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To investigate the levels of hepatitis B virus total DNA (HBV DNA) and covalently closed circular (ccc) DNA in liver transplant recipients who received hepatitis B vaccination, including responders and non-responders, following liver transplantation due to hepatitis B-related diseases and to investigate the efficacy of hepatitis B immune reconstitution against HBV reinfection. Twenty responders and 34 non-responders were enrolled in the present study. The levels of HBV total DNA and ccc DNA in peripheral blood mononuclear cells (PBMCs) and the liver and plasma were detected by real-time polymerase chain reaction (PCR). Fifty-three blood samples and 38 liver allograft tissues were acquired. For the responders, the mean serum titer for anti-HBs (antibodies against hepatitis B surface antigen) was 289 (46.64–1000) IU/ml. Also for the responders, HBV total DNA was detected in PBMCs for one recipient and in the liver for another recipient, but ccc DNA was not detected in either of those 2 recipients. For the non-responders, HBV total DNA was detected in PBMCS for 2 recipients, neither of whom had ccc DNA. Also for the non-responders, HBV total DNA was detected in the livers of 3 recipients, 2 of whom also had ccc DNA. All responders had discontinued hepatitis B immunoglobulin (HBIG), and 13 responders had discontinued antiviral agents. One responder experienced HBV recurrence during the follow-up period. For the majority of liver transplant recipients, no HBV total DNA or ccc DNA was detected in the blood or liver. The lack of HBV total DNA and ccc DNA both in PBMCs and the liver in liver transplant recipients who received hepatitis B vaccination to prevent HBV reinfection should be a prerequisite for the withdrawal of HBIG and/or antiviral agents.
Worldwide, there are approximately 350 million chronic HBV carriers and 1 million deaths from HBV-related liver disease, including cirrhosis, liver failure and hepatocellular carcinoma (HCC).1 Liver transplantation (LT) is the most effective therapeutic option for HBV-infected patients who have acute or chronic liver failure and/or primary liver cancer, and the outcomes of LT have been reported to be as good or even better in HBV-infected patients than in non-HBV patients.2,3 This success has been attributed to the prophylactic strategies against HBV reinfection after LT. The combination prophylaxis with antiviral agents and HBIG reduces the HBV recurrence rate to 5% at 5 years, which is now almost universally adopted by most transplant centers as the golden standard for the prevention of HBV reinfection.4 However, passive immune prophylaxis with long-term administration of HBIG is associated with many issues, including the limited availability of HBIG, possible emergence of HBV envelope protein mutations,5 development of resistance to nucleotide analogs6 and, especially, extremely high costs.7
The ideal prophylactic strategy is to stop prophylaxis with HBIG when HBV infection had been completely cleared. However, it is very difficult to completely eradicate intrahepatic and extrahepatic HBV DNA and ccc DNA in liver transplant recipients, which are potential sources of reinfection.8-11 Active immunization with standard hepatitis B vaccines in these patients is considered a promising substitute to the current treatment strategy, although the results of this new strategy have been controversial.12-22 Some studies have reported that responders to hepatitis B vaccination, defined as patients with anti-HBs, should stop taking HBIG and/or antiviral agents.12,16,17,20–22 However, the safety of discontinuing HBIG and/or antiviral agents in responders remains unclear. In the present study, we investigated intrahepatic and extrahepatic HBV total DNA and ccc DNA in responders to active hepatitis B immune reconstitution who had discontinued HBIG and/or antiviral agents and in non-responders to active hepatitis B immune reconstitution.
Twenty responders and 34 non-responders to active hepatitis B immune reconstitution were enrolled in the study. For the responders and non-responders, the mean age was 51.5 ± 10.24 y and 54.2 ± 8 .26 y (p = 0.381) and the ratio of males to females was 15:5 and 27:7 (p = 0.979), respectively. The mean duration of follow-up after LT for the responders was 6.09 ± 0.49 y and that for the non-responders was 4.66 ± 0.32 years, and this difference was statistically significant (p = 0.014). The samples collected included 53 blood samples (20 from responders and 33 from non-responders) and 38 liver biopsies (18 from responders and 20 from non-responders). For the responders, the median anti-HBs titer was 289 (46.64–1000) IU/ml at enrollment. For the immunohistochemical test, in 16 responders and 19 non-responders, neither intrahepatic hepatitis B surface antigen (HBsAg) nor hepatitis B core antigen (HBcAg) were detected; one non-responder was positive for HBcAg (Table 1).
Total HBV DNA was not detected in the sera of any responders, except one (#19), and in that responder the level was below the lower limit of detection (<2.00E + 1 IU/L), which might indicate the existence of HBV DNA. Only one recipient (#10) was found to have total HBV DNA in PBMCs, and the titer was 2.36E-2 copies/cell, but no HBV ccc DNA was detected in that recipient. Similarly, intrahepatic total DNA was only found in one responder (#16) at 10.29E-2 copies/cell, and no HBV ccc DNA was detected in that responder. In conclusion, 2 out of 20 responders (10%) were found to have HBV total DNA, but no responders were found to have HBV ccc DNA (Table 2).
The titers of serum HBV DNA were less than 2.00E + 1 IU/l in 4 (12.1%) non-responders (#10, #20, #25, and #27). In PBMCs, total HBV DNA was detected in 2 (6.3%) recipients (#4 and #32) (the titers were 1.26E-2 copies/cell and 2.36E-2 copies/cell, respectively). However, HBV ccc DNA was not detected in the sera or PBMCs of any non-responders. Moreover, intrahepatic total HBV DNA was measured in 3 (15%) recipients (#1, #29, and #3), 2 of whom (#1, #29) were found to have ccc DNA. The levels of total HBV DNA in liver allografts were 14.74E-2 copies/cell, 14.48E-2 copies/cell and 6.83E-2 copies/cell for recipients #1, #29, and #3, respectively and levels of ccc DNA in liver allografts were 7.67E-2 copies/cell and 1.55E-2 copies/cell for recipients #1 and #29, respectively. In brief, HBV total DNA was detected in 9 non-responders (26%), 2 of whom also had HBV ccc DNA (Table 3).
The sera of all recipients were negative for HBV DNA, and the liver biopsy only one recipient was positive for HBcAg. HBV DNA was detected in the PBMCs or liver biopsies of 11 recipients (20.7%), and HBV ccc DNA was also detected in liver biopsies of 2 of those recipients (3.8%). No significant differences were found between the responders and non-responders (p = 0.529).
The follow-up period ended on 31th Dec, 2012. The mean durations of follow-up for LT responders and non-responders were 79.75 ± 25.22 months and 65.38 ± 23.84 months, respectively, and this difference was statistically significant (p = 0.041). All responders discontinued HBIG after a mean duration of treatment of 27.7 ± 11.2 months; thirteen (65%) recipients discontinued nucleotide analogs after a mean duration of treatment of 25.7 ± 11.0 months. Recurrent and/or persistent HBV infection after liver transplant was defined as the reappearance of HBsAg after the initial seroclearance or the persistence of HBsAg in the serum after liver transplant, irrespective of the HBV DNA level. During the follow-up, no HBV recurrence occurred in any recipients, except for one responder for whom HBsAg reappeared and for whom HBV DNA was detected in PBMCs. Moreover, the patient with HBV recurrence had the HBV mutation G145R. Other recipients with detectable HBV DNA did not experience HBV reinfection.
Life-long prophylaxis against HBV recurrence is recommended in patients that undergo liver transplant for HBV-related disease. In this study, total HBV DNA, but not HBV ccc DNA, was detected in post-LT liver biopsies of most patients, even though neither HBV DNA nor HBsAg were detected in the sera of those patients.8 Although there were only a few cases of HBV recurrence after 5 years, low-level HBV DNA was detected in 45.4% of patients who did not experience HBV recurrence during 10 y of follow-up.11 HBV total and ccc DNA were detectable in the sera, livers, and PBMCs of almost all patients for up to 15 y post-LT.10 Moreover, Lenci et al. found that most patients with undetectable HBV DNA at transplant who received conventional HBV prophylaxis had no evidence of intrahepatic HBV total or ccc DNA; in that study, only one patient experienced HBV recurrence after transplant and tested positive for intrahepatic HBV total and ccc DNA.9 All patients with HBV reactivation were found to have HBV total and ccc DNA in liver allografts and/or PBMCs.8-10 Recipients with detectable HBV ccc DNA might be considered at high risk of HBV recurrence after liver transplantation. In our study, over one fifth of recipients were found to have HBV DNA in the liver or PBMCs even though they received antiviral agents and HBIG for many years after LT, and this finding was consistent with that of previous studies. Moreover, even for the responders with active hepatitis B immune reconstitution who were able to produce anti-HB antibody, HBV DNA was also found in PBMCs and the liver.
Identifying liver transplant recipients who can discontinue prophylaxis or, conversely, recipients who are at risk of prophylaxis failure is important for long-term graft survival. However, the persistence of HBV DNA in PBMCs and the liver is responsible for HBV recurrence after LT, which suggests that the HBV DNA levels in these tissues should be monitored to determine when HBIG and/or antiviral therapy can be discontinued. Theoretically speaking, recipients who can produce anti-HB antibody after hepatitis B vaccination will not suffer from HBV reactivation after the withdrawal of HBIG and/or antiviral agents. Some studies have demonstrated that HBIg discontinuation with maintenance oral anti-viral monotherapy was safe and effective for HBV liver transplant recipients.21,22 In the present study, 11 recipients retained detectable HBV total and ccc DNA levels even though the standard prophylactic strategy after LT was followed, and those recipients should be considered at high risk of HBV reinfection. For those recipients, it might be necessary to administer long-term therapy of HBIG in combination with antiviral agents. Interestingly, the only recipient with HBV recurrence was one who responded to the HBV vaccine and had stopped taking HBIG for 3four months and antiviral agents for over 2three months; also, no HBV mutation was found in that recipient. The reason for recurrence in that recipient was likely the presence of a detectable level of HBV DNA in PBMCs and the withdrawal of antiviral agents. Clearly, the withdrawal of HBIG is relatively safe for responders with undetectable HBV total and ccc DNA levels. In regards to the timing of cessation of antiviral agents for responders, our study suggested that this decision should be based on the HBV total and ccc DNA levels in the liver and PBMCs and that, at a minimum, responders should be able to produce anti-HB antibody, indicating active immunity against HBV, prior to withdrawal of antiviral agents. Therefore, it is vital to detect intrahepatic and extrahepatic HBV in all recipients, whether they are responders or non-responders, before withdrawal of HBIG and/or antiviral agents in order to avoid HBV recurrence.
Our study had some drawbacks, especially in regards to the method of HBV detection, as no worldwide standard detection method exists. Also, the sample size was not sufficient to allow for valid conclusions to be made based on the results of this study. Therefore, a more sensitive and specific method as a standard for the detection of HBV total and ccc DNA is urgently needed, and further studies must be performed to support our results.
In summary, it is fairly difficult to eliminate HBV total DNA and ccc DNA from liver transplant recipients due to hepatitis B-related diseases that can be maintained for many years after LT. However, it the cessation of HBIG and/or antiviral agents for responders with active hepatitis B immune reconstitution should be relatively safe. It is vital to determine intrahepatic and extrahepatic HBV levels, including the levels of total and ccc DNA before the withdrawing HBIG and/or antiviral agents, rather than relying solely on personal experience.
Two hundred liver transplant recipients received hepatitis B vaccination following liver transplantation due to hepatitis B associated liver diseases from 1999 to 2010. Recipients receiving HBV vaccination should meet the following criteria: (1) at least 18 months out from transplantation; (2) receiving a combined prevention regimen, including nucleoside analogs and HBIG; and (3) liver function normal or near normal. Fifty recipients were defined as responders as follows: an increase in serum anti-HBs titer of more than 100% above the baseline value during any vaccination course lasting more than 3 months or an elevated serum anti-HBs titer that remained high, although it was less than 100% above the baseline level, with regular intramuscular injection of a recombinant vaccine (Engerix-B 20 µg, GSK) or a bivalent vaccine (Twinrix 20 µg, GSK).23 According to the definition of responders, 20 responders and 34 non-responders were voluntarily enrolled to the present study. Prior to the study, the protocol was approved by the Institutional Review Board of Beijing You-An Hospital, Capital Medical University according to the guidelines of the 1975 Declaration of Helsinki. Written informed consent was obtained from all participants.
Ten-milliliter peripheral blood samples were collected for the isolation of plasma and PBMCs. PBMCs were isolated by density gradient centrifugation and diluted to 1 × 106/ml. Percutaneous liver biopsies were performed using a Menghini-derived aspiration needle (Surecut, 16G, TSK Laboratories, Japan). The liver biopsies were not specifically performed for this study but, rather, were performed as part of the routine liver transplant follow-up protocol. Liver specimens were immediately divided into 2 portions; one portion was immediately snap-frozen in liquid nitrogen and rapidly transferred to the laboratory; the other portion was formalin-embedded for histopathologic examination, including hematoxylin eosin (HE) and immunohistochemical staining, by the Pathology Department of our hospital. All samples were stored at −80°C until processing for HBV DNA extraction.
The serum HBV markers included HBsAg, hepatitis B surface antibody (HBsAb), hepatitis B e antigen (HBeAg), hepatitis B e antibody (HBeAb) and hepatitis B core antibody (HBcAb), and they were examined using commercially available assays. Intrahepatic HBV markers, including HBsAg and HBcAg, were examined by immunohistochemical staining. The immunohistochemical staining procedures for HBsAg and HBcAg followed the standard avidin-biotin peroxidase method using goat polyclonal anti-HBs and rabbit polyclonal anti-HBc, respectively.
Serum HBV DNA levels were quantified by real-time PCR using commercial reagents in an automatic nucleic acid detection instrument (Roche, USA). The lower limit of detection was 20 IU/L.
HBV total and ccc DNA in the liver and PBMCs were measured using the LightCycler® 480 system (Roche, USA) as described by Wang et al.24 Total DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions with minor modifications. DNA from liver biopsies was extracted using a QIAamp DNA Micro Kit (Qiagen, Hilden, Germany). The concentration of total DNA was determined at 260 nm with a spectrophotometer (American GE Company GeneQuant pro). Total DNA was digested by Plasmid-safe ATP-dependent DNase (Epicentre Technologies, Madison, WI) for the detection of HBV ccc DNA. The remaining fraction of extracted total DNA was used for the detection of HBV total DNA and β-globin. Each sample was assayed 3 times to determine the mean cycle threshold (Ct) values for HBV total DNA, HBV ccc DNA and β-globin detection. The amount of HBV ccc DNA and HBV total DNA was expressed as copies/cell and were both estimated to be 6.667 pg of hgDNA/cell. Liver tissues from patients that were not infected with HBV were used as negative controls, and the recombinant plasmid pBB4.5HBV1.2 was used as a positive control, as described earlier in this article.
The statistical analysis was performed using the Statistical Program for Social Sciences (SPSS 18.0 for Windows, SPSS, Chicago, IL). Continuous variables were expressed as medians (range) unless otherwise indicated and were compared by the Mann-Whitney U-test. Categorical variables were compared by chi-squared test. Statistical significance was defined as a P-value <0.05.
No potential conflicts of interest were disclosed.
The paper was supported by grants from the Capital Health Development Special Funds (No.2011-2018-03) and Ministry of Science and Technology Support Plan (2012BAI06B01).