Impact of HBV replication in peripheral blood mononuclear cell on HBV intrauterine transmission

Abstract

This study determined the effect of hepatitis B virus (HBV) replication in peripheral blood mononuclear cell (PBMC) from HBsAg-positive mothers on HBV intrauterine transmission. A total of 150 HBsAg-positive mothers and their neonates were recruited in this study. Within 24 h after birth, HBV serological markers, serum HBV DNA, PBMC HBV relaxed circular DNA (rcDNA), and covalently closed circular DNA (cccDNA) were measured in the HBsAg-positive mothers and their neonates before passive-active immune prophylaxis. The relationship between HBV replication in PBMC and HBV intrauterine transmission was examined through Chi-square test and logistic regression. The rate of HBV intrauterine transmission was 8.00% (12/150) in the 150 neonates born to HBsAg-positive mothers. The positivities of PBMC HBV rcDNA and cccDNA in the HBsAg-positive mothers were 36.67% (55/150) and 10% (15/150), respectively. Maternal PBMC HBV cccDNA was a risk factor of HBV intrauterine transmission (OR= 6.003, 95% CI: 1.249–28.855). Maternal serum HBeAg was a risk factor of PBMC HBV rcDNA (OR= 3.896, 95% CI: 1.929–7.876) and PBMC HBV cccDNA (OR= 3.74, 95% CI: 1.186–11.793) in the HBsAg-positive mothers. Administration of hepatitis B immune globulin was a protective factor of PBMC HBV cccDNA (OR= 0.312, 95% CI: 0.102–0.954) during pregnancy. The positivity of PBMC HBV rcDNA was related to that of cccDNA in the HBsAg-positive mothers (c²=5.087, P= 0.024). This study suggests that PBMC is a reservoir of HBV and an extrahepatic site for virus replication and plays a critical role in HBV intrauterine transmission.

Keywords

PBMC / HBV cccDNA / HBV rcDNA / HBV intrauterine transmission

Introduction

Hepatitis B virus (HBV) infection, a major public health problem worldwide, increases the risk of more than 250 million people to developing terminal liver disease [1]. Perinatal transmission, including intrauterine, intrapartum, and puerperal transmissions, is the predominant route of HBV transmission in highly endemic areas [2]. Existing studies suggested that 80%–90% of HBV-infected infants could develop chronic infection [3,4]. Passive-active immunoprophylaxis with hepatitis B immune globulin (HBIG) and hepatitis B vaccine has been administered for many years, but it cannot completely prevent HBV intrauterine transmission, the main cause of infantile HBV infection. Thus, the mechanism underlying HBV intrauterine transmission should be clarified.

Peripheral blood mononuclear cell (PBMC) is an extrahepatic reservoir of HBV, and it facilitates immune response escape. Emerging evidence suggests that PBMC plays an important role in HBV intrauterine transmission [5]. HBV-infected PBMC in pregnant women may be transmitted from the mother to the fetus, which increases the risk of HBV intrauterine infection [6]. Some studies have detected HBV covalently closed circular DNA (cccDNA) in PBMC from patients with active chronic hepatitis B [7]. Our research group has found PBMC HBV cccDNA in HBsAg-positive mothers and their neonates [8]. HBV cccDNA, which is converted from HBV relaxed circular DNA (rcDNA), serves as a template for viral RNAs and subsequent generation of progeny virions [9,10]. HBV cccDNA is the most specific detection index of HBV replication and plays a key role in viral persistence and chronic hepatitis B [9]. Current therapies cannot eliminate cccDNA, and a few copies of cccDNA can reinitiate a full-blown infection [11]. However, whether the PBMC HBV cccDNA of HBsAg-positive pregnant women can increase the risk of neonatal HBV infection remains unresolved. Therefore, the present study aimed to explore the impact of PBMC HBV cccDNA on HBV intrauterine transmission and identify the influencing factors of PBMC HBV cccDNA. This study could guide further research on the mechanism and interruption of HBV intrauterine transmission.

Materials and methods

A total of 182 HBsAg-positive mothers and their neonates in the Third People Hospital of Taiyuan City during June 1, 2001 to December 31, 2002 were recruited. Among them, 31 HBsAg-positive mothers and their neonates were excluded because of incomplete information and lack of blood sample, and a pair of twins was excluded. Hence, 150 HBsAg-positive mothers and their neonates were finally included in this study. The basic information, including maternal demographics, history of disease, and HBV infection details before and during pregnancy, of the HBsAg-positive mothers was collected by well-trained interviewers utilizing standardized and unified questionnaires through face-to-face interview or was abstracted from medical records. All mothers donated 3 mL of anticoagulant and non-anticoagulant peripheral blood before delivery, and 3 mL of femoral venous blood of neonates was obtained within 24 h after birth before administering hepatitis B vaccine and HBIG. Blood samples were processed immediately within 24 h and stored at −80 °C. The research protocol was approved by the Ethics Committee of Shanxi Medical University, and all mothers signed written informed consents. Serum HBsAg and HBeAg levels were measured by enzyme-linked immunosorbent assay (Shanghai Kehua Biotech Co. Ltd., China) in accordance with the manufacturers’ instructions. PBMC was extracted from anticoagulant blood by Ficoll-Paque Plus (Shanghai Sangon Biotech Co. Ltd., China) and washed three times with 10 mL of Hank’s solution. We detected HBV DNA in the last washing solution by polymerase chain reaction (PCR), and the results were negative, which suggested that PBMC was not polluted by plasma HBV.

HBV DNA was tested by nested polymerase chain reaction (n-PCR). The PCR was carried out with 25 mL of template, 5 mL of 10× buffer, 4 mL of 25 mmol/L MgCl₂, 1 mL of primer [primer 1, 5′-CTGCTGGTGGCTCCAGTT-3′ (nt 59−76); primer 2, 5′-CAATACCACATCATCCA-3′ (nt 758−741); primer 3, 5′-CCTGCTCGTGTTACAGGC-3′ (nt 189−206); primer 4, 5′-GGCACTAGTAAACTGAGC-3′ (nt 689−672)], 1mL of 10 mmol/L dNTP, and 0.4 mL of Taq DNA polymerase (5 U/mL). The cycling program was as follows: 94 °C for 5 min; 35 cycles of 94 °C for 50 s, 55 °C for 50 s, and 72 °C for 50 s; and incubation at 72 °C for 5 min to ensure complete synthesis of the expected PCR products. If the detected results of the first round of PCR were negative, then the first-round product of 5mL was taken as a template for the second round of amplification; the amplification components and parameters were the same as those in the first round of PCR. All primers were synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). Primer 1/primer 2 and primer 3/ primer 4 were used in the first (699 bp) and second rounds of PCR (500 bp), respectively.

HBV cccDNA was transformed from HBV rcDNA by repairing the gap of each chain. PBMC HBV rcDNA and cccDNA were tested by selected polymerase chain reaction (s-PCR). The PCR was carried out with 15mL of template, 2 mL of 10× buffer, 0.15 mL of 25 mmol/L MgCl₂, and 0.2 mL of primer [primer 5, 5′-CCGACCACGGGGCGCACCTCTCTTTACG-3′ (nt 1515−1542); primer 6, 5′-CTAATCTCCTCCCCCAGCTCCTCCCAGT-3′ (nt 1758−1731); primer 7: 5′-CAAGGCACAGCTTGGAGGCTTGAACAGT-3′ (nt 1888−1861)], 0.4mL 10 mmol/L dNTP, and 0.2 mL Taq DNA polymerase (5 U/mL).

The cycling program was as follows: 94 °C for 5 min; 30 cycles of 94 °C for 60 s and 72 °C for 3 min; and incubation at 72 °C for 5 min. Primers 5 and 6 were used to amplify HBV rcDNA, which was 373 bp. Primers 5 and 7 were used to amplify HBV cccDNA, which was 243 bp. The PCR products were sequenced and blasted with HBV standard sequences.

Intrauterine transmission was defined as the presence of serum HBsAg and/or HBV DNA positive in the peripheral blood of neonates within 24 h after birth and before passive-active immune prophylaxis [12,13].

In univariate analyses, Chi-square test or Fisher’s exact test was employed to analyze categorical data, and Student’st-test or non-parametric test was employed to analyze continuous data. The unconditional logistic regression model was used to determine the risk factors of maternal HBV PBMC rcDNA positivity and cccDNA positivity as well as HBV intrauterine transmission. All statistical analyses were performed using SAS software (Version 9.2). Statistical significance was considered atP<0.05.

Results

Influencing factors of HBV intrauterine transmission

Of 150 neonates, the rate of HBV intrauterine transmission was 8.00% (12/150), with 10 neonates positive for HBsAg and 5 neonates positive for serum HBV DNA. The maternal and neonatal characteristics are listed in Table 1. HBV intrauterine transmission was significantly associated with neonatal weight (Mann–Whitney U-test, Z=−2.626, P= 0.009), the status of maternal serum HBV DNA (Chi-square test, c² = 5.087, P = 0.024), the status of maternal PBMC HBV cccDNA (Fisher’s exact test, P=0.020), and the status of maternal HBeAg (Fisher’s exact test, P=0.001) (Table 1).

Effect of maternal PBMC HBV infection and replication on HBV intrauterine transmission

After adjusting for covariates, we found that maternal PBMC HBV cccDNA was a risk factor of HBV intrauterine transmission (unconditional logistic regression, OR= 6.003, 95% CI: 1.249–28.855) (Table 2).

Influencing factors of PBMC HBV infection and replication in HBsAg-positive mothers

After adjusting for covariates, we found that HBeAg was the risk factors of HBV infection and replication in PBMC (Table 3). HBeAg was the risk factor of PBMC HBV rcDNA (unconditional logistic regression, OR= 3.896, 95% CI: 1.929–7.876) and PBMC HBV cccDNA (unconditional logistic regression, OR= 3.74, 95% CI: 1.186–11.793). HBIG administration was a protective factor of PBMC HBV cccDNA in the HBsAg-positive mothers (unconditional logistic regression, OR= 0.312, 95% CI: 0.102–0.954).

Relationship between PBMC HBV rcDNA and cccDNA in HBsAg-positive mothers

Of the 150 HBsAg-positive mothers, 36.67% (55/150) and 10% (15/150) were positive for PBMC HBV rcDNA and cccDNA, respectively. The positivity of PBMC HBV rcDNA was related to that of cccDNA in the HBsAg-positive mothers (Chi-square test,c² = 5.087, P = 0.024).

Discussion

HBV is the smallest hepatotropic DNA virus with a partly double-stranded DNA [14]. After HBV infection of the host cell, the rcDNA is released and converted to a cccDNA [9]. HBV cccDNA, as the transcriptional template of HBV, is the intracellular seed for initiation of the viral life cycle [9,10]. Previous work has shown that HBV possesses significant lymphotropic properties even if hepatocytes are considered as its main target [15]. Brind et al. [16] found that HBV with special nucleotide mutations spreads from PBMC to the serum, liver, and PBMC before and after liver transplantation. Stollbeckeret al. [17] detected HBV mRNA in PBMC, and Cabrerizo et al. [7] detected HBV cccDNA in PBMC. These results suggest that HBV can be transcribed and replicated in PBMC. In the present study, we found HBV rcDNA and cccDNA in the PBMC of HBsAg-positive mothers and that PBMC HBV rcDNA was tightly associated with PBMC HBV cccDNA, which suggested that HBV can exist and be transcribed in PBMC. These results indicate that PBMC is a reservoir of HBV and an extrahepatic site for virus replication.

In the present study, 8.00% of the neonates born to HBsAg-positive mothers had HBV intrauterine transmission, which is within the range of reported rates (5%–40%) in other areas of China [18–20]. The mechanism of HBV intrauterine transmission remains unclear. Several mechanisms of HBV intrauterine transmission, such as the placental leakage mechanism [21] and the germ cell transfer mechanism [22], have been proposed. However, our research group observed that PBMC HBV infection exerts a significant impact on HBV persistent infection, immune tolerance, and immune interruption in neonates. We also found several pieces of evidence supporting the PBMC transfer mechanism. Wanget al. [23] detected HBV DNA in 114 pregnant women and neonatal PBMC smear byin situ hybridization, confirming the consistency of PBMC HBV infection between HBsAg-positive pregnant women and their newborns. Shiet al. [8] were the first to report that maternal PBMC cccDNA is related to neonatal PBMC cccDNA. Weiet al. [24] reported that the cell traffic from an HBsAg-positive mother to the fetus contributes to HBV intrauterine infection. Those studies showed that the HBV-infected PBMC of mothers may be transmitted to neonates and that PBMC HBV cccDNA may be related to HBV intrauterine transmission. Other studies reported similar results. Xuet al. [5] reported that the HBV-infected PBMC of mothers may serve as a source of HBV intrauterine transmission. In the current study, we found that maternal PBMC HBV cccDNA was a risk factor of HBV intrauterine transmission. Thus, PBMC might play an important role in HBV intrauterine transmission, and PBMC HBV cccDNA was more likely to be transmitted from HBsAg-positive mothers into their neonates.

Furthermore, maternal HBeAg was a risk factor of maternal PBMC HBV rcDNA and cccDNA, suggesting that HBeAg might be related to PBMC HBV infection and replication. HBeAg was an important marker of HBV actively replicated in the liver and may also serve as an index of HBV actively replicated in PBMC. HBIG serves as a passive measure for interrupting HBV transmission and improving immune ability. The result showed that HBIG administration during pregnancy was a protective factor of PBMC HBV cccDNA, which suggested that HBIG might suppress the replication of PBMC HBV DNA and thus reduce the rate of HBV intrauterine transmission. Zhuet al. [25] reported that HBIG administered during the antepartum stage reduces HBV mother–neonate transmission rates, whereas Yuanet al. [26] reached an opposite conclusion. The effect of HBIG administration on HBV intrauterine transmission needs further exploration. HBV cccDNA, the index of HBV replication, was regarded as a viral persistence reservoir and a key obstacle for the cure of chronic hepatitis B. Recent research has focused on developing treatment approaches that can eliminate cccDNA [27–29]. Several studies showed that CRISPR/Cas9 nuclease efficiently inhibits viral replication [28], but this finding still needs further investigation. This result demonstrated that HBV replication can be effectively interrupted and that the rate of HBV intrauterine transmission can be reduced by suppressing PBMC HBV cccDNA. PBMC is a collection of various cells. Previous studies have shown that the HBV infection rate of different subgroup cells in PBMC is different, but the relationship between HBV infection and HBV intrauterine transmission has not been reported. We were unable to identify the HBV infection in different PBMC subgroups in this study because of the limited sample size. This topic will be explored in future research.

In the present study, we detected HBV rcDNA and cccDNA in the PBMC of HBsAg-positive mothers and explored the relationship between PBMC HBV infection and replication and HBV intrauterine transmission. We also identified the influencing factors HBV rcDNA and cccDNA in PBMC. Although the sample size of this study was not large enough, the results can guide further explorations on the mechanism and interruption method of HBV intrauterine transmission.

In conclusion, PBMC is a reservoir of HBV and an extrahepatic site for HBV replication and plays an important role in HBV intrauterine transmission. Maternal PBMC HBV cccDNA is a risk factor of HBV intrauterine transmission. Maternal HBeAg is a risk factor of PBMC HBV cccDNA, whereas HBIG administration during pregnancy is a protective factor.

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