Impact of timing on protection of combined immunoprophylaxis in preventing mother-to-child transmission of hepatitis B virus: a retrospective study

Abstract Objective: To evaluate the impact of timing combined immunoprophylaxis on mother-to-child transmission (MTCT) of the hepatitis B virus (HBV) in pregnant women living with hepatitis B. Methods: A retrospective cohort study was included HBsAg-positive pregnant women who delivered full-term at Tianjin Third Central Hospital from January 2019 to December 2021. The objective of this study is to determine whether early administration of hepatitis B immune globulin (HBIG) and the first dose of hepatitis B vaccination after birth can further improve protection. Result: A total of 694 pregnant women living with hepatitis B were included; 93 infants from these mothers were lost to follow-up [including moving (n = 21), emigrating (n = 26), changing contact information (n = 27), and other reasons (n = 19)], leaving 601 infants for analysis. The incidence in babies born to mothers with hepatitis B was 1.50% (9/601). Based on the different timing of combined immunoprophylaxis administration after birth, 601 infants were divided into two groups (within 2 h and within 2–12 h). The incidence in babies born to mothers with hepatitis B were 0.32% (1/308) and 2.73% (8/293) for infants who received combined immunoprophylaxis within 2 h and between 2 and 12 h of birth, respectively (p = 0.037). The infection incidence of infants born to HBeAg-positive mothers and HBeAg-positive mothers who did not receive antiviral treatment during pregnancy was lower in the 2-h group compared to the 2–12 h group (0.72% vs. 5.84%, p = 0.04 and 1.20% vs. 9.46%, p = 0.047). Conclusion: Using combined immunoprophylaxis as soon as possible (within two hours of birth) may protect against MTCT of HBV more.


Background
Chronic hepatitis B virus (HBV) infection is a serious global health issue.According to the World Health Organization (WHO), the prevalence of HBsAg in the general population worldwide in 2019 was 3.8%, with approximately 1.5 million new HBV infections, 296 million chronic infections, and 820,000 deaths from liver failure, cirrhosis, hepatocellular carcinoma (HCC), and other HBV-related diseases [1] Mother-to-child transmission (MTCT) of HBV is the main route of chronic HBV infection in China and most of Asia, particularly intrapartum transmission [2].The concomitant use of hepatitis B immunoglobulin (HBIG) and hepatitis B vaccine within 12 h of birth to newborns of HBV surface antigen (HBsAg) positive mothers, known as passive-active or combined immunoprophylaxis, can effectively prevent MTCT of HBV [3,4].Recent research has shown that after combined immunoprophylaxis, the infant's protection rate for hepatitis B e antigen (HBeAg)-positive pregnant women is 90%-97%, with an infection rate of 3%-10% [5][6][7].However, such immunization may not be effective in a proportion of infants born to highly viremic mothers (serum HBV DNA >10 6-7 IU/mL).Despite HBIg and active immunization, these women carry a > 10% risk of vertical HBV transmission [8,9].
Further studies have shown that if combined immunoprophylaxis is administered within 1 h of delivery to neonates of HBeAg-positive pregnant women, the protection rate can be greater than 97% and the infection rate can be less than 3% [10,11].In addition, these studies have demonstrated that the incidence of MTCT in newborns of HBeAg-positive mothers who did not receive antiviral treatment during pregnancy would be as low as 1.28%-2.4% with the administration of both HBIG and the hepatitis B vaccine within 0.5 h after birth [11,12], which is significantly lower than the previously reported 5 to 10% within 12 h.In order to explore if timing combined immunoprophylaxis can further reduce the MTCT of HBV, we conducted a retrospective study on infants of HBVinfected mothers in our hospital.

Study design and population
We retrospectively reviewed the pregnant women living with hepatitis B who gave birth to full-term singleton infants at Tianjin Third Central Hospital between January 2019 and December 2021.The inclusion criteria were as follows: HBsAg positive for more than 6 months; gestational age �37 weeks; newborn birth weight � 2500 g; and antiviral treatment with Terivudine (600 mg/day) or tenofovir disoproxil fumarate (300 mg/day) starting from the 28th to 32nd gestational weeks, which lasted until delivery or four weeks postpartum, was offered and used voluntarily by women with HBV DNA levels greater than 2x10 5 IU/mL [13][14][15][16].
All of the participants got regular prenatal care, and those who met any of the following criteria were not allowed to take part: (1) Positive serological test for human immunodeficiency virus (HIV) or hepatitis A and C viruses; (2) Received or is receiving immunosuppression treatment; (3) History of anti-HBV treatment for more than 6 months; (4) Alanine aminotransferase (ALT) > 10-fold of the upper limit of normal value or total bilirubin > 3-fold of the upper limit of normal value; (5) Severe chronic diseases, including heart and kidney diseases, asthma, liver cirrhosis, diabetes, hyperthyroidism, epilepsy, and any mental disorders; and (6) Preterm birth and severe pregnancy complications, including preeclampsia and intrauterine fetal growth restriction.
All the babies of the recruited pregnant women were tested quantitatively for HBV markers, including HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc, between 7 and 12 months of age using a commercial ELISA kit (Beijing Wantai Biological Pharmacy Enterprise Co., Ltd.) in the clinical laboratory of Tianjin Third Central Hospital.

Combined immunoprophylaxis in infants
All newborns of the recruited pregnant women received an intramuscular injection of HBIG (100 IU) and the first dose of recombinant yeast hepatitis B vaccine (10 lg) within 2 h or 2-12 h after delivery.Infants received the second and third doses of hepatitis B vaccine at 1-month and 6-month intervals, respectively.The yeast-derived recombinant hepatitis B vaccine was provided by Shenzhen Kangtai Biological Products Co. Ltd., China (10 lg/0.5 mL).HBIG was provided by Shandong Taibang Biological Products Co. Ltd., China (100 IU).

Immunoprophylaxis outcome
Babies who tested positive for HBsAg but negative for antibody against HBsAg (anti-HBs) were diagnosed with immunoprophylaxis failure and HBV infection.The seroprotective response to vaccine was defined as an anti-HBs level of �10mIU/mL [17].

Statistical analysis
Data analysis was performed using SPSS 22.0 statistical software (International Business Machines Crop.).The frequencies for categorical variables were compared using the v2 test or Fisher's exact test.Quantitative data with a normal distribution were expressed as mean ± standard deviation and compared using t-tests for two samples.The baseline characteristics for continuous variables are described as the means ± standard deviations or medians (interquartile range, IQRs).The continuous variables were compared using Student's t-test, or the Wilcoxon rank-sum test, depending on the normality of the data.If p < 0.05, a difference was considered statistically significant.

Baseline characteristics
As shown in Figure 1, 694 pregnant women were enrolled in the work, and 93 infants were lost to followup because of moving (n ¼ 21), emigrating (n ¼ 26), changing contact information (n ¼ 27), and other reasons (n ¼ 19).Ultimately, 601 infants born to these mothers were analyzed.Based on the timing of combined immunoprophylaxis administration after birth, there were 308 cases of immunization within 2 h and 293 cases of immunization between 2 and 12 h after birth.Table 1 describes the baseline demographics and virological characteristics of the pregnant women, which were similar.The level of HBV DNA-positive patients in the 2-h group was similar to that of those in the 2-12 h group (p ¼ 0.364).There was no statistical difference in the proportion of high HBV DNA levels (>2 � 10 5 IU/mL) (36.7% vs 39.9%).In addition, the levels of HBsAg and HBeAg were similar between the two groups (p > 0.05).
The group immunizing within 2 h included 139 HBeAg-positive patients and 169 HBeAg-negative pregnant women, among whom 56 HBeAg-positive patients with high viral load (HBV DNA > 2 � 10 5 IU/mL) received antiviral therapy of entecavir or tenofovir disoproxil fumarate orally from 28 to 32  gestational weeks until delivery or 4 weeks postpartum, whereas 83 pregnant women did not receive any intervention during pregnancy.The group immunizing between 2 and 12 h after birth included 137 HBeAgpositive pregnant women and 156 HBeAg-negative pregnant women, among whom 63 HBeAg-positive pregnant women received antiviral therapy during pregnancy, whereas 74 pregnant women did not receive any intervention (Figure 1).The baseline demographics and laboratory characteristics of the pregnant women in the two groups were generally similar (Table 2).

Immunoprophylaxis, infection with HBV and anti-HBs response in infants
The average age of the followed-up infants was 8.6 ± 1.2 months (median 9.0 months, range 7.0-12.0months).One infant in the immunoprophylaxis within 2 h group was HBsAg positive, and eight infants in the 2-12 h group were infected with HBV, with the incidence in babies born to mothers with hepatitis B being 0.32% (1/308) and 2.73% (8/293), p ¼ 0.037.All of the HBV-infected infants were born to HBeAg-positive mothers, while none of the 325 infants born to HBeAg-negative mothers (169 in the 2 h group and 156 in the 2-12 h group) were infected.Further analysis and comparison of the incidence in babies born to mothers with hepatitis B according to different times of combined immunization revealed that the incidence in babies born to mothers with hepatitis B of HBeAg-positive mothers was 3.26% (9/276), with infection rates of 0.72% (1/139) and 5.84% (8/137) in the immunization within 2 h and the 2-12 h groups, respectively, p ¼ 0.040.The infection rates of infants born to untreated HBeAg-positive mothers were 1.20% (1/83) and 9.46% (7/74), respectively, p ¼ 0.047.Among the 119 HBeAg-positive pregnant women with high viral loads who received antiviral therapy during pregnancy, there was no statistically significant difference in the HBV infection rate of neonates between the two immunization time groups, with rates of 0% (0/56) and 1.59% (1/63) respectively (Table 3).

Discussion
In this study, we compared the difference in the MTCT rate of HBV between infants who received immunoprophylaxis within 2 h after birth and those who received it between 2 and 12 h after birth.We revealed that the HBV infection rate of HBsAg-positive mothers, HBeAg-positive mothers, and HBeAg-positive mothers who did not receive antiviral therapy was lower in the 2 h group than in the 2-12 h group, with 8 cases of neonatal infection occurring in the 2-12 h group.We identified delayed immunization as a major factor contributing to the insufficient implementation of neonatal immunoprophylaxis measures, leading to an increased risk of MTCT [18].Therefore, our findings emphasize the significance of early administration of HBIG and vaccinations to minimize the risk of MTCT [19].Early administration of immunoprophylaxis after birth, rather than within 12 h, may enhance the protective effectiveness of current immunoprophylaxis.This study found that the risk of HBV infection was lower in babies born to HBeAg-positive mothers who got combined immunoprophylaxis within 2 h of birth.The efficacy of the combined hepatitis B vaccine and HBIG has been fully confirmed in infants from HBeAgpositive mothers.Therefore, timely administration of the hepatitis B vaccine and HBIG is crucial for interrupting vertical transmission [20].In two prospective studies undertaken in China, transmission rates among newborns born to HBeAg-positive mothers who received immunoprophylaxis within an average time of 0.5 h or 0.17 h after birth were 1.28% (among 156 newborns) and 2.4% (among 375 newborns), respectively [11][12].Other studies have reported similar results: the transmission rate for HBeAg-positive mothers receiving HBIG and the hepatitis B vaccine within 6 h after delivery ranges from 2.84% to 5.62% [13,[21][22][23], which is lower than the 7%-10% [24].In a recent study, Aslam et al. suggested that although HBeAg can cross the placenta and generate specific T cell tolerance to HBV, resulting in failed immune prevention [25], prompt administration of the hepatitis B vaccine and HBIG immediately after birth can break the MTCT chain and significantly reduce the risk of HBV infection in newborns [26].This work indicated that early administration of HBIG and the hepatitis B vaccine to neonates of HBeAg-positive mothers can further increase the protective effect against the MTCT of HBV.
Furthermore, recent studies have revealed that the failure of immune prevention in infants born to HBeAg-positive mothers may be linked to the mother's high viral load [27][28][29].Our study found no statistically significant difference in the rate of HBV infection among newborns delivered by HBeAg-positive pregnant women with HBV DNA > 2 � 10 5 IU/mL who received antiviral therapy during pregnancy and were administered HBIG and the hepatitis B vaccine within 2 h and 2-12 h after birth.The main reasons for the absence of chronic infection may be that the mothers with high viral loads who received antiviral therapy during pregnancy had a significant reduction in maternal viral load at delivery and that HBIG and hepatitis B vaccines were timely administered to newborns.Another possibility is that the sample size of the study is small.
The major findings of this research suggest that the use of HBIG and hepatitis B vaccination within 2 h and 2-12 h after delivery in neonates of HBV-positive mothers who did not receive antiviral therapy reduces the incidence of MTCT in a statistically significant manner.The rate of HBV infection is extremely low (1.2%) when newborns receive combined immunoprophylaxis within 2 h after birth.In a multicenter prospective study in China, the incidence of MTCT was 2.4% among 375 neonates born to HBeAg-positive mothers who did not receive antiviral medication during pregnancy and who received combined immunoprophylaxis within 1 h after delivery [13].Recently, five studies with large sample sizes have demonstrated chronic infection rates of 2.84% (10/352) [12], 3.2% (12/374) [30], 6.37% (21/624) [31], 3.44% (21/473) [32], and 5.19% (18/347) [21] among newborns of HBeAgpositive mothers who did not receive antiviral therapy and who were administered HBIG and the hepatitis B vaccine after 6 h of birth.These rates were lower than the previously reported 7%-10%, indicating that, even in the case of HBeAg-positive mothers with high viral loads who did not receive antiviral therapy during pregnancy, the use of HBIG and vaccine immediately after birth (within 2 h) improves protective efficacy and decreases infection rate with an earlier time of immunization.
In addition, the feasibility of immediate use (within 2 h) of HBIG and hepatitis B vaccine in infants born to HBV-infected mothers faces many challenges.Every effort should be made to ensure the immediate administration of HBIG and hepatitis B vaccine so that the protection efficacy of current immunoprophylaxis against MTCT of HBV can be maximized.First, improving the protection efficacy against MTCT of HBV should be widely recognized.Second, hepatitis B vaccines and HBIG should be available in the delivery room so that the newborn infants can be injected immediately after birth.Third, all pregnant women should be screened for HBsAg in order to detect newborns at risk of HBV infection.
The main limitation of our study was the small sample size, particularly for HBeAg-positive cases and pregnant women with HBV DNA >2 � 10 5 IU/mL who received antiviral medication during pregnancy.Since antiviral therapy during pregnancy was voluntary, and HBeAg-positive mothers were often concerned about the potential adverse effects on their fetuses, they frequently rejected medication, leading to a lack of antiviral treatment for some pregnant mothers with high viral loads.Due to constraints in our hospital's vaccination process for HBsAg-positive mothers after delivery, the combined immunization of newborns after birth could only be limited to within 2 h.It was not feasible to collect sufficient samples in a shorter time frame (within 1 h).A larger sample size, specifically for HBeAg-positive cases, a multicenter investigation, standardized antiviral treatment during pregnancy, and, most significantly, shortening the time for combined immunoprophylaxis after birth (within 1 h) may better verify the findings of this study.
In conclusion, early administration of HBIG and the hepatitis B vaccination can greatly improve the protection against MTCT of HBV in babies born to HBsAgpositive mothers, especially in pregnant women who are not monitored for HBeAg and HBV DNA.Immunoprophylaxis should be administered to all infants born to HBsAg-positive mothers as soon as possible after birth, ideally within 2 h or more.

Table 1 .
Baseline demographic and virological characteristics of HBsAg-positive mother.

Table 2 .
Baseline demographic and virological characteristics of HBeAg-positive mother.

Table 3 .
Characteristics of paired HBV-infected infants and mothers.