The hepatitis B virus (HBV) genome consists of 4 overlapping genes that encode a number of complex and multifunctional proteins. Long-term antiviral therapy directed towards the HBV reverse transcriptase (RT) can lead to the selection of drug-resistant mutants, which over time become the dominant species. The HBV polymerase gene overlaps the envelope genes, including the S gene, which encodes for the hepatitis B surface antigen (HBsAg) in a frame-shifted manner. The result is that drug-resistant mutants may affect the HBsAg's antigenicity. Likewise, mutations in the surface gene selected by HBV vaccination or hepatitis B immune globulins may also produce functional alterations in the viral polymerase.
Recently it has been shown that a triple-mutational pattern causing lamivudine (3TC) resistance (rtV173L + rtL180M + rtM204V) has enhanced HBV replication compared to rtL180M + rtM204V alone.1 This triple mutant causes 2 amino acid changes in the overlapping surface gene (sE164D + sI195M), reducing its anti-HBs binding to levels seen only with the vaccine escape mutant sG145R.2-4 On the other hand, another study has recently shown that patients treated with 3TC and apparently showing clearance of HBsAg actually continued to have detectable circulating plasma HBV-DNA. Subsequent genetic analyses demonstrated that the selection of an sP120A mutation in these patients was associated with the apparent HBsAg seroconversion.5 This mutation produced a reduced anti-HBs binding, which explained the failure to detect HBsAg.
The aim of this study was to identify any possible changes in the HBsAg protein that occur spontaneously or as a consequence of antiviral therapy in a relatively large cohort of chronic HBV-monoinfected and HBV/HIV-coinfected individuals.
PATIENTS AND METHODS
Serum samples from 19 HBV-monoinfected and 52 HBV/HIV-coinfected patients treated for at least 12 months with nucleos(t)ide analogues with anti-HBV activity attending Hospital Carlos III in Madrid, Spain, were identified. Biochemical and virologic characteristics were analyzed at baseline and during therapy. Serum HBV-DNA was quantified using COBAS Taqman (Roche, Barcelona, Spain), which has a lower limit of detection of 12 IU/mL. DNA extraction was carried out using the Qiagen DNA blood kit (Qiagen, Mannheim, Germany) following manufacturer's instructions.
A 720 bp DNA fragment of the RT/envelope gene from HBV was amplified using a nested polymerase chain reaction (PCR), as previously described,6 then sequenced and analyzed for selected mutational patterns in both the RT and the overlapping envelope gene. The sequences from each isolate were compared with all known HBV genotypes (A to H) using the Megalign software (DNAStar, Madison, WI).
Univariate and multivariate analyses were performed using the SPSS v13.0 software (SPSS, Chicago, IL), including as variables HBV genotype, HIV coinfection, and treatment with 3TC. Differences were considered significant when P values were <0.05.
Of the 19 HBV-monoinfected patients, 9 were treated with 3TC plus adefovir (ADV) and the remaining 10 with ADV alone. Of 52 HBV/HIV-coinfected patients, 40 were treated with tenofovir (TDF) plus 3TC. All of them also received either a protease inhibitor (n = 24), a non-nucleoside reverse transcriptase inhibitor (n = 13), another nucleoside analogue (n = 2) or a combination of them (n = 12); 1 patient was additionally treated with enfuvirtide. The distribution of HBV genotypes was as follows: genotype A, 44; genotype D, 23; genotype E, 2; and others, 2. Overall, 41 (58%) patients were positive for HBV e antigen (HBeAg). Other baseline characteristics are shown in Table 1.
HBV Reverse Transcriptase Mutations
Sequences from 16 patients could be analyzed before commencing treatment. In all 16, wild-type virus was observed. 3TC resistance-associated mutations were observed in 3 of 9 3TC-treated, HBV-monoinfected individuals; all 3 patients were treated with 3TC monotherapy at the time of selection (mean treatment duration, 37 months). 3TC-resistance-associated mutations were also seen in 28 of 49 3TC- or emtricitabine (FTC)-treated HBV/HIV-coinfected patients (mean treatment duration, 35 months). Three patients in the coinfected group selected the triple mutation rtV173L + rtL180M + rtM204V. All 3 patients had been treated with 3TC for an extended time period (64, 72, and 77 months) compared to the mean (35 months). Other patterns of HBV resistance-associated mutations observed are shown in Table 2.
Of the 19 HBV-monoinfected patients treated with ADV, 2 harbored the rtN236T mutant and 5 harbored viruses with changes rtQ215S, rtI233V, and rtS213T + rtV214P, all of which have been associated occasionally with ADV resistance in HBV.7-9 Virus in 3 patients treated with TDF developed the A194T mutation,6 and in 2 of them this change appeared along with 3TC-resistance-associated mutations (Table 2), a pattern that has been demonstrated to produce TDF resistance in HBV.5
HBV Envelope Mutations
The 3 patients who harbored the triple HBV polymerase mutant rtV173L + rtL180M + rtM204V demonstrated characteristic changes in the envelope gene, such as sE164D + sI195M, which have recently been demonstrated to produce reduced anti-HBs binding.2,3
In addition, 4 patients had other envelope changes found in the “a” determinant region (aa 90 to 160). These mutations included sP120T, sM133I, and sD144E, all of which have been previously shown to represent potential vaccine or hepatitis B immunoglobulin (HBIg) escape mutations (Table 2), although none of our patients presented detectable surface antigen antibodies (anti-HBs) nor were they transplant patients receiving HBIg. Only 1 patient harboring the sP120T mutant selected this mutation during therapy (12 months of treatment with ADV); the remaining 3 had the mutants at baseline. In this series of patients, none were found to harbor HBV with sG145R or sP120A, which have been associated with vaccine escape mutations.5,10
Most 3TC-resistance-associated mutations produced changes in the envelope C-terminal hydrophilic region. In 3 patients, a premature stop codon was observed. In contrast, none of the mutations conferring resistance to ADV or TDF resulted in changes detrimental for the 2 antigenic loops of the envelope protein.
In a multivariate analysis, C-terminal mutations selected with 3TC treatment were significantly more frequent in HBV genotype A compared to D (28 of 39 versus 3 of 16; P = 0.001), whereas immune-selected “a” determinant vaccine escape mutants were more frequently associated with genotype D than A (4 of 23 versus 0 of 44; P = 0.011), as shown in Table 3. Finally, no association could be found between HBeAg status and RT or S gene mutations.
Vaccine/HBIg-escape mutations sP120T and sG145R, in combination with 3TC-resistance-associated mutations, are often seen in HBV-monoinfected patients following 3TC or HBIg treatment and have been shown to increase viral replication in vitro in the presence of 3TC therapy.3,11 Although in our study we did not detect sG145R, 2 patients carried HBV mutants with the sP120T change (1 in combination with rtL180M + rtM204I). A number of other HBVs with mutations associated with reduced anti-HBs binding have also been recently described, including changes sM133I and sD144E,9 which in our study population appeared more frequently in HBV-monoinfected patients with genotype D.
In contrast, 3TC-resistance-associated mutations rtV173L, rtM204V/I, and rtV207I may simultaneously alter the S gene, causing critical antigenic changes in the C terminal region of HBsAg, as a result of producing mutations sE164D, sI195M, sW196S/L/stop, and sM198I or sW199stop, respectively.10 At least 1 study has shown that an HBV mutant containing rtV173L + rtL180M + rtM204V has reduced HBs antigen-antibody binding.12 We found this same pattern of mutations in 3 of our isolates. Interestingly, this HBV drug-resistance mutation profile was detected only in HBV/HIV-coinfected patients.1
Other 3TC-resistance-associated mutations may cause premature stop codons in the S gene, resulting in impaired secretion of HBsAg.13 Three of our patients had HBV with premature stop codons in the S antigen. Genotypic analyses demonstrated that mutations in the HBV surface antigen selected by 3TC therapy developed more significantly in HBV genotype A compared to genotype D (P = 0.001). Interestingly, whereas genotype D was more prevalent among HBV-monoinfected subjects, genotype A was the predominant variant among HBV/HIV-coinfected individuals, as described in other European cohorts of coinfected patients.14
In summary, our results suggest that HBV genotyping and polymerase gene sequencing may be helpful to appropriately manage patients undergoing antiviral therapy for chronic hepatitis B. This may be particularly helpful in patients coinfected with HIV, because most of them carry HBV genotype A, which is more prone to select the lamivudine-resistant triple mutant, which may cause vaccine escape and diagnostic problems.15 The circulation of HBV encoding envelope mutations selected by antiviral agents requires further investigation to determine whether they may be transmitted and therefore represent a public health concern. This issue may be of particular relevance in populations where genotype A is predominant.
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