Li, Ding-You; Schwarz, Kathleen B.
It is estimated that there are more than 170 million persons infected with the hepatitis C virus (HCV) worldwide (1,2). Approximately 4 million individuals in the United States, or 1.8% of the general population, show evidence of having been infected with HCV (3). The majority of those are persistently viremic (approximately 2.7 million). In the United States, the prevalence of HCV in children is 0.2% (under 12 years) to 0.4% (12 to 19 years) (3). Since blood-donor screening was initiated in 1990, HCV is rarely transmitted by blood transfusion in developed countries (4). Injection drug use currently accounts for most HCV transmission in adults in USA. Following acute infection with HCV, about 10 to 50% of adult patients will clear the virus and the remaining patients will develop chronic infection (5).
According to older published reports on children, which generally describe children referred to university centers in the past, children acquired HCV principally through the transfusion of blood or blood products or through perinatal transmission (6). The rate of mother-to-infant transmission of HCV is approximately 4 to 5% (6,7) but is higher when the mother is co-infected with HIV (8,9). In a recent study of HCV in children who presumably acquired HCV from neonatal blood transfusions and who were then followed for about 20 years, 55% of the anti-HCV-positive patients had detectable HCV RNA in their blood at the time of reevaluation, suggesting a higher rate of spontaneous viral clearance than in adult patients (10). This study also demonstrated that the clinical manifestations of hepatitis C in the infected children were minimal. The reasons for the apparent differences in viral clearance between adults and children are not clear but different host immune response in children may be implicated. Unfortunately, there are no data available to date which demonstrate that infants do indeed mount a better immunologic response to HCV than adults. A recent report of Conte et al. (7) showed that 18 newborns born to HCV-infected mothers had detectable HCV-RNA in cord blood samples, 16 of whom became HCV-RNA-negative at 4 months of age. The authors attributed the HCV-RNA-positivity in the cord blood samples to maternal blood contamination. On the other hand, this same study may imply that infants were able to mount vigorous immune responses against HCV.
The hepatitis C virus (HCV) has been classified as a member of the Flaviviridae family (11). It is an enveloped positive-stranded RNA virus of approximately 9.6 kb, which consists of a 5` non-translated region (NTR), a single uninterrupted open reading frame (ORF) encoding the viral polyprotein and a 3` NTR. The polyprotein encoded by the HCV ORF includes the capsid or core protein C, the envelope glycoprotein E1 and E2, a small polypeptide of unknown function known as p7, and six nonstructural (NS) proteins: NS2, NS3, NS4A, NS4B, NS5A, and NS5B (12). The nucleocapsid (core) protein has the highest sequence homology among all isolates sequenced to date. E1 and E2 form non-covalent heterodimers, which are believed to represent the functional subunit embedded in the lipid envelope of the virion.
Comparison of the HCV sequences has shown two hypervariable regions within E2, called HVR1 (amino acids 390–410) and HVR 2 (amino acids 474–480) (13). Mutations in HVR 1 occur during the course of HCV infection in individual patients and may be involved in HCV escape from antiviral immune response.
It is believed that HCV is not cytopathic for infected cells. Therefore, immune responses must play a central role in the pathogenesis of HCV (14). Extensive research has been focused on the nature of the immune response and the cytokine profile in HCV infection. Much has been learned in the past few years about the mechanism for HCV clearance and persistence. Specifically, a strong T helper type 1 (Th1) response against viral antigens such as NS3 and a robust CD8+ cytotoxic T-cell (CTL) response against multiple HCV epitopes have been associated with viral clearance and better prognosis, while Th2 responses against core antigen have been demonstrated in chronically infected patients (15,16). The purpose of this review is two-fold: (1) to summarize current understanding of the immune response to HCV and (2) to attempt to understand the various clinical presentations of HCV in childhood in the context of present understanding of the immunopathogenesis of HCV. For more in-depth discussion of the immunopathogenesis of HCV infection, readers are referred to excellent reviews (14,16,17).
1. MHC MOLECULES AND ANTIGEN PRESENTATION
There are three genes for MHC class I molecules: HLA-A, HLA-B, and HLA-C, located on chromosome six. MHC class I molecules display fragments of proteins manufactured “endogenously”, which include viral proteins as well as cellular and structural proteins. Almost every cell in the human body expresses class I molecules on its surface. As shown in Figure 1A, “endogenous” proteins are degraded into peptides by proteasomes in the cytosol. Those peptides are transported across the membrane of endoplasmic reticulum (ER) by specific transporter-associated proteins (TAP1 and TAP2). In the ER, suitable peptides (8 to 11 amino acids) bind to MHC class I molecules and are presented on the cell surface as an MHC I-peptide complex, which is recognized by T cell receptor (TCR)/CD3 complexes on the surface of CD8+ T lymphocytes (18).
In contrast to class I molecules, MHC class II molecules bind to invariant chains in the ER (Fig. 1B). The invariant chain is actually acting as a “chaperone” to prevent the class II molecule from picking up other peptides in the ER and as a guide to direct the class II molecule to the endosome in the cytoplasm. Meanwhile, the “exogenous” proteins are taken inside the cell and enclosed in a vesicle called a phagosome, where the proteins are degraded into peptides of variable lengths. Then the phagosome merges with the endosome and the invariant chain is displaced. The viral peptides of 10 to 25 amino acids are loaded into the empty MHC class II molecules and displayed on the cell surface as an MHC II-peptide complex, which is recognized by TCR/CD3 complex on the surface of CD4+ T lymphocytes (18).
2. T HELPER CELLS AND CYTOKINES
Once the TCR-CD3 complex on the surface of CD4+ T helper (Th) cells recognizes a specific MHC II-peptide complex on the surface of the antigen-presenting cell, a co-stimulatory molecule called B7 on the surface of antigen-presenting cells binds to its receptor. This receptor, called CD28, is on the surface of Th cells, which are subsequently activated and secrete cytokines. The cytokines, in turn, stimulate the activated Th cells to proliferate to a clone. Based on the cytokines they secrete, CD4+ T cells are subdivided into at least two subsets: a Th1 subset that secretes interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α and a Th2 subset that secretes IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 (17,19). The determinants of which cytokines Th cells will produce include co-stimulatory molecules, the affinity of TCR for the MHC-peptide complex, doses of viral antigens and the mixture of cytokines in the microenvironment in which the stimulation occurs (17,20–23). In general, Th1 cytokines are involved in activation of macrophages, CD8+ T cells and NK cells, which defend against viral and bacterial invasion, whereas Th2 cytokines induce activation and differentiation of B cells and production of protective antibodies.
IL-12 is a proinflammatory cytokine produced by macrophages, B cells, and other antigen-presenting cells and is the major cytokine that influences Th cells to differentiate into Th1 cells (Fig. 2). On the other hand, IL-4 produced by T or B cells influences Th cells to secrete a Th2 profile cytokine. Once the Th cells are committed to differentiate into Th1 or Th2, they will secrete their own growth factor, IL-2 or IL-4, which drives Th1 or Th2 cells to proliferate, respectively. There is also a negative feedback mechanism, which influences the rate of proliferation of Th1 or Th2 cells. IFN-γ secreted by Th1 cells decreases the rate of Th2 cell proliferation whereas IL-10 secreted by Th2 inhibits Th1 cell proliferation (18).
3. ROLE OF CD8+ CYTOTOXIC T CELLS (CTL) IN HCV INFECTION
CD8+ CTL plays an important role in viral infection. In acute hepatitis C infection, spontaneous viral clearance is associated with an early, vigorous, and multispecific CTL response (15,24). HCV-specific CD8+ CTLs are detectable in the peripheral blood and intrahepatic lymphocytic infiltrates. HCV-specific CTL responses have been identified against all translated proteins of HCV (25). However, the number of HCV-specific CTL in the peripheral blood is very low. To enhance the detection of the HCV-specific CTL, it is necessary to expand this population by repetitive in vitro stimulation with HCV-derived peptides (25,26). In contrast, HCV-specific CTLs can be easily identified from liver-infiltrating lymphocytes without expansion or with nonspecific in vitro stimulation (14,25,27–30).
Recently, a novel and sensitive technique was developed using HCV-derived peptide-MHC tetramers to quantitate HCV-specific CD8+ T cells in peripheral blood and liver (31). HCV NS3-specific CD8+ cells were detected in the blood of HCV infected patients at a frequency from 0.01% to 1.2% of peripheral CD8+ cells. However, the frequency of NS3-specific CD8+ cells in the liver was 1 to 2%, at least 30-fold higher than that in the peripheral blood. Importantly, all of the intrahepatic NS3-specific CD8+ T cells were activated CTLs because they were expressing the activation marker CD69, whereas NS3-specific CD8+ T cells in peripheral blood were predominantly negative for the activation markers CD69, suggesting they are memory T cells. This finding suggests that intrahepatic HCV-specific CD8+ CTL may play a more active role for viral clearance because it is believed that extrahepatic replication contributes little to the viral pool and the liver is the primary site of HCV replication.
It has been observed that a strong polyclonal CTL response in the peripheral blood and liver is associated with viral clearance. In contrast, in chronically infected patients, the HCV-specific CTL responses are weak and unable to clear the virus completely (30,32) (Fig. 3).
Recently Takaki et al. (33) showed a high recovery rate among women infected with HCV 18 to 20 years after documented exposure to accidentally HCV-contaminated human Rh immunoglobulin. There were 43 recovered (negative serum HCV-RNA) and 34 chronically infected patients (positive serum HCV-RNA) based on repeated tests during the last 4 years of follow-up. A high percentage of the recovered patients (43%) also tested negative for HCV antibodies. Interestingly, peripheral blood HCV-specific cytotoxic T cell responses with an IFN-γ-producing phenotype persisted in the patients with viral clearance, whereas they were undetectable in patients with chronic infection.
4. ROLE OF CD4+ TH CELLS IN HCV INFECTION
In patients who present with acute and chronic HCV infection, the CD4+ T cell responses are usually measured by the antigen-specific proliferative responses of peripheral blood mononuclear cells (PBMC) to recombinant HCV-encoded antigenic peptides. Multiple HCV peptides have been defined and are recognized by CD4+ T-cells. Peptides in the regions of core, NS3, and NS4 were found to be more immunogenic and immunodominant than others (34–36). CD4+ T cells from chronically infected patients are characteristically polyclonal and multispecific for more than one viral antigen. Indeed, core, NS3 and NS4 are recognized by CD4+ T cells of most patients, whereas E1, E2/NS1, and NS5 are immunogenic only for a small proportion of patients (34,36,37). After in vitro stimulation with recombinant HCV peptides (core, NS3, and NS4), HCV-specific CD4+ T cells can be detected during the first few weeks of acute infection.
Several studies have consistently demonstrated that the CD4+ T cell responses against the NS3, helicase, and protease domains are much stronger and more frequently found in patients who resolve acute hepatitis C than in patients who develop persistent infection, suggesting that a strong NS3-specific T cell response may be necessary for viral clearance (37,38). Further study characterized the specific site recognized by the NS3-specific Th cells as a short immunodominant region at amino acid position 1248–1261 with the putative minimal epitope at amino acid position 1251–1259 within the NS3 protein (36). Interestingly, this epitope binds to 10 common HLA class II alleles with high binding affinity and is recognized in the context of 5 different HLA alleles. Its sequence is completely conserved within HCV1a, 1b, 1c, 2a, and 2b genotypes (36).
It is still not clear how a strong CD4 T cell response leads to viral clearance. It has been suggested that the CD4+ Th cells mediating the NS3-specific immune response frequently display a Th1 cytokine profile, which has been associated with viral clearance and better clinical prognosis, whereas the activation of Th2 responses seems to be involved in the development of chronic hepatitis C (16,37). Other studies with in vitro stimulation of PBMC and cytokine production with recombinant NS3 antigens showed that the lack of IL-2, low levels of IFN-γ, and high secretion of IL-10 are associated with viremia, whereas production of IL-2, high levels of IFN-γ, and little or no IL-10 correlated with self-limited infection (16,37). In a single-source outbreak of HCV, HCV-specific CD4 responses were much stronger and much more frequently observed in patients who recovered than in those with persistent infection (33).
In summary, both vigorous polyclonal CD4 Th cell responses and a characteristic Th1 cytokine profile have been associated with viral clearance (Fig. 3). However, the precise mechanism by which this is accomplished and the relative importance of the cytokine profile and the CD4 Th cell response is not understood.
5. HUMORAL IMMUNITY IN HCV INFECTION
Following acute infection with most viruses, antibody is produced to limit or even clear the infection by neutralizing free viral particles, interfering with viral entry into host cells, or destroying virally infected cells through complement-mediated cytotoxicity or antibody-dependent cellular cytotoxicity.
The issue of whether anti-HCV antibodies are neutralizing antibodies is complex and still under debate. Re-infection of chimpanzees who had successfully recovered from acute hepatitis C with homologous or heterologous HCV strains resulted in reappearance of viremia in the presence of anti-HCV antibodies, implying the failure of these antibodies to protect against re-infection (39,40). On the other hand, various studies have shown that the HVR1 region of the E2 protein of HCV is the main target for neutralizing antibody production. Indeed, neutralizing antibodies raised against the HVR1 region of E2 protein were demonstrated to protect chimpanzees from infection with the homologous viral strain (41) and to prevent HCV infection in cell culture (42). In addition, E2 antigen binding to hepatocytes in vitro was inhibited by hyperimmune sera from patients with acute and chronic HCV infection (43). There is evidence that antibodies against HVR1 region of E1 can block viral attachment (44) and inhibit viral infectivity in vitro and in vivo (41,45,46).
A possible explanation for persistent viremia rather than viral clearance in the presence of neutralizing antibodies is the emergence of neutralization escape mutant virus (41,47). Indeed, anti-E2 antibodies are constantly detectable in chronic HCV infection. It is well documented that the sequence of HVR1 region varies considerably in HCV isolates (48). The high variability of the HVR1 region in the viral quasi-species could select some resistant strains (escape mutants) (47,49).
Antibodies to HCV may be involved in immunologic aspects of extrahepatic manifestations associated with chronic HCV infection, such as type II mixed cryoglobunemia, glomerulonephritis, cutaneous vasculitis, and arthritis. The recent observation that CD81, a cell-surface protein widely expressed on B and T lymphocytes and other cells including hepatocytes, can bind to E2 of HCV (50) may have important implications for the pathogenesis of the extrahepatic manifestations in HCV infection.
6. MECHANISMS FOR VIRAL PERSISTENCE
Infection with HCV leads to chronicity in 50 to 90% of adult patients despite evidence of active, antiviral immunologic responses (5). However, the mechanisms underlying the viral persistence are still poorly understood.
It is observed that viral quasi-species as well as T cell subpopulations differ profoundly in the liver and the peripheral blood (51,52). Differences in HCV quasi-species may lead to different HCV antigenic presentation in those two compartments. Therefore, T cell responses induced predominantly in the peripheral blood may not recognize and eliminate intrahepatic HCV populations efficiently. Immune escape mutations have been studied extensively for their role in HCV persistence. Considerable sequence variation exists in all HCV isolates. Even within a single patient, HCV circulates as a population of quasi-species and often a patient is infected with more than one strain (16). The wild-type epitope-specific CD8+ cells may not recognize the variant epitopes, resulting in persistence of the variant strains (53,54). Some viral variants may act as an antagonist for the induction of T cells.
Variation in immunodominant epitopes can change an agonist peptide to an antagonist peptide that is able to inhibit CTL recognition of both wild-type and variant virus or block T cell receptor interaction (54,56). Most interestingly, Eckels et al. (16) demonstrated that naturally occurring single point mutations in an immunodominant epitope of HCV NS3 antigen (identified by PCR amplification, cloning and sequencing) were able to transition CD4+ T cells from the Th1 (IL-2-producing) to the Th2 (IL-10-producing) response phenotype. The authors called these viral variants "transition variants". Therefore, HCV may be able to control CD4+ T cells by mutating its immunodominant epitopes that downregulate the strong antiviral Th1 responses and up-regulate Th2 cytokines, which foster host tolerance to HCV (16) in spite of the fact that in infections with other pathogens, Th2 cytokines are generally associated with the production of protective antibodies.
7. VARIOUS CLINICAL PRESENTATIONS OF HCV IN CHILDHOOD
The course of HCV infection in childhood has been studied in different groups, including infants of HCV-infected mothers and children transfused with blood or blood products for various indications including thalassemia, hemophilia, sickle cell anemia, malignancy, immunodeficiency, or major surgery. There are differences in the chronicity rates of HCV infection in various pediatric cohorts, probably due to a number of factors, including the definition of clearance, length of follow-up, number of patients studied, mode of acquisition, age at acquisition, ethnicity, and co-morbidity (Table 1).
Perinatally acquired HCV infection is the major route of new infection for pediatric population, particularly given the lack of effective means of preventing transmission. There are several reports examining the natural history of HCV infection acquired by maternal-infant transmission. Sasaki et al. (57) followed 15 infants born to mothers infected with HCV and found that three infants became HCV-RNA positive in their sera. However, serum HCV-RNA tests in two of the infants became negative within 2 months. In an additional group of 8 children who acquired HCV perinatally, two cleared HCV-RNA. In an Italy multicenter study, only 2 of the 14 infants infected with HCV perinatally cleared HCV-RNA (58). Most recently, Conte et al. (7) showed that of 18 newborns with HCV-RNA in cord blood samples, 16 infants became HCV-RNA-negative by the fourth month after delivery. The Conte data could be interpreted as due to either effective viral clearance or to frequent contamination with maternal blood during cord blood sampling. In contrast, Palomba et al. (59) followed seven children with perinatally acquired HCV infection for a mean period of 65 months. All children remained HCV-RNA positive throughout follow-up. The wide differences in the viral clearance rate in those reports are probably due to different study designs, small sample sizes, differences in HCV transmission risk factors among maternal populations, or to methodological differences (Table 1). For example, co-infection with HIV in the mother increases HCV infection rates in the infant. Papaevangelou et al. (8) reported that the rate of HCV infection was higher among HIV-infected infants (40%) than among HIV-uninfected infants (7.5%).
Matsuoka et al. (60) followed 29 patients (ages 4 to 18 years) who developed HCV infection after blood transfusion for open-heart surgeries for congenital heart disease. Only 14 of the 29 patients (48%) had detectable HCV RNA after 4 to 13 years. Similar findings were confirmed by a recent report of Vogt et al. (10). These authors found that 67 (14.6%) of the 458 patients who had undergone cardiac surgery developed HCV infection. At a mean interval of 19.8 years after the first operation, 37 (55%) of the 67 patients had detectable HCV RNA in their blood. Of the 17 patients who underwent liver biopsies, only 3 had histologic signs of progressive liver damage. Those studies indicated that children who had undergone cardiac surgery before the implementation of blood-donor screening for hepatitis C had a substantial risk of acquiring the infection. However, the virus spontaneously cleared in about 50% of the patients after many years.
HCV infection in children with underlying malignancy or immunodeficiency may have a different outcome than in children who acquired HCV through transfusion related to surgery. Hoshiyama et al. (61) studied 231 children with a history of blood product transfusion; 116 patients had a history of malignant disease and 115 patients had undergone open-heart surgery. HCV infection developed in 35 of 116 patients (30%) with malignancies and 20 of 115 patients (17%) who had undergone cardiac surgery. Moreover, HCV RNA was detectable in 86% of the 35 patients with malignancy and 60% of the 20 patients with heart surgery. Therefore, persistent HCV infection in children with malignant disease was more frequent in those without malignancy, suggesting that immunosuppression associated with malignancy may contribute to the low viral clearance rate. Indeed, HCV infection in children with primary immunodeficiency is associated with a high percentage of chronic infection and worse clinical outcome compared with the usually mild clinical outcome associated with HCV in children (62,63). Of the 20 patients with primary hypogammaglobulinemia who received contaminated immune globulin, 17 (85%) were seropositive for HCV-RNA approximately 10 years after receiving the immunoglobulin. Those patients tended to have a severe and progressive course and responses to interferon therapy were poor (62). Jonas et al. (63) followed 21 patients with primary immunodeficiency (e.g., common variable immunodeficiency, IgG2 deficiency, agammaglobulinemia) who had evidence of HCV infection associated with intravenous immunoglobulin. They found 87.5% of these individuals developed chronic infection. In addition, 5 of the 7 pediatric patients who underwent liver biopsy had chronic active hepatitis.
Prior to blood-donor screening for HCV, children with congenital hematological disorders such as sickle cell anemia, thalassemia, and hemophilia were at significant risk for HCV infection due to frequent transfusions. Two groups (64,65) studied polytransfused thalassemic patients and found HCV prevalence rates of 30 to 60%, with some groups as high as 90%. Zellos et al. (66) showed that children with hemophilia exhibited high HCV viral load but liver histopathology was less severe than in children who acquired HCV by blood transfusion or maternal-fetal transmission. The authors observed that the cytokine profile of peripheral blood lymphocytes in hemophiliac children with HCV is of a Th-2 type whereas the comparable cytokine profile of children with transfusion-acquired HCV was a Th-1 type (67).
In a unique study in which Garcia-Monzon et al. (68) compared a cohort of adults to a cohort of children infected with HCV, the mean duration of viral infection was similar (11 years). The authors showed that children with chronic HCV were characterized by lower ALT levels and viral loads as well as milder histologic and immunohistochemical changes compared to adult patients. Most series of liver histology in children referred to university centers have reported mild to moderate fibrosis and relatively mild clinical manifestations. On the other hand, there are a few reports of children in whom HCV infection had progressed rapidly to end-stage liver disease requiring transplantation (69,70).
In summary, children infected with HCV exhibit a wide range of viral clearance rates, just as do adults. Perinatally acquired HCV emerges as the major route of HCV infection in children, highlighting the importance of clarifying mechanisms of viral clearance in these young subjects. As noted above, there is also a broad range of histologic findings in children who present with HCV. Thus, it will be important in future studies to understand the relationships between immune response, viral clearance, and disease progression in children who present with HCV infection (55,71).
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