Hepatitis B Virus Immunization and Neonatal Acquisition of Persistent Infection in England and Wales

Hepatitis B Virus Immunization and Neonatal Acquisition of Persistent Infection in England and Wales Abstract Background It is believed that between 2% and 5% of infants born to hepatitis B virus (HBV)–infected mothers at a high risk of perinatal transmission will become persistently infected despite immunization starting at birth. We investigated factors associated with breakthrough infections. Methods Sixty-nine samples from HBV-infected infants born between 2003 and 2015 were tested for HBV serological and molecular markers. Sequencing and epitope phenotyping were used to investigate alterations in hepatitis B surface antigen (HBsAg) sequence and antigenicity in infants and in mothers known to have transmitted and not to have transmitted virus to their infants. Results Vaccine/hepatitis B immune globulin uptake was complete in the majority of HBV-infected infants. A minority (8 [12%]) had detectable plasma antibody to HBsAg at 12 months. Twenty-five of 68 (37%) infants harbored a virus with amino acid changes in the HBsAg “a” determinant, of which 13 displayed altered HBsAg antigenicity. Viral load was 30-fold higher in maternal samples from those who transmitted. Conclusions Our data provide evidence to suggest that immune selection drives change at mother–infant transmission, resulting in the alteration of HBsAg antigenicity. These changes may play a role in immunization failure, but other factors including viral load may be more important. Continued monitoring of vaccine efficacy is essential. HBV, immunization, HBsAg mutants, vaccine escape, viral load, infected infants Mother-to-child transmission (MTCT) of hepatitis B virus (HBV) represents an important mechanism for the generation and maintenance of persistent infections within populations. As well as being a reservoir for onward transmission, persistently infected individuals remain at high risk for the development of cirrhosis, liver failure, and/or hepatocellular carcinoma [1]. The World Health Organization’s recommendation for national HBV immunization programs worldwide is aimed at eliminating perinatal transmission through active HBV immunization within 24 hours of birth with the addition of hepatitis B immune globulin (HBIG) where there is a high risk of transmission [2]. Optimally implemented, these actions have had a dramatic impact on reducing MTCT, with an 87% reduction observed in the seroprevalence of hepatitis B surface antigen (HBsAg) in children in countries of previous high endemicity [3]. In the United Kingdom, a selective neonatal HBV immunization program has been adopted and will continue after introduction of universal HBV immunization in 2017. Since 2000, all women are tested antenatally for HBsAg and infants born to HBV-infected mothers are immunized at 0, 1, 2, and 12 months of age. HBIG is also offered in addition to vaccine (passive–active immunization) for infants born to (1) high-transmission-risk mothers (classified as having detectable plasma hepatitis B e antigen [HBeAg], or an HBV DNA level >6 log10 IU/mL); (2) mothers who have an acute HBV infection during pregnancy; and (3) mothers whose booking blood samples are undetectable for both “e” markers. HBIG will also be given to low-birth-weight infants (<1500 g) born to HBV-infected mothers regardless of maternal viral load or “e” status [4]. The National Institute for Health Care Excellence guidelines also recommend antiviral treatment during the third trimester where HBV DNA levels exceed 7 log10 IU/mL [5]. It is recommended that infants born to HBV-infected mothers are tested at 12 months of age for evidence of current HBV infection. There are approximately 3000 pregnancies in HBV-infected women each year in England [6], of which 5%–10% will be in women classified as having a high perinatal transmission risk [7]. The program of National Enhanced Surveillance of High-Risk Infants born to high-transmission-risk mothers shows HBIG administration and vaccine uptake rates for the first 3 doses to be >90%, marginally better than the 90% uptake of 3 doses in all infants at HBV risk [7]. Even though 2%–5% of infants born to high-transmission-risk mothers in England are predicted to acquire persistent infection despite receiving passive–active immunoprophylaxis, only half of the high-risk infants are reported as being tested at 12 months for evidence of HBV infection [7]. A number of reasons have been postulated for these transmissions including possible intrauterine infection [8–14], incomplete or delayed prophylaxis, failure to respond to vaccine [14], and specific virological factors including vaccine escape variants [15–18]. The latter have been reported occasionally, but their overall significance in vaccine failure is not well characterized. As part of the National Enhanced Surveillance of High-Risk Infants program, samples from these infants at 12 months of age are sent to Public Health England (PHE) and screened for evidence of current HBV infection. Comprehensive serological and molecular characterization, together with HBsAg epitope phenotyping, was undertaken on samples from infants found to be HBV infected and, where available, on samples from their mothers. We aimed to inform on why breakthrough infections are occurring and whether additional measures need to be implemented to reduce MTCT of HBV in the United Kingdom. METHODS Infant Samples Oral fluid, venous, or dried blood spot samples from infants aged 12 months are received at PHE Colindale for testing for HBsAg and antibody to hepatitis B core antigen (anti-HBc). For infants with reactive samples tested at PHE and for HBV-infected infants reported to PHE after local testing, further blood samples are requested for confirmation. Venous samples were available from a total of 69 HBV-infected infants identified between 2003 and 2015. Sixty of the infants were aged 12 months; 9 were aged between 3 and 12 years. Infant Immunization/HBIG Uptake Data The National Enhanced Surveillance collects maternal demographic data before birth, and follows up high-risk infants to 12 months to document the issue and administration of HBIG and vaccine at birth and the first year of life and to determine evidence of current HBV infection. Maternal Samples Samples were obtained usually during pregnancy, just after delivery or as close as possible to the pregnancy from 45 of the 69 mothers who transmitted infection (transmitters). Twenty-one additional samples, held at PHE Colindale, from HBV-infected mothers whose samples also contained HBeAg but who did not transmit HBV to their infants (nontransmitters), were available as a comparator group. Serology Samples from the 69 HBV-infected infants were tested for HBeAg, antibody to HBeAg (anti-HBe), and antibody to HBsAg (anti-HBs) using commercial assays (DiaSorin, Dartford, Kent, United Kingdom). DNA Studies Nucleic acid was extracted from 200 µL of maternal and infant plasma/serum using the MagNA Pure 96 DNA viral small volume kit (Roche, West Sussex, United Kingdom). HBV DNA viral load was quantified as previously described [19]. Genotype determination with additional analysis for inferred amino acid changes across HBsAg was undertaken as previously described [20]. In brief, alignments of 1800 sequences representing described HBV genotypes/subgenotypes were obtained from either in-house–generated sequences or from GenBank. The sequences were checked both visually and by position-specific scoring matrix to identify intragenotypic motifs. Amino acid alignments for wild-type (WT) consensus genotype-specific sequences were created for the HBsAg region and used to identify mutations. Genotype determination was based on clustering within phylogenetic trees generated using the 1800 representative HBsAg nucleotide sequences. HBsAg Epitope Ex Vivo Phenotyping The antigenic profile of HBsAg in a sample was determined by epitope mapping using 4 monoclonal antibodies (mAbs) directed against different epitopes of the “a” determinant region. The mAb P2D3 recognizes a first-loop linear epitope between codons 121 and 129; mAbs H3F5, D2H5, and HB04 were raised against a mixture of ad/ay HBsAg and recognize conformational epitopes. Tentative mapping data indicated that H3F5 bound to epitopes between codons 131 and 142, whereas D2H5 and HB04 recognized similar epitopes in the second loop between codons 142 and 147. The Luminex platform (Bio-Rad Laboratories) provides a method of interrogating protein–antibody interactions on discrete and identifiable bead solid phases and was used to measure the interaction of plasma HBsAg with the 4 mAbs, each on an individually identifiable solid phase as previously described [21]. Poor or loss of reactivity against 1 of more mAbs indicated an alteration of epitope phenotype. RESULTS Characterization of the Infant Infection HBV Markers Small sample volumes meant that HBV marker testing could not be completed on all infant samples. The majority (57/59 [96%]) of available infants’ samples were HBeAg positive, 1 was anti-HBe positive, and 1 was negative for both HBeAg and anti-HBe. The majority of available samples (58/66 [88%]) were unreactive for anti-HBs (<10 IU/L). In the 8 reactive samples, anti-HBs ranged from 26.6 to 235 IU/L. HBV DNA quantification was undertaken in 64 samples; HBV DNA levels ranged from 3.55 to 9.78 log10 IU/mL (median, 8.582 [standard deviation, 1.242] log10 IU/mL). The majority (62/64 [97%]) of the infants had a viral load >5 log10 IU/mL. HBsAg Sequence and Epitope Phenotype Analysis There was sufficient plasma for sequence analysis on 68 of 69 infant samples and for epitope phenotyping on 60 samples. The majority of the infants were infected with genotype C (n = 23 [34%]) and D viruses (n = 24 [35%]), with the remainder harboring genotype A (n = 5 [7%]), B (n = 11 [16%]), E (n = 4 [6%]), and CD recombinant (n = 1 [2%]) viruses. All infant genotypes detected were concordant with that of the available paired maternal samples. Sequence analysis indicated that 35 of the 68 (51%) infants harbored viruses with amino acid changes across the HBsAg, of which the majority (25/35 [71%]) had codon changes which lay within the “a” determinant region, between codons 120 and 150 (Figures 1A and 2). Twenty-two of the 25 were available for epitope phenotyping, of which more than half (13/22 [59%]) displayed altered HBsAg antigenicity (Table 1); the remaining 9 displayed a WT HBsAg epitope phenotype. Of the 10 viruses bearing HBsAg codon changes outside the “a” determinant, 9 were available for epitope phenotyping and all displayed a WT HBsAg epitope phenotype. Thirty-three viruses carried no codon changes across HBsAg, 29 samples were available for epitope phenotyping, and all exhibited WT antigenicity (Figure 2). Figure 1. View largeDownload slide Sites of codon change across the entire hepatitis B surface antigen (HBsAg) protein. A, “Hot spot” across HBsAg representing positions of amino acid (AA) changes observed in virus sequences from infants (solid bars) and also indicating where identical amino acid changes in the infant (cross-hatched bars) were also observed in the maternal sample. B, “Hot spot” across HBsAg representing positions of amino acid changes observed in virus sequences from mothers who transmitted (solid bars) vs those who did not transmit (cross-hatched bars) hepatitis B virus to their infants. Figure 1. View largeDownload slide Sites of codon change across the entire hepatitis B surface antigen (HBsAg) protein. A, “Hot spot” across HBsAg representing positions of amino acid (AA) changes observed in virus sequences from infants (solid bars) and also indicating where identical amino acid changes in the infant (cross-hatched bars) were also observed in the maternal sample. B, “Hot spot” across HBsAg representing positions of amino acid changes observed in virus sequences from mothers who transmitted (solid bars) vs those who did not transmit (cross-hatched bars) hepatitis B virus to their infants. Figure 2. View largeDownload slide Hepatitis B surface antigen sequence and epitope phenotype data linkage in 68 infants. Inferred amino acid changes displayed as within or outside the “a” determinant domain. Abbreviations: HBsAg, hepatitis B surface antigen; WT, wild-type sequence. Figure 2. View largeDownload slide Hepatitis B surface antigen sequence and epitope phenotype data linkage in 68 infants. Inferred amino acid changes displayed as within or outside the “a” determinant domain. Abbreviations: HBsAg, hepatitis B surface antigen; WT, wild-type sequence. Table 1. Linked Hepatitis B Surface Antigen Sequence and Epitope Phenotyping Data of 22 Infants’ Samples That Had an “a” Determinant Mutation Genotype  Sequence Data  HBsAg Epitope Phenotyping Data (Expressed as Proportional Percentage)  mAb P2D3  mAb D2H5  mAb H3F5  mAb HB04  B  Wild-type  20.36  24.96  23.41  31.27  B  G145R  68.78  0.27  18.80  12.15  B  G145A  27.68  22.28  24.71  25.33  B  M133L  22.31  22.55  22.96  32.19  C  Wild-type  25.23  23.33  20.84  30.60  C  S132F/Y, G145R  54.63  1.01  29.97  14.38  C  G145G/R  30.42  18.01  21.54  30.03  C  D144D/A  33.63  15.53  23.29  27.55  C  I126F, G145R  46.35  1.34  38.99  13.32  C  G145A  25.95  21.47  25.08  27.50  C  P62P/L, G145G/A, V184A  18.09  19.53  18.73  43.64  C  I128T  23.86  24.36  23.55  28.24  C  T131A  25.71  24.23  23.58  26.48  D  Wild-type  20.63  21.75  25.09  32.53  D  P142L, G145R  43.46  6.68  43.32  6.54  D  G145R  29.78  16.07  29.23  24.91  D  D144A  27.44  15.51  29.11  27.94  D  T143L  31.12  30.89  31.08  6.91  D  G145G/R  25.13  19.20  27.05  28.62  D  D144D/A, G145R, V184A  29.45  19.20  27.29  24.07  D  D144A  29.65  17.97  30.18  22.21  D  P120S  16.81  21.75  20.36  41.09  D  D144E  27.08  23.04  25.87  24.00  D  D144D/E, S174S/N, I208I/T, L213L/I  26.85  24.35  24.88  25.12  D  P120T, D144A, S174N  27.40  21.64  24.90  26.06  Genotype  Sequence Data  HBsAg Epitope Phenotyping Data (Expressed as Proportional Percentage)  mAb P2D3  mAb D2H5  mAb H3F5  mAb HB04  B  Wild-type  20.36  24.96  23.41  31.27  B  G145R  68.78  0.27  18.80  12.15  B  G145A  27.68  22.28  24.71  25.33  B  M133L  22.31  22.55  22.96  32.19  C  Wild-type  25.23  23.33  20.84  30.60  C  S132F/Y, G145R  54.63  1.01  29.97  14.38  C  G145G/R  30.42  18.01  21.54  30.03  C  D144D/A  33.63  15.53  23.29  27.55  C  I126F, G145R  46.35  1.34  38.99  13.32  C  G145A  25.95  21.47  25.08  27.50  C  P62P/L, G145G/A, V184A  18.09  19.53  18.73  43.64  C  I128T  23.86  24.36  23.55  28.24  C  T131A  25.71  24.23  23.58  26.48  D  Wild-type  20.63  21.75  25.09  32.53  D  P142L, G145R  43.46  6.68  43.32  6.54  D  G145R  29.78  16.07  29.23  24.91  D  D144A  27.44  15.51  29.11  27.94  D  T143L  31.12  30.89  31.08  6.91  D  G145G/R  25.13  19.20  27.05  28.62  D  D144D/A, G145R, V184A  29.45  19.20  27.29  24.07  D  D144A  29.65  17.97  30.18  22.21  D  P120S  16.81  21.75  20.36  41.09  D  D144E  27.08  23.04  25.87  24.00  D  D144D/E, S174S/N, I208I/T, L213L/I  26.85  24.35  24.88  25.12  D  P120T, D144A, S174N  27.40  21.64  24.90  26.06  Amino acid changes in HBsAg sequence, compared against genotype consensus listed by codon. The reactivity against the 4 mAbs is expressed as the percentage reactivity of each mAb as part of the total reactivity for that sample. Alterations in HBsAg antigenicity, measured as epitope loss against a range of monoclonal antibodies, are shown in bold type and displayed for each monoclonal antibody. The observed reactivity of wild-type viruses is also given. Abbreviations: HBsAg, hepatitis B surface antigen; mAb, monoclonal antibody. View Large Table 1. Linked Hepatitis B Surface Antigen Sequence and Epitope Phenotyping Data of 22 Infants’ Samples That Had an “a” Determinant Mutation Genotype  Sequence Data  HBsAg Epitope Phenotyping Data (Expressed as Proportional Percentage)  mAb P2D3  mAb D2H5  mAb H3F5  mAb HB04  B  Wild-type  20.36  24.96  23.41  31.27  B  G145R  68.78  0.27  18.80  12.15  B  G145A  27.68  22.28  24.71  25.33  B  M133L  22.31  22.55  22.96  32.19  C  Wild-type  25.23  23.33  20.84  30.60  C  S132F/Y, G145R  54.63  1.01  29.97  14.38  C  G145G/R  30.42  18.01  21.54  30.03  C  D144D/A  33.63  15.53  23.29  27.55  C  I126F, G145R  46.35  1.34  38.99  13.32  C  G145A  25.95  21.47  25.08  27.50  C  P62P/L, G145G/A, V184A  18.09  19.53  18.73  43.64  C  I128T  23.86  24.36  23.55  28.24  C  T131A  25.71  24.23  23.58  26.48  D  Wild-type  20.63  21.75  25.09  32.53  D  P142L, G145R  43.46  6.68  43.32  6.54  D  G145R  29.78  16.07  29.23  24.91  D  D144A  27.44  15.51  29.11  27.94  D  T143L  31.12  30.89  31.08  6.91  D  G145G/R  25.13  19.20  27.05  28.62  D  D144D/A, G145R, V184A  29.45  19.20  27.29  24.07  D  D144A  29.65  17.97  30.18  22.21  D  P120S  16.81  21.75  20.36  41.09  D  D144E  27.08  23.04  25.87  24.00  D  D144D/E, S174S/N, I208I/T, L213L/I  26.85  24.35  24.88  25.12  D  P120T, D144A, S174N  27.40  21.64  24.90  26.06  Genotype  Sequence Data  HBsAg Epitope Phenotyping Data (Expressed as Proportional Percentage)  mAb P2D3  mAb D2H5  mAb H3F5  mAb HB04  B  Wild-type  20.36  24.96  23.41  31.27  B  G145R  68.78  0.27  18.80  12.15  B  G145A  27.68  22.28  24.71  25.33  B  M133L  22.31  22.55  22.96  32.19  C  Wild-type  25.23  23.33  20.84  30.60  C  S132F/Y, G145R  54.63  1.01  29.97  14.38  C  G145G/R  30.42  18.01  21.54  30.03  C  D144D/A  33.63  15.53  23.29  27.55  C  I126F, G145R  46.35  1.34  38.99  13.32  C  G145A  25.95  21.47  25.08  27.50  C  P62P/L, G145G/A, V184A  18.09  19.53  18.73  43.64  C  I128T  23.86  24.36  23.55  28.24  C  T131A  25.71  24.23  23.58  26.48  D  Wild-type  20.63  21.75  25.09  32.53  D  P142L, G145R  43.46  6.68  43.32  6.54  D  G145R  29.78  16.07  29.23  24.91  D  D144A  27.44  15.51  29.11  27.94  D  T143L  31.12  30.89  31.08  6.91  D  G145G/R  25.13  19.20  27.05  28.62  D  D144D/A, G145R, V184A  29.45  19.20  27.29  24.07  D  D144A  29.65  17.97  30.18  22.21  D  P120S  16.81  21.75  20.36  41.09  D  D144E  27.08  23.04  25.87  24.00  D  D144D/E, S174S/N, I208I/T, L213L/I  26.85  24.35  24.88  25.12  D  P120T, D144A, S174N  27.40  21.64  24.90  26.06  Amino acid changes in HBsAg sequence, compared against genotype consensus listed by codon. The reactivity against the 4 mAbs is expressed as the percentage reactivity of each mAb as part of the total reactivity for that sample. Alterations in HBsAg antigenicity, measured as epitope loss against a range of monoclonal antibodies, are shown in bold type and displayed for each monoclonal antibody. The observed reactivity of wild-type viruses is also given. Abbreviations: HBsAg, hepatitis B surface antigen; mAb, monoclonal antibody. View Large Of the 13 infants whose viruses displayed an altered HBsAg epitope phenotype, 3 (23%) had a detectable anti-HBs response (>10 IU/L; range, 26–235 IU/L); in contrast, only 3 of the 47 (6%) infants with a WT HBsAg epitope phenotype had a detectable anti-HBs response. Mother-and-Infant Pairs Viral Evolution Samples were available from only 42 mother-and-infant pairs (Figure 3). Analysis of the matched sequences indicated some concordance between the maternal and infant viruses but also notable differences. Where 3 maternal viruses harbored an amino acid change in the “a” determinant, this was transmitted (Figures 3 and 4). In 1 case, the infant’s virus acquired an additional amino acid change in the “a” determinant (Figure 3). Ab initio selection of variants in the “a” determinant was observed in 14 infants, 9 of 26 (35%) of infants born to mothers harboring a WT HBsAg virus and 5 of 13 (38%) of infants born to mothers with a virus harboring amino acid changes outside of the “a” determinant (Figure 4). Figure 3. View largeDownload slide Hepatitis B surface antigen sequence and epitope phenotype data linkage in 42 mother–infant pairs. Indication of those infants among the original 68 who had paired maternal samples and the linked maternal sample sequence. *Same sequence in maternal and infant samples. **One maternal sample with amino acid changes in the “a” where the corresponding infant sample has an additional amino acid change. Inferred amino acid changes displayed as within or outside the “a” determinant domain. Abbreviations: HBsAg, hepatitis B surface antigen; WT, wild-type sequence. Figure 3. View largeDownload slide Hepatitis B surface antigen sequence and epitope phenotype data linkage in 42 mother–infant pairs. Indication of those infants among the original 68 who had paired maternal samples and the linked maternal sample sequence. *Same sequence in maternal and infant samples. **One maternal sample with amino acid changes in the “a” where the corresponding infant sample has an additional amino acid change. Inferred amino acid changes displayed as within or outside the “a” determinant domain. Abbreviations: HBsAg, hepatitis B surface antigen; WT, wild-type sequence. Figure 4. View largeDownload slide Role of maternal virus hepatitis B surface antigen (HBsAg) sequence and epitope phenotype in the selection of viruses in infants. ***Only 2 samples available for epitope phenotyping. Linkage between the maternal virus and infant virus. Maternal sequence: inferred amino acid changes displayed as within or outside the “a” determinant domain, maternal HBsAg epitope phenotype; and the linked infant sequence: inferred amino acid changes displayed as “Out.” (outside “a” determinant) or “In” (within the “a” determinant) and determined HBsAg epitope phenotype. Abbreviation: WT, wild-type. Figure 4. View largeDownload slide Role of maternal virus hepatitis B surface antigen (HBsAg) sequence and epitope phenotype in the selection of viruses in infants. ***Only 2 samples available for epitope phenotyping. Linkage between the maternal virus and infant virus. Maternal sequence: inferred amino acid changes displayed as within or outside the “a” determinant domain, maternal HBsAg epitope phenotype; and the linked infant sequence: inferred amino acid changes displayed as “Out.” (outside “a” determinant) or “In” (within the “a” determinant) and determined HBsAg epitope phenotype. Abbreviation: WT, wild-type. With the exception of 1 maternal sample with an altered HBsAg epitope phenotype which harbored a T143L amino acid change in the “a” determinant, all 41 remaining maternal samples displayed a WT HBsAg epitope phenotype (Figure 4). HBV Immunoprophylaxis Uptake Prophylaxis data were available for 55 of the 69 infants. Forty-seven had received HBIG and completed the full immunization course of 4 doses of vaccine. Of the remaining 8 infants, 2 did not receive HBIG and all but 1 had documented receipt of 3 doses of vaccine. Sequence and HBsAg epitope phenotype data were available for 46 of the 47 viruses from infants with complete immunization. Seventeen viruses harbored changes in the “a” determinant, 11 of which displayed phenotypically altered HBsAg (Table 1). The remaining 29 viruses carried WT sequences, all of which expressed a WT epitope phenotype. Data for the 8 viruses from infants with incomplete immunization showed a similar distribution. Three harbored amino acid changes in the “a” determinant but displayed a WT epitope phenotype. Five carried WT sequences and displayed WT epitope phenotype. Virological Factors in Mothers Who Transmitted and Those Who Did Not No difference in the genotype distribution was seen between viruses from those mothers who transmitted virus to their infants compared to those who did not. Although the majority of samples from both maternal groups harbored WT sequence virus, there was a difference in distribution of amino acid changes observed in mothers who transmitted HBV vs those who did not (Figure 1B). Amino acid changes in the “a” determinant were noted in 3 of 42 (7%) and 7 of 21 (33%) of the transmitters and nontransmitting mothers, respectively (Figure 1B). Only a single phenotypically abrogated virus was identified by HBsAg epitope phenotyping and this was in a transmitting mother whose virus carried an “a” determinant single-nucleotide polymorphism T143L; all remaining 9 “a” determinant sequence variations in any mother were serologically silent. A comparison of HBV DNA viral loads between the 2 maternal groups, however, showed that the transmitter group had a 30-fold higher median viral load (8.64 log10 IU/mL; interquartile range [IQR], 7.80–9.00; range, 1.38–9.92 log10 IU/mL) compared with the group of nontransmitters (7 log10 IU/mL; IQR, 5.10–7.80; range, 3.39–9.35 log10 IU/mL). DISCUSSION Follow-up of infants born to the specific group of high-transmission-risk mothers has previously shown that up to 5% of their children become persistently infected despite a high uptake of perinatal HBIG and vaccine [7, 22]. Understanding why breakthrough infections occur when interventions are implemented correctly is essential to inform whether, and if so how, management of the HBV affected pregnancies should be altered. Surveillance implemented by Public Health England provides an invaluable national platform by which to evaluate the effectiveness of current national strategies aimed at reducing MTCT of HBV. Assuming that adequate and timely prophylaxis is provided to infants born to HBV-infected mothers, 2 aligned hypotheses can be invoked to explain continuing perinatal transmission. The first is that the maternal virus escapes from both passively acquired anti-HBs and the developing neonatal response to vaccine through being innately different and not susceptible to antibody blockage, as would be the case of an infant acquiring a G145R variant directly from the mother. The second is that under the immunological pressure of both HBIG and vaccine, the infant virus escapes through the acquisition of sequence changes which render it ab initio to escape antibody blockade. It is entirely possible that both hypotheses apply in the maternal/infant interface and the virus that escapes may indeed have been present in the maternal viral quasispecies but as a minor population, undetectable by consensus sequencing. The samples from 69 HBV-infected infants identified between 2003 and 2015 included in this study probably represent less than half of the expected number of breakthrough infections leading to viral persistence in England and Wales over this period. The broad genotype distribution in the infants reflects the wide diversity of the HBV-infected population in the United Kingdom [20]. Data from Japan have suggested an association between genotype C and transmission when compared to other genotypes [23]. These findings have not been supported by others [16], and our data showed no differences in genotype distribution between those mothers who transmitted HBV to their infants and those that did not. As expected, most of the infected children had detectable plasma HBeAg, displaying high viral loads and having the future potential for onward transmission. These vaccine failures may be facilitated by factors in the host, the virus, or both. A primary failure of an adequate response to the vaccine in the neonate is plausible. A previous study described an absence of a detectable response to HBV vaccine in 5% of infants, which was associated with premature birth and suboptimal timing of immunization doses [14]. However, consistent with earlier studies, the majority of infants in our study received HBIG and a complete vaccine course and therefore it seems unlikely that a poor response would have been a major issue in our cohort [22]. The absence of detectable anti-HBs in the majority of infants is predictable as both passively acquired and vaccine-induced anti-HBs would be removed by the saturating quantity of infant-derived HBsAg and cannot be invoked to indicate primary vaccine unresponsiveness. Preexisting intrauterine infection resulting from transplacental transmission could also account for an unsuccessful outcome following postnatal immunization in this setting. Although rates of intrauterine infection of approximately 5% have been previously reported [12], well-structured studies are needed to better define the role of in utero transmission in HBV “breakthrough” infections. In addition to infants who develop persistent infection, it is estimated that approximately 25% of children born to HBV-infectious mothers develop anti-HBc, suggesting that many infections are cleared under the influence of active/passive postnatal immunization. What are currently termed as breakthrough infections might better be considered as failures to clear infection, thus potentially providing a broader view on the processes involved in viral selection during MTCT of HBV. Twenty-five of the 68 HBV-infected infants harbored viruses carrying amino acid changes in the “a” determinant, the majority of which clustered in the second loop. Epitope phenotyping data indicated that just over half of these viruses have altered HBsAg antigenicity, suggesting immune escape from the vaccine response. What influences selection of these specific variants is not clear. There was no convincing evidence that variations in the maternal virus predisposed for the selection of HBsAg mutants in their respective infants. Indeed, the majority of maternal viruses were genetically and phenotypically WT across the HBsAg. In the 3 instances where the maternal virus harbored an amino acid change in the “a” determinant, this was always transmitted. Paradoxically, amino acid changes were noted more commonly in those mothers who did not transmit their virus compared to those who did transmit to their infants, perhaps scarring from an increasingly suppressive cytotoxic T cell (CTL) response, evidenced by their lower HBV DNA levels. The distribution of these changes across the surface gene was different; viruses from the nontransmitting mothers were more likely to carry amino acid changes in the first loop of the “a” determinant and the Cʹ terminal domain (Figure 1B). It is plausible that selection in the neonate leading to HBsAg sequence and phenotypic change could be influenced by anti-HBs in the neonate generated in response to the vaccine, or passively acquired from HBIG. It is intriguing that 3 of the 12 infants whose virus displayed an altered HBsAg epitope phenotype had a detectable anti-HBs response, compared with 3 of 48 samples with a WT epitope phenotype. It is well described that HBIG and vaccine pressure will select for viruses with altered HBsAg antigenicity [15, 24–31] and also recognized that coexistence of anti-HBs with antigenemia is an indicator for variant HBsAg. In the absence of a control group of infants who had not been immunized, it is difficult to define the precise role of neonatal immunization in driving vaccine escape and to balance this against the likely transmission rate in the absence of this intervention. If, as we have seen in this small group of infants, the acquisition of “a” determinant mutations was as common in mother-to-child transmission globally, one would expect to have commonly observed such mutations in populations where HBV MTCT has previously not been subjected to immune prophylaxis. Given that MTCT produces the next generation of persistent infections, changes acquired at the time of transmission would be expected to persist. Clearly all expressed proteins of HBV, including HBsAg, remain targets for the immune response. Thus it would not be surprising if vaccine-induced antibody and a CTL response were acting in concert on the surface gene in the infected infant. This would lead both to selection of phenotypically altered HBsAg and to selection of phenotypically silent changes perhaps representing CTL escape, both of which we see in infants. Whatever role the immune system may play at this time, the most important maternal factor in our study was the viral load of the mother. We accept that as the maternal samples in this investigation were not always at the optimal time, there remains uncertainty over the maternal viral load at the time of delivery. However, our data are supported by other previously published reports [25–27]. A higher maternal viral load may also make intrauterine infections more likely [8, 9, 12] and, in these cases, even an optimally delivered HBIG and vaccine intervention may be ineffective. Antiviral treatment during pregnancy decreases maternal HBV DNA levels and has been shown to reduce the occurrence of MTCT of HBV when used with standard interventions [32–35]. In England, it is currently recommended that HBV-infected pregnant women with DNA levels of >7 log10 IU/mL are treated in the third trimester of pregnancy. The resulting reduction in viral load during the third trimester is likely to lower the risk of perinatal transmission and potentially also intrauterine infections. Earlier intervention with antivirals could further reduce MTCT of HBV as has been demonstrated by the use of early administration of telbivudine [35]. Studies from across Europe have demonstrated the success of HBV programs in reducing HBsAg prevalence rates; however there remain few data on breakthrough infections and why these occur. The PHE-led surveillance system has suggested that HBsAg mutants are selected for and some of these mutations alter the antigenicity of the HBsAg protein. There are limited data on the disease progression and outcome in patients infected with viruses carrying these escape variations and on the onward transmission and potential for further acquisition of these variations (as was seen in 1 infant). It is important to continue to monitor these mutations with the aim of understanding their impact on existing and future immunization programs. Vaccine-induced immunity is not sterilizing, and repeated passing of variant viruses through future human generations may lead to accumulation of an altered genetic background of HBV in the dwindling population of persistently infected persons. With the introduction of universal infant immunization in 2017, it is critical that timely intervention with vaccine to protect all newborn infants, effective surveillance programs to determine the outcome of HBV-affected pregnancies, and the appropriate use of antiviral treatment in infected mothers to reduce viral burden at the time of delivery are maintained. Notes Acknowledgments. The authors thank colleagues both in PHE and in the various National Health Service Trusts who facilitated this study by assisting in the identification of mothers and children and in the acquisition of relevant patient samples. Disclaimer. The Immunisation, Hepatitis and Blood Safety Department has provided pharmaceutical manufacturers with postmarketing surveillance reports (none related to hepatitis B), which the companies are required to submit to the UK Licensing authority in compliance with their risk management strategy. A cost recovery charge is made for these reports. Financial support. This work was supported by PHE and its predecessor, the Health Protection Agency. Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Presented in part: Annual Clinical Virology Network/Society of General Microbiology Conference, Birmingham, United Kingdom, 31 March–1 April 2015; European Society for Clinical Virology, Edinburgh, United Kingdom, 9–12 September 2015; International Meeting on Molecular Biology of Hepatitis B Viruses, Bad Nauheim, Germany, 4–8 October 2015. References 1. Liaw YF, Chu CM. Hepatitis B virus infection. Lancet  2009; 373: 582– 92. Google Scholar CrossRef Search ADS PubMed  2. World Health Organization, Western Pacific Region. Introduction of hepatitis B vaccine into childhood immunisation services. Management guidelines including information for health workers and parents . http://www.wpro.who.int/hepatitis/whovb0131.pdf?ua=1. Accessed 25 September 2015. 3. Wait S, Chen DS. Towards the eradication of hepatitis B in Taiwan. Kaohsiung J Med Sci  2012; 28: 1– 9. Google Scholar CrossRef Search ADS PubMed  4. Public Health England. Hepatitis B: the green book, chapter 18 . https://www.gov.uk/government/publications/hepatitis-b-the-green-book-chapter-18. Accessed 17 July 2017. 5. UK National Institute for Health Care Excellence. Hepatitis B (chronic): diagnosis and management of chronic hepatitis B in children, young people and adults . https://www.nice.org.uk/Guidance/cg165. Accessed 25 September 2015. 6. Public Health England. National antenatal infections screening monitoring. Available at: https://www.gov.uk/government/publications/national-antenatal-infections-screening-monitoring-annual-data-tables. Accessed 25 September 2015. 7. Department of Health. Cover of vaccination evaluated rapidly (COVER) programme 2014 to 2015: quarterly data. https://www.gov.uk/government/statistics/cover-of-vaccination-evaluated-rapidly-cover-programme-2014-to-2015-quarterly-data . Accessed 25 September 2015. 8. Chen Y, Wang L, Xu Y, et al.   Role of maternal viremia and placental infection in hepatitis B virus intrauterine transmission. Microbes Infect  2013; 15: 409– 15. Google Scholar CrossRef Search ADS PubMed  9. Yin YZ, Chen XW, Li XM, Hou HY, Shi ZJ. Intrauterine HBV infection: risk factors and impact of HBV DNA [in Chinese]. Nan Fang Yi Ke Da Xue Xue Bao  2006; 26: 1452– 4. Google Scholar PubMed  10. Yin YZ, Zhang J, Wu LL, Zhou J, Zhang PZ, Zhou SS. Development of strategies for screening, predicting, and diagnosing intrauterine HBV infection in infants born to HBsAg positive mothers. J Med Virol  2013; 85: 1705– 11. Google Scholar CrossRef Search ADS PubMed  11. Zhang SL, Yue YF, Bai GQ, Shi L, Jiang H. Mechanism of intrauterine infection of hepatitis B virus. World J Gastroenterol  2004; 10: 437– 8. Google Scholar CrossRef Search ADS PubMed  12. Zhang Z, Li A, Xiao X. Risk factors for intrauterine infection with hepatitis B virus. Int J Gynaecol Obstet  2014; 125: 158– 61. Google Scholar CrossRef Search ADS PubMed  13. Zhu YY, Mao YZ, Wu WL, Cai QX, Lin XH. Does hepatitis B virus prenatal transmission result in postnatal immunoprophylaxis failure? Clin Vaccine Immunol  2010; 17: 1836– 41. Google Scholar CrossRef Search ADS PubMed  14. Ko SC, Schillie SF, Walker T, et al.   Hepatitis B vaccine response among infants born to hepatitis B surface antigen-positive women. Vaccine  2014; 32: 2127– 33. Google Scholar CrossRef Search ADS PubMed  15. Ho M, Mau Y, Lu C, et al.   Patterns of circulating hepatitis B surface antigen variants among vaccinated children born to hepatitis B surface antigen carrier and non-carrier mothers. A population-based comparative study. 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Liu Y, Wang M, Yao S, et al.   Efficacy and safety of telbivudine in different trimesters of pregnancy with high viremia for interrupting perinatal transmission of hepatitis B virus. Hepatol Res  2016; 46: E181– 8. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Hepatitis B Virus Immunization and Neonatal Acquisition of Persistent Infection in England and Wales

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
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0022-1899
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1537-6613
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10.1093/infdis/jiy209
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Abstract

Abstract Background It is believed that between 2% and 5% of infants born to hepatitis B virus (HBV)–infected mothers at a high risk of perinatal transmission will become persistently infected despite immunization starting at birth. We investigated factors associated with breakthrough infections. Methods Sixty-nine samples from HBV-infected infants born between 2003 and 2015 were tested for HBV serological and molecular markers. Sequencing and epitope phenotyping were used to investigate alterations in hepatitis B surface antigen (HBsAg) sequence and antigenicity in infants and in mothers known to have transmitted and not to have transmitted virus to their infants. Results Vaccine/hepatitis B immune globulin uptake was complete in the majority of HBV-infected infants. A minority (8 [12%]) had detectable plasma antibody to HBsAg at 12 months. Twenty-five of 68 (37%) infants harbored a virus with amino acid changes in the HBsAg “a” determinant, of which 13 displayed altered HBsAg antigenicity. Viral load was 30-fold higher in maternal samples from those who transmitted. Conclusions Our data provide evidence to suggest that immune selection drives change at mother–infant transmission, resulting in the alteration of HBsAg antigenicity. These changes may play a role in immunization failure, but other factors including viral load may be more important. Continued monitoring of vaccine efficacy is essential. HBV, immunization, HBsAg mutants, vaccine escape, viral load, infected infants Mother-to-child transmission (MTCT) of hepatitis B virus (HBV) represents an important mechanism for the generation and maintenance of persistent infections within populations. As well as being a reservoir for onward transmission, persistently infected individuals remain at high risk for the development of cirrhosis, liver failure, and/or hepatocellular carcinoma [1]. The World Health Organization’s recommendation for national HBV immunization programs worldwide is aimed at eliminating perinatal transmission through active HBV immunization within 24 hours of birth with the addition of hepatitis B immune globulin (HBIG) where there is a high risk of transmission [2]. Optimally implemented, these actions have had a dramatic impact on reducing MTCT, with an 87% reduction observed in the seroprevalence of hepatitis B surface antigen (HBsAg) in children in countries of previous high endemicity [3]. In the United Kingdom, a selective neonatal HBV immunization program has been adopted and will continue after introduction of universal HBV immunization in 2017. Since 2000, all women are tested antenatally for HBsAg and infants born to HBV-infected mothers are immunized at 0, 1, 2, and 12 months of age. HBIG is also offered in addition to vaccine (passive–active immunization) for infants born to (1) high-transmission-risk mothers (classified as having detectable plasma hepatitis B e antigen [HBeAg], or an HBV DNA level >6 log10 IU/mL); (2) mothers who have an acute HBV infection during pregnancy; and (3) mothers whose booking blood samples are undetectable for both “e” markers. HBIG will also be given to low-birth-weight infants (<1500 g) born to HBV-infected mothers regardless of maternal viral load or “e” status [4]. The National Institute for Health Care Excellence guidelines also recommend antiviral treatment during the third trimester where HBV DNA levels exceed 7 log10 IU/mL [5]. It is recommended that infants born to HBV-infected mothers are tested at 12 months of age for evidence of current HBV infection. There are approximately 3000 pregnancies in HBV-infected women each year in England [6], of which 5%–10% will be in women classified as having a high perinatal transmission risk [7]. The program of National Enhanced Surveillance of High-Risk Infants born to high-transmission-risk mothers shows HBIG administration and vaccine uptake rates for the first 3 doses to be >90%, marginally better than the 90% uptake of 3 doses in all infants at HBV risk [7]. Even though 2%–5% of infants born to high-transmission-risk mothers in England are predicted to acquire persistent infection despite receiving passive–active immunoprophylaxis, only half of the high-risk infants are reported as being tested at 12 months for evidence of HBV infection [7]. A number of reasons have been postulated for these transmissions including possible intrauterine infection [8–14], incomplete or delayed prophylaxis, failure to respond to vaccine [14], and specific virological factors including vaccine escape variants [15–18]. The latter have been reported occasionally, but their overall significance in vaccine failure is not well characterized. As part of the National Enhanced Surveillance of High-Risk Infants program, samples from these infants at 12 months of age are sent to Public Health England (PHE) and screened for evidence of current HBV infection. Comprehensive serological and molecular characterization, together with HBsAg epitope phenotyping, was undertaken on samples from infants found to be HBV infected and, where available, on samples from their mothers. We aimed to inform on why breakthrough infections are occurring and whether additional measures need to be implemented to reduce MTCT of HBV in the United Kingdom. METHODS Infant Samples Oral fluid, venous, or dried blood spot samples from infants aged 12 months are received at PHE Colindale for testing for HBsAg and antibody to hepatitis B core antigen (anti-HBc). For infants with reactive samples tested at PHE and for HBV-infected infants reported to PHE after local testing, further blood samples are requested for confirmation. Venous samples were available from a total of 69 HBV-infected infants identified between 2003 and 2015. Sixty of the infants were aged 12 months; 9 were aged between 3 and 12 years. Infant Immunization/HBIG Uptake Data The National Enhanced Surveillance collects maternal demographic data before birth, and follows up high-risk infants to 12 months to document the issue and administration of HBIG and vaccine at birth and the first year of life and to determine evidence of current HBV infection. Maternal Samples Samples were obtained usually during pregnancy, just after delivery or as close as possible to the pregnancy from 45 of the 69 mothers who transmitted infection (transmitters). Twenty-one additional samples, held at PHE Colindale, from HBV-infected mothers whose samples also contained HBeAg but who did not transmit HBV to their infants (nontransmitters), were available as a comparator group. Serology Samples from the 69 HBV-infected infants were tested for HBeAg, antibody to HBeAg (anti-HBe), and antibody to HBsAg (anti-HBs) using commercial assays (DiaSorin, Dartford, Kent, United Kingdom). DNA Studies Nucleic acid was extracted from 200 µL of maternal and infant plasma/serum using the MagNA Pure 96 DNA viral small volume kit (Roche, West Sussex, United Kingdom). HBV DNA viral load was quantified as previously described [19]. Genotype determination with additional analysis for inferred amino acid changes across HBsAg was undertaken as previously described [20]. In brief, alignments of 1800 sequences representing described HBV genotypes/subgenotypes were obtained from either in-house–generated sequences or from GenBank. The sequences were checked both visually and by position-specific scoring matrix to identify intragenotypic motifs. Amino acid alignments for wild-type (WT) consensus genotype-specific sequences were created for the HBsAg region and used to identify mutations. Genotype determination was based on clustering within phylogenetic trees generated using the 1800 representative HBsAg nucleotide sequences. HBsAg Epitope Ex Vivo Phenotyping The antigenic profile of HBsAg in a sample was determined by epitope mapping using 4 monoclonal antibodies (mAbs) directed against different epitopes of the “a” determinant region. The mAb P2D3 recognizes a first-loop linear epitope between codons 121 and 129; mAbs H3F5, D2H5, and HB04 were raised against a mixture of ad/ay HBsAg and recognize conformational epitopes. Tentative mapping data indicated that H3F5 bound to epitopes between codons 131 and 142, whereas D2H5 and HB04 recognized similar epitopes in the second loop between codons 142 and 147. The Luminex platform (Bio-Rad Laboratories) provides a method of interrogating protein–antibody interactions on discrete and identifiable bead solid phases and was used to measure the interaction of plasma HBsAg with the 4 mAbs, each on an individually identifiable solid phase as previously described [21]. Poor or loss of reactivity against 1 of more mAbs indicated an alteration of epitope phenotype. RESULTS Characterization of the Infant Infection HBV Markers Small sample volumes meant that HBV marker testing could not be completed on all infant samples. The majority (57/59 [96%]) of available infants’ samples were HBeAg positive, 1 was anti-HBe positive, and 1 was negative for both HBeAg and anti-HBe. The majority of available samples (58/66 [88%]) were unreactive for anti-HBs (<10 IU/L). In the 8 reactive samples, anti-HBs ranged from 26.6 to 235 IU/L. HBV DNA quantification was undertaken in 64 samples; HBV DNA levels ranged from 3.55 to 9.78 log10 IU/mL (median, 8.582 [standard deviation, 1.242] log10 IU/mL). The majority (62/64 [97%]) of the infants had a viral load >5 log10 IU/mL. HBsAg Sequence and Epitope Phenotype Analysis There was sufficient plasma for sequence analysis on 68 of 69 infant samples and for epitope phenotyping on 60 samples. The majority of the infants were infected with genotype C (n = 23 [34%]) and D viruses (n = 24 [35%]), with the remainder harboring genotype A (n = 5 [7%]), B (n = 11 [16%]), E (n = 4 [6%]), and CD recombinant (n = 1 [2%]) viruses. All infant genotypes detected were concordant with that of the available paired maternal samples. Sequence analysis indicated that 35 of the 68 (51%) infants harbored viruses with amino acid changes across the HBsAg, of which the majority (25/35 [71%]) had codon changes which lay within the “a” determinant region, between codons 120 and 150 (Figures 1A and 2). Twenty-two of the 25 were available for epitope phenotyping, of which more than half (13/22 [59%]) displayed altered HBsAg antigenicity (Table 1); the remaining 9 displayed a WT HBsAg epitope phenotype. Of the 10 viruses bearing HBsAg codon changes outside the “a” determinant, 9 were available for epitope phenotyping and all displayed a WT HBsAg epitope phenotype. Thirty-three viruses carried no codon changes across HBsAg, 29 samples were available for epitope phenotyping, and all exhibited WT antigenicity (Figure 2). Figure 1. View largeDownload slide Sites of codon change across the entire hepatitis B surface antigen (HBsAg) protein. A, “Hot spot” across HBsAg representing positions of amino acid (AA) changes observed in virus sequences from infants (solid bars) and also indicating where identical amino acid changes in the infant (cross-hatched bars) were also observed in the maternal sample. B, “Hot spot” across HBsAg representing positions of amino acid changes observed in virus sequences from mothers who transmitted (solid bars) vs those who did not transmit (cross-hatched bars) hepatitis B virus to their infants. Figure 1. View largeDownload slide Sites of codon change across the entire hepatitis B surface antigen (HBsAg) protein. A, “Hot spot” across HBsAg representing positions of amino acid (AA) changes observed in virus sequences from infants (solid bars) and also indicating where identical amino acid changes in the infant (cross-hatched bars) were also observed in the maternal sample. B, “Hot spot” across HBsAg representing positions of amino acid changes observed in virus sequences from mothers who transmitted (solid bars) vs those who did not transmit (cross-hatched bars) hepatitis B virus to their infants. Figure 2. View largeDownload slide Hepatitis B surface antigen sequence and epitope phenotype data linkage in 68 infants. Inferred amino acid changes displayed as within or outside the “a” determinant domain. Abbreviations: HBsAg, hepatitis B surface antigen; WT, wild-type sequence. Figure 2. View largeDownload slide Hepatitis B surface antigen sequence and epitope phenotype data linkage in 68 infants. Inferred amino acid changes displayed as within or outside the “a” determinant domain. Abbreviations: HBsAg, hepatitis B surface antigen; WT, wild-type sequence. Table 1. Linked Hepatitis B Surface Antigen Sequence and Epitope Phenotyping Data of 22 Infants’ Samples That Had an “a” Determinant Mutation Genotype  Sequence Data  HBsAg Epitope Phenotyping Data (Expressed as Proportional Percentage)  mAb P2D3  mAb D2H5  mAb H3F5  mAb HB04  B  Wild-type  20.36  24.96  23.41  31.27  B  G145R  68.78  0.27  18.80  12.15  B  G145A  27.68  22.28  24.71  25.33  B  M133L  22.31  22.55  22.96  32.19  C  Wild-type  25.23  23.33  20.84  30.60  C  S132F/Y, G145R  54.63  1.01  29.97  14.38  C  G145G/R  30.42  18.01  21.54  30.03  C  D144D/A  33.63  15.53  23.29  27.55  C  I126F, G145R  46.35  1.34  38.99  13.32  C  G145A  25.95  21.47  25.08  27.50  C  P62P/L, G145G/A, V184A  18.09  19.53  18.73  43.64  C  I128T  23.86  24.36  23.55  28.24  C  T131A  25.71  24.23  23.58  26.48  D  Wild-type  20.63  21.75  25.09  32.53  D  P142L, G145R  43.46  6.68  43.32  6.54  D  G145R  29.78  16.07  29.23  24.91  D  D144A  27.44  15.51  29.11  27.94  D  T143L  31.12  30.89  31.08  6.91  D  G145G/R  25.13  19.20  27.05  28.62  D  D144D/A, G145R, V184A  29.45  19.20  27.29  24.07  D  D144A  29.65  17.97  30.18  22.21  D  P120S  16.81  21.75  20.36  41.09  D  D144E  27.08  23.04  25.87  24.00  D  D144D/E, S174S/N, I208I/T, L213L/I  26.85  24.35  24.88  25.12  D  P120T, D144A, S174N  27.40  21.64  24.90  26.06  Genotype  Sequence Data  HBsAg Epitope Phenotyping Data (Expressed as Proportional Percentage)  mAb P2D3  mAb D2H5  mAb H3F5  mAb HB04  B  Wild-type  20.36  24.96  23.41  31.27  B  G145R  68.78  0.27  18.80  12.15  B  G145A  27.68  22.28  24.71  25.33  B  M133L  22.31  22.55  22.96  32.19  C  Wild-type  25.23  23.33  20.84  30.60  C  S132F/Y, G145R  54.63  1.01  29.97  14.38  C  G145G/R  30.42  18.01  21.54  30.03  C  D144D/A  33.63  15.53  23.29  27.55  C  I126F, G145R  46.35  1.34  38.99  13.32  C  G145A  25.95  21.47  25.08  27.50  C  P62P/L, G145G/A, V184A  18.09  19.53  18.73  43.64  C  I128T  23.86  24.36  23.55  28.24  C  T131A  25.71  24.23  23.58  26.48  D  Wild-type  20.63  21.75  25.09  32.53  D  P142L, G145R  43.46  6.68  43.32  6.54  D  G145R  29.78  16.07  29.23  24.91  D  D144A  27.44  15.51  29.11  27.94  D  T143L  31.12  30.89  31.08  6.91  D  G145G/R  25.13  19.20  27.05  28.62  D  D144D/A, G145R, V184A  29.45  19.20  27.29  24.07  D  D144A  29.65  17.97  30.18  22.21  D  P120S  16.81  21.75  20.36  41.09  D  D144E  27.08  23.04  25.87  24.00  D  D144D/E, S174S/N, I208I/T, L213L/I  26.85  24.35  24.88  25.12  D  P120T, D144A, S174N  27.40  21.64  24.90  26.06  Amino acid changes in HBsAg sequence, compared against genotype consensus listed by codon. The reactivity against the 4 mAbs is expressed as the percentage reactivity of each mAb as part of the total reactivity for that sample. Alterations in HBsAg antigenicity, measured as epitope loss against a range of monoclonal antibodies, are shown in bold type and displayed for each monoclonal antibody. The observed reactivity of wild-type viruses is also given. Abbreviations: HBsAg, hepatitis B surface antigen; mAb, monoclonal antibody. View Large Table 1. Linked Hepatitis B Surface Antigen Sequence and Epitope Phenotyping Data of 22 Infants’ Samples That Had an “a” Determinant Mutation Genotype  Sequence Data  HBsAg Epitope Phenotyping Data (Expressed as Proportional Percentage)  mAb P2D3  mAb D2H5  mAb H3F5  mAb HB04  B  Wild-type  20.36  24.96  23.41  31.27  B  G145R  68.78  0.27  18.80  12.15  B  G145A  27.68  22.28  24.71  25.33  B  M133L  22.31  22.55  22.96  32.19  C  Wild-type  25.23  23.33  20.84  30.60  C  S132F/Y, G145R  54.63  1.01  29.97  14.38  C  G145G/R  30.42  18.01  21.54  30.03  C  D144D/A  33.63  15.53  23.29  27.55  C  I126F, G145R  46.35  1.34  38.99  13.32  C  G145A  25.95  21.47  25.08  27.50  C  P62P/L, G145G/A, V184A  18.09  19.53  18.73  43.64  C  I128T  23.86  24.36  23.55  28.24  C  T131A  25.71  24.23  23.58  26.48  D  Wild-type  20.63  21.75  25.09  32.53  D  P142L, G145R  43.46  6.68  43.32  6.54  D  G145R  29.78  16.07  29.23  24.91  D  D144A  27.44  15.51  29.11  27.94  D  T143L  31.12  30.89  31.08  6.91  D  G145G/R  25.13  19.20  27.05  28.62  D  D144D/A, G145R, V184A  29.45  19.20  27.29  24.07  D  D144A  29.65  17.97  30.18  22.21  D  P120S  16.81  21.75  20.36  41.09  D  D144E  27.08  23.04  25.87  24.00  D  D144D/E, S174S/N, I208I/T, L213L/I  26.85  24.35  24.88  25.12  D  P120T, D144A, S174N  27.40  21.64  24.90  26.06  Genotype  Sequence Data  HBsAg Epitope Phenotyping Data (Expressed as Proportional Percentage)  mAb P2D3  mAb D2H5  mAb H3F5  mAb HB04  B  Wild-type  20.36  24.96  23.41  31.27  B  G145R  68.78  0.27  18.80  12.15  B  G145A  27.68  22.28  24.71  25.33  B  M133L  22.31  22.55  22.96  32.19  C  Wild-type  25.23  23.33  20.84  30.60  C  S132F/Y, G145R  54.63  1.01  29.97  14.38  C  G145G/R  30.42  18.01  21.54  30.03  C  D144D/A  33.63  15.53  23.29  27.55  C  I126F, G145R  46.35  1.34  38.99  13.32  C  G145A  25.95  21.47  25.08  27.50  C  P62P/L, G145G/A, V184A  18.09  19.53  18.73  43.64  C  I128T  23.86  24.36  23.55  28.24  C  T131A  25.71  24.23  23.58  26.48  D  Wild-type  20.63  21.75  25.09  32.53  D  P142L, G145R  43.46  6.68  43.32  6.54  D  G145R  29.78  16.07  29.23  24.91  D  D144A  27.44  15.51  29.11  27.94  D  T143L  31.12  30.89  31.08  6.91  D  G145G/R  25.13  19.20  27.05  28.62  D  D144D/A, G145R, V184A  29.45  19.20  27.29  24.07  D  D144A  29.65  17.97  30.18  22.21  D  P120S  16.81  21.75  20.36  41.09  D  D144E  27.08  23.04  25.87  24.00  D  D144D/E, S174S/N, I208I/T, L213L/I  26.85  24.35  24.88  25.12  D  P120T, D144A, S174N  27.40  21.64  24.90  26.06  Amino acid changes in HBsAg sequence, compared against genotype consensus listed by codon. The reactivity against the 4 mAbs is expressed as the percentage reactivity of each mAb as part of the total reactivity for that sample. Alterations in HBsAg antigenicity, measured as epitope loss against a range of monoclonal antibodies, are shown in bold type and displayed for each monoclonal antibody. The observed reactivity of wild-type viruses is also given. Abbreviations: HBsAg, hepatitis B surface antigen; mAb, monoclonal antibody. View Large Of the 13 infants whose viruses displayed an altered HBsAg epitope phenotype, 3 (23%) had a detectable anti-HBs response (>10 IU/L; range, 26–235 IU/L); in contrast, only 3 of the 47 (6%) infants with a WT HBsAg epitope phenotype had a detectable anti-HBs response. Mother-and-Infant Pairs Viral Evolution Samples were available from only 42 mother-and-infant pairs (Figure 3). Analysis of the matched sequences indicated some concordance between the maternal and infant viruses but also notable differences. Where 3 maternal viruses harbored an amino acid change in the “a” determinant, this was transmitted (Figures 3 and 4). In 1 case, the infant’s virus acquired an additional amino acid change in the “a” determinant (Figure 3). Ab initio selection of variants in the “a” determinant was observed in 14 infants, 9 of 26 (35%) of infants born to mothers harboring a WT HBsAg virus and 5 of 13 (38%) of infants born to mothers with a virus harboring amino acid changes outside of the “a” determinant (Figure 4). Figure 3. View largeDownload slide Hepatitis B surface antigen sequence and epitope phenotype data linkage in 42 mother–infant pairs. Indication of those infants among the original 68 who had paired maternal samples and the linked maternal sample sequence. *Same sequence in maternal and infant samples. **One maternal sample with amino acid changes in the “a” where the corresponding infant sample has an additional amino acid change. Inferred amino acid changes displayed as within or outside the “a” determinant domain. Abbreviations: HBsAg, hepatitis B surface antigen; WT, wild-type sequence. Figure 3. View largeDownload slide Hepatitis B surface antigen sequence and epitope phenotype data linkage in 42 mother–infant pairs. Indication of those infants among the original 68 who had paired maternal samples and the linked maternal sample sequence. *Same sequence in maternal and infant samples. **One maternal sample with amino acid changes in the “a” where the corresponding infant sample has an additional amino acid change. Inferred amino acid changes displayed as within or outside the “a” determinant domain. Abbreviations: HBsAg, hepatitis B surface antigen; WT, wild-type sequence. Figure 4. View largeDownload slide Role of maternal virus hepatitis B surface antigen (HBsAg) sequence and epitope phenotype in the selection of viruses in infants. ***Only 2 samples available for epitope phenotyping. Linkage between the maternal virus and infant virus. Maternal sequence: inferred amino acid changes displayed as within or outside the “a” determinant domain, maternal HBsAg epitope phenotype; and the linked infant sequence: inferred amino acid changes displayed as “Out.” (outside “a” determinant) or “In” (within the “a” determinant) and determined HBsAg epitope phenotype. Abbreviation: WT, wild-type. Figure 4. View largeDownload slide Role of maternal virus hepatitis B surface antigen (HBsAg) sequence and epitope phenotype in the selection of viruses in infants. ***Only 2 samples available for epitope phenotyping. Linkage between the maternal virus and infant virus. Maternal sequence: inferred amino acid changes displayed as within or outside the “a” determinant domain, maternal HBsAg epitope phenotype; and the linked infant sequence: inferred amino acid changes displayed as “Out.” (outside “a” determinant) or “In” (within the “a” determinant) and determined HBsAg epitope phenotype. Abbreviation: WT, wild-type. With the exception of 1 maternal sample with an altered HBsAg epitope phenotype which harbored a T143L amino acid change in the “a” determinant, all 41 remaining maternal samples displayed a WT HBsAg epitope phenotype (Figure 4). HBV Immunoprophylaxis Uptake Prophylaxis data were available for 55 of the 69 infants. Forty-seven had received HBIG and completed the full immunization course of 4 doses of vaccine. Of the remaining 8 infants, 2 did not receive HBIG and all but 1 had documented receipt of 3 doses of vaccine. Sequence and HBsAg epitope phenotype data were available for 46 of the 47 viruses from infants with complete immunization. Seventeen viruses harbored changes in the “a” determinant, 11 of which displayed phenotypically altered HBsAg (Table 1). The remaining 29 viruses carried WT sequences, all of which expressed a WT epitope phenotype. Data for the 8 viruses from infants with incomplete immunization showed a similar distribution. Three harbored amino acid changes in the “a” determinant but displayed a WT epitope phenotype. Five carried WT sequences and displayed WT epitope phenotype. Virological Factors in Mothers Who Transmitted and Those Who Did Not No difference in the genotype distribution was seen between viruses from those mothers who transmitted virus to their infants compared to those who did not. Although the majority of samples from both maternal groups harbored WT sequence virus, there was a difference in distribution of amino acid changes observed in mothers who transmitted HBV vs those who did not (Figure 1B). Amino acid changes in the “a” determinant were noted in 3 of 42 (7%) and 7 of 21 (33%) of the transmitters and nontransmitting mothers, respectively (Figure 1B). Only a single phenotypically abrogated virus was identified by HBsAg epitope phenotyping and this was in a transmitting mother whose virus carried an “a” determinant single-nucleotide polymorphism T143L; all remaining 9 “a” determinant sequence variations in any mother were serologically silent. A comparison of HBV DNA viral loads between the 2 maternal groups, however, showed that the transmitter group had a 30-fold higher median viral load (8.64 log10 IU/mL; interquartile range [IQR], 7.80–9.00; range, 1.38–9.92 log10 IU/mL) compared with the group of nontransmitters (7 log10 IU/mL; IQR, 5.10–7.80; range, 3.39–9.35 log10 IU/mL). DISCUSSION Follow-up of infants born to the specific group of high-transmission-risk mothers has previously shown that up to 5% of their children become persistently infected despite a high uptake of perinatal HBIG and vaccine [7, 22]. Understanding why breakthrough infections occur when interventions are implemented correctly is essential to inform whether, and if so how, management of the HBV affected pregnancies should be altered. Surveillance implemented by Public Health England provides an invaluable national platform by which to evaluate the effectiveness of current national strategies aimed at reducing MTCT of HBV. Assuming that adequate and timely prophylaxis is provided to infants born to HBV-infected mothers, 2 aligned hypotheses can be invoked to explain continuing perinatal transmission. The first is that the maternal virus escapes from both passively acquired anti-HBs and the developing neonatal response to vaccine through being innately different and not susceptible to antibody blockage, as would be the case of an infant acquiring a G145R variant directly from the mother. The second is that under the immunological pressure of both HBIG and vaccine, the infant virus escapes through the acquisition of sequence changes which render it ab initio to escape antibody blockade. It is entirely possible that both hypotheses apply in the maternal/infant interface and the virus that escapes may indeed have been present in the maternal viral quasispecies but as a minor population, undetectable by consensus sequencing. The samples from 69 HBV-infected infants identified between 2003 and 2015 included in this study probably represent less than half of the expected number of breakthrough infections leading to viral persistence in England and Wales over this period. The broad genotype distribution in the infants reflects the wide diversity of the HBV-infected population in the United Kingdom [20]. Data from Japan have suggested an association between genotype C and transmission when compared to other genotypes [23]. These findings have not been supported by others [16], and our data showed no differences in genotype distribution between those mothers who transmitted HBV to their infants and those that did not. As expected, most of the infected children had detectable plasma HBeAg, displaying high viral loads and having the future potential for onward transmission. These vaccine failures may be facilitated by factors in the host, the virus, or both. A primary failure of an adequate response to the vaccine in the neonate is plausible. A previous study described an absence of a detectable response to HBV vaccine in 5% of infants, which was associated with premature birth and suboptimal timing of immunization doses [14]. However, consistent with earlier studies, the majority of infants in our study received HBIG and a complete vaccine course and therefore it seems unlikely that a poor response would have been a major issue in our cohort [22]. The absence of detectable anti-HBs in the majority of infants is predictable as both passively acquired and vaccine-induced anti-HBs would be removed by the saturating quantity of infant-derived HBsAg and cannot be invoked to indicate primary vaccine unresponsiveness. Preexisting intrauterine infection resulting from transplacental transmission could also account for an unsuccessful outcome following postnatal immunization in this setting. Although rates of intrauterine infection of approximately 5% have been previously reported [12], well-structured studies are needed to better define the role of in utero transmission in HBV “breakthrough” infections. In addition to infants who develop persistent infection, it is estimated that approximately 25% of children born to HBV-infectious mothers develop anti-HBc, suggesting that many infections are cleared under the influence of active/passive postnatal immunization. What are currently termed as breakthrough infections might better be considered as failures to clear infection, thus potentially providing a broader view on the processes involved in viral selection during MTCT of HBV. Twenty-five of the 68 HBV-infected infants harbored viruses carrying amino acid changes in the “a” determinant, the majority of which clustered in the second loop. Epitope phenotyping data indicated that just over half of these viruses have altered HBsAg antigenicity, suggesting immune escape from the vaccine response. What influences selection of these specific variants is not clear. There was no convincing evidence that variations in the maternal virus predisposed for the selection of HBsAg mutants in their respective infants. Indeed, the majority of maternal viruses were genetically and phenotypically WT across the HBsAg. In the 3 instances where the maternal virus harbored an amino acid change in the “a” determinant, this was always transmitted. Paradoxically, amino acid changes were noted more commonly in those mothers who did not transmit their virus compared to those who did transmit to their infants, perhaps scarring from an increasingly suppressive cytotoxic T cell (CTL) response, evidenced by their lower HBV DNA levels. The distribution of these changes across the surface gene was different; viruses from the nontransmitting mothers were more likely to carry amino acid changes in the first loop of the “a” determinant and the Cʹ terminal domain (Figure 1B). It is plausible that selection in the neonate leading to HBsAg sequence and phenotypic change could be influenced by anti-HBs in the neonate generated in response to the vaccine, or passively acquired from HBIG. It is intriguing that 3 of the 12 infants whose virus displayed an altered HBsAg epitope phenotype had a detectable anti-HBs response, compared with 3 of 48 samples with a WT epitope phenotype. It is well described that HBIG and vaccine pressure will select for viruses with altered HBsAg antigenicity [15, 24–31] and also recognized that coexistence of anti-HBs with antigenemia is an indicator for variant HBsAg. In the absence of a control group of infants who had not been immunized, it is difficult to define the precise role of neonatal immunization in driving vaccine escape and to balance this against the likely transmission rate in the absence of this intervention. If, as we have seen in this small group of infants, the acquisition of “a” determinant mutations was as common in mother-to-child transmission globally, one would expect to have commonly observed such mutations in populations where HBV MTCT has previously not been subjected to immune prophylaxis. Given that MTCT produces the next generation of persistent infections, changes acquired at the time of transmission would be expected to persist. Clearly all expressed proteins of HBV, including HBsAg, remain targets for the immune response. Thus it would not be surprising if vaccine-induced antibody and a CTL response were acting in concert on the surface gene in the infected infant. This would lead both to selection of phenotypically altered HBsAg and to selection of phenotypically silent changes perhaps representing CTL escape, both of which we see in infants. Whatever role the immune system may play at this time, the most important maternal factor in our study was the viral load of the mother. We accept that as the maternal samples in this investigation were not always at the optimal time, there remains uncertainty over the maternal viral load at the time of delivery. However, our data are supported by other previously published reports [25–27]. A higher maternal viral load may also make intrauterine infections more likely [8, 9, 12] and, in these cases, even an optimally delivered HBIG and vaccine intervention may be ineffective. Antiviral treatment during pregnancy decreases maternal HBV DNA levels and has been shown to reduce the occurrence of MTCT of HBV when used with standard interventions [32–35]. In England, it is currently recommended that HBV-infected pregnant women with DNA levels of >7 log10 IU/mL are treated in the third trimester of pregnancy. The resulting reduction in viral load during the third trimester is likely to lower the risk of perinatal transmission and potentially also intrauterine infections. Earlier intervention with antivirals could further reduce MTCT of HBV as has been demonstrated by the use of early administration of telbivudine [35]. Studies from across Europe have demonstrated the success of HBV programs in reducing HBsAg prevalence rates; however there remain few data on breakthrough infections and why these occur. The PHE-led surveillance system has suggested that HBsAg mutants are selected for and some of these mutations alter the antigenicity of the HBsAg protein. There are limited data on the disease progression and outcome in patients infected with viruses carrying these escape variations and on the onward transmission and potential for further acquisition of these variations (as was seen in 1 infant). It is important to continue to monitor these mutations with the aim of understanding their impact on existing and future immunization programs. Vaccine-induced immunity is not sterilizing, and repeated passing of variant viruses through future human generations may lead to accumulation of an altered genetic background of HBV in the dwindling population of persistently infected persons. With the introduction of universal infant immunization in 2017, it is critical that timely intervention with vaccine to protect all newborn infants, effective surveillance programs to determine the outcome of HBV-affected pregnancies, and the appropriate use of antiviral treatment in infected mothers to reduce viral burden at the time of delivery are maintained. Notes Acknowledgments. The authors thank colleagues both in PHE and in the various National Health Service Trusts who facilitated this study by assisting in the identification of mothers and children and in the acquisition of relevant patient samples. Disclaimer. The Immunisation, Hepatitis and Blood Safety Department has provided pharmaceutical manufacturers with postmarketing surveillance reports (none related to hepatitis B), which the companies are required to submit to the UK Licensing authority in compliance with their risk management strategy. A cost recovery charge is made for these reports. Financial support. This work was supported by PHE and its predecessor, the Health Protection Agency. Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Presented in part: Annual Clinical Virology Network/Society of General Microbiology Conference, Birmingham, United Kingdom, 31 March–1 April 2015; European Society for Clinical Virology, Edinburgh, United Kingdom, 9–12 September 2015; International Meeting on Molecular Biology of Hepatitis B Viruses, Bad Nauheim, Germany, 4–8 October 2015. References 1. Liaw YF, Chu CM. Hepatitis B virus infection. Lancet  2009; 373: 582– 92. Google Scholar CrossRef Search ADS PubMed  2. World Health Organization, Western Pacific Region. Introduction of hepatitis B vaccine into childhood immunisation services. 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Journal

The Journal of Infectious DiseasesOxford University Press

Published: Apr 24, 2018

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