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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Hepatitis B virus (HBV) mutants resistant to treatment with nucleoside or nucleotide analogs and those with the ability to escape from HBV-neutralizing antibody have the potential to infect HBV-vaccinated individuals. To address this potential serious public health challenge, we tested the efficacy of immunity induced by a commercial hepatitis B vaccine against a tissue culture-derived, clonal HBV polymerase mutant in HBV seronegative chimpanzees. The polymerase gene mutant contained a combination of three mutations (rtV173L, rtL180M, rtM204V), two of which resulted in changes to the overlapping viral envelope of the hepatitis B surface antigen (sE164D, sI195M). Prior to the HBV mutant challenge of vaccinated chimpanzees, we established virologic, serologic, and pathologic characteristics of infections resulting from intravenous inoculation of the HBV polymerase gene mutant and the sG145R vaccine-escape surface gene mutant. Cloning and sequencing experiments determined that the three mutations in the polymerase gene mutant remained stable and that the single mutation in the surface gene mutant reverted to the wild-type sequence. Immunological evidence of HBV replication was observed in the vaccinated chimpanzees after challenge with the polymerase gene mutant as well as after rechallenge with serum-derived wild-type HBV (5,000 chimpanzee infectious doses administered intravenously), despite robust humoral and cellular anti-HBV immune responses after hepatitis B vaccination. Conclusion: Our data showing successful experimental infection by HBV mutants despite the presence of high anti-HBs levels considered protective in the vaccinated host are consistent with clinical reports on breakthrough infection in anti-HBs-positive patients infected with HBV mutants. In the absence of a protective humoral immunity, adaptive cellular immune responses elicited by infection may limit HBV replication and persistence. (HEPATOLOGY 2009.)

Hepatitis B is a global health problem; an estimated 350 million people worldwide are chronically infected with hepatitis B virus (HBV) and are at risk of developing chronic active hepatitis, liver cirrhosis, and primary liver cancer.1 Vaccination against HBV prevents new infections and efforts toward the control of chronic disease have involved the therapeutic inhibition of viral replication using analogs of nucleotides or nucleosides.2 Lamivudine was the first drug in this class to be licensed for the treatment of chronic hepatitis B and remains in widespread use. Antiviral drug-resistant mutations selected during treatment with lamivudine in the HBV polymerase gene (Pol) clustered within its B domain (rtV173L, rtL180M) and in the C domain in the conserved YMDD motif (rtM204V).3, 4 It has been reported that these mutant viruses are transmissible5 and may have the potential to cause breakthrough infections among recipients of hepatitis B vaccine.6, 7 The development of neutralizing antibody escape in those individuals may result from the changed amino acid composition of the HBV envelope protein, as the envelope and polymerase open reading frames overlap in the circular HBV genome.7 Consequently, alterations in the neutralization of the “a” determinant of the hepatitis B surface antigen (HBsAg) protein have been reported in patients with mutations in the Pol-gene receiving therapy with lamivudine.3, 4, 8 A markedly reduced binding of anti-HBs antibodies to HBsAg with mutations corresponding to Pol-protein changes (rtV173L, rtL180M, rtM204V) has been demonstrated in vitro.8 These findings raise the possibility of the selection of lamivudine-resistant HBV mutants with antigenically modified HBsAg proteins that could act as vaccine escape mutants and infect vaccinated individuals in whom anti-HBs exerts a further positive selection pressure.

Mutations in the “a” determinant of HBV surface gene (S-gene) have been identified in infants receiving postexposure prophylaxis (hepatitis B immunoglobulin [HBIG] or hepatitis B vaccination) for prevention of perinatal HBV infection, in liver transplant patients receiving HBIG, and persons receiving pre-exposure hepatitis B immunization. The most common mutation in the S-gene product is glycine to arginine at position 145 (sG145R), although the other amino acid substitutions have also been described at positions 124, 126, 137, 141, 143, and 144.9

It has been suggested that the immunity induced by existing vaccines may not be protective against various HBV mutants with altered surface proteins because of the conformational nature of the “a” determinant.10–12 Furthermore, the existence of such HBV isolates and the potential of these mutants to infect hepatitis B-vaccinated individuals may lead to occult HBV infection5, 13, 14 and, consequently, have serious public health implications. Breakthrough infections, despite the presence of high levels of anti-HBs antibodies, have been observed in various patient populations harboring mutations in the S-gene.7, 9

In this study we evaluated the efficacy of immunity induced by a commercial hepatitis B vaccine against a clonal Pol-gene HBV mutant containing a combination of three mutations (rtV173L, rtL180M, rtM204V) and describe the pathobiologic characteristics of the infection induced by antiviral drug-resistant HBV clonal mutants in naïve chimpanzees. We used genetically engineered Pol-gene and S-gene mutants and wild-type HBV (wt-HBV) generated in vitro because clinical isolates usually contain a heterogeneous mixture of HBV composed of the mutated species of the virus, other quasispecies resulting from error rate of HBV polymerase, as well as the wild-type virus. The use of genetically engineered mutants ensures that the infectivity and pathogenetic profile observed after an experimental inoculation with these viruses is attributed to the mutants per se.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Tissue Culture-Derived Mutant and Wild-Type HBV Inocula.

Point mutations were introduced by site-directed mutagenesis into a 1.5-times genome-length wt-HBV belonging to genotype A, subtype adw 2 (GenBank Access. No. X02763), which was inserted into the plasmid pBluescript KS+, according to instructions provided with the Quickchange mutagenesis kit (Stratagene). Primers (Table 1) were designed containing the desired nucleotide change in the HBV Pol-gene and synthesized by GeneWorks, (Hindmarsh, Australia). The sG145R mutation was produced in the S-gene with overlapping rtW153Q mutation in the Pol-gene. A combination of three HBV Pol-gene mutations, rtV173L, rtL180M, and rtM204V, was produced that correspond to sE164D and sI195M mutations in the S-gene (rtL180M does not result in an amino acid change in the S-gene). Successive mutagenesis reactions were performed to generate clones containing more than one nucleotide change. Automated DNA sequencing in both directions verified the sequences of each mutant.

Table 1. Sequences of Primers Used for Incorporating Mutations in the Surface and Polymerase Genes of Hepatitis B Virus and for PCR for Detection of HBV DNA
rtW153Q (sG145R) Fw5′-GTACAAAACCTACGGACAGAAATTGCACCTG-3′
rtW153Q (sG145R) Rv5′-CAGGTGCAATTTCTGTCCGTAGGTTTTGTAC-3′
rtV173L Fw5′-GCAAAATACCTATGGGACTGGGCCTCAGTC-3′
rtV173L Rv5′-GACTGAGGCCCAGTCCCATAGGTATTTTGC-3′
rtL180M Fw5′-CCTCAGTCCGTTTCTCATGGCTCAGTTTACTAG-3′
rtL180M Rv5′-CTAGTAAACTGAGCCATGAGAAACGGACTGAGG-3′
rtM204V Fw5′-GGCTTTCAGCTATGTGGATGATGTGGTATTGGG-3′
rtM204V Rv5′-CCCAATACCACATCATCCACATAGCTGAAAGCC-3′
SK-1985′-CGAGAATTCAGAGTCTAGACTCGTGGTGGACTT-3′
SK-1995′-CGCGAATTCTAGACTCGTGGTGGACTTCTCTCA-3′
SK-2005′-TGAGGATCCTACGAACCACTGAACAAATGGCAC-3′
SK-2015′-CTAGGATCCACTGAACAAATGGCACTAGTAA-3′
AA-7RI5′-CAGCGGTATAAAGGGACT C-3′

HepG2 cells (from the American Type Culture Collection, Manassas, VA) were grown in minimum essential medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2 and seeded to semiconfluence for transfections. Transient transfections were carried out with FuGene 6 (Roche Diagnostics) according to the manufacturer's instructions. Extracellular supernatants were harvested 5 days after transfection and tested for HBsAg. The extracellular supernatant was subjected to ultracentrifugation in an SW41 rotor on a sucrose cushion (1 mL of 20% [wt/vol] in Tris-HCl, pH 7.4) for 6 hours at 12°C at 35,000 rpm. The resuspended pellets (in 50 μL of nuclease free water) were aliquoted and stored at −70°C. HBV DNA was extracted from the pooled samples, quantified by a real-time polymerase chain reaction (PCR) kit (Artus HBV LC PCR ASR) (Qiagen), and used for sequencing analysis to confirm the presence of the mutated sequences. Tissue culture-derived HBV was examined by direct electron microscopy following partial purification through the sucrose cushion. The grids were washed 3 times with phosphate-buffered saline (PBS) before drying, stained with 3% phosphotungstic acid (pH 7.4), and viewed using a Tecnai 12 electron microscope (FEI, OR) at 120 kV. Immune electron microscopy was performed using rabbit polyclonal anti-HBs exactly as described15 detecting typical virions and subviral particles of HBV (Fig. 1).

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Figure 1. (A) Direct electron micrograph of partially purified wild-type HBV showing a typical 42 nm virus-like particle. (B) Immune electron micrograph of the sample shown in panel A following incubation with polyclonal anti-HBs and processed for electron microscopy as described in Materials and Methods. Filamentous forms, 22 nm subviral particles as well as HBV virions can be readily observed.

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Chimpanzee Infectivity Experiments.

Eleven colony-born chimpanzees (Pan troglodytes) (six females and five males, ranging in age from 3 to 11 years and weighing between 11 to 37 kg), determined to be seronegative for markers of HBV infection, were used for infectivity, vaccination, and challenge studies. The housing, maintenance, and care of the animals met all requirements of the Institutional Animal Care and Use Committee of the Centers for Disease Control and Prevention.

Chimpanzee identification numbers, type of inocula used, and sizes of infectious doses of the eight chimpanzees inoculated with tissue culture derived inocula are listed in Table 2. Of the three animals inoculated with the HBV Pol-gene mutant, CH257 and CH6407 received an inoculum from one batch, which contained 6.5 × 109 copies of HBV DNA, and the third animal, CH1611, received an inoculum prepared in a second batch that contained 9.8 × 109 copies of HBV DNA. All chimpanzees inoculated with the wild-type and mutant HBV inocula were followed up for 180 to 200 days after inoculation.

Table 2. Virological and Serological Markers of HBV Infection in Chimpanzees Inoculated with Tissue Culture-Derived HBV Inocula
Inoculum [Copies HBV DNA]Chimpanzee IDPositive (Days After Inoculation)
HBV DNAHBsAgHBeAgIgM anti-HBcTotal anti-HBcAnti-HBeAnti-HBs
  • Neg, negative.

  • Anti-HBs, anti-HBc, and anti-HBe markers were detected until the end of the 180-day follow-up period unless otherwise indicated.

  • *

    CH10301 was hepatitis A-vaccinated control chimpanzee

Wild-type HBV [3.14 × 107]CH102737–2929–56Neg70–92675664
 CH1027410–4221–42Neg49–7042764
sG145R [2.06 × 108]CH159998–168133–168138–148145–159145154172
 CH1600NegNegNegNegNegNegNeg
rtV173L+L180M+M204V [6.5 × 109]CH2573–3531NegNeg314952
 CH640710–31NegNeg49–604214–2142
rtV173L+L180M+M204V [9.8 × 109]CH161177–10282–110105113–158110113113
 CH10301*42–91NegNeg87–10887Neg84

Chimpanzee HBV Immunization and Challenge Experiments.

Two chimpanzees (CH10364 and CH10369) were given a pediatric dose (10 μg/0.5 mL) of a licensed recombinant hepatitis B vaccine (ENGERIX-B, GlaxoSmithKline) injected into the deltoid muscle at 0, 28, and 56 days. One control chimpanzee (CH10301) was vaccinated with hepatitis A vaccine (HAVRIX, GlaxoSmithKline) (720 Elisa units/0.5 mL). All three vaccinated chimpanzees were challenged with the clonal HBV Pol-gene mutant containing 9.8 × 109 copies of HBV DNA 42 days after the third vaccine dose; the control chimpanzee was challenged 35 days after the hepatitis A vaccination. The two hepatitis B-vaccinated chimpanzees were further cross-challenged with 5,000 chimpanzee infectious doses (CID) of human serum-derived wt-HBV inoculum (HLD1, genotype D, serotype ayw) 216 days after the Pol-gene mutant challenge. A naïve chimpanzee, CH10270, served as a control for the wt-HBV challenge and was also inoculated with 5,000 CIDs of the HLD1 inoculum.

Serial serum specimens were collected from all the chimpanzees twice weekly and whole blood samples for peripheral blood mononuclear cells (PBMCs) isolation were collected every 2 weeks. Serum alanine aminotransferase (ALT) levels were measured in fresh serum specimens using a colorimetric method on a Prochem-V Clinical Analyzer (Drew Scientific). Individual cutoff values (mean + 3 SD) for ALT levels were established for each animal using at least 10 preinoculation measurements.

HBV DNA in Serum.

Serum samples were tested for HBV DNA by an in-house nested PCR. DNA was extracted from serum using 200 μL of the sample by the QIAamp DNA Mini kit or QIAamp UltraSens Virus kit (Qiagen). The primers used in the first round of PCR were SK-198 and SK-200 and in the second round were SK-199 and SK-201 (Table 1). The resulting fragment of 453 basepairs comprised an S-gene segment that encompasses the “a” determinant region. For amplification of a 553-bp fragment of Pol-gene encompassing all three mutation sites, the same PCR protocol as described above was followed with the exception that an antisense primer, AA-7RI, was used in both PCR rounds (Table 1). For quantitative determination of HBV DNA, samples were further tested by a real-time PCR kit (Artus HBV LC PCR ASR).

HBV DNA Sequence Analysis.

The amplicons derived from HBV DNA-positive samples collected from chimpanzees and from inocula used in the infectivity and challenge studies were cloned using the Topo TA Cloning Kit (Invitrogen). From each cloned specimen, 20 to 29 clones were selected and a portion of the S-/Pol-gene overlap containing the mutation sites was directly amplified with the set of primers used in the second PCR round. The PCR products were sequenced in both directions using BigDye Terminator 3 with an ABI 3100 genetic analyzer (Applied Biosystems). Sequences were analyzed using LaserGene software (DNA Star).

Serological Markers of HBV Infection.

All serum samples were stored at −70°C until use. The samples were tested for HBsAg by AUSZYME Monoclonal, antibody to HBsAg (anti-HBs) by AUSAB EIA, total antibody to hepatitis B core antigen (anti-HBc) by CORZYME, IgM anti-HBc by CORZYME-M (Abbott), hepatitis B e antigen (HBeAg) by ETI-EBK Plus, and total anti-HBe by ETI-AB-EBK Plus (DiaSorin, Stillwater, MN). The levels of anti-HBs in samples collected during vaccination and challenge periods were also quantitatively measured by AUSAB Quantitation Panel (Abbott).

T-Cell Responses.

PBMCs from the chimpanzees were enriched on Ficoll-Hypaque gradients and tested in an interferon-γ ELISpot assay for reactivity to HBV surface and polymerase antigens. Briefly, 96-well microtiter plates (Millipore) were precoated with antibodies to human interferon-γ (U-Cytech, Utrecht, Netherlands) and 2 × 105 chimpanzee PBMCs in AIM V medium containing 2% heat-inactivated human serum were added to each well. Duplicate wells received pools of overlapping synthetic peptides at a concentration of 1 μM (15 amino acids in length overlapping by 11 residues) that spanned either amino acids 67–237 of the HBsAg or amino acids 1–344 of the reverse transcriptase domain of the polymerase protein. Plates were incubated at 37°C for 36 hours and developed for spot formation using a second antibody to interferon-γ conjugated to enzyme followed by substrate. Spot-forming cells (SFCs) in the microtiter wells were quantified using a CTL analyzer (CTL, Cleveland, CT). Fewer than 10 SFC/well were observed with pooled peptides from an irrelevant antigen (the hepatitis C virus NS3 protein). Responses were considered positive when an average of 10 SFC over background was detected in the duplicate wells.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Wild-Type HBV.

The dynamics of HBV infection observed in CH10273 and CH10274 were similar (Table 2; Fig. 2A). Infection was characterized by the presence of HBV DNA, HBsAg, and IgM anti-HBc, and seroconversion to total anti-HBc, anti-HBe, and anti-HBs. HBV DNA levels did not exceed 1 log IU/mL in any of the HBV DNA-positive samples. The time of appearance and duration of all the markers are shown in Table 2. A mild elevation in the ALT levels was observed on day 77 in both chimpanzees. Sequencing studies showed that all clones from the inoculum and samples collected from the two infected chimpanzees at the beginning, during, and at the end of viremia maintained the wild-type sequence.

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Figure 2. Biochemical, serological, and virologic markers of HBV infection in three chimpanzees, CH10274 (A), CH1599 (B), and CH1611 (C) inoculated with tissue culture-derived HBV wild-type, S-gene sG145R mutant, and Pol-gene mutant, respectively.

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HBV Pol-Gene Mutant (rtV173L, rtL180M, rtM204V).

Both CH257 and CH6407 (inoculated with 6.5 × 109 copies of HBV DNA) developed similar profiles of HBV infection. HBV DNA, at levels that did not exceed 1 log IU/mL, was detected from days 3 to 35 and 10 to 31 in CH257 and CH6407, respectively. HBsAg was detected only in CH257 on day 31; IgM anti-HBc was detected only in CH6407. The chimpanzees seroconverted to total anti-HBc, anti-HBs, and anti-HBe (Table 2). No elevation in ALT levels was observed in the two chimpanzees. In CH1611, inoculated with 9.8 × 109 copies of HBV DNA, HBV DNA was detected from days 77 to 102 and its levels did not exceed 1 log IU/mL during the viremia. HBsAg in serum was detected from days 82 to 110, HBeAg on day 105, and IgM anti-HBc from days 113 to 158. CH1611 seroconverted to total anti-HBc, anti-HBe, and anti-HBs from day 113 after inoculation (Table 2; Fig. 2C). Sequencing studies showed that all clones from the two inocula and samples collected at the beginning, during, and at the end of viremia in the three infected chimpanzees showed the preservation of the rt173L, rt180M, and rt204V mutations.

HBV S-Gene Mutant (sG145R).

The inoculation of sG145R mutant in two chimpanzees resulted in the infection of CH1599 only (Table 2; Fig. 2B). HBV DNA was first detectable on day 98 and remained positive until day 168, with the highest HBV DNA level of 2.7 log IU/mL observed on day 141. As shown in Table 2, all the markers of acute infection, HBsAg, HBeAg, and IgM anti-HBc were detected in the serum of the chimpanzee followed by seroconversion to anti-HBc, anti-HBe, and anti-HBs. The chimpanzee developed hepatitis with elevated serum ALT levels from days 145 to 161 with a peak of 473 IU/L (cutoff, 91 IU/L) observed on day 151, just after the HBV DNA reached the highest level. Sequencing studies of 24 clones from the first HBV DNA-positive serum sample postinoculation (day 98) showed the preservation of the inoculum sequence in only 33% of the clones, whereas 67% had the wild-type sequence. A complete reversion to the wild-type sequence had occurred by day 105 after inoculation (29 clones sequenced) and on day 165 after inoculation (20 clones sequenced).

Protective Efficacy of HBV Vaccine.

The ability of a licensed hepatitis B vaccine to provide protection against challenge with the HBV Pol-gene mutant was evaluated in two chimpanzees (CH10364 and CH10369) that received the ENGERIX-B hepatitis B vaccine and a control chimpanzee (CH10310) that received the HAVRIX hepatitis A vaccine. Both ENGERIX-B recipients seroconverted to anti-HBs 1 week after the first dose of the vaccine, and anti-HBs levels were boosted to steady-state levels of >75 mIU/mL after the administration of the second and third vaccine doses (Fig. 3). Interferon-γ producing T-cells were detected in the blood of both chimpanzees within 4-6 weeks of the first vaccine dose and stable frequencies were observed through 8 weeks of follow-up until they were challenged with the Pol-gene mutant virus (Fig. 4B). HBsAg-specific T-cell lines derived from the blood of these chimpanzees were exclusively CD4+ (data not shown). CD8+ T cells specific for HBsAg were not detected. As expected, T-cell responses to polymerase peptides were not detected after ENGERIX-B vaccination. Neither humoral nor cellular immunity to HBV antigens was detected in the control chimpanzee CH10301 vaccinated with HAVARIX.

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Figure 3. Anti-HBs levels in CH10364 and CH10369 after hepatitis B vaccination, challenge with Pol-gene mutant, and cross-challenge with human derived wild-type HBV (HLD1, 5,000 CIDs). The small arrowheads indicate hepatitis B (ENGERIX-B) vaccine administration and vertical gray bars indicate the time when the chimpanzees were challenged.

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Figure 4. T-cell immunity measured by INF-γ responses against peptide pools, HBsAg 67–237 (A) and Pol 62–272 (B) in CH10364 and CH10369 after hepatitis B vaccination, challenge with Pol-gene mutant and cross-challenge with human derived wild-type HBV (HLD1, 5,000 CIDs). The small arrowheads indicate hepatitis B (ENGERIX-B) vaccine administration and vertical gray bars indicate the time when the chimpanzees were challenged.

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All three chimpanzees were challenged with the Pol-gene mutant to determine if coding changes in the corresponding S-gene (sE164D and sI195M) compromised protective immunity elicited by hepatitis B vaccination. After challenge the hepatitis B-vaccinated chimpanzees, CH10364 and CH10369, were negative for serum HBV DNA and HBsAg during the entire 6-month follow-up period. Serum ALT also remained within normal limits. A substantial immune response to the virus was nonetheless observed in both chimpanzees. On the day of the challenge, CH10364 and CH10369 had baseline serum anti-HBs antibody titers of 125 mIU/mL and 81 mIU/mL, respectively. Within 7 days of challenge these anti-HBs antibody titers increased more than 100-fold (Fig. 3). Anti-HBc, which is a marker of exposure or infection, was detected on day 7 in CH10364 and days 7, 10, and 17 in CH10369. Analysis of cellular immunity in chimpanzee CH10364 also provided evidence of productive infection with the Pol-gene mutant virus. Specifically, a transient T-cell response targeting the polymerase protein was detected on day 28 postinfection by the interferon-γ ELISpot assay (Table 3; Fig. 4A). T-cell lines specific for the HBV polymerase protein derived from the peripheral blood of the chimpanzee on day 28 were CD4+, suggesting that the response was mediated in part by T-helper cells (Fig. 5A,B).

Table 3. Peak IFN-γ ELISpot Responses to the Surface and Polymerase Gene-Derived Synthetic Peptides After Hepatitis B Vaccination and Challenge with the HBV Polymerase Mutant and Serum-Derived Wild-Type Virus
ChimpanzeeAntigenAmino Acid CoordinateIFN-γ (SFC/106 PBMCs)
PreimmuneAfter VaccinationHBV pol MutantHBV Wild-Type (HLD1)
10364HBsAg67–23701981351865
 Pol(rt)1–720000
 Pol(rt)62–272007025
 Pol(rt)262–344000228
10369HBsAg67–2370328601327
 Pol(rt)1–720 00
 Pol(rt)62–272000320
 Pol(rt)262–3440000
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Figure 5. Polymerase-specific T-cells from chimpanzee CH10364. Peripheral blood mononuclear cells (PBMCs) from chimpanzee CH10364 were first used in an ELISpot assay as described in Fig. 4. At termination of the ELISpot assay PBMCs were recovered from the cultures and expanded to derive HBV-specific T-cell lines. Data shown in Fig. 5 were generated by rescue and expansion of T-cells from the ELISpot assay at day 28 after challenge with the Pol-gene mutant when a transient interferon-γ response was detected. (A) The expanded T-cell line responded in an interferon-γ ELISpot assay when stimulated with overlapping peptides spanning HBV Pol residues 62-272 but not a control peptide pool representing the HCV NS3 gene. (B) Phenotype analysis of the cell line revealed that ≈90% were positive for CD4. The percentage of cells in each quadrant is indicated.

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The hepatitis A-immunized control chimpanzee CH10301 was infected; HBV DNA was detected in serum from days 42 to 91, not exceeding 1 log IU/mL (Table 2). The chimpanzee seroconverted to total anti-HBc from day 87 and anti-HBs from day 84 postinfection. Despite replication of the Pol-gene mutant virus in this chimpanzee, peripheral blood T-cell responses against the surface or polymerase proteins were not detected. No ALT elevation was observed during the follow-up period.

The presence of a humoral immune response to the HBV core antigen in both ENGERIX-B-vaccinated chimpanzees and of circulating T-cells against the HBV polymerase antigen in one chimpanzee (CH10364) indicated that sterilizing immunity against the HBV Pol-gene mutant was lacking. Therefore, chimpanzees CH10364 and CH10369 were challenged with human-derived wt-HBV to determine if they were protected against challenge with a virus encoding intact (that is, unmutated) HBsAg. Challenge with the wt-HBV inoculum (HLD1, 5,000 CIDs) was carried out when anti-HBs levels were 63,610 mIU/mL and 18,868 mIU/mL in CH10364 and CH10369, respectively, and after loss of HBV mutant-stimulated T-cell responses in both chimpanzees. Two weeks after challenge with HLD1, strong T-cell responses were measured against surface antigen by ELISpot in both chimpanzees (Fig. 4,A,B). High frequencies of circulating T-cells against HBsAg suggested an anamnestic response primed by vaccination and perhaps the prior infection with the HBV Pol-gene mutant. Responses to the polymerase protein were delayed by comparison (Fig. 4A,B). Chimpanzee CH10369, who had an undetectable polymerase response after Pol-gene mutant challenge, had a robust polymerase-specific T-cell activity after wt-HBV challenge that peaked at week 6. Chimpanzee CH10364, which had a polymerase-specific CD4+ T-cell response after Pol-gene mutant challenge, displayed a novel polymerase response at weeks 4 and 6 after wt-HBV challenge (Table 3). Polymerase-specific T-cell lines expanded from the blood of both chimpanzees expressed CD4 and not CD8 (data not shown). Anti-HBs titers increased 6-fold in chimpanzee CH10369 and 10-fold in chimpanzee CH10364 (Fig. 3). However, HBV DNA, HBsAg, IgM anti-core, and HBeAg were not detected in any of the chimpanzees during the follow-up period after the challenge.

A markedly different profile of wt-HBV infection was observed in a naïve unvaccinated chimpanzee CH10270. The infection was characterized by the presence of HBV DNA, which reached the peak levels of 4.1 log IU/mL on day 66, and of HBsAg, HBeAg, and IgM anti-HBc. The chimpanzee subsequently developed acute hepatitis with elevated serum ALT levels from days 66 to 129, with the peak of 2,500 IU/L (cutoff 73 IU/L) observed on day 91. This chimpanzee subsequently seroconverted to total anti-HBc, anti-HBe, and anti-HBs. Circulating T-cells specific for the surface and polymerase antigens were not detected in chimpanzee CH10270 by the ELISpot assay (data not shown), an unexpected result given the strong responses to these proteins after infection with the Pol-gene mutant and then wt-HBV of the two hepatitis B-vaccinated chimpanzees

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Breakthrough infections etiologically linked to S-gene HBV mutants have been reported to occur among HBV-immunized populations, despite the presence of seroprotective levels of anti-HBs in the infected individuals.6, 16 In this experimental study, we evaluated the protective efficacy of a commercial hepatitis B vaccine against the Pol-gene HBV mutant (rtV173L, rtL180M, rtM204V) encoding the surface protein mutations (sE164D, sI195M) that was previously shown to have significantly reduced anti-HBs binding properties.8 The Pol-gene mutant inoculum used in naïve and vaccinated chimpanzees was prepared in vitro to ensure their purity from wt-HBV or from HBV with other mutations that may be present in clinical isolates. The inoculation of naïve chimpanzees with clonal HBV mutants allowed studying the virologic, immunologic, and pathologic findings exclusively related to the mutant infection. The subsequent challenge experiment provided an opportunity to determine whether HBV mutants are infectious to vaccinated chimpanzees. Because our cellular immunity data indicated that the commercial vaccine did not induce sterilizing immunity against the Pol-gene HBV mutant, we further challenged two immunized chimpanzees with the serum-derived wt-HBV.

In naïve nonimmunized chimpanzees we observed variations in the infectivity profile following injection with inocula carrying various viral titers of HBV mutants. The length of the incubation period, measured by first appearance of HBV DNA in serum, ranged from 1 week (clonal Pol-gene HBV mutant and wt-HBV) to 14 weeks (sG145R mutant) and did not correlate with the number of HBV DNA copies in the inocula. The inoculation of the Pol-gene mutant HBV, which contained 6.5 × 109 copies of DNA, resulted in an incubation period of 1 or 2 weeks compared to 11 weeks in a chimpanzee inoculated with 9.8 × 109 copies of HBV DNA of the same mutant. These differences, which are at variance with data from previous experimental infectivity studies,17 may be indicative of a lack of correlation between calculated number of HBV DNA copies and infectivity titer. Also of interest were differences in the stability of mutations between Pol-gene and S-gene mutants during the experimental infection of chimpanzees. Whereas mutations in the Pol-gene remained stable throughout the period of viremia in all four chimpanzees, the sG145R mutant in one infected chimpanzee readily reverted to wild-type. In a previously reported infectivity study in chimpanzees, inoculation with the sG145R mutant, which also contained wt-HBV, stability of the mutation was observed throughout the infection18; however, the inoculum was serum obtained from a 7-year-old child born to a carrier mother and who was given HBIG and hepatitis B vaccination.19 The differences in the stability of mutations, the duration and magnitude of viremia and of HBs antigenemia, and liver pathology between chimpanzees inoculated with Pol-gene and S-gene mutants may be attributed to the mutation site, replication fitness of the mutants, the size of the inocula, or a combination of these.

Vaccinated chimpanzees in this study developed a robust T-cell immunity measured by the interferon-γ ELISpot assay and had pronounced anti-HBs serum antibody titers (>75 mIU/mL) that significantly exceeded those considered protective (10 mIU/mL).20, 21 Detection of anti-HBc antibodies after challenge with the Pol-gene HBV mutant in one serum sample from CH10364 and three consecutive samples from CH10369 provided immunological evidence of the HBV replication despite high anti-HBs levels. Anti-HBc responses have been considered presumptive evidence of the breakthrough infection in other studies.22, 23 Further evidence of the breakthrough infection in our challenge experiment using the clonal Pol-gene HBV mutant was provided by priming of T-cell responses against the polymerase protein in at least one vaccinated chimpanzee. This protein is not a component of the vaccine and it is unlikely that sufficient quantities are physically associated with the Pol-gene mutant virus particles in the original inoculum to prime T-cell immunity in the absence of virus replication. Indeed, this latter possibility seems remote, as we did not observe polymerase specific responses in unvaccinated chimpanzees that received high doses of Pol-gene mutant or wild-type particles that resulted in infection. High serum titers of anti-HBs antibodies are a reliable marker of vaccine-induced protective immunity against HBV infection. It is conceivable that virus-specific T cells provide protection when mutant or wild-type viruses breakthrough sterilizing humoral immunity. However, cellular responses have not been studied in vaccinated chimpanzees or humans under these conditions. Our observation that Pol-specific T-cell responses are elicited in hepatitis B-vaccinated animals challenged with mutant and wild-type viruses is clearly consistent with this possibility. Antibody-mediated depletion of CD4+ or CD8+ T-cells from chimpanzees has provided direct evidence of their involvement in control of acute HBV and HCV infection.24, 25 A similar experimental approach in animals receiving hepatitis B vaccine prior to HBV challenge may help define the full scope of protective immunity afforded by vaccination.

The efficacy of anti-HBV immunity induced by the commercial vaccine was further tested by rechallenging the two chimpanzees, previously vaccinated and challenged with the Pol-gene mutant, with serum-derived wt-HBV. This experiment showed a subsequent boost of anti-HBs levels (6-fold to 10-fold increase) in both chimpanzees and a surge of T-cell responses against both surface-derived and polymerase-derived peptides. This pattern of cellular and humoral immune responses against HBV antigens strongly implicated viral replication despite very high levels of anti-HBs suggesting breakthrough HBV infection after wt-HBV challenge. It is possible, however, that a dose of 5,000 CIDs used in the challenge experiment may have been large enough to overcome the seroprotective barrier. A further challenge inoculum titration study would be needed to evaluate the relationship between the size of inoculum and protective level of anti-HBV immunity induced by a vaccine and measured by the dynamics of cellular immunity to HBV polymerase protein and the level of anti-HBs antibody.

In conclusion, we have demonstrated that in vitro-generated HBV clonal Pol-gene mutant, bearing the lamivudine-resistance-associated mutations (rtV173L, rtL180M, rtM204V) and the prototype vaccine-escape S-gene mutant (sG145R), are infectious in naïve chimpanzees. The S-gene mutant readily reverted to the wild-type sequence but the Pol-gene mutant was stable during the course of infection. Vaccination of naïve chimpanzees with a commercial hepatitis B vaccine that resulted in the induction of both humoral and cellular immune responses did not seem to confer sterilizing immunity against challenge with the Pol-gene mutant and subsequent challenge with a serum-derived wt-HBV. Our data showing successful experimental infection by HBV mutants despite the presence of high anti-HBs levels considered protective in the vaccinated host are consistent with clinical reports on breakthrough infection in anti-HBs-positive patients infected with HBV mutants and warrant further studies of efficacy of HBV vaccine.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References