Genomic characterization of hepatitis B virus from a vaccinated child in Nigeria suggests delayed neonatal immunoprophylaxis rather than vaccine escape: a case report

Highlights

• •Whole-genome sequencing characterized hepatitis B virus from a vaccinated Nigerian child with persistent hepatitis B surface antigen positivity.
• •The isolate belonged to genotype E, the predominant hepatitis B virus genotype circulating in West Africa.
• •No vaccine escape or antiviral resistance mutations were identified.
• •Delayed birth-dose vaccination rather than vaccine escape was the most plausible explanation for infection.
• •Molecular surveillance complements the vaccination program by identifying mechanisms underlying suspected breakthrough infections.

Abstract

Background

Breakthrough hepatitis B virus (HBV) infections continue to be reported among vaccinated children in endemic settings. Although viral escape mutations are often suspected, delayed neonatal immunoprophylaxis and vertical transmission remain important but under-investigated mechanisms. Whole-genome sequencing provides an opportunity to distinguish between these competing explanations and strengthen molecular surveillance.

Objectives

This case report describes the molecular characterization of HBV isolated from a vaccinated Nigerian child with breakthrough hepatitis B surface antigen (HBsAg) positivity, with particular emphasis on viral genotype, HBsAg variation, recognized vaccine-escape mutations and antiviral resistance-associated mutations.

Methods

Children participating in a hepatitis B vaccine impact study in Oyo State, Nigeria, were screened for HBsAg. Positive samples underwent quantitative polymerase chain reaction, whole-genome amplification and Oxford Nanopore sequencing. Consensus genomes were analyzed using phylogenetic and mutation analyses to determine genotype, vaccine-escape mutations and antiviral resistance profiles.

Results

Among 336 vaccinated children, four were HBsAg positive (1.2%). Two samples had detectable HBV deoxyribonucleic acid, but only one yielded sufficient viral load for complete genome sequencing. Phylogenetic analysis classified the isolate as genotype E. No recognized vaccine-escape mutations or antiviral resistance-associated mutations were detected. Review of immunization records showed that the child received the first hepatitis B vaccine dose at 2 weeks of age and subsequently completed the vaccination schedule. This approximately 14-day delay in birth-dose vaccination provides a plausible explanation for the breakthrough infection in the absence of recognized vaccine-escape mutations.

Conclusion

Whole-genome sequencing demonstrated that HBV infection in this vaccinated child was unlikely to have resulted from recognized vaccine-escape mutations. Instead, delayed birth-dose vaccination provides a more plausible explanation for the breakthrough infection, although perinatal transmission could not be confirmed because maternal HBV status was unavailable.

Introduction

The hepatitis B virus (HBV) remains one of the world's leading causes of chronic liver disease, with approximately 254 million people living with chronic infection and more than 1 million deaths annually [1]. In Nigeria, the prevalence of HBV is about 5.4% (intermediate endemicity), translating to about 14.4 million persons infected with HBV. The highest rates are obtained in regions of the northern states, particularly in the Northwest (12.1%), mainly as a result of poor sanitation and restricted vaccination [2]. Transmission occurs through percutaneous or mucosal exposure to infected blood/body fluids, including mother-to-child transmission, sexual contact, and unsafe procedures [1].

Vaccination for all infants, introduced in the 1990s, has greatly reduced global pediatric carriage by more than 80% in vaccinated regions [1,3].

Despite substantial progress achieved through infant vaccination programs, hepatitis B continues to pose a major public health challenge in sub-Saharan Africa, where mother-to-child transmission remains an important driver of chronic infection and genotype E predominates [4,5].

Universal infant vaccination has dramatically reduced childhood HBV infection worldwide. Nevertheless, HBV infection continues to be detected among fully vaccinated children. Such infections are frequently described as vaccine breakthrough infections, although the underlying mechanisms are heterogeneous [5,6]. Proposed explanations include delayed or incomplete neonatal immunoprophylaxis, high maternal viral load, failure to administer hepatitis B immunoglobulin where indicated, impaired host immune responses and, less commonly, viral mutations capable of escaping vaccine-induced immunity [4,7].

Whole-genome sequencing is a valuable approach for comprehensive investigation of suspected vaccine breakthrough infections because it enables characterization of viral genetic variation across the genome, including regions associated with vaccine escape and antiviral resistance, while providing high-resolution information for phylogenetic analysis [3,8]. However, molecular investigations of HBV breakthrough infections remain scarce in West Africa despite the predominance of genotype E and the region's high burden of chronic HBV infection [[9], [10], [11], [12]].

The features of genotype E in terms of virology, clinical aspects, and epidemiology are still under scrutiny. Studies have reported differences in clinical characteristics across HBV genotypes, including HBeAg positivity, viral load, the risk of chronic infection following childhood acquisition, and liver disease severity [8], [11], [13]. So-called "vaccine escape mutants" still occur less frequently in genotype E, but ongoing molecular surveillance remains critical, especially in hyperendemic settings where the virus continues to circulate during mass immunization [4,8].

Traditional Sanger, next-generation, and third-generation sequencing methods have transformed the molecular epidemiology and surveillance of HBV. Unquestionably, the Oxford Nanopore technology, with long-read capacity, has changed the dynamics by enabling rapid, near-complete HBV genome sequencing directly from clinical samples, giving way to high-resolution phylogenetic analysis, characterization of transmission networks, and detection of vaccine-escape or drug resistance mutations, among other clinically relevant mutations [14,15]. Such advances are prioritized in the end to characterize the strains associated with vaccine breakthroughs and provide local prevention strategies [16].

Despite increased vaccine coverage in Nigeria [17], mother-to-child transmission continues to be a constant pathway for HBV infection, exacerbated in cases of missed birth doses, no maternal screening during antenatal care, and limited catch-up vaccination [4,5].

In a previous vaccine impact study conducted among vaccinated children in Oyo State, Nigeria, four participants tested positive for hepatitis B surface antigen (HBsAg) despite documented vaccination. While this finding suggested possible breakthrough infection, the underlying molecular mechanism remained unknown [10,12,13].

The present study therefore performed whole-genome sequencing and molecular characterization of HBV isolated from one vaccinated child with detectable viremia. Although analysis of the S gene is sufficient for assessing established vaccine-escape mutations and can support genotype assignment, whole-genome sequencing was employed to provide broader molecular characterization of the isolate, including phylogenetic analysis and assessment of the surface and polymerase regions for vaccine-escape and antiviral resistance-associated mutations.

Methods

Study participants and sample collection

Hepatitis B vaccination was introduced into Nigeria’s routine childhood immunization program in 2003-2004, with monovalent hepatitis B vaccine administered at birth and subsequent doses during infancy [18], [19]. The study participant, who was 14 years old at enrollment in 2023, was therefore born in approximately 2009, before the introduction of hepatitis B-containing pentavalent vaccine into Nigeria’s routine immunization program in 2012 [20]. Review of the child’s vaccination card showed that the first hepatitis B vaccine dose was administered at 2 weeks of age, followed by completion of the vaccination schedule. The commercial brand/manufacturer of the vaccine was not documented on the available vaccination record. Hepatitis B immunoglobulin (HBIG) was not part of the universal routine infant immunization schedule, and there was no documentation that HBIG was administered to this child [21].

The sample collection procedure was previously described [4]; this is a continuation of the study. From the vaccine impact study in Oyo State, four HBsAg-positive participants were selected for quantitative polymerase chain reaction (qPCR) analysis, but only two samples from participants aged 11 and 14 years tested positive by qPCR with viral loads of 1000 and 1.98 × 10⁸ IU/mL, respectively.

HBV deoxyribonucleic acid (DNA) extraction

Viral DNA was extracted from 200 µL of plasma using the Total deoxyribonucleic acid (DNA) extraction kit (Norgen Biotek; cat. no. 17200; 3430 Schmon Parkway, Thorold, ON, Canada L2V 4Y6). Following the manufacturer's protocols, samples underwent proteinase K digestion, lysis buffer treatment, ethanol precipitation, spin column purification, and elution with preheated buffer at 72°C to obtain high-purity HBV DNA. Extracted DNA was stored at −20°C until further analysis.

qPCR for viral load

Quantification of HBV DNA was carried out using a highly sensitive qPCR assay (TEL-HBV-Viral Load Test Kit v5; cat. no. TEL-300M9; Evancare Medical (Nantong) Co., Ltd, China) designed to amplify a 110-bp region in the HBV precore/core genomic region of the HBV genome. Reactions contained 15 µL of master mix and 5 µL of extracted DNA in a 20-µL total volume. Thermal cycling was performed on the Rotor-Gene 3000 real-time PCR instrument (Corbett Research, Mortlake, NSW, Australia). with an initial activation at 95°C for 1 min followed by 45 cycles at 95°C for 10 s and 60°C for 30 s. Amplification was detected in real time using probes labelled with 6-carboxyfluorescein (FAM), with positive samples defined by cycle threshold values ≤45. Viral loads were calculated by interpolation from a standard curve provided in the kit. The assay detection limit was <41.5 IU/mL [22].

PCR product documentation by agarose gel electrophoresis

The integrity and specificity of HBV PCR amplification were verified by agarose gel electrophoresis before sequencing.

HBV genome organization and primer scheme

To enable complete genome sequencing, six overlapping nested PCR amplicons were designed to span the entire HBV genome (GenBank accession NC_003977; genome length 3215 bp) [15,23]. Figure 1 [24] presents the circular HBV reference genome, showing nucleotide coordinates and the mapped primer sets that provide full genomic coverage. Table 1 [25] shows the primer pairs and the sequences used for HBV detection and sequencing in the study.

Figure 1.

Organization of the HBV genome and tiled nested PCR amplicons. Circular map derived from the HBV reference genome (NC_003977; 3215 bp) shows the positions and coverage of the six PCR products for full sequencing. The primer binding sites, as described, ensure overlap and complete genomic coverage (Chook et al. [24]). HBV, hepatitis B virus; PCR, polymerase chain reaction.

Table 1.

Sequences of primers used for HBV sequencing.

Primer set	Primer	Sequence (5′–3′)	Position (nt)	Typical amplicon size (bp)	Sequencing primer
Set 1a	251f	GAC TYG TGG TGG ACT TCT C	251–269	940	Yes
	1190r	TCA GCA AAY ACT YGG CA	1190–1174		Yes
Set 1b	595f	CAC HTG TAT TCC CAT CCC A	595–613	1,203	Yes
	1797r	CCA ATT TMT GCY TAC AGC CTC	1797–1777		No
	1190f	AYG CAA CCC CCA CTG G	1190–1205		Yes
Set 2a	2300f	CCA CMW AAT GCC CCT ATC	2300–2317	1,131	Yes
	215r	AGR AAM ACM CCG CCT GT	215–200		Yes
Set 2b	2819f	ACC WTA TWC YTG GGA ACA A	2819–2837	1,032	Yes
	617r	GAY GAY GGG ATG GGA ATA CA	617–598		Yes
	654r	GSC CCA MBC CCA TAG G	652–637		No
Set 3	1859f	ACT NTT CAA GCC TCC RAG CTG	1859–1879	959	No
	1877f	CTG TGC CTT GGR TGG CTT	1877–1894		Yes
	2835r	GTT CCC AVG WAT AWG GTG AYC C	2835–2814		Yes
Set 4	1584f	ACT TCG MBT CAC CTC TGC ACG T	1583–1604	748	No
	2331r	GGA AGY GTK GAY ARG ATA GGG GCA TT	2331–2306		Yes
	2396r	GTC KGC GAG GYG AGG GAG TT	2396–2377		No

These tiled amplicons provided overlapping coverage across the HBV genome, facilitating accurate sequence assembly and high-quality data generation for Oxford Nanopore sequencing [25].

Nested PCR for whole-genome amplification

The PCRs were performed using MyFi 2 × premix (Bioline, UK). Each 50-µL PCR reaction contained MyFi 2 × premix, primers, and template DNA. Amplification was performed under the following cycling conditions: denaturation at 95-97°C, annealing tailored per primer set, and extension at 72°C. Amplification products were verified by agarose gel electrophoresis as described above. Sensitivity was confirmed by serial dilution as previously described [26].

Whole-genome sequencing

Nested PCR products with the correct target size were purified and sequenced following Oxford Nanopore Technology protocols [25].

Bioinformatics and phylogenetic analysis

Consensus sequences were edited and aligned with GenBank reference genomes using BioEdit and ChromasPro. Phylogenetic analysis was performed using the Neighbor-Joining method in MEGA version 12.0.14 [27]. Reference sequences representing HBV genotypes A–H, together with additional Nigerian genotype E sequences, were included for comparison and genotype assignment. HBV genotype assignment was further assessed using the NCBI Virus Genotyping Tool. Mutation analysis focused on the hepatitis B surface antigen (S gene) and polymerase regions of the whole-genome consensus sequence. The polymerase/reverse-transcriptase region was analysed using HBVseq, Stanford HIV Drug Resistance Database (https://hivdb.stanford.edu/HBV/), to identify recognized antiviral resistance-associated mutations. The HBsAg amino-acid sequence was separately compared with the genotype A2/adw2 reference sequence X02763.1 to assess amino-acid variation and identify recognized vaccine-escape-associated substitutions, as described in the following section.

HBsAg amino acid sequence comparison

To assess antigenic differences between the study isolate and the HBsAg background represented in recombinant hepatitis B vaccines, the HBsAg amino acid sequence of Sample 386 (genotype E) was aligned with the genotype A2/adw2 reference sequence X02763.1 retrieved from GenBank. Comparative analysis focused on amino-acid positions 100–161, covering part of the major hydrophilic region (MHR; amino acids 99–169) and encompassing the entire immunodominant “a” determinant (amino acids 124–147). Amino acid differences between the study isolate and the A2/adw2 reference were identified and evaluated in the context of known genotype/serotype-associated variation and reported immune-escape substitutions [8].

Validity and quality control

Internal positive and negative controls were included for all laboratory assays, which were performed in accredited facilities participating in external quality assurance (Babcock University Molecular Laboratory, Ilisanremo, Ogun State, and Nigerian Institute of Medical Research, Yaba, Lagos, Nigeria).

Results

Identification of HBsAg-positive children and selection for molecular analysis

A total of 336 vaccinated children aged 1-14 years were screened for HBsAg as part of a hepatitis B vaccine impact study conducted in Ogbomoso, Oyo State, Nigeria.

HBsAg screening was performed using the BIO-INTECO HBsAg enzyme-linked immunosorbent assay (ELISA) (INTECO Diagnostics (UK) Ltd., London, UK), according to the manufacturer’s instructions, as previously described [4]. The assay is a solid-phase ELISA, with manufacturer-reported diagnostic sensitivity and specificity of 100% and 99.8%, respectively. No rapid diagnostic test was used. Four children (1.2%) tested positive for HBsAg despite documented hepatitis B vaccination.

All four HBsAg-positive samples were subjected to qPCR to determine HBV DNA levels. Detectable viremia was confirmed in two samples, while the remaining two samples had undetectable viral DNA. However, only one sample contained sufficient viral load for successful whole-genome amplification and sequencing and was therefore selected for subsequent molecular characterization.

Figure 2 summarizes the workflow from participant recruitment to whole-genome sequencing.

Figure 2.

Participant selection and sequencing workflow. DNA, deoxyribonucleic acid; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; PCR, polymerase chain reaction.

Quantification of HBV DNA

Quantitative PCR demonstrated detectable HBV DNA in two of the four HBsAg-positive samples. Of these, the sample from the 14-year-old participant had a markedly higher HBV DNA concentration of 1.98 × 10⁸ IU/mL and yielded sufficient viral material for complete genome recovery by Oxford Nanopore sequencing. This participant was positive for HBsAg, antibodies to hepatitis B core antigen (anti-HBc), and HBeAg, as previously reported [4]. Quantitative HBsAg testing was not performed. The participant appeared clinically healthy at enrollment, with no known chronic or current illness reported. However, systematic clinical evaluation for liver disease, including liver-function testing, imaging or fibrosis assessment, was not undertaken as part of the study protocol. As previously reported [4], another HBsAg-, anti-HBc-, and HBeAg-positive participant identified in the original study was a sibling of this child, born to the same mother. The mother’s HBV serological and virological status was not assessed; therefore, mother-to-child transmission could not be confirmed. The second HBV DNA-positive sample had a substantially lower viral load and yielded insufficient DNA for complete genome assembly despite repeated amplification attempts. The remaining HBsAg-positive samples had undetectable HBV DNA, most likely reflecting low circulating viral loads below the assay detection threshold or resolved infection with persistent HBsAg positivity.

Whole-genome sequencing and genome assembly

Whole-genome sequencing of the selected HBV isolate was successfully performed using the Oxford Nanopore sequencing platform. Following quality control, read filtering, and consensus sequence generation, a near-complete HBV genome was assembled with high sequence coverage, enabling comprehensive molecular characterization. The assembled genome was deposited in GenBank under accession number PV021398.

Genome annotation identified all expected HBV open reading frames, including the polymerase, surface, core, and X genes, providing sufficient sequence information for downstream phylogenetic and mutation analyses.

Phylogenetic analysis

Neighbor-joining phylogenetic analysis demonstrated that the HBV isolate clustered within genotype E, the predominant genotype circulating across West Africa (Figure 3 [8,28]). The isolate grouped closely with previously reported Nigerian genotype E strains, indicating high genetic similarity with endemic HBV variants rather than the introduction of a genetically distinct lineage.

Figure 3.

Phylogenetic analysis of the HBV whole-genome sequence obtained in this study. Neighbor-joining phylogenetic tree showing the relationship between the HBV whole-genome sequence generated from the study participant (Sample 386; ●) and reference HBV sequences retrieved from the NCBI GenBank database. Reference sequences representing HBV genotypes A-H, together with additional Nigerian genotype E sequences, were included for comparison and are identified by their GenBank accession numbers and isolate designations. Sample 386 represents the only sequence generated in the present study and is specifically highlighted (●) for ease of identification. The study isolate clustered within the genotype E lineage and closely with Nigerian genotype E reference sequences. Branch lengths represent genetic distance (Ingasia et al. [8]; Egbe et al. [28]).

HBV, hepatitis B virus; MEGA, Molecular Evolutionary Genetics Analysis; NCBI, National Center for Biotechnology Information; WHV, woodchuck hepatitis virus.

HBsAg amino acid comparison with genotype A2/adw2

Comparison of the HBsAg amino acid sequence of Sample 386 (genotype E) with the genotype A2/adw2 reference sequence X02763.1 identified six amino acid differences within positions 100-161: K122R, P127L, I140S, T143S, A159G and Y161F (Figure 4). Three of these differences, P127L, I140S and T143S, occurred within the immunodominant “a” determinant (amino acids 124-147). The observed differences are consistent with established genotype/serotype-associated variation between genotype E/ayw4 and genotype A2/adw2 rather than, in themselves, demonstrating vaccine escape [8].

Figure 4.

Amino acid comparison of HBsAg between the genotype E study isolate and genotype A2/adw2 reference sequence. Alignment of HBsAg amino acids 100-161 from Sample 386 (genotype E) and the genotype A2/adw2 reference sequence X02763.1. Six amino acid differences were identified: K122R, P127L, I140S, T143S, A159G and Y161F. MHR (amino acids 99-169) and immunodominant “a” determinant (amino acids 124-147) are indicated. Three differences (P127L, I140S and T143S) occurred within the “a” determinant.

aa, amino acids; anti-HBs, antibodies to hepatitis B surface antigen; HBsAg, hepatitis B surface antigen; MHR, major hydrophilic region.

Analysis of vaccine escape and antiviral resistance mutations

Detailed analysis of the HBV surface (S) gene identified no recognized vaccine-escape mutations, including mutations within the MHR or the "a" determinant known to reduce vaccine-induced antibody recognition. Similarly, examination of the polymerase gene detected no established antiviral resistance-associated substitutions linked to nucleos(t)ide analog therapy. These findings indicate that the sequenced HBV isolate retained the characteristic molecular features of wild-type genotype E viruses and provided no molecular evidence to suggest immune escape or antiviral drug resistance.

Summary of key findings

Overall, molecular characterization of the HBV isolate demonstrated the following:

Four of 336 vaccinated children (1.2%) were HBsAg positive.

HBV DNA was detected in two HBsAg-positive samples.

One sample yielded a complete genome suitable for sequencing.

The isolate belonged to HBV genotype E.

No vaccine-escape mutations were identified.

No antiviral resistance mutations were detected.

Delayed administration of the hepatitis B birth dose may have contributed to the infection, although the timing and route of transmission could not be established.

Discussion

The present study used whole-genome sequencing to characterize HBV infection in a vaccinated child identified during a hepatitis B vaccine impact study in southwestern Nigeria. Our findings demonstrated that the infecting virus belonged to HBV genotype E, the predominant genotype circulating in West Africa, and importantly, no recognized vaccine-escape or antiviral resistance mutations were identified. Direct comparison of the HBsAg amino acid sequence of Sample 386 with the genotype A2/adw2 reference sequence X02763.1 identified six amino acid differences within positions 100-161: K122R, P127L, I140S, T143S, A159G and Y161F. Three of these differences (P127L, I140S and T143S) occurred within the immunodominant “a” determinant (amino acids 124-147). These differences should be interpreted within the context of the established genotype- and serotype-associated diversity of HBsAg, particularly between genotype E/ayw4 and genotype A2/adw2 [8]. Thus, amino acid differences from the A2/adw2 reference sequence do not, by themselves, constitute evidence of vaccine escape. Consistent with this interpretation, no recognized vaccine-escape mutations were identified in the study isolate.

These molecular findings suggest that the observed infection was unlikely to represent true vaccine failure mediated by viral immune escape. Instead, the delayed administration of the hepatitis B birth dose provides a more plausible explanation for persistent infection, highlighting the critical importance of timely neonatal immunoprophylaxis in preventing mother-to-child transmission [29,30].

Identification of HBV genotype E is consistent with previous molecular epidemiological studies that have reported genotype E as the dominant HBV genotype throughout Nigeria and much of West Africa [9]. The relatively low genetic diversity observed within genotype E has led to the hypothesis that this lineage emerged comparatively recently and expanded rapidly across the region. Although genotype E is generally associated with favorable responses to antiviral therapy, its continued widespread circulation reflects ongoing transmission within highly endemic populations. Our finding therefore reinforces existing evidence that genotype E remains the principal circulating lineage in Nigeria and provides additional genomic data that contribute to the limited number of publicly available HBV whole-genome sequences from West Africa [9,31].

A major objective of this study was to determine whether persistent HBV infection following vaccination resulted from viral immune escape. Vaccine-escape mutations, particularly within the MHR and the "a" determinant of the HBsAg, have been associated with reduced recognition by vaccine-induced antibodies and occasional breakthrough infections. However, comprehensive analysis of the sequenced isolate identified no recognized vaccine-escape mutations, indicating that viral immune escape was unlikely to explain infection in this child. This finding is important because suspected vaccine breakthrough infections are frequently attributed to vaccine failure without molecular confirmation. Our study demonstrates the value of genomic sequencing in distinguishing true vaccine escape from alternative mechanisms of infection [32,33].

The child’s vaccination history revealed that the first dose of hepatitis B vaccine was administered at 2 weeks of age, representing an approximately 14-day delay in birth-dose vaccination.

Numerous studies have demonstrated that the birth dose represents the most critical component of hepatitis B prevention, particularly in interrupting mother-to-child transmission during delivery. Administration within 24 h substantially reduces the risk of chronic infection, whereas delays significantly compromise vaccine effectiveness, especially among infants born to mothers with active HBV infection. In the absence of maternal HBV screening data, mother-to-child transmission cannot be confirmed in the present study. However, the documented delay in birth-dose vaccination, together with the absence of recognized vaccine-escape mutations, suggests that inadequate timely neonatal prophylaxis represents a plausible explanation for infection in this child [5,[34], [35], [36]].

These findings have important implications for hepatitis B control programs in Nigeria and other highly endemic settings. While routine infant vaccination has substantially reduced HBV transmission, gaps in timely birth-dose administration remain an important challenge in many low- and middle-income countries. Our findings support strengthening maternal HBV screening during antenatal care, ensuring universal administration of the hepatitis B birth dose within 24 h of delivery, and providing hepatitis B immunoglobulin to infants born to highly infectious mothers where indicated. Integrating molecular surveillance into routine hepatitis B control programs would further improve understanding of breakthrough infections and facilitate early detection of emerging vaccine-escape variants should they arise [37,38].

The successful application of Oxford Nanopore sequencing for whole-genome analysis from a clinical sample with a high viral load emphasizes the utility of this technology in resource-limited settings for detailed HBV surveillance [25].

A major strength of this study is the application of whole-genome sequencing to investigate a suspected vaccine breakthrough infection, providing comprehensive molecular characterization beyond conventional serological testing. Integration of epidemiological, vaccination, molecular and phylogenetic data enabled a robust assessment of the likely mechanism underlying infection. Nevertheless, some limitations are acknowledged. Only one complete viral genome was successfully recovered because of low viral loads in the remaining HBsAg-positive samples. In addition, maternal HBV infection status and viral load were unavailable, preventing direct confirmation of vertical transmission. Despite these limitations, the genomic findings provide valuable molecular evidence supporting the interpretation presented in this study.

Future studies involving larger numbers of vaccinated children with persistent HBV infection are needed to determine the prevalence of vaccine-escape mutations in Nigeria and to better understand the molecular epidemiology of HBV vaccine breakthrough infections across sub-Saharan Africa. Expanding genomic surveillance alongside routine immunization programs will provide important evidence to guide hepatitis B elimination strategies and monitor the long-term effectiveness of vaccination programs as countries progress toward the World Health Organization 2030 hepatitis elimination targets.

In conclusion, this study demonstrates the value of genomic characterization in investigating HBV infection occurring despite documented vaccination. The absence of recognized vaccine-escape mutations, together with the documented 2-week delay in administration of the first hepatitis B vaccine dose, suggests that delayed neonatal immunoprophylaxis may have contributed to infection in this child. However, the route of transmission could not be established because maternal HBV status was unavailable. These findings reinforce the importance of timely hepatitis B birth-dose vaccination, maternal HBV screening and continued molecular surveillance of breakthrough infections, particularly in HBV-endemic settings.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Ethics approval and consent to participate

Statement of ethical approval: This study was ethically approved by the Research and Ethical Committee of the Bowen University Health Research Ethics Committee with approval number BUTH/REC-852. Caregivers were appropriately educated on the significance of the research to get their optimal support. The study adhered to the Declaration of Helsinki.

Statement of informed consent: Written informed consent was obtained from the parents or guardians, while assent was obtained from participants aged 10 years and older.

Consent for publication: Written informed consent for study participation was obtained from the child’s parent or legal guardian. The consent form informed participants that the study findings would be published for scientific purposes. No identifying personal information about the child is disclosed in this report.

Studies in human: This study was performed in compliance with relevant laws, regulatory frameworks and guidelines where the research took place.

This study was approved by Research and Ethical Committee of the the Bowen University Health Research Ethics Committee.

(Approval No. BUTH/REC-852).

Case reports: This research follows the CARE guidelines and the CARE checklist.

Availability of data and materials

The whole-genome sequence obtained from this study has been deposited in the NCBI GenBank database under the accession number PV021398.

The data supporting the findings of this study are available from the corresponding author, Dr. Busayo Kayode Akomolafe, upon reasonable request, subject to applicable ethical and confidentiality requirements. Dr. Akomolafe can be contacted at akomolafe.kayode@ncdc.gov.ng or via phone at +2348035156816.

Clinical trial number

Not applicable.

CRediT authorship contribution statement

Busayo Kayode Akomolafe: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Etuvie Favour Akomolafe: Writing – review & editing, Visualization, Methodology, Investigation, Data curation. Olawale Sunday Animasaun: Writing – review & editing, Investigation, Formal analysis. Bamidele Temidayo Fasanmi: Writing – review & editing, Investigation, Conceptualization. Nengak Precious Danladi: Investigation, Formal analysis. Rosemary Ajuma Audu: Writing – original draft, Supervision, Conceptualization.

Declaration of competing interest

The authors have no competing interests to declare.