Effects of hepatitis B vaccination on hepatitis B surface antigen in neonates and its change in vivo

We conducted a retrospective analysis of data from newborns at the Affiliated Hospital of North Sichuan Medical College born from July 2017 to July 2020 using electronic medical records. The inclusion criteria were as follows: the patients were aged 1–30 d; hepatitis B vaccine (10 μg) was administered within 24 h after birth; hepatitis B immunoglobulin (200 μg) was administered at the same time if the mother was HBsAg positive, and hepatitis B markers were measured during hospitalization. The exclusion criteria were as follows: patients with multiple congenital hereditary and immunodeficiency diseases and those with admission causes such as premature delivery, neonatal jaundice, neonatal pneumonia, or septicemia. The following data were sought and recorded on clinical data forms: name, sex, birth weight, date and time of administration of hepatitis B vaccine, date and time of collection of blood specimens, and HBV infection history of the mother.

The HBsAg test used in this study was the Cobas HBsAg assay (Cobas E602, Roche). The results showed that HBsAg >0.06 IU/mL was positive, anti-HBs <10 mIU/mL was negative, 10 mIU/mL ≤ anti-HBs < 100 mIU/mL indicated a low level of antibodies, and anti-HBs ≥ 100 mIU/mL indicated a high level of antibodies.

The serum specimens of HBsAg (−)/anti-HBs (−) healthy subjects were numbered and an online random number generator was used to randomly select corresponding specimens to simulate newborns whose mothers were negative for anti-HBs. The sera of HBsAg (−)/anti-HBs (+) healthy subjects were chosen to simulate newborns whose mothers were positive for anti-HBs. Then, the recombinant hepatitis B vaccine (derived from Chinese Hamster Ovary cells) (North China Pharmaceutical Jintan Biotechnology) (20 μg/1.0 mL/dose) was added to the selected samples to adjust the concentration of the vaccine in the serum to 0.5 ng/mL, 1.0 ng/mL, 2.0 ng/mL, 5.0 ng/mL, and 10.0 ng/mL. Finally, we measured hepatitis B quantitatively.

Statistical analyses were conducted using IBM SPSS Statistics for Windows, version 22.0. According to the normal test of the Q-Q chart, the positive results of HBsAg in newborns were distributed in a skewed way, and the mean value was represented by the median (quartile). A Fisher test was used to compare HBsAg results after vaccination of neonates born to antigen-negative and antigen-positive mothers. A chi-square test was used to compare the distribution of newborn HBsAg for mothers negative for surface antigen, but with different antibody concentrations. A rank sum test was used to compare the detection of HBsAg results in different serum groups after the recombinant hepatitis B vaccine was added to the serum. When the vaccine in serum was at 10 ng/mL, the linear correlation between HBsAg and anti-HBs results in the anti-HBs (+) group was determined by their Pearson correlation coefficient. P < 0.05 was considered significant.

The 1417 neonates (783 male and 634 female) had been vaccinated with the hepatitis B vaccine within 24 h after birth, and among them, 306 (21.6%) were HBsAg positive. The average HBsAg-positive result was 0.186 IU/mL (range: 0.104–0.339 IU/mL). The neonatal HBsAg and anti-HBs results are shown in Table 1. In 306 cases of HBsAg-positive newborns, 66.7% of the neonates were negative for anti-HBs, 22.6% of the neonates had low levels of anti-HBs, and 10.8% of the neonates had high levels of anti-HBs.

Through electronic medical records and an examination report inquiry system, 716 maternal prenatal hepatitis B quantitative test results were collected, among which 59 mothers were HBsAg positive, accounting for 8.2%. The results of HBsAg analysis for newborns and mothers are shown in Figure 1. There were 205 (31.20%) cases with positive HBsAg in newborns born to mothers with negative HBsAg. By contrast, there were only 2 (3.4%) positive cases among the newborns whose mothers were positive for HBsAg. Because P < 0.001, we concluded that the distribution difference of HBsAg in neonates according to the mother’s HBsAg group is significant.

Figure 1.

Neonatal cases with negative maternal HBsAg were selected (HBV immunoglobulin was not injected after birth) and were grouped according to the concentration of maternal anti-HBs. The results for neonatal HBsAg are shown in Table 2. When the HBsAg of the mother was negative, the level of positive HBsAg of the newborn was significantly inversely correlated with the increase in the concentration of anti-HBs of the mother (χ² = 156.51, P < 0.001), indicating that the distribution of HBsAg detected after the newborn was inoculated in different maternal anti-HBs groups was significant. In the negative anti-HBs maternal group, 193 newborns without anti-HBs accounted for 85.8%; in the maternal group with a low level of anti-HBs, 133 newborns had a low level of anti-HBs, accounting for 79.5%; and in the maternal group with a high level of anti-HBs, 256 newborns had a high level of anti-HBs, accounting for 96.6%. This distribution trend of neonatal anti-HBs is consistent with that of maternal anti-HBs.

The groups indicated above were further analyzed according to the days between vaccination and blood collection, and the results are shown in Figure 2. The figure shows that the overall trend of the positive rate of neonatal HBsAg increased 2 d before and then gradually decreased 2 d after vaccination, regardless of the maternal anti-HBs concentration. When the mother had no anti-HBs, the positive rate of HBsAg in the newborn was higher than that in mothers with normal and lower concentrations of anti-HBs.

Figure 2.

Among the 1417 neonates for whom HBsAg was measured quantitatively after receiving the hepatitis B vaccine, HBsAg positivity was mainly concentrated at 8 d after inoculation and was negative 9 d after vaccination (Table 3). The proportion of newborns positive for HBsAg was highest on the 2nd day after inoculation, at 42.4%, and then decreased gradually with time. The mean HBsAg-positive concentration varied with time on the days between vaccination and blood collection. It reached 0.222 (0.110–0.405) IU/mL on the 2nd day after the injection of the hepatitis B vaccine and then gradually decreased with time, and the average HBsAg-positive concentration was <0.05 IU/mL on the 9th day after vaccination (Figure 3).

Figure 3.

In the anti-HBs negative sample group, positive HBsAg was detected when the recombinant hepatitis B vaccine concentration in the serum was 0.5 ng/mL, the HBsAg positive was 15.4% (2/13), and the average HBsAg-positive concentration was 0.064 IU/mL. When the concentration of recombinant hepatitis B vaccine was 5 ng/mL, all samples were positive for HBsAg, and the median HBsAg concentration was a mean of 0.132 (range: 0.122–0.143) IU/mL. However, in the anti-HBs-positive sample group, HBsAg positivity was not observed until the concentration of recombinant hepatitis B vaccine was 5 ng/mL, the positive proportion was 84.6% (11/13), and the median HBsAg concentration was a mean of 0.103 (0.079–0.111) IU/mL (Table 4; P < 0.05 was considered significant). When the serum concentration of the recombinant hepatitis B vaccine was 5 ng/mL and 10 ng/mL (P < 0.05), there was a significant difference in the distribution of HBsAg results between the 2 groups. The HBsAg concentration was detected after different concentrations of the hepatitis B vaccine were added to the serum samples of the 2 groups, as shown in Figure 4.

Figure 4.

When the serum concentration of the recombinant hepatitis B vaccine was 10 ng/mL, all HBsAg results in the anti-HBs (+) group were positive. The anti-HBs results in the samples gave a Pearson correlation coefficient of r = −0.658, P = 0.014, indicating that the concentration of original anti-HBS in plasma was negatively correlated with the concentration of HBsAg after adding the same amount of hepatitis B vaccine (Figure 5).

Figure 5.