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Perinatal Transmission and Viral Evolution of Hepatitis C Virus Quasispecies in Infants Coinfected With HIV
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JAIDS Journal of Acquired Immune Deficiency Syndromes: Volume 36(4) 1 August 2004
Pollack, Henry MD; Hou, Zhiying PhD; Hughes, Austin L. PhD*; Borkowsky, William
From the Department of Pediatrics, The Saul Krugman Division of Infectious Diseases and Immunology, New York University School of Medicine, New York, NY, and *Department of Biological Sciences, University of South Carolina, Columbia.
"...Transmission rates of HCV to their children are 5- to 7-fold higher in women who are also HIV coinfected...Some studies suggest that as many as 50% of HCV-infected children (all HIV uninfected) will resolve infection by the end of the 2nd decade of life, although it is not clear at what age resolution occurs. We have not seen any HIV-coinfected infants who have resolved their HCV infection in a decade of follow-up. HIV coinfection in HCV-infected adults results in a more rapidly evolving HCV infection with faster progression to cirrhosis than in HIV-uninfected individuals... This could result from higher HCV viral loads or greater viral diversity in HIV-coinfected women or could be a function of the infant's ability to clear infection. HCV viremia is 10-fold higher in HIV-infected individuals. Although HCV transmission has been correlated with the maternal viral load, 28 we have previously reported 26 that the increase in HCV transmission was limited to HIV infected and not related to maternal viral load..."
Abstract
Objectives: Three HIV/hepatitis C virus (HCV)-coinfected children and the mothers of 2 were studied to examine the nature of perinatal HCV infection in HIV-coinfected infants and to assess the evolution of viral quasispecies thereafter. Sequences of the hypervariable region in the N terminus of the E2/NS1 region (HVR-1) of the children and their mothers were compared. HCV quasispecies changes in the infants were tracked over several years.
Methods: Sequence similarity comparisons and phylogenetic trees were derived from cDNA of plasma isolates. Quantitation of plasma HCV and HIV was performed in the children, as well as CD4 T-cell percentage and liver transaminases.
Results: Phylogenetic analysis of the mother-child pairs suggested that transmission of multiple dominant and nondominant variants identified in the mother were seen. HCV diversification in the children was seen as early as 2 months of life. The child with the best immune status and HIV control demonstrated the most diversification throughout.
Conclusion: Multiples HCV variants transmitted from mother to child and their early changes in the child may be related to maternal antibody. Variation after the 1st year of life may reflect immunologic pressure from the child. There was no trend suggesting that the presence or absence of selective immunologic pressure affected HCV load or liver transaminase values.
BACKGROUND
Hepatitis C virus (HCV) infects >4 million persons in the United States. In young children, perinatal transmission accounts for new infections, with rates between 4-7%. HCV will produce swarms of closely related clones or quasispecies; the most extensively documented area is an 81-bp region in the N-terminus of the env 2 region called the hypervariable region 1 (HVR-1), a major target for neutralizing antibodies. Analysis of HVR-1 allows establishment of linkage between different sequences. Perinatal transmission can be verified by demonstrating close linkage between maternal and infant HCV isolates. Weiner et al reported a case of an HCV-infected newborn whose unique isolate was different from those of the mother and raised the issue of whether the transmitted virus was an escape mutant or whether selection occurred at the time of transmission. Subsequent studies in children have demonstrated diverse patterns of transmission including transmission of multiple clones, transmission of dominant or subdominant clones, or a mixture of these.
Evolution of the quasispecies is hypothesized to be due to ongoing selection of viruses that are most fit for a particular host. Selective pressure can be related to several factors but host immune pressure is thought to be a main factor driving diversification. Failing to detect a correlation between HCV evolution and the strength of the host immune response to HVR-1 epitopes, Allain et al have suggested that variation can result from genetic drift occurring independently of immune pressure. Comparison frequencies of synonymous nucleotide substitutions per synonymous site (dS) and nonsynonymous nucleotide substitutions per nonsynonymous site (dN) can be used to evaluate the process of natural selection. In the absence of selection, dS exceeds dN in most protein-coding genes. A pattern of dN > dS suggests positive selection.
There have been relatively few studies of viral diversification in perinatally HCV-infected children, mainly in a handful of immune-competent infants and in 1 child with hypogammaglobulinemia. Manzin et al have suggested that HCV diversification in immunocompetent infants occurs as a result of selective pressure. Viral diversification resulting from host immunologic pressure is thought to be a factor that contributes to viral persistence. 15 In immunocompromised hosts, diminished host immune pressure is more likely to result in less HCV diversification, as has been observed in HIV-coinfected adults and in hypogammaglobulinemia.
This report evaluates the relationship of HCV transmitted from 2 HIV/HCV-coinfected mothers to their offspring and the quasispecies changes that occur in these and an additional infected infant.
Patients
Three perinatally HIV- and HCV-coinfected infants and the coinfected mothers of 2 of these infants were enrolled. They represent an example of our HCV-infected population selected by virtue of having samples near birth. All were infected with HCV genotype 1b. Infants C1 and C2 were treated with nucleoside reverse transcriptase inhibitors soon after birth with nevirapine added at 4 months and 1 month of age, respectively. C3 was treated with reverse transcriptase inhibitor at 5 months and a protease inhibitor was added 3 years later. C1 and C2 had detectable antibody to HCV that developed in the 1st year of life after the attrition of transplacentally transmitted antibody. C3 had no antibody detected to HCV until well after his 2nd birthday. HCV quantitation was performed using the Roche COBAS Monitor v.2.0 assay (Indianapolis, IN). HIV quantitation was performed using the Roche Amplicor PCR assay.
AUTHOR DISCUSSION
These results, obtained in children coinfected with HIV and HCV, confirm the findings of other studies suggesting that infants of HCV-infected mothers may be infected with multiple HCV clones from the mother, representing either dominant or minor populations of variants. The appearance of new variants in the child that were closely related to variants found in the mother at other timepoints suggests transmission of an even larger number of maternal variants in low frequency and not identifiable at earlier timepoints. The number of transmitted variants in these children is considerably higher than reported for children with only HCV infection. Whether this is merely a sampling issue or whether it reflects transmission of greater numbers of variants or less initial selection of transmitted variants when the infant is also HIV infected is unclear. Transmission rates of HCV to their children are 5- to 7-fold higher in women who are also HIV coinfected. This could result from higher HCV viral loads or greater viral diversity in HIV-coinfected women or could be a function of the infant's ability to clear infection. HCV viremia is 10-fold higher in HIV-infected individuals. Although HCV transmission has been correlated with the maternal viral load, we have previously reported that the increase in HCV transmission was limited to HIV infected and not related to maternal viral load. In both children with paired maternal results, dominant and minor variants were transmitted; in one case (C1) predominantly minor variants were transmitted, whereas in the 2nd child (C2) the large majority of isolates corresponded to the dominant maternal variant.
Diversification of HCV occurred over time but with different rates and evolution in each of the 3 children. Differential HCV phylogenetic evolution of common source-infected individuals suggests that individual host selective pressures are at play in determining quasispecies transmission and evolution. The effect of immune pressure, or adaptive selection, not only increases viral diversification but may also result in escape from humoral or cellular immunity. A number of studies have documented that the hypervariable region (HRV-1) in the E2 region of the virus is a dominant neutralization epitope. The carboxyterminal end of HRV-1 also contains epitopes for both T-helper and cytotoxic responses. The presence of immune pressures could play a role in determining which viral clones are passed from mother to child and could also influence the evolution of the viruses in both the mother and child over time. Additional factors that have been evoked have been the mode of transmission (ie, intrauterine vs. perinatal) and potentially different quasispecies populations (blood, placenta vs. blood, vaginal fluid, and the amount of inoculum). The high rate of changes in the 1st year of life, a time when infant immunity is generally diminished, reflects possible viral escape as a result of declining titers of passively acquired maternal HCV antibody rather than newly acquired HCV-specific immune responses in the infant. After the 1st year of life, changes in infant quasispecies populations are less dramatic and suggest a more slowly evolving process.
In each infant consistent early changes were noted in the amino acid residues of the particular regions of HVR-1, especially after amino acid 390, that may correspond to critical anti-HCV epitopes. Zibert et al have shown that in chronic HCV infection, antibodies are directed mainly against the C-terminus of the HVR-1, whereas antibodies to the N terminus of HVR-1 are associated with acute self-limited HCV infection. A major binding site has been mapped at position 390-405. Antibodies binding to the 390-410 region were found to correlate with neutralizing activity.
The dramatic early shifts in these particular variants, especially the rapid alternating changes in C1 and C2 within the 1st year of life, might be explained by changes expected in the levels of transplacental maternal antibody followed by the appearance of the child's humoral immune response. In the case of C3, HCV antibody production was delayed beyond the age of 2 years and may account for the absence of the alternation in viral variants seen in the other 2 infants. Viral diversification became more prominent only after antibodies to HCV developed and his HIV infection was under better control.
It would be impossible to accurately correlate viral diversification and immune status studying only 3 individuals. However, in these HIV-coinfected children, a trend was observed between HCV viral diversity and evolution over time and the degree of HIV-associated immunosuppression. C2, the child who was aggressively treated within the 1st month of life resulting in the maintenance of normal CD4 counts and complete and durable HIV suppression, had the most diversification of HCV. C3, who was treated at a much older age and with poor HIV suppression and greater immunoattrition, demonstrated the least amount of HCV diversification. In part this may have been related to his failure to produce HCV antibody until after 2 years of age. When finally begun on more effective antiretroviral agents, he achieved excellent HIV suppression and evidence of more extensive HCV diversification. No direct effect of HIV therapy on HCV viral load that could account for these differences was observed. In a study of quasi-species diversification (QSD) in HIV/HCV-coinfected adults, no contribution was noted by HIV except in patients with CD4 counts <50 cells/uL in whom the QSD index was one-quarter of that seen in those with higher CD4 counts or in those without HIV infection. 17 Moreover, those with the lowest CD4 had both lower cell-mediated immunity (CMI) and decreased antibody to the C100-3 antigen of HCV, underscoring the fact that CMI and humoral immunity play a role in quasispecies selection of regions in the HRV-1 region. A report of 10 adults with chronic HCV infection who had recently become coinfected with HIV showed a trend toward lower QSD and lower nonsynonymous substitutions in 7 of the individuals. The overall magnitude of the effect was small but was most marked in the 5 individuals with rapid HIV disease progression.
Some studies suggest that as many as 50% of HCV-infected children (all HIV uninfected) will resolve infection by the end of the 2nd decade of life, although it is not clear at what age resolution occurs. We have not seen any HIV-coinfected infants who have resolved their HCV infection in a decade of follow-up. HIV coinfection in HCV-infected adults results in a more rapidly evolving HCV infection with faster progression to cirrhosis than in HIV-uninfected individuals, but there is little information available in a comparable population of children. More direct measures of humoral and cell-mediated immune responses to the HVR-1 region will be necessary to understand sequence changes that occurred in these children. In all 3 infants, HCV-specific cytotoxic T-lymphocyte responses to other regions of the HCV genome, outside of the HVR-1 region, were previously examined by tetramer analysis and were found to be relatively weak.
In conclusion, the analysis of HVR-1 changes is particularly complicated in perinatally infected children because of the frequent occurrence of multiple founder clones and the confounding effect of passively acquired maternal anti-HCV antibody, which dissipates over the first 12-15 months of life, on viral selection. Additionally, the presence of multiple transmitted maternal clones makes it difficult, at times, to distinguish new variants that arise in the infant from variants transmitted at birth at low frequency that only emerge at later time-points. Despite these difficulties we were able to demonstrate evidence of the selection and evolution of HCV variants even among HIV-coinfected infants before and after a year of age.
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