigens (WHsAg, rWHcAg, rWHeAg, and rWHxAg), respectively. The drug treatment phase (0 to 32 weeks) is demarcated by a vertical dotted line. Arrowheads represent the four immunizations using 50-µg doses of an alum-adsorbed WHsAg vaccine at weeks 32, 36, 40, and 48.

 
The main goal of the present study is to show the increasing degree to which humoral and cellular immunologic tolerance can be broken in WHV carriers undergoing the monotherapy versus the combination immunotherapies. Such results have important implications for the application of HBV immunotherapies as well as for understanding the pathogenesis of chronic HBV and WHV infections. Further details regarding additional case studies and the follow-up through later time points of the group-specific antiviral responses and therapeutic efficacy of the treatments against disease progression will be described elsewhere.

Chronic WHV carriers develop anti-WHs following WHsAg vaccination alone or vaccination following therapy with L-FMAU. Anti-WHs responses were not routinely detected in the L-V- control group during either the drug treatment or vaccine phase of the study (Fig. 2, Table 1). Anti-WHs responses were more evident during the vaccine phase than during the drug treatment phase in the L+V+, L-V+, and L+V- groups. During the vaccine phase, the percentages of L+V+ and L-V+ carriers that developed anti-WHs in response to vaccine were significantly higher than those of the L-V- control carriers. The frequencies of anti-WHs-positive serum samples were also significantly higher in the L+V+ and L-V+ carriers than in the L-V- carriers (Table 1). The L+V+ and L-V+ carriers had similar anti-WHs responses based on these two parameters. After drug treatment was discontinued, about half of the L+V- carriers developed anti-WHs responses (Fig. 2). The frequency of anti-WHs-positive serum samples in this group (L+V-) was intermediate (P < 0.01) between the L-V- control carriers and the L+V+ and L-V+ carriers (Table 1).



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  		FIG. 2. Kinetics of serum anti-WHs antibody responses of chronic WHV carrier woodchucks from the four experimental groups during the drug treatment and vaccine phases of the study. L+V+, L-FMAU/vaccine group; L-V+, placebo/vaccine group; L+V-, L-FMAU/injection control group; L-V-, placebo/injection control group. The drug treatment phase (0 to 32 weeks) is demarcated by a vertical dotted line. Arrowheads represent the four immunizations using 50-µg doses of an alum-adsorbed WHsAg vaccine at weeks 32, 36, 40, and 48.

 

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  		TABLE 1. Cumulative frequencies of woodchucks with anti-WHs responses and of serum samples positive for anti-WHs in experimental groups during the drug treatment and vaccine phases of the studya

 
The anti-WHs titers of vaccinated carriers (Fig. 2) were about 10-fold lower on average than those usually observed in WHV-susceptible woodchucks given the same vaccine regimen (data not shown). One of the L+V+ carriers (woodchuck 5478) had consistent evidence of low-level anti-WHs response during the drug treatment phase. This one woodchuck was highly responsive to vaccination in terms of its anti-WHs response. Overall, the anti-WHs titers induced by vaccination were similar through week 60 in the L+V+ and L-V+ carriers.

Chronic WHV carriers develop enhanced vCMI to WHsAg when vaccinated following therapy with L-FMAU. The anti-WHs responses observed in treated carriers (Fig. 2) were usually associated with vCMI to WHsAg (Fig. 1 and 3). The vCMI to WHsAg in treated carriers was considered robust and specific compared to that of the L-V- control carrier group, which remained negative for vCMI to WHsAg throughout the entire 60-week observation period (Fig. 3). vCMI to WHsAg was essentially undetectable in all groups during the 32-week drug treatment phase (Fig. 3), including the drug-treated carriers that achieved marked reductions in viral and antigen load (Fig. 1). The lack of detectable vCMI during this period was not due to a general unresponsiveness of carrier PBMC or to drug-induced lymphotoxicity, since the samples from all carriers were highly responsive to polyclonal activators (ConA and rIL-2; data not shown). vCMI to viral antigens other than WHsAg was observed sporadically and at low frequency during the drug treatment phase of the study. Significant vCMI to WHsAg and even to other viral antigens was more evident during the vaccine phase (Fig. 3). Accordingly, in order to simplify the numbers of statistical comparisons, subsequent references will be made only to the results obtained during the vaccination and immediate follow-up phases of the study (i.e., weeks 32 to 60).



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  		FIG. 3. Kinetics of vCMI to WHV antigens of chronic WHV carrier woodchucks from the four experimental groups during the drug treatment and vaccine phases of the study. The frequencies of responding woodchucks (percent of woodchucks at each time point with SI values of >=3.1) to WHsAg, rWHcAg, rWHeAg, and rWHxAg are shown as vertical bars. The group means of SI values are presented as circles. L+V+, L-FMAU/vaccine group; L-V+, placebo/vaccine group; L+V-, L-FMAU/injection control group; L-V-, placebo/injection control group. The horizontal bar represents the drug or placebo treatment phase (weeks 0 to 32). Vertical arrowheads represent the four immunizations using 50-µg doses of an alum-adsorbed WHsAg vaccine or saline at weeks 32, 36, 40, and 48. The mean cpm ± SD of the unstimulated PBMC from L+V+ carriers, from L-V+ carriers, from L+V- carriers, and from L-V- carriers were, respectively, 2,826 ± 1,203, 2,803 ± 1,192, 2,811 ± 1,231, and 2,795 ± 1,249.

 
During the vaccine phase, a high proportion of the carriers that were immunized with the WHsAg vaccine developed a sustained vCMI to WHsAg (Fig. 3, Table 2; L+V+ and L-V+). All L+V+ carriers developed vCMI to WHsAg following vaccination, with 71% of the PBMC samples positive between weeks 32 and 60 (P < 0.01 versus L-V-, Table 2). There was a noticeable increase in the mean SI for this group between weeks 36 and 60 (Fig. 3). Most (88%) of the carriers in the L-V+ group also developed vCMI to WHsAg during vaccination (P < 0.01 versus L-V-; Table 2). However, while their frequency of positive PBMC samples (40%, Table 2) was significantly higher than in L-V- controls (P < 0.01), it also was significantly less than in the L+V+ carriers (P < 0.01). This related to the virtual lack of detectable vCMI in the L-V+ carriers following the fourth dose of vaccine (i.e., with the exception of week 56; see Fig. 3).


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  		TABLE 2. Cumulative frequencies of responding woodchucks and of PBMC samples positive for vCMI to WHV antigens and core peptides during the vaccine phase of the studya

 
Regarding the drug monotherapy group, 43% of the L+V- carriers developed vCMI to WHsAg after drug treatment was discontinued (Fig. 3, Table 2). This frequency was not significantly different from that of the L-V- and L-V+ groups, but was significantly less than in the L+V+ group (i.e., 100%; P < 0.05, Table 2). The corresponding frequency of PBMC samples positive for vCMI to WHsAg was clearly intermediate (P < 0.01) between those of the L-V- and L+V+ carriers and not significantly different from that of the L-V+ carriers. The trend for frequency of PBMC samples positive for vCMI to WHsAg was L+V+ (71%), L-V+ (40%), L+V- (28%), and L-V- (0%). This suggested that the greater (i.e., more sustained) vCMI to WHsAg in the combination therapy group (L+V+) resulted from a possible additive effect of the monotherapies.

vCMI of chronic WHV carriers following WHsAg vaccination was broadened to include other viral antigens when preceded by L-FMAU therapy. There was a significant (P < 0.05 to P < 0.01) collateral effect of WHsAg vaccination on the vCMI to other WHV antigens that was most remarkable in the L+V+ carriers (Fig. 3, Table 2). This appeared to be accentuated in part as a result of the discontinuation of drug treatment. For example, the discontinuation of drug treatment in the L+V- carriers resulted coincidentally in endogenous vCMI in some of the treated woodchucks, mainly to WHsAg and to rWHcAg (Fig. 3, Table 2). The L-V+ carriers responded primarily to WHsAg, with only a small number developing sporadic vCMI to the other WHV antigens (one L-V+ carrier was consistently responsive to rWHeAg for unknown reasons). In these comparisons, the frequencies of PBMC samples positive for vCMI to rWHcAg, rWHeAg, and rWHxAg were each significantly higher in the L+V+ carriers than in all other carrier groups (P < 0.05 to P < 0.01, Table 2). The posttreatment frequencies of PBMC samples positive for vCMI to WHsAg and rWHcAg in the L+V- carriers were significantly higher than in the L-V- control carriers (P < 0.01, Table 2). vCMI to WHsAg was similar, however, between the L+V- and L-V+ carriers.

Previous epitope mapping of the PBMC proliferative responses in woodchucks with acute self-limited WHV infection identified several key epitopes of WHcAg (25, 27, 28) that were also recognized by some of the treated carriers (Fig. 4, Table 2). This included a broadly recognized epitope in core peptide C100-119 that was demonstrated to be an immunogen capable of protecting against experimental challenge with WHV (28). Following vaccination with WHsAg, vCMI to core peptide C100-119 became evident in 63% of L+V+ carriers and in 38% of L-V+ carriers (Table 2). Vaccination induced vCMI to core peptides C1-20 and C112-131 in more L+V+ carriers than L-V+ carriers (P < 0.05; Fig. 4, Table 2). Overall, the frequencies of PBMC samples positive for vCMI to core peptides C100-119, C1-20, and C112-131 were each significantly higher in L+V+ carriers than in L-V+ carriers (P < 0.01, Table 2). This difference correlated with the significant broadening of the vCMI to rWHcAg and rWHeAg in L+V+ carriers (Fig. 3). Core peptide C100-119 was also recognized by a few of the L+V- carriers during the post-drug treatment period (Fig. 4, Table 2). Here, the frequency of positive PBMC samples was higher than in L-V- control carriers (P < 0.01), but lower than in L+V+ carriers (P < 0.05) and not significantly different from that of L-V+ carriers.



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  		FIG. 4. Kinetics of vCMI to selected core peptides of chronic WHV carrier woodchucks from the four experimental groups during the vaccine phase of the study. The frequencies of responding woodchucks (percentage of woodchucks at each time point with SI values of >=3.1) to core peptides C1-20, C100-119, and C112-131 are shown as vertical bars. The group means of SI values are presented as circles. L+V+, L-FMAU/vaccine group; L-V+, placebo/vaccine group; L+V-, L-FMAU/injection control group; L-V-, placebo/injection control group. Vertical arrowheads represent the four immunizations using 50-µg doses of an alum-adsorbed WHsAg vaccine or saline at weeks 32, 36, 40, and 48. For mean cpm ± SD of the unstimulated PBMC, see the legend to Fig. 3.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The rational treatment of chronic HBV infection ultimately may combine the use of antiviral drugs and immunotherapy. The present study demonstrates that immunization of chronic WHV carrier woodchucks with WHsAg vaccine after treatment with L-FMAU breaks humoral and cell-mediated immune tolerance and also enhances the vCMI profile beyond the monotherapies alone. Accordingly, the results improve the understanding of humoral and cellular immune responses to therapeutic vaccination after marked reduction in the viral and antigen load induced by antiviral drug therapy. Results of the present study indicated remarkably different cell-mediated immune responses to viral antigens among WHV carriers vaccinated with WHsAg alone versus in combination with L-FMAU. This has further implications regarding the immunopathogenic mechanisms that initiate and maintain the carrier state.

Influence of antiviral drug treatment on the immune response to therapeutic vaccination. This study confirms the humoral and cellular immunologic tolerance of chronic WHV carrier woodchucks to endogenously produced WHsAg. This tolerance was disrupted by administration of multiple doses of an inactivated, serum-derived WHsAg-alum vaccine alone or after treatment with L-FMAU. The vCMI to WHsAg was enhanced, especially in duration, when the vaccinations were preceded by L-FMAU treatment, which markedly reduced viral and antigen load (Fig. 1) (34). The combination of L-FMAU and vaccine therapy was associated further with a collateral enhancement of vCMI to other viral antigens. Such effects appeared to result from an additive effect of drug and vaccine. The expected inhibition of viral replication and of WHsAg production by L-FMAU was abolished, in part, when drug treatment was discontinued, which may have provided an additional endogenous stimulant for the subsequent humoral and cellular immune responses to vaccine. The timing of such responses in the drug monotherapy group (L+V-) was consistent with an effect of discontinuation of drug treatment. However, the present study design does not exclude the possibility that such effects might have occurred if drug treatment had been continued.

Combination therapy with L-FMAU and vaccine did not enhance the measured anti-WHs responses beyond those of vaccine alone. This similarity in the anti-WHs response was not predictive of the marked differences in vCMI between these groups. However, additional tests of the anti-WHs antibodies might uncover differences in immune complex composition or in epitope specificity that correlate with the individual treatments. The relatively low anti-WHs levels detected in the vaccinated carriers may be explained, in part, by the presence of excess WHsAg in serum. Even when WHs antigenemia decreased to below the detectable limit, the levels could still be as high as 109 to 1010 antigen particles per ml of serum, based on the sensitivity of the assay.

The vCMI to WHsAg in L+V+ carriers was rapid and sustained. Carriers may be primed in part from the infection, and therefore responded sooner to vaccine than WHV-susceptible recipients (Menne et al., unpublished data). Carriers were continually exposed to WHsAg between vaccinations, and this might facilitate sustained responses once they are elicited. However, such responses were better sustained through four vaccine immunizations in the carriers with low viral and antigen load (L+V+) compared to the carriers with high viral and antigen load receiving the same vaccine regimen (L-V+). Whether this difference might also relate to group differences in the trafficking of responding cells is not known.

Collateral vCMI to rWHcAg were not very frequent in carriers that received vaccine alone (L-V+), and these carriers had no significant vCMI to the core peptides, including the protective core peptide C100-119 (28). This may partly explain the absence of a collateral effect of vaccine alone on viral and antigen load in such carriers (Fig. 1, L-V+). The enhanced collateral vCMI to core gene products (i.e., rWHcAg and rWHeAg) in carriers receiving the combination therapy (L+V+) appeared to be partly dependent on drug and the subsequent discontinuation of drug treatment, in that rWHcAg and core peptide C100-119 were recognized frequently posttreatment by some of the carriers that received drug monotherapy (L+V-). The mechanism(s) for induction of collateral vCMI by WHsAg vaccine is not known. In any case, the result of combination therapy (L+V+) was to significantly modulate the humoral and cellular immune response profiles of carriers toward that observed in self-limited WHV infection (7, 25, 27-29).

Immunologic tolerance in the onset and maintenance of chronic WHV infection. Reduction of serum and hepatic viral and antigen load during L-FMAU treatment (34) may facilitate emergence of the host immune system from a tolerant state, depending possibly on the duration of treatment. That L-FMAU treatment alone followed by withdrawal was associated with an interruption of tolerance provides evidence for the presence of endogenous, virus-specific immune responses in some carriers and for the negative influence of high viral and antigen load on these responses in the untreated carrier. In fact, carriers vaccinated under conditions of high viral and antigen load (L-V+) may have become refractory to later immunizations. That vCMI was enhanced and broadened by combination therapy (L+V+) suggests further that high viral and antigen loads contribute to cellular immune tolerance and maintenance of the chronic carrier state in woodchucks.

Regarding the onset of chronic WHV infection, approximately half of neonatally infected woodchucks that become chronic WHV carriers develop a mild, transient acute hepatitis (5, 33) and transient, acute-phase vCMI to WHV antigens (25). Such early-responding carriers may ultimately represent the 40 to 50% of established carriers that can generate endogenous vCMI and anti-WHs responses after the cessation of L-FMAU monotherapy (as in L+V- carriers after week 32). The remaining half of neonatally derived carriers develop little or no transient acute hepatitis (5, 33) and no vCMI (25). Such woodchucks may develop chronic WHV infection as a result of a more efficient tolerogenic mechanism that is maintained throughout infection. However, this study shows that therapeutic vaccination can recall vCMI to WHsAg in a substantial proportion of carriers, independent of their viral and antigen load, including all carriers that were first treated with L-FMAU.

There is reasonable evidence to assume that the cells responding in the in vitro PBMC proliferation assay are T cells (7, 14, 27, 28, 33). In a hypothetical model of central tolerance (30, 31), treatment with L-FMAU reduces the load of antigen that induces tolerance (i.e., WHcAg, WHeAg, and/or WHsAg), thus decreasing the negative selection of precursor T cells by thymic deletion. This enables a time-dependent and thymus-dependent regeneration of WHV antigen-specific T cells from bone marrow stem cells. In a hypothetical model of peripheral tolerance (30, 31), WHV antigen-specific T cells escape early negative selection in the thymus and later are rendered unresponsive in the periphery by anergy or exhaustion caused by increased antigen load. Such cells could become responsive to the antigen after the antigen load was reduced.

Regarding WHsAg, vCMI to WHsAg in this study was not unmasked upon initial reduction of viral and antigen load during L-FMAU treatment, suggesting that central tolerance may represent a predominant mechanism of tolerance in chronic WHV carrier woodchucks. However, the rapid development of vCMI to WHsAg in L-V+ carriers with high viral and antigen loads suggests that some WHsAg-specific T cells may escape negative selection in the thymus and become tolerant to endogenously produced WHsAg due to peripheral mechanisms. There are no established assays for serum WHeAg or WHcAg. However, both antigens are most likely reduced considerably in serum, since L-FMAU produces such a profound inhibition of viral replication in the liver. This results in decreased covalently closed circular WHV DNA, WHV mRNA transcription, and WHV protein expression, as evidenced by the decreased serum WHsAg in this study and decreased WHcAg staining in liver in another study (34). In fact, the endogenous vCMI to WHcAg and core peptide C100-119 in L+V- carriers in particular following drug withdrawal may represent a consequence of the prior reduction of WHeAg and/or WHcAg load by drug treatment. This could provide a further rational explanation for the improved collateral effect of WHsAg vaccination on vCMI to WHcAg in the carriers that received the combination therapy (L+V+).

Implications for immunotherapy of chronic HBV infection. Therapeutic vaccination of chronic HBV carriers with commercial HBsAg vaccine alone or before and after treatment with IFN-{alpha} elicited transient anti-HBs antibody responses, associated T-cell responses in some patients, and modest reductions in viremia in about 50% of patients. However, clearance of HBsAg from serum was not reported in any of the patients (9, 12, 20). Comparable reductions in viremia were not observed in the chronic WHV carrier woodchucks given vaccine alone (L-V+) in this study. T-cell responses to HBcAg are not often improved in chronic HBV patients during treatment with lamivudine alone or in combination with IFN-{alpha} (23, 35).

The present study in L-FMAU-treated chronic WHV carrier woodchucks (L+V+ and L+V-) is consistent to the extent that vCMI to WHV antigens was not observed (i.e., unmasked) during the drug treatment phase. In one study (2), however, chronic HBV patients developed strong and sustained T-cell responses to HBeAg, HBcAg, and several HBV core peptides shortly after the initiation of lamivudine treatment. Most of the lamivudine-treated patients who responded immunologically were believed to have adult-acquired chronic infections (2). Most of the patients with T-cell responses also had increased serum enzyme levels indicative of liver disease on entry, whereas nonresponders had little or no evidence of existing liver disease. One nonresponder patient may have been tolerant as a result of infection early in life (2). The chronic WHV carrier woodchucks in this study were infected neonatally, and they were selected for minimal liver disease on entry in the drug treatment phase. Thus, these carriers may not have had sufficient existing chronic liver injury to unmask vCMI immediately after L-FMAU treatment was initiated.

The results presented in the woodchuck model open the way for the development of new therapeutic strategies for the immunologic control of chronic HBV infection and its disease sequelae. In contrast to the immunologic tolerance of chronic infection, recovery from HBV infection involves a curative immunologic process that significantly reduces the lifetime risk of serious liver disease. Immunotherapies that elicit humoral and cellular response patterns that resemble those of recovery could provide an important adjunct to other forms of antiviral therapies. When combined with a potent antiviral drug that reduced both viral and surface antigen load, therapeutic vaccination in the carrier woodchuck led to an immune response profile that resembles that observed during recovery from acute, self-limited WHV infection. The experimental results described in this report demonstrate the feasibility and potential safety of using such an approach in humans with chronic HBV infection.


   ACKNOWLEDGMENTS
 
This work was supported by contracts NO1-AI-05399 and NO1-AI-35164 to the College of Veterinary Medicine, Cornell University, with the National Institute of Allergy and Infectious Diseases (NIAID) and contracts NO1-AI-95390 and NO1-AI-45179 to the Division of Molecular Virology and Immunology, Georgetown University School of Medicine, with the NIAID.

We gratefully acknowledge the expert assistance of Betty Baldwin, Lou Ann Graham, Richard Moore, and Chris Bellezza (Cornell University) and of Karen Gaye, Francis Wells, and Christine Ferrar (Georgetown University). We also gratefully acknowledge Triangle Pharmaceuticals, Inc., for providing the generous supply of L-FMAU (clevudine).


   FOOTNOTES
 
* Corresponding author. Mailing address: Gastrointestinal Unit, Department of Clinical Sciences, College of Veterinary Medicine, Room C-2005 VMC, Cornell University, Ithaca, NY 14853. Phone: (607) 253-3280. Fax: (607) 253-3289. E-mail: sm119{at}cornell.edu. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Virology, June 2002, p. 5305-5314, Vol. 76, No. 11
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.11.5305-5314.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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