Perinatal transmission of hepatitis c antigens: envelope 1, envelope 2 and non-structural 4
ABDElFATTAH M. ATTAllAH1, SANAA O. ABDAllAH2, MOHAMED El-FAR3, MOHAMED M. OMRAN4, ASHRAF A. TABll5, MOHAMED F. gHAly6, SAID M. EZZAT6, MANAl O. ElHAMSHARy7, ZEINAB M. El-gOHARy3, ATTIyA H. MOHAMEDIN3, ADEl A. El-MORSI3, AHMED A. ASkORA6, MOHAMED A. ABDElRAZEk1, HAlA M. El-kAFRAwy1, MOHAMED H. kENEBER1, MOHAMED R. kHAlIl1, MOSTAFA M. AggAg1, MOHAMED S. ElBENDARy1, MENATAllAH M. El-DEEB1, MAHMOuD S. ABuZAID1, AHMED T. MANSOuR1 & AHMED A. ATTAllAH1
From the 1Biotechnology Research Center, New Damietta City, 2Faculty of Science, Cairo University, Giza, 3Faculty of Science, Mansoura University, Mansoura, 4Faculty of Science, Helwan University, Cairo, 5National Research Center, Giza, 6Faculty of Science, Zagazig University, Zagazig, and 7Genetic Engineering and Biotechnology Institute, Menoufiya University, Sadat City, Egypt
abstractBackground: Perinatal exposure to hepatitis C virus (HCV) antigens during pregnancy may affect the developing immune system in the fetus. we aimed to study the perinatal transmission of HCV structural and non-structural antigens. Methods: Sera from 402 pregnant mothers were tested for anti-HCV antibody and HCV RNA. HCV antigens were determined in sera from 101 HCV-infected mothers and their cord blood. Results: In both serum and cord blood samples, HCV NS4 (non-structural 4) at 27 kDa, E1 (envelope 1) at 38 kDa and E2 (envelope 2) at 40 kDa were identified, purified and quantified using western blotting, electroelution and ElISA. Maternal sera and neonate cord blood samples had similar detection rates for NS4 (94.1%), E1 (90.1%) and E2 (90.1%). The mean maternal serum levels (optical density, OD) of HCV NS4 (0.87 0.01), E1 (0.86 0.01) and E2 (0.85 0.01) did not differ significantly (p 0.05) from those of neo-natal cord blood (0.83 0.01, 0.87 0.01 and 0.85 0.01, respectively). Also, strong correlations (p 0.0001) were shown between sera and cord blood sample levels of HCV NS4, r 0.77; E1, r 0.76 and E2, r 0.80. The vertical transmis-sion of these antigens in vaginal delivery did not differ significantly (p 0.05) from those in caesarean section. Conclusions: These findings indicate that vertical transmission of HCV NS4, E1 and E2 antigens was very high. Thus, exposure to these antigens may influence the developing immune responses to natural infection or future vaccination.
Keywords: HCV; NS4; E1; E2; placental transfer
introduction
worldwide, viral hepatitis is the most common cause of hepatic dysfunction in pregnancy. It is associated with high risk of maternal complications and has become a leading cause of fetal death [1]. Because of vaccination programs against hepatitis B, hepatitis C virus (HCV) has become the primary cause of chronic viral hepatitis in children, with vertical transmission becoming the leading source of infection [2]. The prevalence of HCV infection in pregnant women is
estimated to be between 1 and 8% [3]. unfortunately, to this day, most cases of hepatitis C in children from resource-limited settings can be traced to transfusion of inadequately screened blood products and/or par-enteral transmission [4]. Consistent with this, mother-to-child (vertical) transmission of HCV has been clearly documented, with reported rates averaging 5–10% [5].
The majority of reported vertically transmitted HCV infections have occurred from anti-HCV-positive
mothers with detectable HCV RNA levels during pregnancy, but other reports describe HCV-infected children born to mothers who had no HCV RNA detected in their serum. Indeed, in the European Pediatric HCV Network study, vertical HCV transmis-sion occurred in 3.3% of women classified as not virae-mic during their pregnancy [6]. unfortunately, because of these findings, non-viraemic women cannot be assured that they have no chance of infecting their neo-nates, although the risk appears to be very low [7].
There is no safe and effective intervention known to prevent perinatal transmission of HCV. An effective hepatitis C vaccine has not been developed and the drugs used most commonly to treat HCV in both chil-dren and adults are not recommended for use in pregnancy [3,8]. HCV contains a positive-sense single-stranded RNA genome that is 9600 nucleotides in length. The genomic order of precursor protein has been shown to be C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B [9]. The use of these HCV pro-teins as a vaccine candidate has been evaluated with positive safety and immunogenicity profiles [10,11]. In the present study, we evaluated the placental delivery of the HCV NS4 (non-structural 4), E1 (envelope 1) and E2 (envelope 2) viral proteins from infected moth-ers to their newborns. The fetus has its own developing yet already active immune system that can modulate to the antigenic environment [12]. Placental transfer of a maternal schistosome antigen was reported to pre-dispose a child to an immunoregulated response [13]. However, newborns have limited adaptive memory, it may be primed by exposure to antigens in utero [14]. Thus, the delivery of such maternal HCV antigens may influence the developing immune responses to natural infection or vaccination of children born in endemic areas [15].
Materials and Methods
Subjects
Pregnant Egyptian women (n 402), attending the antenatal clinic of the gynecology and Obstetrics Department of kafr Saad Central Hospital, Damietta, Egypt, were included. They were screened for anti-HCV antibody (Biotec laboratories ltd, Ipswich, Suffolk, uk) and HCV RNA (COBAS Ampliprep/COBAS TaqMan; Roche Diagnostics, Pleasanton, CA, uSA). At the time of delivery, venous blood and cord blood samples were collected from mothers and their offspring. They were classified according to the mode of delivery: 241 (60%) by vaginal delivery and 161 (40%) by caesarean delivery. There were 101 HCV-infected women (positive for anti-HCV antibody and HCV RNA, with mean age SD of 27.7 4.9 years) and 301 HCV-non infected women (negative for anti-HCV antibody and HCV RNA, with mean age of
26.9 3.9 years; used as controls). Each blood sample was allowed to clot; the serum that was subsequently obtained was stored at – 20°C until analysed. Informed consent was obtained from all individuals participating in the present study, and they were fully informed concerning the nature of the disease and the diagnostic procedures involved. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki.
Identification of HCV NS4, E1 and E2 antigens using SDS and Western blot
Sodium dodecyl sulfate polyacrylamide gel electro-phoresis (SDS-PAgE) was carried out in 0.75 mm thick, 12% vertical slab gels according to the method of laemmli [16]. Serum samples were mixed with the sample buffer (0.125 M Tris base, 4% sodium dodecyl sulfate, 20% glycerol, 10% mercaptoetha-nol, and 0.1% bromophenol blue as a tracking dye) and immediately boiled for 3 min. A mixture of reference proteins (BioRad laboratories, Hercules, CA, uSA) was run in parallel. gels were then stained with Coomassie blue. Resolved samples separated by SDS-PAgE were electro-transferred onto 0.45 mm pore size nitrocellulose (NC) sheet (Sigma Chemical Co., St louis, MO, uSA) according to the method of Towbin et al. [17]. The NC sheet was blocked using 5% (w/v) non-fat dry milk in 0.05 M Tris-buffered saline (TBS) containing 200 mM NaCl (pH 7.4). The NC sheet was rinsed in TBS and incubated separately with the antibody to HCV NS4 [18], E1 or E2 (ABC Diagnostics, New Damietta, Egypt). The NC sheet was washed three times (30 min each) in TBS, followed by incubation for 2 h with goat anti-rabbit Igg (Sigma Chemical Co.) for the NS4 antibody or goat anti-mouse Igg alkaline phos-phatase conjugate (Sigma Chemical Co.) for E1 and E2 antibodies. After washing three more times with TBS (15 min each), the NC sheet was exposed to alkaline phosphatase substrate [5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT)] in 0.1 M Tris buffer, pH 9.6 (Sigma). Then 10 min later the reaction was stopped using distilled water and the development of colour was observed.
Detection of HCV NS4, E1 and E2 antigens using ELISA
Target HCV NS4, E1 or E2 bands were cut from preparative polyacrylamide gels and electroeluted from the gels in a dialysis bag (Sigma) as described by Attallah et al. [18]. The protein content of the purified antigen band was determined [19] before storage at –20°C. Detection of HCV target antigens in serum and cord blood samples was carried out using ElISA according to Attallah et al. [18]. In brief,
serum and cord blood samples were diluted and incubated overnight at 4°C in ElISA polyethylene micro plates (Costar, Corning life Sciences, Acton, MA, uSA). After blocking of free binding sites with 0.5% bovine serum albumin (BSA), specific anti-HCV NS4, E1 or E2 antibodies diluted in phosphate-buffered saline (PBS, pH 7.2), with 0.05% Tween were added (50 ml/well) and incubated at 37°C for 2 h. After washing, 50 ml/well, anti-rabbit Igg alkaline phosphatase conjugate (Sigma Chemical Co.) for NS4 or goat anti-mouse Igg alkaline phosphatase conjugate (Sigma Chemical Co.) for E1 and E2 diluted in 0.2% (w/v) BSA in PBS-T20, was added and incubated at 37°C for 1 h. The amount of cou-pled conjugate was determined by incubation with nitrophenyl phosphate substrate for 30 min at 37°C. The reaction was stopped using 3 M NaOH. The optical density (OD) at 405 nm was read using the 960 microplate autoreader (Metertech Inc., Taipei, Taiwan). Colour intensity was proportional to the amount of bound conjugate and therefore is a func-tion of the target antigen concentration present in the serum sample. The cut-off OD for ElISA positivity was set at 0.31 for NS4, 0.32 for E1 and E2 – the mean OD plus 3 SD for sera from HCV-non-infected mothers.
Statistical analysis
All statistical analyses were done using Statistical Package for the Social Sciences (SPSS) version 15.0 on Microsoft windows XP (SPSS Inc., Chicago, Il, uSA) and the graphPad Prism package (v.5.0, graphPad Software, San Diego, CA, uSA). Continu-ous variables were expressed as mean standard deviation (SD) or mean standard error of the mean (SEM), whereas categorical variables were expressed as numbers (percentages). Statistically significant dif-ferences between the groups were determined by the Student’s t test. The correlations between level of serum and cord blood antigens were evaluated by Pearson’s correlation coefficient. The diagnostic val-ues of HCV NS4, E1 and E2 antigens were assessed by receiver operating characteristics (ROC) analysis. The area under the ROC (AuC) equal to 1.0 is char-acteristic of an ideal test, whereas 0.5 indicates a test of no diagnostic value. The nearer a curve shifts to the top left-hand corner of the graph, the more useful the marker is for the diagnosis [20].
results
Identification of HCV NS4, E1 and E2 antigens
Eight randomly selected serum and cord blood sam-ples from pregnant mothers and their neonates were
subjected to western blotting. At 27 kDa, a single immunoreactive band corresponding to HCV NS4 appeared in sera of mothers infected with HCV and cord blood samples of their offspring. No reactive band was observed in serum from healthy mothers or their neonates under this condition (Figure 1A). As shown in Figure 1B, HCV E1 migrates at approxi-mately 38 kDa as a single immunoreactive band. No reactive band was observed in samples from healthy controls. In contrast to healthy individuals, the specific monoclonal antibody to HCV E2 identified a single immunoreactive band at 40 kDa molecular weight in sera of pregnant mothers infected with HCV and cord blood samples of neonates (Figure 1C).
Vertical transmission of HCV NS4, E1 and E2 antigens
ROC analysis was used to evaluate the ability of HCV NS4, E1 and E2 antigens to differentiate HCV-infected from non-infected mothers. The AuC was
Figure 1. western blots of serum and cord blood samples from pregnant mothers and their neonates showing the detection of (A) HCV NS4, (B) E1 and (C) E2 antigens. No reaction was observed in samples from healthy controls. Molecular weight markers were ovalbumin (60.0 kDa), carbonic anhydrase (39.2 kDa), trypsin inhibitor (28.0 kDa) and lysozyme (18.3 kDa).
0.956, 0.945 and 0.939 (p 0.0001) for HCV NS4, E1 and E2, respectively (Figure 2). ElISA screening for HCV NS4 showed that serum and cord blood samples have the same detection rate (95/101, 94.1%). The mean HCV NS4 levels (OD) in serum of HCV-infected mothers and cord blood samples of their neonates were almost similar to each other with no significant difference (p 0.06; Table Ι). No false-positive results were found in serum and cord blood samples of 301 uninfected controls. A strong correla-tion was shown between the OD levels of HCV NS4 antigen in sera of infected mothers and cord blood samples from their neonates (r 0.77; p 0.0001; Figure 3A). Overall, the above findings indicated that vertical transmission of HCV NS4 antigen from HCV-infected mothers to offspring was 100%.
Vertical transmission of HCV E1and E2 did not differ from that of HCV NS4 (100%). using ElISA, the detection rates for HCV E1 and E2 were the same for both serum and cord blood samples
Figure 2. ROC analysis for (A) HCV NS4, (B) E1 and (C) E2 to discriminate HCV-infected from non-infected mothers. AuC, area under curve; ROC, receiver operating characteristic.
Figure 3. Correlation between levels of (A) HCV NS4, (B) E1 and (C) E2 in mother’s serum and their neonate’s corresponding cord blood samples. For each antigen, there is a strong correlation between levels in serum and cord blood samples (p 0.0001).
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Perinatal transmission of HCV E1, E2 and NS4 5
(91/101, 90.1%). There was no significant difference (p 0.18) between the mean of HCV E1 (OD) or E2 (p 0.17) levels in serum of infected mothers and in cord blood samples of their neonates (Table I). levels of HCV E1 in serum samples were correlated strongly (r 0.76; p 0.0001; Figure 3B) with those in cord blood samples. Also, there was a strong correlation (r 0.80; p 0.0001; Figure 3C) between levels of HCV E2 in serum and cord blood samples.
Impact of mode of delivery on vertical transmission of HCV antigens
The mean of HCV NS4, E1 or E2 concentration in maternal serum and cord blood samples from their neonates did not differ significantly (p 0.05; Table I) between vaginal and caesarean delivery. So, our results revealed that the mode of delivery has no significant impact on the vertical transmission of the aforemen-tioned antigens.
Discussion
Although the rate of HCV transmission from mother to child during delivery is low, with reported average transmission rates of 5–10%, it has a great health impact. Approximately 75% of children vertically infected with HCV will develop chronic hepatitis C; 80% with asymptomatic inactive infection and 20% with active infection indicated by persistent viraemia, elevated alanine aminotransferase (AlT) and hepato-megaly [21,22].
An effective hepatitis C vaccine has not been developed and the drugs used most commonly to treat hepatitis C in both children and adults, interferon and ribavirin, are not recommended for use in pregnancy [23,24]. Thus effective prevention strategies are badly needed as the global burden of chronic hepatitis C increases. The identification of the targeted viral anti-gens facilitated the rational development of a vaccine for the prevention of chronic HCV infection [25]. HCV vaccine studies directed at the induction of virus neutralizing antibodies have focused on the use of the E1E2 heterodimer. Interestingly, E1-neutralizing anti-bodies together with recruited Th responses appear to have facilitated HCV 1b clearance [26]. Other
mechanisms may have contributed to viral clearance, such as antibody-dependent cellular cytotoxicity, recruited innate responses in the liver or late induction of cytotoxic T-lymphocyte activity [26]. Recombinant adenovirus as a vaccine vector expressing NS4 antigen in an HCV-infected mouse model induces high T-cell proliferation, proinflammatory cytokines such as inter-feron (IFN)-g, tumour necrosis factor (TNF)-a, inter-leukin (Il)-2 and Il-6, and antibody responses that can significantly reduce the HCV viral load [12]. Exposure to maternal antigens might predispose a child to develop an immunoregulated clinical presen-tation of the infection [15]. In this respect, the present study was designed to investigate the possibility of pla-cental transmission of HCV NS4, E1 and E2 and thus examined their probability to influence the developing immune responses to natural infection. At the same molecular mass, the HCV NS4 (27 kDa), E1 (38 kDa) and E2 (40 kDa) antigens were identified in umbilical cord serum, as well as in serum samples from HCV-infected mothers. Other evidence that these antigens transfer through the mother’s placenta to their neo-nate is based on ElISA screening; HCV NS4, E1 and E2 were detected in mother’s serum and their corre-sponding neonate’s cord blood with the same detec-tion rate (vertical transmission rate of 100%). Similarly, however, some viral antigens like hepatitis B e antigen totally cross the human placenta [27], others, due to partial degradation, like human immunodeficiency virus-1 p24 antigen cross the placenta at a low rate [28]. Here, there is no significant difference (p 0.05) between antigen levels in serum and those of cord blood samples. Some biochemical treatments indi-cated that the nature of the epitopes of these HCV antigens did not change with the transfer via placenta (data not shown). Other infectious antigens that can cross the human placenta are hepatitis B surface anti-gen [29], glycoprotein H of cytomegalovirus [30] and Schistosoma mansoni antigen [13]. High transmission rates for anti-HCV and HCV RNA were reported [31,32].
Our findings suggest that these HCV antigens can traverse the placental barriers efficiently. Babik et al. estimated that 1 1013–1 1014 HCV virions reach the placental during gestation, making it highly prob-able that some particles would cross the placenta even
Table I. Comparison between serum and cord levels (optical density, OD) of HCV NS4, E1 and E2 in relation to mode of delivery.
HCV antigen All HCV-infected pairs Vaginal delivery Caesarean section
Serum Cord p value Serum Cord p value Serum Cord p value
cytotoxic T-cell response against HCV leads to chronic infection with this virus [46,47].
Collectively, we have identified the placental transfer of intact HCV NS4, E1 and E2 antigens. The use of these HCV proteins as a vaccine candidate has been evaluated with positive safety and immunoge-nicity profiles [10,11]. On the other hand, in utero exposure to such antigens can result in a state of immunologic tolerance in the neonate [48]. Thus, further studies to fully establish the mechanisms involved in the influence of placental delivery of these HCV antigens on immunoregulation are needed.
acknowledgments
we would like to thank the staff of the Biotechnology Research Center for their involvement in the experi-mental work. This work was completely supported financially and carried out at the Biotechnology Research Center, New Damietta, Egypt.
Declaration of interest: The authors report no conflicts of interest. The authors alone are respon-sible for the content and writing of the paper.
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