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 2-4 5
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
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.
RESULTS
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 .
TABLE 1: Main Characteristics Of The Study Population
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 .
TABLE 2: Mutations in the HBV Reverse Transcriptase and Envelope Genes Corresponding to Antiviral Resistance or Immune Escape
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 6 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.
TABLE 3: Univariate and Multivariate Analysis of HBV Polymerase and Envelope Genotypic Changes According to HBV Genotypes A and D and Mono-/Coinfection With HIV
DISCUSSION
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 9
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 12 1
Other 3TC-resistance-associated mutations may cause premature stop codons in the S gene, resulting in impaired secretion of HBsAg.13 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
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