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Hepatitis C Virus (HCV) Diversity in HIV-HCV Coinfected Subjects Initiating Highly Active Antiretroviral Therapy
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The Journal of Infectious Diseases 2004;189:1472-1481
Jason T. Blackard,1 Yijun Yang,2 Paola Bordoni,1 Kenneth E. Sherman,3 and Raymond T. Chung,1 for the AIDS Clinical Trials Group 383 Study Team
1Gastrointestinal Unit, Massachusetts General Hospital, and 2Center for Biostatistics in AIDS Research, Harvard School of Public Health, Boston, Massachusetts; 3Department of Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio
ABSTRACT
Because of increased mortality and reduced treatment response rates in subjects coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV), understanding the selection pressures underlying the evolution of HCV is important for the development of strategies to control both viruses.
We therefore investigated diversity of HCV in 11 HIV-HCV coinfected subjects initiating highly active antiretroviral therapy (HAART). Distinct categories of HCV virologic response to suppression of HIV were identified. The diversity of quasi species at several genomic regions was characterized over the course of a 48-week period.
Consensus data suggested a shift in the virus population at all loci except the 5 untranslated region (UTR) after initiation of HAART.
Intrasubject genetic distance and entropy were highest in hypervariable region (HVR)1. In contrast, variation in the 5 UTR was limited. Positive immune selection pressure directed against HVR-1, but not other protein-coding regions, was also detected.
These data suggest that there are several mechanisms by which suppression of HIV replication and a reconstituted immune system influence diversity of HCV in HIV-HCV coinfected subjects.
INTRODUCTION
In the United States, an estimated 150,000-300,000 subjects are infected with both HIV and hepatitis C virus (HCV) Since its introduction, the use of highly active antiretroviral therapy (HAART) to treat HIV infection has led to a remarkable reduction in the number of AIDS-related deaths. However, because of their shared routes of transmission, chronic HCV infection has become a major cause of morbidity and mortality among HIV-positive subjects. Several studies have demonstrated that coinfection with HIV and HCV adversely affects liver fibrosis, HCV loads, progression of HCV disease, and response rates to current HCV treatments. The mechanisms by which these 2 viruses influence each other are unclear. To date, no direct virus-virus interactions have been demonstrated; thus, an impaired adaptive immune response may partially explain the hastened clinical progression of HCV observed among HIV-HCVcoinfected subjects.
Previous studies have demonstrated increased HCV loads during HAART, although these results have not always been confirmed. AIDS Clinical Trial Group study 383 (ACTG 383) was a prospective trial designed to evaluate virologic trends among HIV-HCVcoinfected subjects initiating HAART . After receiving HAART for 16 weeks, the vast majority of subjects achieved undetectable HIV loads. Surprisingly, mean HCV loads increased and were maintained through week 48, indicating that decreased HIV replication and successful immune reconstitution in HIV-HCVcoinfected subjects is associated with increased HCV replication.
A hallmark of both HIV and HCV is their extreme genetic diversity. At the population level, HCV can be divided into multiple genotypes with markedly different global distributions. Within an individual, HCV exists as a population of distinct but closely related viruses termed the "quasi species." The association between HCV heterogeneity and outcome of liver transplantation, progression of disease, response to treatment, and chronicity of infection has been addressed previously. However, few studies have assessed diversity of HCV during HIV-HCV coinfection; those studies performed thus far have focused mainly on hypervariable region (HVR)1. Two cross-sectional studies found more HVR-1 diversity in HIV-HCVcoinfected subjects than in monoinfected subjects or HIV-HCVcoinfected subjects with higher CD4 cell counts. Decreased diversity of HVR-1 was also found in subjects with chronic HCV infection who then experienced HIV seroconversion. A single study has addressed diversity of HCV in HIV-HCVcoinfected subjects receiving HAART; Babik et al. found no significant changes in diversity of HCV over time among HIV-HCVcoinfected subjects, regardless of their use of HAART. However, the inclusion of multiple HCV genotypes, the analysis of a small portion of the HCV genome, and the variable use of HAART among diverse subject populations limits the generalizability of their findings.
We were therefore interested in comprehensively assessing longitudinal diversity of HCV in a group of HIV-HCV coinfected subjects initiating HAART as part of a prospective ACTG treatment study. We hypothesized that suppression of HIV with HAART would result in restored immune selection pressure against HCV and lead to increased diversity and that these selection pressures would have a differential impact on the virus quasi species, depending on the genomic region analyzed. We also tested the hypothesis that increased HCV loads were the result of outgrowth of a particular virus variant with enhanced replicative capacity.
Sample population. All subjects were previously enrolled in ACTG 383. Written informed consent was obtained from subjects as part of the original ACTG 383 study. The human-experimentation guidelines of the US Department of Health and Human Services and Massachusetts General Hospital were followed in the conduct of this clinical research. Plasma HCV loads were determined by use of the Roche Amplicor Monitor kit (lower limit of detection, 600 IU/mL; Roche Diagnostic Systems). Plasma HIV loads were also determined by use of the Roche Amplicor Monitor kit (lower limit of detection, 500 copies/mL). The HCV genotype was initially determined by use of the LiPA assay (Innogenetics). In ACTG 383, 88% of HIV-HCVcoinfected subjects achieved HIV RNA levels <500 copies/mL after 16 weeks of HAART. However, mean HCV loads increased by 0.43 log10 IU/mL by week 48.
We were interested in further characterizing diversity of HCV in ACTG 383 participants whose HCV loads responded differentially to initiation of HAART. Therefore, subjects experiencing a >0.5 log10 increase in HCV load between weeks 0 and 48 (D0-48) were selected, referred to hereafter as the "HCV increaser group." Subjects with a <0.2 log10 increase or a decrease in HCV load, between weeks 0 and 48, are referred to hereafter as the "HCV stable group." Because of the potential for short-term fluctuations in HCV loads and inherent variability in current HCV load assays, D0-48 HCV loads were considered to be more likely to reflect sustained alterations in HCV viremia. Using these stringent criteria, 22% of ACTG 383 participants were in the HCV increaser group, and 15% were in the HCV stable group; the remainder had decreased or variable HCV loads at the 3 time points reported in the original ACTG 383 study. Because of the potential for divergent evolution among differing HCV genotypes, we selected only HCV genotype 1infected participants in the prospective arm of the study (24 of 29) for whom samples corresponding to weeks 0, 16, and 48 were available (16 of 24). Five subjects were excluded because they did not fit the definition of either HCV increaser or HCV stable. This left 11 subjects7 in the HCV increaser group and 4 in the HCV stable groupwho were selected for further study. Nine subjects achieved HIV loads of <500 copies/mL by their last available time point, whereas the remaining 2 subjects had HIV loads of 2000 copies/mL, which is consistent with significant decreases in HIV RNA levels from baseline.
RESULTS
Subject characteristics. In the ACTG 383 cohort, it was found that, despite an overall increase in HCV load after initiation of HAART, HCV loads did not increase in all subjects. We therefore examined changes in clinical parameters between the HCV increaser and HCV stable groups. Eleven HCV genotype 1 HIV-HCVcoinfected subjects were selected on the basis of changes in HCV loads after initiation of HAART. These participants did not differ significantly from the overall cohort with regard to sex, race, intravenous drug use, age, alanine aminotransferase (ALT) levels, CD4 cell count, or HCV load; however, they did have a higher mean baseline HIV load (5.1 vs. 4.3 log10 copies/mL; data not shown). By week 48, in the 11 subjects, the mean HIV load decreased by 3.1 log10 copies/mL, and mean CD4 cell counts increased by 200 cells/mm3, results similar to those obtained from the parent cohort. By week 48, mean HCV loads increased by 0.7 log10 IU/mL.
Sex, race, intravenous drug use, ALT levels, HIV load, and CD4 cell count were not different between groups, for the HCV increaser and HCV stable groups. However, the HCV increaser group was older than the HCV stable group (48.3 vs. 35.0 years) and had lower baseline HCV loads (5.6 vs. 6.3 log10 IU/mL). As would be expected on the basis of our selection criteria, the HCV increaser group showed a 1.0 log10 IU/mL increase in HCV load from week 0 to week 48, whereas the HCV stable group showed no change.
Intersubject diversity of HCV, by genomic region. Phylogenetic analysis of 4 genomic regions, including the 5 UTR (nt 70341), core (342729), E1 (13291489), and HVR-1 (14911571), was performed to assess diversity of quasi species before and during HIV treatment. Week 0, 16, and 48 consensus sequences were generated for each region.
For all subjects except INC9, the 5 UTR consensus sequences at each time point were identical, whereas intersubject diversity was <1% at each time point (data not shown). HCV genotype was inferred from consensus core or E1 sequences. Ten of 11 subjects were infected with HCV genotype 1a, and 1 subject (S3) was infected with HCV genotype 1b. There was no apparent clustering of sequences, according to HCV increaser or HCV stable classification. In core, the week 0, 16, and 48 sequences were identical for subjects INC5, INC7, and S4. In E1, consensus sequences were identical for subjects INC5, INC9, S2, and S4, whereas HVR-1 consensus sequences were identical for subjects INC5, INC8, S2, and S4.
As a measure of immune selection pressure, dN : dS ratios were calculated for each protein-coding region. dN : dS ratios >1 are consistent with positive immune selection pressure and have been used to explore HIV and HCV evolution. Here, the dN : dS ratios were consistently <1 for core and E1, indicating neutral selection pressure at these loci. In contrast, the dN : dS ratio for HVR-1 increased from 0.78 at week 0 to >1 at week 16 and week 48, suggesting that positive selection pressures act on this region only after initiation of HAART.
Further phylogenetic analysis of clones was performed to investigate the potential for preferential expansion of viral variants representing highly adapted viruses responsible for increased HCV loads. However, outgrowth of particular variants was rarely observed among the HCV increaser or HCV stable groups, although only 10 sequences/time point were analyzed (data not shown).
Changes in diversity of quasi species by HCV response group. Although mean HCV load increased after initiation of HAART, this was not observed for all subjects. We therefore investigated potential changes in diversity between HIV-HCV coinfected subjects with increased HCV loads and those with stable HCV loads. For 5 UTR and E1, no significant changes in quasi species parameters between weeks 0 and 16 (016) or weeks 0 and 48 (D0-48) were found between the 2 groups. However, for HVR-1, D0-16 entropy (P = .030), 016 dN : dS (P = .047), and D0-48 entropy (P = .047) were all significantly different between the 2 groups. For core, D0-16 dN : dS (P = .030), D0-16 entropy (P = .029), and D0-48 genetic distance (P = .030) were also different between the 2 groups.
Correlation of virologic and diversity parameters. We also analyzed potential correlations among various virologic and quasi species parameters across the HCV genome. When all time points for all subjects were analyzed, absolute values of CD4 cell count, HCV load, and HIV load were not consistently associated with multiple measures of quasi species diversity for any genomic region analyzed (data not shown). However, changes (016 or 048) in CD4 cell count and HCV load from baseline were correlated with changes in several measures of diversity. D0-16 HCV load was highly correlated with HVR-1 entropy (P = .009) and UTR genetic distance (P = .041). However, the same variables were not correlated with D0-48 HCV load. Core dN : dS (P = .016) and core entropy (P = .036) were correlated with D0-48 CD4 cell count, but not D0-16 CD4 cell count. These data suggest that short-term bursts in replication of HCV (D0-16 HCV load) may contribute significantly to the generation of nucleotide diversity in the 5 UTR and HVR-1. Of interest, sustained changes in CD4 cell count (D0-48 CD4) were associated with core, but not HVR-1 diversity, implying that adaptive immune selection pressures differentially impact these distinct genomic loci.
DISCUSSION
Because of increased morbidity and mortality of chronic HCV infection and reduced treatment response rates in subjects coinfected with HIV and HCV, understanding the selection pressures underlying the evolution of HCV is important for the development of strategies to control both viruses. We therefore undertook an investigation of diversity of HCV in HIV-HCVcoinfected subjects initiating HAART. Several prior studies have reported conflicting results with regard to the effect of HAART on HCV loads. However, in a recently completed prospective clinical trial, distinct categories of HCV virologic response to immune reconstitution were identified, including those with increased HCV load after suppression of HIV, and those maintaining relatively stable HCV loads throughout HIV therapy. Thus, we were interested in characterizing differences in the HCV quasi species across several genomic regions in 2 groups of subjects, to provide a more comprehensive understanding of the complex interactions between the HCV quasi species and the reconstituted immune system.
The present study has uniquely characterized the effect of diversity of HCV on HIV suppression and differs from previous studies in several significant ways. First, because of the potential for genotype-specific differences in diversity, we restricted our analyses to subjects infected with HCV genotype 1, the most prevalent genotype in the United States. Indeed, a study of HIV-HCVcoinfected subjects found that complexity of HVR-1, but not core, differed according to HCV genotype. Similarly, other researchers have noted higher virus loads and increased genetic distance in subjects infected with HCV genotypes 2 or 3. Because such subjects represented 38% of their study cohort, these data may not be representative of the general HIV-HCVcoinfected population in the United States. Second, our subjects were derived from a longitudinal clinical trial of those initiating HAART, rather than from distinct HIV-HCVcoinfected populations with variable duration of HAART. Thus, we could directly compare virologic and diversity parameters among subjects at fixed intervals and assess changes in these parameters during immune reconstitution. Third, the availability of this treatment population allowed us to investigate potential quasi species variables associated with divergent HCV virologic responses to immune reconstitution. Finally, because diversity is not restricted to a single viral locus, we analyzed variation within the 5 UTR, as well as protein-coding regions.
We found longitudinal diversity within all loci analyzed, although diversity was not constant across the genome. For instance, the 5 UTR demonstrated limited intersubject or intrasubject diversity and evolved more slowly over the course of the 48-week study period. In contrast, we noted a significant increase in HVR-1 genetic distance and dN : dS ratios during HIV treatment that was not observed in the adjacent E1 nonHVR-1. These data suggest the existence of highly conserved motifs that are critical for HCV replication, in the case of the UTR, and potent immunologic pressure directed against HVR-1.
Our initial correlation analysis showed no significant association between absolute CD4 cell count and HCV load, as noted elsewhere. Nonetheless, we did confirm previous results, finding a highly significant association between HCV loads and HVR-1 diversity (as measured by 016 entropy) and UTR genetic distance. In addition, we extended these observations to include a highly significant positive correlation between CD4 cell count and core diversity (as measured by 048 dN : dS and 048 entropy). Our study design also allowed us to assess potential differences in diversity of quasi species between subjects experiencing significant increases in HCV load after initiating HAART and subjects maintaining stable HCV load. When comparing these 2 groups, differences in several core and HVR-1 quasi species parameters were noted; however, such differences were not found within E1 nonHVR-1 or the 5 UTR. These differential interactions between immune selection pressures and core or HVR-1 diversity may reflect the relative number and strength of immunologic epitopes in these regions, cell typespecific immunologic pressures, or the higher baseline HCV loads in the HCV stable group.
Surprisingly, the observed increase in HCV loads noted in the initial ACTG 383 report was not likely due to expansion of a more fit viral variant. Several explanations may underlie these findings. First, immune reconstitution after suppression of HIV may impose renewed immunologic selection pressures targeting several genomic regions, leading to the expansion of multiple variants rather than to the selection and expansion of a restricted number of variants. Second, HIV suppression may allow for efficient replication of HCV in an extrahepatic reservoir, such as peripheral blood mononuclear cells (PBMCs). At present, several cross-sectional studies have demonstrated replication of HCV in PBMCs.
Although protein-coding regions may be under significant selection pressure after immune reconstitution, this is not the case with the 5 UTR. The 5 UTR was well conserved among HIV-HCVcoinfected subjects over the 48-week study period, highlighting secondary structural constraints within the UTR that are likely to be critical for viral replication and translation. Nonetheless, we did note intrasubject diversity within the 5 UTR, albeit at levels lower than for other regions analyzed. Of interest, single nucleotide changes result in UTRs with different translational efficiencies, as well as cell type specificities. These data suggest that selection pressures other than those imposed directly by the adaptive immune system also drive HCV evolution.
Although our data may, theoretically, be influenced by PCR-generated mutations, this possibility seems unlikely for 2 reasons. First, the various measures of quasi species diversity used here suggest extensive genomewide diversity in excess of the Taq polymerase error rate of 1 x 10-5. Second, multiple amplifications of the same template yielded similar estimates of quasi species diversity (data not shown). It is also important to note that our data encompass changes between time points such that the week-0 time point represents an internal control (i.e., before HAART), compared with the week-16 and week-48 time points (i.e., after HAART).
One limitation of the present study is the general lack of functional immunologic data. Although CD4 and CD8 cell counts may provide a quantitative estimate of immune restoration after initiation of HAART and successful suppression of HIV, at least 1 study has found a general lack of CD4 cell responses to HCV and weaker CD8 cell responses to HCV than to HIV in HIV-HCV coinfected subjects. In ACTG 383, HCV-specific CD4 ELISPOT responses were not detected in most subjects examined (D. Anthony, personal communication), and CD8 cell counts were not measured. Therefore, we chose to use suppression of HIV load and increased CD4 cell counts as surrogate markers of immune reconstitution. Further investigations of quasi species evolution in conjunction with HCV-specific cellular responses will be necessary when more-sensitive immunologic assays are available.
In some instances, treatment of HIV may result in sustained disappearance of HCV viremia; however, it appears that this phenomenon is not common in the majority of HIV-HCVcoinfected subjects. At the population level, many HIV-HCVcoinfected subjects experience a sustained increase in HCV viremia. We found that HAART is associated with evolution of the HCV quasi species, particularly HVR-1, indicating that reconstituted immune selection pressures are indeed acting on HCV. However, the finding of increased HCV loads in most HIV-HCVcoinfected subjects suggest that this renewed immunological pressure alone is insufficient to control HCV replication.
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