Differences between Chronically Hepatitis B Virus-Infected Pregnant Women with and without Intrafamilial Infection: From Viral Gene Sequences to Clinical Manifestations

Approximately 250 million people worldwide are infected with the hepatitis B virus (HBV) [1, 2], and more than 90 million individuals in China are chronically infected [3]. Moreover, the clinical course and long-term outcomes of HBV infections are affected by the viral genetic sequence, structure, diversity, and status of the host immune system [1, 4‒9]. Therefore, an in-depth study of viral gene sequences is important to understand its etiology, develop new therapeutic strategies, and cure HBV infection. HBV infection is prone to the familial aggregation phenomenon [10‒12]. Most studies suggest that HBV transmission is mainly intrafamilial, particularly via mother-to-child transmission (MTCT) [13‒15]. HBV genotyping and phylogenetic analysis, followed by next-generation sequencing (NGS) comparisons of different viral strains, may help differentiate between patients with and without intrafamilial infections [16]. Pregnancy alters the natural process of chronic HBV infection and the immune status of pregnant women differs from that of others. There is limited evidence regarding the differences between chronic hepatitis B (CHB) pregnant women with and those without intrafamilial infections. Further investigations may help improve the postpartum prognosis of pregnant women with CHB and reduce vertical transmission between mothers and children. Using viral gene sequencing and clinical information, this study aimed to investigate the differences between pregnant women with CHB and intrafamilial infections and those without intrafamilial infection.

Serum samples were collected from 22 patients with CHB for viral deoxyribonucleic acid (DNA) sequencing. There were 4 pregnant women and 6 their family members in the intrafamilial infection group. In these families, at least one infector was the mother or daughter of a pregnant woman. The remaining 12 pregnant women with infections were not affected by intrafamilial infections. The average age of these pregnant women with CHB was 29.3 ± 3.3 years. The mean number of pregnancy times in the pregnant women was 2.1 ± 1.2. Another cohort of 74 HBsAg and HBeAg dual-positive pregnant women was followed up from the second trimester to 3 months postpartum to study maternal viral markers (i.e., HBV-DNA, HBeAg, and HBsAg), other laboratory examinations (alanine transaminase [ALT], aspartate transaminase, hemoglobin, albumin, blood urea nitrogen, and creatinine [Cr]), and maternal adverse events. 13 pregnant women whose serum samples were collected for viral sequencing were included in the cohort. The other three pregnant women only cooperated with blood sampling for virus sequencing test and did not complete the subsequent follow-up study. Therefore, they were not included in the 74 samples. Seventy-four pregnant women were divided into two groups: pregnant women with CHB with intrafamilial infection and without intrafamilial infection. The mean age was 31.3 ± 5.4 and 28.8 ± 3.1 years in pregnant women with and without intrafamilial infection, respectively. The mean number of pregnancy times in the pregnant women with and without intrafamilial infection was 2.3 ± 1.2 and 1.7 ± 0.8. In pregnant women with CHB and intrafamilial infection, HBsAg-positive mothers are the source of HBV infection.

All pregnant women with CHB were recruited from the First Affiliated Hospital of Xi’an Jiaotong University. They did not receive HBV vaccination when they were born. They received tenofovir disoproxil fumarate treatment from the second trimester until 1 month postpartum. The exclusion criteria were as follows: (1) a history of HBV treatment within the last 6 months; (2) Cr clearance rate <100 mL/min, ALT level >5 times the upper limit of normal, bilirubin level >2 mg/dL, or evidence of hepatocellular carcinoma, renal, or hepatic dysfunction; (3) the presence of coinfections with HIV, hepatitis C virus, or hepatitis D virus; and (4) the requirement for treatment with special medicines during pregnancy. Infected family members were recruited from each pregnant woman’s household if their HBsAg was positive. This study protocol was reviewed and approved by the Biomedical Ethics Committee of the Medical Department of the Xi’an Jiaotong University (Approval No: 2021-574). Written informed consent was obtained from all patients before the study.

Viral and host DNA were extracted directly from 200 μL aliquots of serum from 22 patients with CHB, and a nuclease-treated commercial DNA Kit (Magen Biotechnology Co., Cat. No. D3191), according to the manufacturer’s instructions. For DNA quantification, a biophotometer was used to determine the quality of viral DNA (A260/280 and A260/230). A Qubit 3.0 Fluorometer (Life Technologies, Thermo Fisher, Stockrick, CA, USA) with a One-step DNA Lib Prep Kit for Illumina V2 (Abclonal, Cat. No. RK20237) was used according to the manufacturer’s instructions to obtain more accurate quantification.

The Illumina sequencing workflow is divided into four steps, including library preparation, cluster generation, sequencing, and data analysis. The viral DNA was fragmented using the NEBNext dsDNA Fragmentase, and the DNA fragments were inputted into the NEB “NEBNext^® Ultra^™ II DNA Library Prep Kit” according to the Illumina^® protocol. An Agilent 2100 Bioanalyzer with a high-sensitivity DNA kit was used to quantify the library. The library followed the Illumina protocol: “Prepare DNA libraries for sequencing on NovaSeq 6000.” Paired-end 150 nt reads were generated.

Read-quality trimming was performed using Skewer, with an additional trimming filter for unreliable sequences and a user-specified quality score [17]. Host read subtraction with read mapping was performed using the BWASW program [18], bacterial genome sequences, and latest host organism genome sequences. De novo assembly followed the A5-miseq pipeline [19]. The final scaffolds were subjected to BWASW read mapping and a MegaBLAST homology search was performed using the NCBI Biotechnology Information NT database.

Individual sequences were aligned to the NCBI nucleotide sequence database using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_645910295) and the best-fit HBV reference genome (AB206817.2) with the smallest e-value was identified. The physical positions of the alignment and the corresponding genotypes were identified using an in-house Perl script. We combined all individual genotypes into a matrix and excluded SNP with missing genotypes at a threshold >3. A total of 2,849 loci were used to construct a phylogenetic tree. FastTree software was used to build a phylogenetic tree based on nucleotide sequence alignments using default settings [20]. A reference sequence (AB206817.2) was used as an outgroup in the phylogenetic tree. The structure of the phylogenetic tree was displayed using the ggtree R package [21].

Clinical information was compared between pregnant women with CHB and intrafamilial infections and pregnant women without intrafamilial infections in a cohort of 74 pregnant women. Baseline characteristics and laboratory results are reported as means ± standard deviation for normally distributed continuous variables, median (interquartile range [Q3–Q1]) for non-normally distributed continuous data, and percentages for categorical variables. The HBV-DNA levels were logarithmically transformed (log₁₀ IU/mL). Pearson’s χ² test modified by Fisher’s exact test was used to compare categorical variables between the two groups. The t test or Mann-Whitney U test for nonparametric analysis was performed to compare continuous variables between the two groups. A mixed model with repeated-measures analysis was used to evaluate group, time, and group-by-time effects when measuring serum HBV-DNA levels and other serum parameters. Time was represented as the follow-up time from baseline to 3 months postpartum. Changes in each indicator from baseline to delivery and from delivery to 3 months postpartum were compared between pregnant women with CHB and intrafamilial infection and those without intrafamilial infection. Multiple linear regression analysis was used to analyze the relationship between the type of pregnant women and changes in serological indicators from delivery to 3 months postpartum, while adjusting for age; type of delivery; and HBV-DNA, HBsAg, HBeAg, ALT, and hemoglobin levels at delivery. The regression coefficients and 95% confidence intervals (CIs) were calculated. An overview of the follow-up period and analyses is shown in online supplementary Figure 1 (for all online suppl. material, see https://doi.org/10.1159/000539994). Statistical significance was set at p < 0.05 indicated statistical significance. All data analyses were conducted using R 4.1.2 and SPSS 24.0 (SPSS, IBM, Chicago, IL, USA).

The 22 HBsAg-positive patients underwent viral gene sequencing. The participants were from 16 families. Of these, 10 had intrafamilial infections and 12 did not. Three patients were HBeAg-negative, and 19 were HBeAg-positive. Sixteen patients were HBsAg-positive pregnant women, and the others were their families.

Another cohort of 74 pregnant women with CHB was included in this study. The clinical characteristics of pregnant women with and without intrafamilial infections were compared (Table 1). CHB pregnant women with intrafamilial infection were older than CHB pregnant women without intrafamilial infection (31.3 ± 5.4 vs. 28.8 ± 3.1, p = 0.036). Moreover, pregnant women with intrafamilial infection had a lower Cr level than women without intrafamilial infection (40.7 ± 6.1 vs. 45 ± 8.6, p = 0.035). No significant differences were observed among the other indicators.

The phylogenetic tree of the full-length HBV genome (Fig. 1) shows that the HBV lines in the mother-spread pedigree (intrafamilial infection) were similar. However, the HBV lines in patients without intrafamilial infections were separated into different branches. The HBV lines in the same family shared a node, indicating that the genetic background of the HBV lines was identical to that of unrelated HBV lines in patients without intrafamilial infection. A phylogenetic tree of the four HBV gene regions is shown in online supplementary Figure 2. For the P, S, and X genes, the HBV lines from the corresponding families had similar genetic backgrounds. This result revealed that the spread model of the mother-spread pedigree with intrafamilial infection differed from that of the pedigree without intrafamilial infection.

The dynamic alteration process of HBV-DNA from baseline to 3 months postpartum in patients with and without intrafamilial infection is shown in Figure 2. The results indicated that there was no significant effect of HBsAg-positive mothers on the change in HBV-DNA levels from baseline to 3 months postpartum (p1 = 0.848), whereas follow-up time was a significant variable that influenced the level of HBV-DNA (p2 < 0.001). Changes in serum HBV infection indicators (HBsAg, HBeAg, HBeAb, and HBcAb) are shown in Figure 3. Among the four indicators, there were no significant differences between patients with and those without intrafamilial infections (all p1 > 0.05). The follow-up time was not a significant variable, except for HBeAb (p2 < 0.001). The other laboratory markers are shown in Figure 4. Intrafamilial infection had no significant effect on any of the indicators. All laboratory parameters showed significant time-varying effects throughout the follow-up period in both groups (all p2 < 0.05).

The changes of indicators in HBV-infection parameters from baseline to delivery and from delivery to 3 months postpartum in the two groups are listed in Table 2. With regard to the changes from baseline to delivery, there were no significant differences in all of the indicators between patients with and without intrafamilial infection, except for HBcAb (−0.88 ± 1.46 vs. 0.39 ± 1.74, p = 0.005). In terms of changes from delivery to 3 months postpartum, there were significant differences between the patients with and without intrafamilial infection (−5.33 ± 0.55 vs. −4.76 ± 0.93, p = 0.009), HBeAg (−0.27 ± 0.27 vs. −0.12 ± 0.25, p = 0.029), HBcAb (0.37 ± 1.05 vs. −0.39 ± 0.91, p = 0.005), AST (0.15 ± 12.47 vs. −14.48 ± 29.93, p = 0.010), ALT (−4.75 ± 13.51 vs. −38.85 ± 53.37, p < 0.001), HB (−19.4 ± 10.3 vs. −8.70 ± 12.85, p = 0.002), ALB (−4.99 ± 3.58 vs. −7.21 ± 2.22, p = 0.017).

To verify the significant differences in viral markers and other laboratory indicators after delivery between the two types of pregnant women, multiple linear regression analysis was conducted after covariate adjustment; the results are listed in Table 3. We found that pregnant women with intrafamilial infection had a greater HBV-DNA increase (β = −0.43, 95% CI: −0.76 to −0.12, p = 0.009), greater HBeAg increase (β = −195.15, 95% CI: −366.35 to −23.96, p = 0.027), and greater hemoglobin increase (β = −8.09, 95% CI = −15.54 to −0.64, p = 0.035). Pregnant women with intrafamilial infection had a lower ALT increase (β = 73.9, 95% CI: 38.92−108.95, p < 0.001) and lower albumin increase (β = 2.73, 95% CI: 0.23−5.23 p = 0.033).

Maternal adverse events and laboratory abnormalities in the infected pregnant women are shown in Table 4. The percentage of amniotic fluid pollution in patients without intrafamilial infection was higher than that in patients with intrafamilial infection (28.6 vs. 8%, p = 0.042). There were no other significant differences between the two groups (all p > 0.05). Regarding laboratory abnormalities, the proportion with an elevated ALT level 1.1 to 5 times the upper limit of the normal range in patients without intrafamilial infection was higher than that in patients with intrafamilial infection (65.3 vs. 32%, p = 0.006). The frequency of ALT flares during the follow-up period was significantly higher in patients without intrafamilial infections than in those with intrafamilial infections (22.4 vs. 0%, p = 0.010). This difference was mainly observed during the postpartum period (18.4 vs. 0%, p = 0.022), whereas no significant difference was observed before delivery (6.1 vs. 0%, p = 0.206).

Using viral genetics and clinical indicators, we compared the differences between pregnant women with chronic HBV infection and intrafamilial infection and those without intrafamilial infection. Although family history is a well-known risk factor for HBV infection, there is sparse evidence of different HBV transmission routes in intrafamilial and non-intrafamilial infections based on viral sequences using NGS technology. McNaughton et al. [9] and Tong and Revill [22] summarized the characteristics of the HBV genome, including its structure, replication cycle, and variability. Other two studies reported the characteristics of HBV in MTCT using NGS [23, 24]. They found that a proportion of the HBV strains in the mother were transferred to the child [9, 23, 24]. The present study provides evidence that, compared with non-intrafamilial infections, the HBV sequences of infected patients within the family are more similar, revealing that they come from the same ancestor.

Another important finding of the present study was that ALT levels increased after delivery, and the frequency of ALT flares was much higher in CHB patients without intrafamilial infection than in those born to HBsAg-positive mothers. Meanwhile, the increase in HBV-DNA and HBeAg levels after delivery in patients with CHB born to HBsAg-positive mothers was higher than that in those without intrafamilial infection. This may be related to alterations in immune status during pregnancy [25, 26]. Previous studies have demonstrated that maternal HBeAg can condition hepatic macrophages in the offspring to suppress the immune response, leading to impaired CD8+ T cells [27, 28]. Accordingly, inflammatory reactions and increased ALT levels may be weakened in patients with CHB infection and MTCT [26]. In contrast, in the absence of conditioning by maternal HBeAg, HBeAg-induced hepatic macrophages display a pro-inflammatory phenotype that contributes to viral clearance [27]. Therefore, in patients without MTCT, it may be easier to clear HBV and lower the antigen burden during pregnancy, leading to the recovery of exhausted T cells and improved immune responses transitorily [25]. Therefore, patients with CHB without intrafamilial infection tend to develop ALT flare-ups after delivery.

Furthermore, pregnant women with CHB without intrafamilial infection were more sensitive to increased ALB levels after delivery than those born to HBsAg-positive mothers. ALB is a critical marker that decreases with the progression of chronic liver disease. ALB biosynthesis is affected by pro-inflammatory cytokines and excessive oxidative agents released by mitochondria from the injured liver [29]. This finding demonstrates that patients without intrafamilial infections may have more evident inflammatory reactions after delivery than those born to HBsAg-positive mothers.

Our study found that maternal HBV infection may cause sharp alterations in HBV infection and other biochemical indicators from delivery to 3 months postpartum. Thus, it can be inferred that delivery is an important time point. Since the immune response is unique during pregnancy, there is an interaction between immune adaptation and modulation by chronic HBV infection [2, 30, 31]. Reversal of immunotolerance to semi-allogeneic fetal antigens following parturition was observed, as well as substantial changes in HBV-related infection markers after delivery [25]. This finding may be helpful for further understanding the immunopathogenesis of chronic HBV infection during pregnancy.

The strength of this study is that we provided dynamic follow-up data on HBsAg and HBeAg dual-positive pregnant women from the second trimester to 3 months postpartum, including HBV-DNA levels, HBV serological markers, and other biochemical indicators. The main limitation was the limited data on viral sequences in HBsAg-positive mothers and their offspring. Further studies with larger sample sizes are needed to investigate the differences between pregnant women with CHB and intrafamilial infections and those without intrafamilial infections.

In conclusion, the findings of this study suggest that the mother-spread pedigree spread model (intrafamilial infections) differs from that for non-intrafamilial infections. Pregnant women with chronic HBV infection due to non-intrafamilial infection were more likely to have hepatitis flares and impaired liver function than those with intrafamilial infection. HBV-DNA and HBeAg levels in pregnant women with chronic HBV infection and intrafamilial infection rebound faster after delivery than in those without intrafamilial infection.

This study protocol was reviewed and approved by the Biomedical Ethics Committee of the Medical Department of the Xi’an Jiaotong University (Approval No. 2021-574). Written informed consent was obtained from all patients before the study.

The authors have no conflict of interest to declare.

This work was supported by Shaanxi Provincial Key Industry Innovation Chain Project (2022ZDLSF04-02) and Shaanxi Provincial Key Innovation Team Project (2019TD-031). The funder had no role in the design, data collection, data analysis, and reporting of this study.

F.G. participated in the project design, experimental analysis, and statistical analysis and drafted the manuscript; X.L. contributed to experiment, interpretation of the results and drafted the manuscript; X.W. contributed to data and sample collection and reviewed the final manuscript; H.L. contributed to experimental analysis and reviewed the final manuscript; W.Z. contributed to data collection and reviewed the final manuscript; Y.Z. contributed to experimental analysis and drafted the manuscript; Y.J. and Z.Z. and participated in data analysis and review of the final manuscript; G.B. participated in the design and coordination of the project and review of the final manuscript.

Fan Gao and Xia Li contributed equally to this work.

The data that support the findings of this study are not publicly available because their containing information that could compromise the privacy of research participants but are available from the corresponding author G.B. (baigq@126.com, tel: 86-18991232517) upon reasonable request.