Virological Response, Resistance And Dynamics To Adefovir For The Treatment Of Chronic Hepatitis B

Worldwide,an estimated 350 million people are actively infected with hepatitis B virus.At present,nucleoside analogues are the most widely used pharmacotherapy. Lamivudine,a deoxycytidine analogue,can quickly suppress the replication of HBV both in vivo and in vitro.However,drug resistance may develop as the treatment goes on,the rate of lamivudine resistance is 14-32%,38%-58%,49%-53%,70%at the year of 1,2,3 and 4 respectively.Adefovir has been proved to be effective both for the wild-type variants and lamivudine resistant viriants,while its resistant rate is 0,3%,4%-11%,18%,29%at the year of 1,2,3,4 and 5 respectively.Clinically,we have observed that some patients receiving adefovir therapy have a "suboptimal" virological response.Those patients can not achieve satisfactory virological response even undergo a long-term therapy and no evidence of adefovir-resistant mutations are detected,which indicate that some other factors may affect the antiviral therapy.Viral quasispecies study has been a hot spot in virology in recent few years. Quasispecies are not simply a collection of diverse mutants but a group of interactive variants,which together contribute to the characteristics of the population.The quasispecies distribution of HBV implies that any newly generated mutation conferring a selective advantage to the virus in a given replicative environment will allow the corresponding viral population to overtake the other variants,and adapt to new environments and challenges during infection.The main target of the nucleoside analogues locate in the HBV reverse transcriptase region,mutations in this region will affect the antiviral activity of the drug. Lamivudine-resistant mutations mainly locate in YMDD motif within this region,including rtM204I(YIDD),rtM204V(YVDD)and rtM204S(YSDD).It has been conferred that the replication capacity of above-mentioned variants are lower compared to the wild type variants in vitro,but can be compensatively improved by mutations locate outside YMDD motif,such as rtL180M and rtV173L.The mechanism for adefovir resistance is more complicated than lamivudine.The main resistant mutations(rtN236T and rtA181V) only confer low level of resistance to adefovir in vitro,while may cause different types of HBV DNA dynamics in different patients.In the infected hosts,the components,complexity of virus quasispecies will change with the immune status of the host and the pressure of the antiviral drugs.Variants with higher fitness will overtake the lower ones and induce a drug resistance which may directly cause the failure of antiviral therapy.In view of that,we enrolled 34 naive and 57 lamivudine-resistant chronic hepatitis B patients,the efficacy and factors correlating with it were monitored.LightCycler-PCR and Amplicor COBAS HBV Monitor assays were compared for HBV DNA detection.Direct PCR sequencing were used to detect drug mutations in 7 lamivudine-resistant patients who received adefovir for continued therapy and quasispecies dynamics were studied in one of them.Our research demonstrated the dynamic changes of quasispecies of HBV RT during the clinical course of a CHB patient and provided some theoretical clues for antiviral therapy.Results:1.As for the naive patients:Patients who achieve undetectable HBV DNA at the end of the therapy have a lower baseline HBV DNA level,higher ALT and higher ratio of initial virologic response(IVR) compared with those who have not(P=0.034,0.011,＜0.001); HBeAg seroconversion were observed more often in patients with lower baseline HBV DNA and those who achieved IVR(P=0.034,＜0.001);and lower baseline HBV DNA and higher ALT predict a higher probability for winning IVR(P=0.006,0.003).Linear regression identified negative correlations between body weight and the reduction level of HBV DNA at the week of 52,104 and 132 respectively(P=0.026,0.016,0.035).2.For the lamivudine-resistant patients:Patients who achieve undetectable HBV DNA at the end of the therapy have a higher baseline ALT and higher ratio of IVR compared with those who have not(P=0.012,＜0.001);HBeAg seroconversion were observed more often in patients with lower baseline HBV DNA and those who achieved IVR(P=0.018,＜0.001); and lower baseline HBV DNA and higher ALT predict a higher probability for winning IVR(P=0.006,0.004).3.In the three lamivudine-resistant patients,the switch from 10 mg to 20 mg of adefovir daily significantly improved antiviral efficacy and ALT normalized in all patients. No signs of renal dysfunction occurred.4.Linear regression identified positive correlations between the results of the HBV LightCycler-PCR assay and those of the Amplicor COBAS HBV Monitor assay for the evaluation of clinical samples(P＜0.001).5.Direct PCR sequencing identified lamivudine-resistance mutation in 6 out of 7 patients at the time of virological breakthough.6.The dynamic changes of quasispecies of a CHB patient who underwent lamivudine and adefovir were demonstrated as follow:variants bearing M204I and M204V+L180M substitutions were detected 36 and 48 weeks respectively.The YMDD population were replaced by selected viruses bearing both the M204V and L180M substitutions at the week of 60 and were wholly replaced by the latter at week 72,at which time a virological breakthrough was monitored.When switching to adefovir,the variants bearing both the M204V and L180M still remained present as a major population until the end of follow-up and wild-type variants and M204I variants reemerged in later stage.Conclusions:1.Patients with lower baseline HBV DNA level,higher ALT and lower body weight were more prone to achieve virologic response.2.We demonstrated a negative correlation between the body weight and the reduction of HBV DNA level.Thus,a higher dosage of adefovir dipivoxil is recommended for the patients with higher body weight.Increasing the adefovir dose to 20 mg daily is beneficial and safe in patients with lamivudine-resistant HBV and a suboptimal response to adefovir 10 mg daily.3.The results of the HBV LightCycler-PCR assay were found to be in good agreement with those of the Amplicor COBAS HBV Monitor test for the evaluation of clinical samples.4.Virological breakthrough was preceded by months by the detection of quasispecies variants bearing amino acid substitutions within the YMDD motif that are known to confer lamivudine resistance.Gradually,these resistant variants,which initially represented a minority of the quasispecies,fully replaced the wild-type YMDD variants.5.The replication capacity of YVDD variants could be compensatively improved by L180M substitutions.6.The wild-type YMDD variants displayed a higher fitness level as it reemerged in later stage of adefovir therapy.Thus,combination therapy is recommended rather than monotherapy.