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Discussion
In this prospective study, we found that an increase in viral load invariably occurred in the course of hepatitis B exacerbation, which peaked before serum ALT level in 93% and coincided with serum ALT peak in 7% of the patients. By investigating the sequential full-length viral nucleotide sequences, we found that the viral genome at the HBV surge stage, whether developing before or coinciding with the onset of AE, remained almost identical to that at baseline (mean nucleotide changes, 0.2 per genome). During AE, the proportion of patients harboring an HBV strain different from that at baseline increased to 36%. After AE, about 50% of the viral genomes remained identical and 50% changed, predominantly possessing multiple point variations or subgenome deletion. In contrast, the serum dominant viral genome remained unchanged during 1-year follow-up in 2 HBeAg carriers. By in vitro transfection assay, we found that the replication potential of the emerged HBV strain was not enhanced compared with that of the corresponding viral strain at baseline.
The development of AE in chronic hepatitis B is suggested to be related to the break of balance between viral replication and host immune responses.4 Nevertheless, whether this break was due to the emergence of variants escaping host immune surveillance or due to the inadequate host control over viral replication remained unclarified.4, 19 Our study clearly showed that changes of viral nucleotide sequences accompanying an upsurge of serum viral load rarely occurred before the onset of AE in most patients. This finding suggested that such an increase in viral load was not due to new variants but due to reactivation of a preexisting HBV strain in most clinical situations. This implied that the break of tolerance between virus and host leading to the onset of exacerbation was more closely related to the change of host immune surveillance rather than the change of viral genome. Consistent with this speculation, about 50% of the viral genome remained unchanged during the entire course of AE. It implied that HBV replication and population in the liver, as a result of fluctuating host immune control over the same virus, might become the main cause of AE.
However, AE resulted in selection of viral variant in half of our patients. Although the mechanisms of HBV variant emergence remain controversial, certain speculations can be inferred from the chronological studies in chimpanzees and humans acutely infected with HBV.21, 30, 31, 36 In chimpanzees, HBV DNA upsurged and then largely disappeared from the liver and blood in the incubation phase, before the peak of T-cell infiltration and maximal liver damage. Noncytopathic mechanisms, possibly via activation of cytokines such as tumor necrosis factor α and interferon gamma derived from an innate immune system, contributed to a rapid viral clearance in this phase.30 This noncytolytic viral clearance phase usually preceded the clinical hepatitis and went unnoted in both humans and chimpanzees.30, 31 Afterward, when viral load decreased substantially, adaptive HBV-specific immune responses (such as T cell) took over, cytolytic processes ensued, and clinical hepatitis became apparent.30, 31 In keeping with these observations, in our patients with AE of chronic hepatitis B, a significant reduction in virus level likewise occurred before maximal liver injury. Noncytolytic antiviral mechanisms thus likely contributed much to viral clearance before the hepatitis phase. After the decrease in viral load, lysis of infected hepatocytes by virus-specific adaptive immunity occurred. Because HBV-related hepatitis activity is HLA class I restricted and T-cell mediated,3 immune escape variant (if any) most likely appeared at or after the clinical hepatitis stage, as noted in previous studies.7, 8, 9 In this study, we consistently found that most nucleotide substitutions or subgenomic deletions occurred at or after the ALT peak. Furthermore, about half of the substituted nucleotides in the postexacerbation viral genomes were located within core or surface gene, in which resides several important immunogenic epitopes. Our findings support the speculation that HBV genomic differences frequently represent variations escaping from the adaptive cytolytic host immunity in the clinical hepatitis phase.23 To clarify whether the postexacerbation viral variants could escape cytotoxic T-cell recognition, functional assays targeting HLA class I–restricted T-cell epitopes are needed. Nevertheless, only a few HLA class I–restricted T-cell epitopes for HBV have been identified until now, and most are HLA-A2 restricted.3 The HLA profiles of the 7 patients harboring altered viral genome after exacerbation were all not HLA-A2 (data not shown). We thus did not perform such in vitro immunologic assays in current studies. Future prospective immunologic studies in suitable subjects are required to address these important questions.
Although the occurrence of hepatitis B exacerbation was closely preceded by an increase in serum viral load, how these 2 events were linked together remained unknown. The dynamics of viral resurgence were determined by multiple factors, including replication efficiency of individual viral strains, host immune responses, and the availability of replication space.37 An imbalance in the host immune response might bring about the enhanced viral replication if replication space within liver was available, which in turn triggered additional immune response and caused a flare of hepatitis. Nevertheless, we did not investigate the complex interplay between host immune responses and changes in viral load because the enrolled subjects were not immunologically homogeneous. On the other hand, serial liver biopsies for immunohistologic studies were not feasible in humans. The chronological relationships between changes in viral load, changes in HBV-specific and -nonspecific T-cell responses, and ALT activity should be clarified in suitable models such as woodchuck hepatitis. From the clinical point of view, the onset of hepatitis B exacerbation was almost preceded by an upsurge of viral load. If this surge is essential to trigger AE, then we speculate that if the viral load can be controlled, there would be few and even no flares of hepatitis. Previous studies comparing interferon monotherapy with the combination of lamivudine and interferon to treat chronic hepatitis B showed that the frequency of hepatitis flares indeed decreased if lamivudine was added.38 Chemotherapy-induced exacerbation of hepatitis B is another clinical condition worthy of attention in that the risk of developing liver failure increases.39 Serum viral load likewise resurges during the course of exacerbation. Whether prophylactic treatment with lamivudine can prevent the resurge of viral load and subsequently decrease the risk of hepatitis flares is now being studied in our hospital.
The true pathogenic HBV strains involved in the development of hepatitis B exacerbation were within the liver. We now evaluated the changes of the dominant viral genomes obtained from the sera in this study. As reported previously, some potential pathogenic strains might replicate actively within the liver but not be released into the circulation.40, 41 Therefore, the HBV genomic variations correlated to the development of hepatitis B exacerbation might be retained within the liver and not be detected in the specimens obtained from the circulation. Future comparisons between circulating HBV genomes and intrahepatic genomes could be helpful to answer this question.
Finally, from a viral evolution point of view, we need to consider the origin of the HBV strains at baseline. Some viral strains may exist and remain inactive in the host for a long time before reactivation, as is the case in chemotherapy-induced AE. The other baseline viral strains, especially those from repeated spontaneous AE, may just represent survivors from previous episodes of AE. These HBV survivors may either be new viral variants selected from host immune surveillance in previous exacerbations or may remain the same strain as before preceding AE and again trigger the current episode of exacerbation. In this study, we show that the replication potential of the viral variants that emerged after AE was not enhanced. Our findings suggest that these viral variants might result from immune selection. In the long run, we need to monitor the viral evolution from the individuals experiencing multiple episodes of AE to truly understand the impact of viral variations on AE.
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发表于 2016-1-4 19:59
