HBV DNA Integration and Clonal Hepatocyte Expansion in Chronic Hepatitis B Patients Considered Immune Tolerant

Abstract

Background & aims: Chronic infection with hepatitis B virus (HBV) progresses through different phases. The first, called the immune-tolerant phase, has been associated with a lack of disease activity. We examined HBV-DNA integration, clonal hepatocyte expansion, HBV antigen expression, and HBV-specific immune responses in patients in the immune-tolerant phase to assess whether this designation is appropriate or if there is evidence of disease activity.

Methods: We studied HBV-DNA integration, clonal hepatocyte expansion, and expression of hepatitis B surface antigen and core antigen in liver tissues from 26 patients with chronic HBV infection (ages, 14-39 y); 9 patients were positive for hepatitis B e antigen (HBeAg) in the immune-tolerant phase and were matched for age with 10 HBeAg-positive patients with active disease and 7 HBeAg-negative patients with active disease. Peripheral blood samples were collected and HBV-specific T cells were quantified for each group.

Results: Detection of HBV antigens differed among groups. However, unexpectedly high numbers of HBV-DNA integrations, randomly distributed among chromosomes, were detected in all groups. Clonal hepatocyte expansion in patients considered immune tolerant also was greater than expected, potentially in response to hepatocyte turnover mediated by HBV-specific T cells, which were detected in peripheral blood cells from patients in all phases of infection.

Conclusions: We measured HBV-specific T cells, HBV-DNA integration, and clonal hepatocyte expansion in different disease phases of young patients with chronic hepatitis B, with emphasis on the so-called immune-tolerant phase. A high level of HBV-DNA integration and clonal hepatocyte expansion in patients considered immune tolerant indicated that hepatocarcinogenesis could be underway-even in patients with early stage chronic HBV infection. Our findings do not support the concepts that this phase is devoid of markers of disease progression or that an immune response has not been initiated. We propose that this early phase be called a high-replication, low-inflammation stage. The timing of therapeutic interventions to minimize further genetic damage to the hepatocyte population should be reconsidered.

Keywords: Antiviral Immunity; HBV Replication; HBsAg; Hepatocyte Proliferation.

Figure 1:. Inverse PCR detection of integrated HBV DNA.

A) Strategy for detection of integrated HBV DNA and clonal hepatocyte expansion. Inverse PCR, as used by Summers et al.,^, was designed to detect the right hand junction of integrations of HBV dslDNA, the predominant precursor for integration, into host DNA.^, Following cleavage and ligation (Figure 1A), the DNA samples were serially diluted and subjected to nested PCR using the indicated forward and reverse primers (Figure 1B). Primers are indicated in Supplementary Table 1 and Materials and Methods. (Figure 1A modified from reference by Mason et al.). B) Gel electrophoresis of inverse PCR products. Samples from nested PCR, carried out in a 96 well tray, were subjected to gel electrophoresis in a 1.3% agarose gel. PhiX phage DNA digested with HaeIII was used as a size marker (M). The fraction of the initial DNA sample distributed across each row of 12 wells is indicated. Bands were picked from the last 5 rows, not including the negative control, and subjected to DNA sequencing to identify virus/cell DNA junctions. For instance, the circled bands arise from a single hepatocyte clone; other clones were also identified by DNA sequencing (not highlighted).

Figure 2:. Profile of HBV-specific T cell responses in all patient groups.

Patient PBMC were analyzed by ELISPOT and intracellular cytokine staining (ICS) for IFN-γ. (A) Evidence of HBV-specific T cell responses by ELISPOT and ICS against the Core, Envelope and Polymerase proteins, for each patient in the groups studied; Shaded black – positive HBV-specific T cell response; unshaded squares – negative HBV-specific T cell response, shaded grey – sample not done. (B) Comparison of spot forming units (SFU) by ELISPOT, in each patient, in the different groups; immune tolerant (IT) (shaded black), HBeAg(+) IA (shaded grey) and HBeAg(−) IA (unshaded). Bars represent the number of SFU cells in response to HBV core, envelope, and polymerase peptide pools. (C) Number of HBV peptide pools recognized by HBV-specific T cells obtained in the indicated patients.

Figure 3:. Differential nuclear core antigen staining but similar fibrosis and inflammatory indices between CHB phases.

Formalin-fixed and paraffin-embedded tissue was analyzed with immunohistochemistry for HBcAg and HBsAg positive hepatocytes, along with quantification of fibrosis and histological activity indices for each patient. (A) Percentage of HBcAg positive hepatocytes (left panel) and HBsAg positive hepatocytes (right panel) in each group; IT (open circles), HBeAg(+) IA (open squares) and HBeAg(−) IA (open triangles). Each point represents 1 patient, data shown as mean with SEM, as error bars. (B) Immunostaining identifying HBcAg positive hepatocytes (brown) and HBsAg positive hepatocytes (pink) from representative patients from each patient group (Table 1) (100x); IT (left panel), HBeAg(+) IA (middle panel) and HBeAg(−) IA (right panel). Inset shows magnified image (400x). (C) Ishak Fibrosis stage (left panel) and collagen proportionate area (right panel) of patients studied in each phase of CHB, data shown as mean with SEM, as error bars. (D) Sirius red staining of liver tissue from representative patients in each phase; IT (left panel), HBeAg(+) IA (middle panel) and HBeAg(−) IA (right panel). (E) Histological activity index scores; (from left to right – Interface hepatitis, Confluent necrosis score, Focal lytic necrosis, apoptosis & focal inflammation score and Portal inflammation score) of patients studied in each phase of CHB, data shown as mean with SEM, as error bars, (F) Identification of the inflammatory infiltrate as shown in (E) from representative patients in each phase of CHB; IT (left panel), HBeAg(+) IA (middle panel) and HBeAg(−) IA (right panel). Significant changes marked with asterisks, *P<.05; **P<.01; ***P<.001; ns=not significant

Figure 4:. Sites of HBV DNA integration on human chromosomes.

A) Integration sites are summarized from all three patient groups (Table 1) by vertical lines. Results include the 208 from IT disease patients (Group 1), 195 from HBeAg(+) IA disease (Group 2), and 97 from HBeAg(−) IA disease (Group 3). Groups 1 (IT) and 2 [HBeAg(+)]; integrations were found on all chromosomes except Y. The single Y chromosome integration was from a patient from group 3. No group 3 patient integration sites were mapped to chromosomes 15 and 16. B) Integration sites in Group 1 patients - IT phase. Integration site details are shown in Supplementary Table 3. Clone sizes: *>5,000 and #>20,000.

Figure 5:. Hepatocyte clones detected in all patient groups

Hepatocyte clones in (A) IT disease (Group 1), (B) HBeAg(+) IA disease (Group 2) and (C) HBeAg(−) IA disease (Group 3). Clone sizes were estimated as described. (Figure 1, Materials and Methods and Supplementary Materials & Methods). The point estimates for clone size were calculated using the program Sim19 (Supplementary Materials & Methods). Clones are grouped by increasing size for each patient, and patients within a group are arranged by increasing age from left to right. D) Mean of the maximum clone size for each patient within a group. Geometric means were calculated using the point estimates in Supplementary Table 4. HCC data are from a published analysis of clone sizes in non-tumorous liver from a group of 5 non-cirrhotic HCC patients. (E) Predicted maximum clone sizes vs. age. These were calculated using the Csize8 program (Materials and Methods and Supplementary Materials & Methods), for 3 different daily rate constants for hepatocyte turnover; k=0.0015/day (0.15%) - (black dashed line); k=0.004/day (0.40%) – (grey dashed line) and k=0.01/day (1.00%) - (solid black line). The adjacent corresponding bars indicate the geometric mean hepatocyte clone size, for each patient group in (D), for comparison against the predicted maximum clone size.