hepatitis b virus- ( hbv- ) related end stage liver diseases ( esld ) account for over 80% of orthotopic liver transplantations ( olts ) in china , and active hbv replicative status prior to olt is present in over 50% of patients .
although hbv graft reinfection and hepatitis b ( hb ) recurrence have been controlled to an acceptable level after the adoption of antiviral drugs such as the nucleoside analogue lamivudine ( lam ) combined with hepatitis b immunoglobulin ( hbig ) [ 13 ] , drug resistance requiring the lifelong use of antiviral agents and virus escape under long - term use of hbig may increase the risk of graft reinfection and hb recurrence [ 36 ] .
additionally , lifelong use of these drugs is associated with significant financial burden and inconvenience .
induction of active immunity against hbv has become a potential alternative in posttransplant patients who have undergone olt for hbv - related liver diseases .
to date , a few pilot studies have confirmed the feasibility of this method [ 711 ] .
however , its effectiveness and response rate require further improvement [ 1215 ] . based on basic and clinical research of the induction of active immunity against hbv after olt during the past decade
, we have conducted this prospective clinical study regarding active immunization against hepatitis b virus graft reinfection in our center .
this study was a prospective clinical study and was approved by the ethics committee of beijing you - an hospital ( on january 4 , 2006 ) and was performed according to the ethical guidelines of the 1975 declaration of helsinki .
study subjects were posttransplant patients who underwent olt for hbv - related esld from 1999 to 2010 .
all organ donations or transplants were approved by the institutional review board of beijing you - an hospital , capital medical university , under the guidelines of the ethics committee of the hospital , the current regulations of the chinese government , and the declaration of helsinki .
specific inclusion criteria were as follows : ( 1 ) at least 18 months following transplantation ; ( 2 ) receiving a combined prevention regimen using nucleoside analogues and hbig ; and ( 3 ) liver function being normal or near normal .
the study was clearly explained to all participants , and they all provided written signed informed consent .
the two vaccines , twinrix and engerix - b , are officially approved for clinical use and are commercially available worldwide and in china .
the participant recruitment period was from january 1 , 2005 , to january 1 , 2012 .
follow - up period ranges from june 10 , 2006 ( from the first successful vaccination ) , to december 31 , 2013 .
the vaccines used were a recombinant hepatitis b surface antigen ( hbsag ) vaccine containing hbsag 20  g per vial ( engerix - b ; gsk ) and a bivalent vaccine which contained inactivated hepatitis a virus antigen and recombinant hbsag 20  g per vial ( twinrix , gsk ) .
one round of inoculation consisted of intramuscular injections of the vaccine ( 20  g for each inoculation ) in the region of the triceps muscle at 0 , 1 , 2 , and 6 months .
all participants received at least one round of inoculation and were given multiple rounds of inoculation according to response status and anti - hbs level .
serum hbv markers including anti - hbs were detected quantitatively with an electrochemiluminescence immunoassay ( roche diagnostics gmbh , mannheim , germany ) using a cobas e 601 ( roche diagnostics gmbh , mannheim , germany ) immunoassay analyzer .
hbv dna was detected with a real - time quantitative pcr diagnostic kit for quantification of hepatitis b virus dna ( shanghai kehua bio - engineering co. , ltd . ,
china ) using an abi 7500  pcr instrument ( applied biosystems , usa ) .
the detection limit was 1010  copies / ml . the combined regimen using nucleoside analogues and hbig was maintained during the inoculation period .
participants received intramuscular injections of hbig ( 400  iu / injection ) regularly in order to maintain a stable baseline level of hbig .
each vaccine inoculation was carried out 14 days after scheduled hbig , and serum anti - hbs concentrations were measured on the day prior to the inoculation .
the patient was defined as a responder if the sera anti - hbs titer increased more than 100% above the baseline value during any vaccination course lasting more than 3 months or the elevated serum hbsab titer remained high although it is less than 100% of baseline level .
as soon as a patient was defined as a responder , exogenous hbig administration was discontinued .
another 3-month observation after exogenous hbig was withdrawn , nucleoside analogues were then withdrawn subsequently if the patients were willing to stop nucleoside analogues . to maintain sustainable and spontaneous high anti - hbs production after discontinuation of hbig ,
a booster vaccination was administered ( usually every 3 to 6 months ) in the early phase for successful responders .
regular surveillance of sera hbv dna and hbv antigens was performed every 3 months . if it was found that the hbsag turned to be positive during the follow - up period , nucleoside analogues were reused and/or intravenous injection of hbig ( 2000  iu ) was performed .
intergroup variations were analyzed with t - test or analysis of variance ( anova ) , and the percentage variation was analyzed with  test .
until december 31 , 2013 , two hundred olt recipients who met the inclusion criteria were enrolled in this study , and all received at least one round of inoculation .
fifty patients , including 41 males ( 82% ) and 9 females ( 18% ) , achieved successful reestablishment of active immunity against hbv and were classified as responders . among the 50 recipients , with a mean postoperative time of 35.8  19.12 months ( median : 27 months ;
range : 12 to 85 months ) , 19 cases were hbv active replicative ( sera hbv dna  10  copies / ml ) and 31 cases were hbv nonactive replicative ( sera hbv dna < 10  copies / ml ) before olt . among the 50 successful cases , 20 received the engerix - b vaccine and 30 received the twinrix vaccine .
the baseline anti - hbs titer of the 50 patients was 87.71  38.82  iu / l ( median : 83.61 
the mean anti - hbs titer was 264.91  197.66  iu / l ( median : 198.64 
the average number of doses administered at the time of establishment of immunity was 5.06  2.39 ( median : 5 ; range : 1 to 11 ) , with 22 cases requiring one round of inoculations , 22 requiring two rounds , and six requiring three rounds .
the highest anti - hbs titer in the follow - up period was 488.07  322.52 
iu / l , which was higher than that at the time of successful response ( 264.91  197.66 
the lowest anti - hbs titer in the follow - up period was 111.82  74.53 
iu / l , which was higher than the baseline anti - hbs titer ( 87.71  38.82 
the mean number of booster vaccinations administered was 1.61  0.79 ( median : 1 ; range : 1 to 4 ) , and the anti - hbs titer increased to 438.09  296.96 
iu / l in these patients , which was similar to the highest sera anti - hbs titer ( 488.07  322.52 
iu / l , t = 0.751 , and p = 0.455 ) in the follow - up period .
the interval of time between successful establishment of immunity and withdrawal of hbig was 3.53  4.00 months ( median : 2 months ; range : 1 to 22 months ) , and the interval of time between withdrawal of hbig and withdrawal of both hbig and antiviral agent was 5.57  3.93 months ( median : 3 months ; range : 3 to 17 months ) .
when hbig was withdrawn , the mean anti - hbs titer was 257.72  160.22  iu / l ( median : 194.50 
iu / l ) , which was higher than the mean baseline anti - hbs titer of 87.71  38.82  iu / l ( median : 83.61 
iu / l ) ( t = 7.273 , p = 0.000 ) but lower than the highest mean anti - hbs titer of mean 488.07  322.52  iu / l ( median : 388.15 
there was no hbv graft reinfection or hb recurrence in the 24 cases who discontinued hbig during the follow - up period of 26.13  7.05 months ( median : 24.5 months ; range : 19 to 52 months ) , and 21 cases discontinued both hbig and nucleoside analogues during the follow - up period of 39.86  15.47 months ( median : 34 months ; range : 20 to 87 months ) .
the mean anti - hbs titer of the 45 cases at the end of the followup was 341.36  262.56  iu / l ( median : 286.55 
iu / l ) , which was higher than that when hbig was withdrawn ( t = 1.829 , p = 0.071 ) , although the difference was not significant . there were 20 cases in engerix - b group and 30 cases in twinrix group in which active immunity against hbv was established .
there was no difference between the two groups in baseline titers , titer at success of immunization , the highest titer , the lowest titer before booster vaccination , the highest titer after booster vaccinations , titer when hbig was withdrawn , and the titer at the end of followup ( figure 2 ) .
the number of inoculation cycles required for success ( 1.75  0.64 versus 1.63  0.72 , t = 0.587 , and p = 0.560 ) , dosage number ( 4.95  2.14 versus 5.13  2.57 , t = 0.264 , and p = 0.793 ) , and number of booster vaccinations ( 1.86  1.03 versus 1.46  0.59 , t = 1.528 , and p = 0.135 ) were similar between the two groups .
the number of cases requiring booster vaccinations ( 15 cases in the engerix - b group and 24 cases in the twinrix group ) was also similar (  = 0.175 , p = 0.467 ) .
however , the drug withdrawal rate of the twinrix group was greater than that of engerix - b group (  = 13.923 , t = 0.001 ; table 3 ) . in the 50 patients that had a successful response ,
there were no differences in those who had liver failure , hepatocellular carcinoma ( hcc ) , liver cirrhosis ( lc ) , and hcc combined with lc in success rate , titer at successful response , the highest titer , the lowest titer before booster vaccination , the highest titer after booster vaccination , titer when hbig was withdrawn , and the titer at the end of followup ( figure 3 ) .
the influence of the donor anti - hbs status ( positive or negative ) on the response of the recipients to vaccination is shown in figure 4 .
there were no differences between the positive and negative groups in titer at successful response , the highest titer , the lowest titer before booster vaccination , the highest titer after booster vaccination , titer when hbig was withdrawn , and titer at the end of followup .
in addition , the round of inoculation when successful immunization occurred , the number of doses , the number of booster vaccinations , and the booster vaccination rate of the two groups were similar ( data not shown ) . recipients ' preoperative sera hbv dna level ( 10  copies / ml or < 10  copies / ml ) did not influence the response to vaccination . there were no differences between the 10  copies / ml group and < 10  copies / ml group in baseline titer , titer at successful immunization , the highest titer , the lowest titer before booster vaccination , the highest titer after booster vaccination , titer when hbig was withdrawn , and the titer at the end of followup ( figure 5 ) .
in addition , the round of inoculation when successful immunization occurred , the number of doses , the number of booster vaccinations , and the booster vaccination rate of the two groups were similar ( data not shown ) . in the 21 patients who discontinued hbig and nucleoside analogues , 4 patients appeared hbsag positive .
the interval of time between withdrawal of nucleoside analogues and hepatitis b virus reinfection was 14.25 months ( 6 to 23 months ) .
the four patients reused nucleoside analogues and one patient whose hbsag and hbv dna were negative was treated with hbig ( 2000  iu ) .
the gene mutations of hbv were detected by nested pcr assay and gene sequence analysis and the clinical characteristics of these 4 patients were reviewed ( table 4 ) .
additionally , we notified other patients withdrawn from hbig and nucleoside analogues regimen that they had the risk of hbv reinfection , although the risk was unclear .
whether hbig and nucleoside analogues administered for the prevention of hbv graft reinfection and hb recurrence after olt for hbv - related esld can be withdrawn or not has yet to be determined . based on the latest findings   and the results from our previous studies [ 1719 ] ,
long - term or lifelong use of these drugs raises a series of issues , including resistance to antiviral drugs , hbig - induced hbv immune escape , and high monetary costs .
thus , an alternative protocol which is more rational , economical , and effective is needed . since active immunization is the gold standard for preventing hbv infection among the general population , induction of active immunity against hbv after olt appeared to be an effective alternative to hbig and nucleoside analogues . while there are multiple clinical reports on the induction of active immunity against hbv in patients undergoing olt for hbv - related esld [ 715 ]
, most studies only included a small number of cases , and all were nonactive hbv replicative .
in addition , participants stopped or did not use exogenous hbig in order to facilitate a response to the vaccine [ 7 , 8 , 1113 ] .
our study included 200 patients who had completed at least one vaccination round , and our results showed that establishment of active immunity against hbv after olt caused by hbv - related esld is feasible , especially including recipients with hbv active replication before surgery . according to literature reports , adoptive immunity is helpful for the reestablishment of active immunity against hbv after olt .
the primed lymphocytes from hbsab - positive donor 's liver graft are beneficial to the induction of positive immunity against hbv .
memory t lymphocytes and memory b lymphocytes play the most important role in this process ; they are activated by hbv antigens such as hbsag in the donors .
after olt , they are stimulated by the hbv vaccine in the recipients and lymphocyte proliferation occurs , which consequently leads to the active immunity against hbv .
unfortunately , reports showed that the adoptive immunity is generally ineffective , and the response intensity to hbv vaccination is relatively weak and short - term .
similarly , we did not find significant adoptive immunity in the present study as there was no significant difference when comparing hbsab - positive donors and hbsab - negative donors .
we also observed that the response intensity of the hcc group was greater than that of the other disease groups , especially after booster vaccinations were performed , though the differences were not significant ( figure 2 ) . to prevent hcc recurrence , immunosuppressive agents such as tacrolimus
were reduced to the possible lowest dosage , which was the reason why the response intensity was greater for the hcc group than other groups .
previous study has indicated that the hbv vaccine type could affect the final immune response outcome .
the vaccination efficiency of recombinant hbsag is only 7.7% , but vaccination efficiency is increased to 47% when the sci - b - vac complex vaccine containing pre - s antigen is used .
these results indicate that improvements in vaccines can enhance the immune response and suggest that a combination vaccine has the potential to improve the vaccination response rate . however , the adjuvant vaccine mentioned above is still not commercially available , and its safety requires clarification . in our study ,
the twinrix group exhibited a greater response intensity than the engerix - b group , and the drug withdrawal rate of the twinrix group was greater than that of the engerix - b group .
the reason for these differences may be that twinrix is a bivalent vaccine which contains inactivated hepatitis a virus antigen and recombinant hbsag , while engerix - b is a monovalent vaccine which only contains recombinant hbsag .
this difference could result in a bystander effect caused by hepatitis a virus antigen , which facilitates the immune response for hbv vaccination .
it is possible that the presence of the hbsag - hbig complex further enhanced the effectiveness of the vaccines . in this study , most patients with successful vaccination had a successful reaction after 5 to 11 doses .
this is because all of the transplant recipients were immunocompromised , whose immune systems had different levels of native / innate tolerance or acquired tolerance to hbsag .
their immune responses could only be restored through long - term and continuous antigenic stimulation .
this is another example illustrating that the immune response can be improved along with the improvement of general conditions after liver transplantation .
this suggests that inoculations should be continued until successful vaccination is achieved among immunocompromised subjects . in this study ,
long - term and repetitive vaccine stimulation was an important method to create and cultivate an enhanced immune response in the immunocompromised individuals .
intermittent vaccination reinforcement ( booster vaccination ) was also an important means to maintain a spontaneous anti - hbs production .
the average length of time after transplantation was 35.80  19.12 months in the patients with successful vaccinations in this study .
our previous study   confirmed that the general condition of olt recipients and the antigen - presenting ability of the primary immune cells ( dendritic cells ) exhibited a tendency towards improvement with elongation of the postoperative time and minimization of the immunosuppression , which was essentially the immunological basis of this phenomenon .
therefore , from an immunological perspective the longer the postoperative time and the better the recovery of immune function , the higher the success rate of vaccination .
however , the risk of viral resistance to drugs and immune escape are higher with the elongation of the postoperative prevention regimen .
we believe that patients should be vaccinated before the occurrence of those aforementioned phenomena . we have named this time period the  opportunity window  to reconstruct active immunity . therefore , timely vaccination is the wisest choice .
our results also suggest that it is reasonable to initiate reconstruction of active immunity 18 months after transplantation .
similarly , the reaction intensity was different in patients with responses . as discussed above , responses were defined based on the reaction intensity . in immunocompromised individuals with responses ,
antibody production was characterized by low intensity , short duration , and the need of regular vaccination reinforcement .
we observed that , during the process of hbig and antiviral drug withdrawal , it was necessary to give one reinforcing inoculation regularly in the early stage in order to maintain a sustainable and spontaneous anti - hbs production over baseline level , which was especially important after entering the withdrawal protocol .
the reasons for hbv reinfection in patients who withdrew hbig and nucleoside analogues were complex .
first , it was shown that mutation for hbsag escape under immune pressure or drug resistance mutations have occurred before liver transplantation .
second , traces of hepatitis b virus still existed in liver , myeloid element , or other tissues .
third , the antibodies , by which the patients reestablished active immunity against hbv generated , could not cover all subtypes of hepatitis b virus .
we will evaluate the status of immunization and hepatitis b virus before and after vaccination in future research . in conclusion ,
induction of active immunity against hbv in patients who have undergone olt for hbv - related esld is feasible . for long - term posttransplant survivors ,
withdrawal of hbig with induction of active immunity against hepatitis b is reasonable , effective , dependable , and economical ; however , discontinuation nucleoside analogues should be cautious .
but because of the complexity of the immune response and the molecules involved in hbv immunology further study is needed ; moreover , the indication for withdrawal of nucleoside analogues needs further exploration .
 
 background . 
 the aim of this study was to establish a hepatitis b virus ( hbv ) vaccination protocol among orthotopic liver transplantation ( olt ) recipients under the coverage of a low - dose hepatitis b immunoglobulin ( hbig ) combined with an antiviral agent prophylaxis protocol . 
 method . 
 two hundred olt recipients were included in this study . 
 the vaccine was injected at months 0 , 1 , 2 , and 6 . 
 low - dose hbig combined with antiviral agent prophylaxis protocol was continued before reestablishment of active immunity against hbv in order to maintain a steady anti - hbs titer . 
 results . active immunity against hbv was reestablished in 50 patients , for an overall response rate of 25% . 
 of the 50 patients , 24 discontinued hbig without any hbv graft reinfection during a follow - up period of 26.13  7.05 months . 
 21 patients discontinued both hbig and antiviral agents during a follow - up period of 39.86  15.47 months , and 4 patients among them appeared to be hbsag positive . 
 there was no recipient death or graft loss because of hbv reinfection . conclusions . 
 vaccination preventing hbv reinfection for olt recipients is feasible . 
 the strategy withdrawal of hbig with induction of active immunity against hepatitis b is reasonable for long - term survivors of olt ; however , discontinuation nucleoside analogues should be cautious . 
1. Introduction
2. Patients and Methods
3. Results
4. Discussion