Wen, Jessica W.*; Haber, Barbara A.†
Chronic hepatitis C infection is a major health concern worldwide. It is the most common blood-borne infection and the leading indication for liver transplantation in adults (1). It has been known that children who acquire perinatal hepatitis C virus (HCV) infection differ from adults regarding the rate of viral clearance, duration of infection, and the progression to cirrhosis. With the adoption of effective blood screening practices since early 1990s, vertical transmission, from mother to infant, is now the major route of HCV infection in children (2). Whereas adults with acute hepatitis C may be symptomatic with jaundice, infants who acquire the infection via vertical transmission are virtually asymptomatic, without signs and symptoms of liver disease, other than possible elevated transaminase levels (3). Nonetheless, cirrhosis, and rarely, hepatocellular carcinoma have been reported during childhood (4,5). At present, there are no effective methods to prevent perinatal transmission (6).
In the present review, we explore the factors that may influence the natural history of HCV infection in children who acquire the infection through maternal–fetal transmission. In particular, how viral diversity and the infant immune system may affect viral transmission are highlighted. With the recent arrival of direct-acting antiviral medications and the attempts at developing successful HCV vaccines, these are important areas to understand, to formulate future strategies in the prevention of maternal–fetal transmission.
NATURAL HISTORY OF MATERNAL–FETAL TRANSMISSION
The prevalence of HCV infection among pregnant women has been difficult to evaluate because of geographical variables and testing methods. It is estimated that the prevalence of antibody to HCV (anti-HCV) in pregnant women is 0.1% to 2.4%. The proportion of women with anti-HCV who have active infection with viremia is approximately 70% (7). Despite this, mother-to-infant transmission of HCV has been shown to be relatively low, approximately 5% in HIV-negative, HCV-infected mothers (8,9). The transmission rate goes up significantly to about 25% in women who are co-infected with HIV (10), highlighting the importance of the immune environment for transmission.
Mother-to-infant transmission of HCV infection can occur by one of 3 ways, in utero, intrapartum, and perinatal. In general, there is a higher rate of transmission when maternal viral load is >106 IU/mL. In a US study of 244 mother–infant pairs, the HCV infection transmission rate was 1.6% in mothers who had viral load of ≤200,000 IU/mL (106 copies/mL), 2.3% in those with viral load between 200,000 and 2 million IU/mL (106–107 copies/mL), and 11.8% in those with viral load ≥2 million IU/mL (107 copies/mL) (10). When maternal HCV RNA level was <1 million IU/mL, the rate of transmission was significantly higher in the case of HCV/HIV co-infection compared with that in HCV monoinfection (10,11). It is, however, important to note that viral load can fluctuate during pregnancy and the transmission of HCV infection can occur even in those with lower viral load. Furthermore, variation in viral load threshold because of different methodologies of measuring viral RNA and maternal HIV status makes it difficult to counsel women regarding their risk of transmission based on viral load.
In addition to maternal HIV status and maternal viral load, other risk factors predisposing to transmission include prolonged rupture of membrane >6 hours before delivery and fetal scalp monitoring (10). Elective cesarean section does not reduce the rate of HCV transmission (12,13) and breast-feeding does not promote transmission (10).
HCV INFECTION IN EARLY INFANCY
HCV is frequently detected in infant cord blood; however, few infants develop subsequent chronic HCV infection. Cord blood testing can be unreliable because contamination with maternal blood may occur. It is also possible that some of these infants are exposed to the virus but have an enhanced ability to effectively resolve their infection. In most infected infants, HCV RNA is detected at or soon after delivery, suggesting in utero or intrapartum transmission (14). It has also been hypothesized that transfer of HCV RNA can occur from mother to child without infective viral particles, such as neutralizing antibody–bound viral RNA that have lower infectivity or naked nucleocapsids (15–17).
Although some infants born to mothers with HCV infection may have detectable HCV RNA early in life, and some even with evidence of biochemical liver injury, the majority will clear the infection by 18 months of age, leading to the low overall transmission rate. In a prospective Spanish study, 20% of children born to HIV−/HCV RNA+ mothers were found to have vertical transmission, defined as positive HCV RNA from serum sampling on at least 2 occasions that were more than 2 months apart (18). Sixty-five percent of those children, however, ultimately cleared the infection by 18 months of age, leading to persistent infection in only 7% of children, with mostly genotype 1 infection, which is consistent with prior data (8,9). Because HCV has a relatively short circulating half-life of 2.5 hours (19,20), these infants likely had true infection with subsequent viral clearance, rather than passive transmission of noninfectious maternal viral RNA. Similar findings were demonstrated in an Italian cohort with predominant genotype 2 and 3 infection, as well as a Japanese cohort, presumably of mostly genotype 1b infection (21,22). Clearance of HCV infection in vertically acquired older children has been reported as late as 7 years of age, although most children clear the infection within the first 18 months of life (21,23).
Because of transient viremia, spontaneous viral clearance, and inconsistent detection of serum HCV RNA in young infants, HCV RNA screening is generally not recommended in infants <1 month of age. Sensitivity of HCV RNA by polymerase chain reaction for diagnosing HCV infection when compared with anti-HCV status at or beyond 18 months of age is low at birth (22%) and becomes 85% when tested at 6 months of life (15). Diagnosis of vertically acquired HCV infection is made with positive RNA testing on 2 occasions between the age of 2 and 6 months, followed by confirmation of chronic hepatitis C infection with RNA testing after 12 months of age (24); however, given the lack of treatment options during infancy, and to avoid repeated testing with its associated costs, the current preferred method for screening at-risk infants is anti-HCV testing at age >18 months followed by HCV RNA testing, if positive (25).
HCV GENOME DIVERSITY
HCV is a single-stranded enveloped RNA virus that belongs to the family Flaviviridae. During evolution of the virus, there is a significant sequence variation resulting in genotypes that vary as much as by 33% (26). Like all of the RNA viruses, there is an inherent infidelity of the HCV RNA polymerase leading to continuous diversification in the viral genome termed quasispecies (27). It is approximated that each HCV viral progeny acquires 0.1 to 1 mutation per viral genome during replication. The viral envelope glycoprotein E2 contains a small region of 27 amino acids termed hypervariable region 1 (HVR1) and it is considered the most diverse domain within the HCV genome. It is the major neutralizing epitope for anti-HCV antibodies, and therefore it is under selective pressure by the host immune system (28). Other than HVR1, the highly conserved 5′-untranslated region, and the core region, sequence variability is distributed equally throughout the rest of the HCV viral genome (29).
The various quasispecies may have different degree of fitness to replicate, and the host environment may also lead to selection of dominance in certain quasispecies (30). It has been suggested that the emergence of quasispecies is promoted by a delay in adaptive immune response during primary infection. Quasispecies also act as a critical mechanism for the virus to escape the host immune system, leading to viral persistence. With the development of newer direct antiviral agents, quasispecies will also play an important role in response to therapy and the selection of drug resistance.
In adults with acute infection, the early evolution of the viral quasispecies has been shown to predict the clinical outcome of acute hepatitis C infection. Those with increased viral heterogeneity are more likely to demonstrate persistent infection, whereas those with stable genome are more likely to be associated with the resolution of infection (31). In contrast, Curran et al (32) showed that in a small cohort of patients with untreated chronic hepatitis C infection, viral diversity gradually increased in those with nonprogressive milder disease compared with relative stasis in viral diversity seen in the patients who had severe progressive cirrhosis. Similar results were seen in a cohort of patients with hemophilia and HCV infection (33). Therefore, long-term viral evolution appears to correlate with the severity of liver disease, either causal or perhaps by association; however, conflicting reports of viral diversity and progression of liver disease exist (34), likely because of differences in mean duration of follow-up, small sample size, phase of the infection, and sequencing techniques. It is also important to note that the present quasispecies analysis does not account for the proportion of clonal representation; therefore, minor clones are weighted equally as major clones in these analyses, although newer techniques have been developed to account for such differences (35).
In children who acquire the infection vertically, limited information is available on the early evolution of viral quasispecies. It has been suggested that children with vertically acquired HCV infection have less viral diversity than their mothers, particularly during infancy (36). By sequencing only the E2 hypervariable region, infants appear to have mostly single dominant variant within the region, whereas their chronically infected mothers possess multiple variants (37). The HCV strain in the infant does not always correspond to the dominant strain in the mother, and may actually represent a minor but transmissible maternal strain. Viral diversity of the infant strain begins to increase at 6 to 7 months of age, and the pattern of diversification is different from that of their mothers (36).
In a study by Farci et al (38) in children with vertical transmission, HCV was found to replicate rapidly, reaching peak titers of viremia within the first months of life, before anti-HCV seroconversion. Similarly, the viral diversity in the E1 and E2 genes, including HVR1, was generally low early in life; however, a small subset of children did have high levels of genetic diversity thought to reflect maternal HCV quasispecies. Interestingly, around the time of anti-HCV seroconversion, children with mild liver enzyme elevation had more complex quasispecies with gradual increase in genetic diversity, whereas those with more evidence of liver injury with higher alanine aminotransferase elevation were associated with homogenous viral population, thought to be related to more effective T-cell response with immune selection pressure.
HCV INFECTION TRANSMISSION AND INFANT IMMUNE SYSTEM
In addition to viral quasispecies, the infant immune system also likely modulates the persistence of HCV infection. In an analysis of maternal–infant human leukocyte antigen (HLA) type pairing, HLA mismatch between the pair was found to be a protective factor for HCV infection transmission, likely because of alloreactive immune responses, preventing vertical transmission of HCV infection (39). There has also been a significant association between HLA-DR13 and the likelihood of viral clearance in infants born to HCV-infected mothers (40). Other HLA factors have been implicated in vertical transmission of HCV as well (41).
In a genome-wide association study, a single nucleotide polymorphism 3 kilobases upstream of the IL28B gene, encoding for interferon-λ-3, was shown to associate with both a 2-fold increase in response to interferon/ribavirin treatment and spontaneous viral clearance (42). In a vertical transmission study, neither the mother's nor infant's IL28B polymorphism had any effect on the rate of HCV transmission; however, a favorable IL28B CC polymorphism was associated with spontaneous clearance of HCV infection in infected children, including genotype 1 HCV (18).
INFANT IMMUNE RESPONSE TO HCV INFECTION
Clearance of HCV infection is mediated by successful host adaptive immune responses, which include both HCV-specific T-cell response and neutralizing antibody. Additionally, the innate immunity also plays an important role in the recognition and clearance of HCV infection (43).
The innate immune system contributes to immune surveillance, neutralizes infection as well as triggering inflammatory response, and it plays a critical role in inducing adaptive immunity. It is also believed to promote hepatic fibrosis in the setting of chronic HCV infection (44). The hallmark of innate immune response to viral infection is rapid production of interferon and other proinflammatory cytokines. The innate immunity is immature in newborns (45); however, in an in vitro study of primary human fetal liver cells, HCV infection led to induction of λ-interferon and increased interferon-induced gene expression (46). Therefore, it is likely that innate immune system contributes to the clearance of HCV in early infancy.
The antibody response to HCV does not follow classical pattern of immunoglobin M response (47). If antibodies are reactive with the hypervariable region E2, they possess neutralizing activities; however, HCV virus can escape the neutralizing antibody effect by mutating within this hypervariable domain. Furthermore, in chimps, reinfection is common even with the homologous HCV strain. This indicates that HCV infection does not elicit protective immunity, and this poor immunity against HCV is not attributed solely to antigenic variation among different strains (48). In infants, the antibody response is immature and does not reach full capacity until the teenage years (49). Memory response occurs starting around 4 months of age (50). The ability to produce immunoglobulin in response to antigen stimulation gradually increases in 2 to 8 months of life, although never reaches adult level until much later in life. This suggests that the low viral diversity within the HVR region of the HCV genome in extremely young infants may be because of a lack of immune selective pressure from neutralizing antibodies.
In addition to the innate immunity and neutralizing antibody response, successful HCV immune response is mostly mediated by T-cell response. In the past, neonates were believed to have extremely immature adaptive immune response, unable to mount good immune responses. Recent studies, however, suggest that neonates are capable of mounting effective T-cell response, but the reaction is highly variable, depending on the condition of antigen exposure (50). This is believed to be a protective mechanism because there is a surge in antigenic exposure after birth and a heightened state of inflammation may be detrimental to the developing infants.
CD8+ cytotoxic T-lymphocyte response shortly after inoculation is important for HCV viral clearance because the emergence of escape mutants of cytotoxic T lymphocytes is associated with persistent viral infection (51, http://links.lww.com/MPG/A292). In acute HCV infection, onset of hepatitis correlates with CD8+ T-cell response. Those with a dampened and limited CD4+ and CD8+ T-cell response subsequently developed chronic infection (52, http://links.lww.com/MPG/A292). In a study by Della Bella et al, children born to HCV-infected mothers had more frequent and vigorous proliferation of HCV-specific CD4+ T cells compared with their mothers (53, http://links.lww.com/MPG/A292). The lymphocytes from these children produced lower level of a suppressive cytokine, interleukin-10, which normally acts to dampen the activation of immune response; however, of the few children who subsequently developed chronic HCV infection, none had lymphocyte proliferation in response to HCV core or envelope peptide challenge. Therefore, intrinsic differences in induction of viral-specific CD4+T-cell–mediated immune response may provide protective function against HCV infection and contribute to the low rate of vertical transmission of HCV infection. Interestingly, a large number of children born to HCV-infected mothers in this study had HCV-specific T-cell activation despite persistently negative HCV RNA. Therefore, it is likely that more children have been exposed to HCV vertically than reported HCV vertical transmission rate based on HCV polymerase chain reaction or anti-HCV antibody testing.
Maternal viral load, HIV co-infection, and high-risk behaviors such as intravenous drug use have been associated with higher transmission rate in epidemiologic studies (10,11,21); however, maternal neutralizing antibodies may also play an important role in maternal–fetal transmission (16). Anti-HCV immunoglobin G is known to cross the placenta (54, http://links.lww.com/MPG/A292). Manzin et al (37) showed that most of the HCV RNA from nontransmitting mothers was detected in heavy sucrose density gradient, presumably bound to neutralizing antibodies. This was in contrast to HCV RNA molecules detected in the light density gradient from HCV-transmitting mothers, which were not antibody bound and may indicate higher infectivity. In another small study, however, the presence of detectable maternal HCV-neutralizing antibodies did not correlate with the development of chronic HCV infection in children (55, http://links.lww.com/MPG/A292).
Amniotic fluid rarely has detectable virus, so in utero HCV transmission is directly through the placenta (56, http://links.lww.com/MPG/A292). In the placentas of HCV-infected mothers, there are an increased number of natural killer cells compared with that in control from non-HCV pregnant women and these cells have increased cytotoxicity (57, http://links.lww.com/MPG/A292). Additionally, HCV-infected pregnant women have a surge of HCV-specific T-cell population during the third trimester and the immediate postpartum period, as well as increased production of endogenous interferon (58,59, http://links.lww.com/MPG/A292). These factors perhaps partially contribute to the low rate of vertical transmission of HCV infection.
In addition to hepatocytes, HCV can also infect peripheral blood mononuclear cells, such as T cells, B cells, macrophages, and dendritic cells. These cells can then act as a reservoir of HCV replication and increase rate of maternal–fetal HCV transmission (60–62, http://links.lww.com/MPG/A292).
WHERE TO GO FROM HERE?
Chronic hepatitis C infection often leads to a silent liver disease with minimal symptoms until the development of cirrhosis or hepatocellular carcinoma. It is, however, a significant cause of morbidity and mortality in the United States and a leading indication for liver transplantation in the adult population. With the arrival of new direct-acting antiviral therapies, most patients can achieve an optimal rate of virologic clearance if the infection is recognized before the development of cirrhosis (63,64, http://links.lww.com/MPG/A292); however, most individuals infected with HCV, including children, are often unaware and undiagnosed (65, http://links.lww.com/MPG/A292). This has led to the augmentation of the Centers for Disease Control and Prevention screening recommendation to test all of the people born during 1945–1965 for HCV without prior ascertainment of HCV risk factors (66, http://links.lww.com/MPG/A292). There have been debates regarding targeted versus universal screening of HCV in pregnancy; however, owing to lack of means for the prevention of vertical transmission and the fact that interferon-based treatments are not feasible during pregnancy or infancy, targeted screening remains the recommended method for HCV screening during pregnancy (59, http://links.lww.com/MPG/A292). With current development of effective interferon-free regimens and availability of increasing numbers of direct-acting antiviral medications in the pipeline, we may in the future see a change in the paradigm of screening and treatment strategies in pregnant women with HCV infection. As an example, oral antivirals have been successfully used in women with hepatitis B infection who are highly viremic during the third trimester of pregnancy, in combination with standard-of-care administration of hepatitis B immunoglobulin and vaccinations to the newborns to prevent transmission (67,68, http://links.lww.com/MPG/A292).
Despite the reported low rate of vertical HCV infection transmission and relatively mild disease during the first 5 to 20 years of life in many infected children, significant pathology can occur. Bridging fibrosis has been reported in up to 12% of children infected with HCV, with mean infection duration of 13.4 years, and some patients went on to require liver transplantation (69, http://links.lww.com/MPG/A292). Hepatocellular carcinoma has also been reported in adolescents with HCV infection (5). Furthermore, the social stigma and parental guilt associated with chronic hepatitis C infection can be difficult for families. HCV infection remains an important health issue for the pediatric population, and prevention of perinatal transmission is a vital area for further research.
The nature of HCV infection in early infancy remains elusive. Early viral diversification, in concert with infant immune system, likely plays an important role in the pathogenesis of HCV infection. There have been reports of lower viral diversification in young infants; however, because of limitation in techniques, the results are ambiguous. Therefore, the process of early HCV viral genome diversification and the host immune responses should be further explored in young infants to better understand HCV infection in this special population. An enhanced understanding of the potential mechanisms and immunological basis of transmission in infants has the potential to affect effective drug and vaccine development for both children and adults.
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