The Mode Of Adefovir Resistance And Its Immunoselection Characteristic

Background and objectiveChronic hepatitis B (CHB) infection is a major public health problem, and about400million people worldwide infected hepatitis B virus chronically. High prevalent areas ofHBV are mainly in China, South Africa, Southeast Asia, and the Mediterranean region.Antiviral treatment is the key in the treatment of chronic hepatitis B to control viralreplication at a minimum level. Thus it may stop the process of liver disease and preventcomplications. There are two main types of Anti-viral drugs currently, IFN-γ and hepatitisB virus (HBV) nucleoside reverse transcriptase inhibitors (NRTIs). The effect of IFN-γ islimited, about40%of patients were with low response to it. Long-time use of NRTIs canlead to resistant mutants and withdrawal rebound. HBV inhibitors are currently divided intothree types according to structure: L-nucleoside analogues, acyclic adenosine phosphonate,deoxyguanosine analogues. There are five drugs of NRTIs in the United States and Europeapproved for HBV treatment: lamivudine (LAM), adefovir (ADV), entecavir (ETV),telbivudine (LDT), and tenofovir (TDF). LAM and ADV are the first-line drugs in thetreatment of CHB. A single NRTI can quickly inhibit HBV replication in CHB treatmentwithin a short term mostly, but with a long-term treatment, poor therapeutic effect willoccur due to the emergence of drug resistant HBV strains. Drug resistance is the mostimportant factor in the failure treatment. The failure of NRTIs treatment brings aconsiderable clinical challenge, and it limits the choice of the follow-up treatment. Thusantiviral drug resistance was concerned widely.ADV is an important nucleotide analogue with activity against wild strains andLAM-resistant mutants. Resistance to ADV is less common and usually occurs later in thetreatment course in comparison with LAM. But in the patients with LAM failure, adefovirresistance develops rapidly. Single amino acid substitution, such as rtN236T and/orrtA181V/T, can lead to the failure of clinical treatment of ADV. They are the typical aminoacid substitutions related to ADV-resistance. Other amino acid substitutions in the reverse transcriptase sequence, including rtL80V/I, rtV84M, rtS85A, rtV214A, rtQ215S, rtE218G,rtP237H, and rtN238T/D/H, have also been suggested to be associated with reducedsusceptibility to ADV antiviral action. They can appear alone or in combination withrtN236T or rtA181V/T. The variation of HBV polymerase has been detected only in aminority of patients with ADV treatment failure. There may be more unknown factorsleading to clinical resistance to ADV.The causes that influence response and resistance ofNRTIs have not been clear, and HBV genotype was reported to be one probable factor. Thereare many different findings. Most recognized that HBV genotypes have an impact on theemergence of resistance and the type of resistant mutation.Many studies have shown that theYVDD/YIDD amino acid variation is related to HBV genotypes. Further researchers haveshown that the incidence of the resistant strains in genotypes C and B is the same, but HBVgenotypes correlate with the type of YMDD/YIDD variation. In the ADV treatment, someresearchers believed that the incidence of resistant mutations had no significant differencein patients with genotype A and D,but some researchers thought that ADV resistance mayoccurred mainly in patients with genotype B and D. The views are still controversial.The natural variation of HBV is lower than RNA, and the intervention of nucleosideanalogues can accelerate virus mutation, especially in reverse transcriptase region. Whetherthe HBV variation appearing after ADV treatment can break the immune balance with thehost, and lead to changes in cytotoxic T lymphocyte (CTL) epitopes, which is related withthe onset of hepatitis. Now some reports showed that the variation of RT region could bringchanges in the immune response, for example, rtM204I+rtL180M may lead to hepatitisafter breakthrough. But the studies about them were lacking, especially the immune effectsof ADV resistant mutations.The patients with genotypic resistance to ADV are rare, thus, the sample of thesestudies was small. We took a long time to collect a larger sample in this study, and analyzedthe relationship between the types of ADV resistant mutations and HBV genotype or theother clinical factors, particularly including the impact factor of rt181variation types andits effect on HBsAg, which have not been reported at home and abroad. On this basis ofabove, the study further analyed the epitope drift of ADV resistant strains.Result1. Of the161patients with clinical resistant to ADV,135cases were with viral reboud and26cases were with primer nonresponse. In the patients with viral rebound, the age andduration of ADV treatment in the ADV genotypic resistant group (42.20±7.99years and30.01±11.03months, respectively) were obviously higher than those in the ADVnon-genotypic resistant group (33.57±9.69years and19.97±10.86months)(both P=0.000),and the rate of HBeAg positive in the ADV non-genotypic resistant group85.2%was higherthan the ADV genotypic resistant group56.8%(P=0.000). Most of the patients with primarynonresponse (24/26) to ADV treatment were failed to detect ADV genotypic resistancemutations (P=0.000).In74patients with viral rebound and ADV genotypic resistance,19patients had anrtA181+rtN236substitution and the other55patients had a single mutation in either thertA181or rtN236position. In the multiple analyses, the HBV genotype and cirrhosis showedstatistical significance in the selection of the mutation positions (P=0.002and P=0.026,respectively). In patients with the rtN236T, most of which were genotype B. It was differentfrom the distribution of HBV genotypes in Chongqing aera, in ADV clinical/genotyperesistant group or in rt181variation goups (P＜0.05).Of the48patients with viral rebound expressing the rtA181mutation, in multipleanalysis, the rtN236T, time resistant strains occurred had statistical significance in the type ofrt181variation (P=0.007, P=0.024, respectively). RtN236Ts with rtA181T emerged morefrequently than with rtA181V. The rtA181T occurred earlier than rtA181V.2. Of the161patients with clinical resistant to ADV, there were46cases (28.6%) withrtN238,105cases (65.2%) with rtN238H,9cases (5.6%) with rtN238T, and1case with rarertN238Q.The types of amino acid in rt238position had no statistical difference in patientswith genetypic resistance or not (P=0.709). The three types of amino acid in rt238positionhad significant difference in HBV genotypes (χ~2=83.874, P=0.000). RtN238H mainlyoccured in genotype B, rtN238occured more frequently in genotype C, and rtN238T wasonly found in genotypes C.The amino acid types of rt238had significant difference in thethree ADV resistant models(rt181, rt236and rt181+rt236)(χ~2=25.640, P=0.000). Usingpartial correlation analysis, with the control of HBV genotypes, it had no significance(P=0.347).In the ADV monotherapy and LAM-ADV sequential therapy groups,the difference ofrt238amino acid was not statistically significant (χ~2=1.262, P=0.532). In6patients,the amino acid of rt238had no change with ADV treatment.As control,52cases with LAM resistance and26cases with untreatment, the amino acidtypes of rt238had been related to HBV genotypes in the two groups respectively (P=0.000).The difference of HBV genotype in untreated group and LAM resistant group werestatistically significant (χ~2=8.751, P=0.003), but it weren't significant in untreated group andADV resistant group (χ~2=0.573, P=0.449). The distribution of rt238types had no significantdifference in untreated group, LAM resistant group and ADV resistant group (P=0.326).3. Of7patients with rt181variation induced by LAM single drug, rtA181T were themain (71.4%). Of15patients with rt181variation induced by ADV single drug, there were8cases with rtA181V, and7cases with rtA181T. The difference between the two groups was notstatistically significant (χ~2=4.139, P=0.067). Aligning the sequences in the two groups, rt223was found to be related to the treatment groups (χ~2=5.455, P=0.020). In ADV group, therewere12patients (80%) with rtA223, and in the LAM treated group, there were5patients(71.4%) with rtS223. Using partial correlation analysis, with the control of HBV genotype,the relationship between rt223and the treatment groups was still significant (P=0.013). Indifferent genotypes, the distributions of amino acid types in rt221, rt223, rt224and rt238were statistical different (P=0.001, P=0.010, P=0.035, P=0.011).4. In15serum samples collected when rt181variation occurred, in different genotypes,HBsAg had significant difference (P=0.037). HBsAg quantification in7patients withgenotype B was8341.57±1510.01S/COI, and in8patients with genotype C was5688.05±2651.79S/COI. The level of HBsAg in serums with a concurrent rtN236T wassignificantly higher than that without rtN236T (P=0.008).In two patients with rt181variation, serums in different times were analyzed. HBsAgincreased less in the time of mutation occurred than in the other times in the patients withrtA181T. In the patients with rtA181V, the logarithm value of HBV DNA correlated withHBsAg titer in each phase point (P=0.001). When the mutation occurred, HBV DNA andHBsAg had a reverse trend.5.2patients with HLA_A2and ADV resistance were investigated before and after thetreatment, and the epitopes of HBV sequences were predicted. The amino acid changesappeared in strong antigenic regions of LHB346-354, LHB382-390, LHB388-396both inthe two patients.This study, using HLA-A2computer molecular models from protein crystal structure database, analyzed the combination of three epitope peptides on LHB346-354before and after the variation and HLA-A2by molecular docking, and showed a strongerhydrogen bond between WFSLLVPFV and MHC molecule. The affinity of the threepeptides and MHC molecular as follows from high to low: WFSLLVPFV＞WLSLLVPFV＞RLSLLVPFV.The epitope peptides on LHB346-354, LHB382-390and LHB388-396before and aftervariation had CTL cell functional verification. In this test, the expressions of IFN-γ in theepitope peptides were all higher than those in negative control. LHB346-1epitope of No.2patient is even higher than that of the positive control.In2HLA_A11patients with ADV resistance, X87-95was predicted as CTL epitope.LHB346-354epitope was not predicted, but WFSLLVPFV was found to have strongaffinity with MHC molecule by molecular docking.Conclusion1. Genotypic resistance was not detected in the majority of clinical resistance to ADV,which suggested other factors except resistant mutation influence the response to ADV.2. The different HBV genotypes influence the selection of ADV-resistant mutationpositions by comparing the patients with rt181or rt236mutation.3. It was showed that ADV treatment duration and rt236mutation both had an importantpart to select the type of rt181variation. The rtA181T occurs more frequently in patients withthe rtN236T and it occurs earlier when compared to the rtA181V.4. RtH238strains were prevalent in Chongqing, and unrelated to treatment, butgenotype B.5. RtA181T was the most common type in patients with rt181variation induced byLAM monotherapy, and rtA181V was more common with ADV monotherapy.6. The amino acid types on rt223were associated with therapeutic drugs in patientswith rt181variation induced by different drugs, which was not found before.7. The loci of rt221, rt223, rt224, and rt238were dependent sites of HBV genotypes.8. HBsAg titer was related to HBV DNA level, and rt181mutation may influenceHBsAg expression.9. LHB346-354, LHB382-390, LHB388-396were CTL cell epitopes of HLA_A2byprediction, in which amino acid substitutions occurred frequently with ADV treatment. They may influence the immue response.10. LHB346-354(coinciding with rt181mutation) was CTL cell epitope of HLA_A11by molecular docking.