Worldwide, an estimated 170 million people are infected with hepatitis C virus (HCV) (2
). Following acute infection, which is usually asymptomatic, 60 to 80% of infected individuals develop persistent infection, which is still the leading cause of hepatocellular carcinoma and liver transplantation in the United States (33
). Only pegylated alpha interferon and ribavirin are approved for treating this virus, and no successful vaccine has been developed (48
The extraordinary evolution and diversity of HCVs are major challenges for vaccine design and drug development. HCV replicates to high levels using an error-prone polymerase (36
), thereby generating in each host a spectrum of closely related but distinct viral variants called quasispecies (6
). Quasispecies distributions containing variants with a range of characteristics facilitate viral escape from selective pressure, balanced by fitness constraints that drive reversion to restore fitness, as has been demonstrated in controlled experiments involving simian immunodeficiency virus (SIV) under immune selective pressure and HCV under selective pressure from small-molecule inhibitors (17
). Similarly, HCV has been observed to escape selective pressure of the host immune response, demonstrating evidence of both escape and reversion and suggesting that intrinsic viral fitness also constrains HCV escape from immune selection in vivo
). However, available data are based on relatively short follow-up, single study subjects, or short amplicons (preventing accurate estimates of the relative rates of evolution of viral genes). As a result, little is known about HCV evolution in humans and about relative rates of change in structural and nonstructural proteins during the transition from acute to chronic infection.
The transition from acute to chronic HCV infection is poorly understood. During the acute phase, HCV RNA levels fluctuate, cellular responses to HCV reach a peak and then begin to wane and become dysfunctional, and neutralizing antibody (NAb) responses become detectable (9
). Established chronic infection is associated with consistently weak cellular immune responses, the presence of antibodies that neutralize a wide variety of HCV isolates, and relatively stable HCV RNA levels between 500,000 IU/ml and 50 million IU/ml in 80% of individuals (3
). The time between these two phases has rarely been studied even though it is likely that important viral adaptations occur during this transition from acute to chronic infection.
Studies of humoral immunity in HCV have advanced significantly due to development of model systems for studying neutralization, including retroviral pseudoparticles bearing HCV glycoproteins (HCVpp) and culture systems that support infection of a small number of HCV isolates (cell-cultured HCV, or HCVcc) (3
). These systems generally give similar results though HCVcc-based assays tend to yield lower reciprocal neutralizing titers (47
). Recent studies have suggested an important role for NAb in driving the evolution of HCV envelope proteins though their role in determining the outcome of acute HCV infection remains controversial (12
). Thus, understanding the interaction between NAb response and HCV evolution is of great importance in defining the role of NAb on HCV control and in developing novel immune interventions for HCV-infected patients.
To better address these issues, we studied viral evolution across multiple genes subject to a variety of selective pressures from the onset of viremia in the acute phase to the early chronic phase over the first 3 years of infection in humans. We found discordant rates of evolution in envelope versus nonenvelope genes and evidence for immunologically driven evolution in the envelope genes. These results provide the first systematically obtained rates of evolution for the core, E1, E2, p7, NS2, and NS3 genes during the first 3 years of HCV infection in vivo.
In this study, we conducted sequential analysis of HCV hemigenomic sequences for six well-characterized human subjects who declined treatment as their infections progressed from initial viremia to early chronicity. Hemigenomic sequences and annual sampling allow, for the first time, direct comparison of evolutionary rates for different genes and different subjects during the transition from acute to chronic infection. Markedly nonlinear evolutionary rates in E2 were investigated mechanistically using autologous HCVpp to measure NAb titers, revealing that evolutionary stasis of HVR1 was associated with a lack of neutralization, whereas the onset of rapid evolution was explained by rising NAb titers.
In HCV-infected individuals who progress to chronicity, neutralizing antibody responses do not seem to initiate until the chronic phase is established (12
), whereas cellular responses are readily detected during the acute phase but lose function during prolonged infection for reasons that are poorly understood (9
). Across the HCV genome, CD8 T-cell epitopes have been mapped, and all genes seem to be potential targets of cellular immune pressure (9
). Although T-cell epitopes have been mapped within the envelope gene, CD8 T-cell responses appear to have minor relative impact on driving sequence evolution in this region (25
). In contrast, the envelope gene is a well-accepted target for humoral immune selection, which appears to be the main selective force driving envelope gene evolution (5
). Therefore, the differential timing and targeted genomic locations of humoral and cellular pressure are the most likely mechanisms of uneven evolutionary rates in envelope and nonenvelope genes during the transition from acute to chronic infection.
The current study demonstrates for the first time in a systematic way in humans that HCV envelope and nonenvelope genes have significantly different trends in nonsynonymous evolution (P
= 0.006), with increasing dN
values in envelope genes and stable or decreasing dN
values in nonenvelope genes during the transition from acute to chronic HCV infection. These findings are consistent with our current understanding of the host immune response to HCV, coupled with immune-driven evolution. Previously, Kuntzen et al. reported similar findings showing that there are more T-cell-driven mutations outside the envelope gene during the acute phase of HCV infection than during the chronic phase (25
). That they did not observe a trend for envelope evolution may have been due to irregular sampling intervals and a smaller number of subjects. The current findings are also consistent with prior studies of hypogammaglobulinemic humans and with our study of chimpanzees that have poor humoral responses to E2, which revealed evolutionary stasis in HVR1 (4
In HIV (16
), SIV (17
), and HCV infection (23
), viral immune escape may incur a fitness cost, reducing replication capacity (17
) or infectivity (23
). Transmission to a new host or reduction in immune pressure in the same host may be associated with reversion to a sequence associated with higher intrinsic fitness, which is generally represented by the population consensus (17
); such changes are called centripetal in the current study. Though there are exceptions in which the immune escape variant has become dominant in a population (37
), immune selection is highly individual, as reflected by the evolutionary divergence of sisters S29 and S30 in the current study and in a pair of concurrently HIV-infected monozygotic twins in a prior study (13
). In the current report, with well-characterized subjects from whom hemigenomic sequences were obtained annually, we have demonstrated that both centrifugal and centripetal substitutions emerge frequently and contribute to viral evolution during the transition from acute to chronic infection in all six subjects, regardless of the phase of infection or the region of the HCV genome (Fig. 4
). This finding is consistent with evidence that escape and reversion both occur during acute infection (25
It is still controversial whether humoral response plays an important role in controlling HCV infection (7
). Earlier studies, primarily of chimpanzees, detected delayed and weak humoral responses, and incomplete protection in animal studies as well as evidence of cell-to-cell spread of HCV in tissue culture has been reported (7
). In contrast, a recent human study suggested that spontaneous resolvers tend to have early induction of NAb responses, whereas chronically evolving subjects have delayed initiation of NAb responses (40
), arguing for a positive role (or at least correlation) for NAbs in HCV control. With a more sensitive autologous HCVpp method, continual neutralization escape during chronic infection was demonstrated in a single individual (55
), supporting an impact of NAbs on viral sequence evolution in vivo
. However, due to limited availability of samples, virological data were missing for a gap of 14 years, and viral evolution could not be investigated during this crucial period in this study (55
). Using the same method, we have recently demonstrated in an acutely infected cohort that virus-specific NAbs drive sequence evolution and correlate with the outcome of infection (12
). Here, we further demonstrated the detailed interaction between NAb response and HCV quasispecies evolution during the transition from acute to chronic infection. With autologous HCVpp and sequencing of population HCV clones for each visit, we showed that HVR1 remained stable for 21 months without NAb pressure but kept changing away from the initial sequence after the initiation of a NAb response. These data strongly support the hypothesis that HVR1 evolution is largely driven by the NAb response.
This study is limited by the number of people studied, the lack of information regarding the source (i.e., transmitter) sequence, and inclusion of only half of the HCV genome. Though we are confident that more can be learned by addressing these limitations in the future, we do not believe these limitations biased our results; in fact, our prospective sampling, stringent inclusion criteria, and detailed analysis of hemigenomic clone sequences at standardized intervals make this the largest and most representative study of viral evolution in humans during acute HCV infection to date. It is unfortunate that detailed immunological results are not available for most subjects because inclusion criteria for this study were focused on annual sampling rather than on the availability of a large volume of blood draws (9
In summary, we determined the first three annual rates of evolution of the core, E1, E2, p7, NS2, and NS3 genes during acute infection in humans. Envelope and nonenvelope genes had distinctly different evolutionary trends, consistent with temporal patterns of immune selection. The accelerating rate of envelope evolution was striking, which is consistent with mounting pressure from neutralizing antibodies, with the nonsynonymous rate ratio of envelope to nonenvelope genes increasing from 2 in year 1 to 5 in years 2 and 3. These findings extend our current knowledge and quantitative understanding of host-HCV interaction during the establishment of chronicity.