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New Research Exploring How HCV Evades the Immune System: can a vaccine be found?
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This report contains first, the press release from Johns Hopkins explaining the findings of their research. This is followed first by an Editorial/Commentary in the journal & then excerpts from the three research articles. The excerpts from the three research articles are dense as they are scientifically written. But if you read the Hopkins press release and the doubts raised by the Commentary/Editorial you can understand the research and the questions about it raised by the Commentary.
The Editorial says:
".....Although it is clear that CTL escape mutations occur in HCV genomes, the relevance of this mechanism to viral persistence is an open question......Although recent data confirms the generation of escape mutations in HCV, it is clear that this field is in its infancy. As is becoming increasingly apparent from the immunodeficiency virus literature, studies comparing circulating viral genomes to consensus sequences are extremely difficult to interpret. Further detailed prospective studies of acute and evolving HCV infection are required to delineate the role of escape mutations in HCV persistence, as well as to define both the immunological and virological factors governing viral evolution. In particular, careful dissection of these factors will be critical to the design of any effective CTL-based vaccine.
JOHNS HOPKINS TEAM FINDS "ANCESTRAL" HEPATITIS-C VIRUS AT THE ROOT OF EVOLUTION IN ACUTE AND CHRONIC INFECTIONS
Press release from Hopkins
Scientists discover how virus evades immune system in acute and chronic infections; new vaccines may result
Researchers at Johns Hopkins have uncovered how a majority of the genetic changes in the hepatic-C virus, the most common cause of liver disease, allow it to evade the body’s immune system during infection. Hepatitis C infection can lead to cirrhosis, cancer and even death. In a series of experiments that describe the virus's transition from an acute to chronic infection, the Hopkins team found that one-half of the virus's changes in its genome are in sites under attack by the body's immune system. As the virus evolves and these changes weaken the body's immune response, a second set of changes at other sites in the genome are reverting back to an "ancestral" set of amino acids.
"We think this piecemeal exchange is helping the virus evade the body’s immune system," says study investigator and infectious disease specialist Stuart Ray, M.D., an associate professor at The Johns Hopkins University School of Medicine. "In a newly infected person, the virus may need to adopt new mutations to escape recognition by the immune system's T cells, which fight infection, but it may need to lose the mutations that had protected it in someone else. Despite pressure to change, the virus is always is restoring its shape."
The Hopkins findings, published in a pair of studies in the Journal of Experimental Medicine this week, are believed to be the first description of the precise genetic changes taking place in the virus during the acute phase of infection, when hepatitis C initially escapes the body's defenses and establishes itself in the body. As the infection moves into the chronic stage, the immune response becomes weak and less effective, but until now, no one could explain exactly why.
A second, related experiment produced similar findings when the Hopkins team partnered with researchers in Ireland to perform what is believed to be the first comparison of genetic changes across multiple genes in strains from chronically infected people to the original strain that infected them.
Ray, who served as senior investigator on the first study and led the second, believes the newly identified ancestral component of the viral genome, called a consensus sequence, could serve as the basis for development of a vaccine that is effective against both acute and chronic infections, thereby stemming the epidemic that currently afflicts more than 170 million people worldwide, including 3 million Americans.
"Hepatitis C is extremely difficult to treat if it becomes chronic," says infectious disease specialist Andrea Cox, M.D., Ph.D., an assistant professor at Hopkins who was lead author of the first study. "While approximately 30 percent of patients have a strong enough immune response to rid themselves of the virus during the acute phase, and current treatments are 90 percent effective at treating any remaining acute infections, these treatments are only 50 percent effective against chronic infections, which otherwise persist for life and can cause death."
According to Cox, the hepatitis C virus naturally mutates, or alters its genome, very rapidly. Its strains have two to three times more genetic variability, for example, than HIV, the virus that causes AIDS, and hepatitis C reproduces more than 100 billion times per day, 100 times faster than HIV. Compounding the problem, the infection is asymptomatic in the acute stage, making it less likely that diagnosis will be made early, when it is easiest to treat.
Conventional wisdom, the researchers say, was that the large numbers of mutations were simply random in the virus's ever-changing genome, but the new study suggests that Darwinian genetic selection is at play. That is, the virus's genome changes in ways that make it more reproductively "fit" in the face of each immune system it encounters, changing what is must to evade the immune system in one host, then restoring itself when the pressure is off.
What Ray's team found when the immune response weakens was that the virus naturally mutates toward a set of 3,000 common amino acids, what the researchers considered the virus's most preferred state. During the acute phase, Ray says, the virus is under severe pressure from the immune response and forced to drift away from the consensus sequence, using mutations to evade the immune response. However, the drift was reversible and, once the virus successfully evaded a particular immune cell, its amino acids reverted back to the consensus set.
To assess the genetic changes in the early stages of infection, the researchers decoded, or sequenced, the virus's genome, made up of RNA, which is very similar to the more widely known DNA that makes up the genome of most organisms. The RNA was gathered from eight newly infected patients in Baltimore, Md., all of whom were offered treatment and were participants in a larger study of infectious diseases in intravenous drug users. The sample group was unusual, allowing analyses before and during the early stages of infection. One patient self-recovered, while the rest proceeded to chronic infection.
Using advanced blood-sorting techniques, the Hopkins team extracted millions of immune system cells, including the system's principal fighters, called T cells, from blood samples taken between 30 days and six months after infection, when the body's initial immune response kicks in and subsequently peaks.
Immune responses were mapped using a series of more than 500 overlapping synthetic peptides, or strings of amino acids whose code was already known. This allowed the researchers to compare changes observed in the RNA sequence to corresponding shifts in the body's immune response to the infection.
When specifically recognized by T cells, the peptides trigger production of interferon gamma, a protein that acts as a signal to many other immune cells to respond to a new infection. Reductions in the production of interferon gamma would indicate, the scientists say, that the immune system was weakening in its response to the virus's mutations.
After analyzing the genetic changes in the sites, called epitopes, where the T cells specifically bind to the virus, the researchers found no changes had occurred during the one year of follow-up in the one patient who self-recovered. However, in the remaining seven patients, there were changes in 69 percent of T-cell epitopes, showing that the virus had mutated at key locations necessary for chronic infection to proceed.
Additional analysis showed that changes in T-cell epitopes were 13 times more frequent than changes in the remaining genome of the virus. The researchers examined the binding ability of T cells obtained early in infection to recognize 10 viral peptides known to have changed during the first six months of infection. Eight showed severely reduced capacity to stimulate production of interferon gamma, offering confirmation that the virus was mutating to evade the immune system.
Analysis of the viral RNA in the blood of seven patients with chronic infections revealed that eight of 16 changes in genome matched to the consensus sequence, confirming the presence of selective evolutionary pressure toward restoration of an ancestral form of the virus.
In the second study, using blood samples collected in Cork, Ireland, the researchers compared the genetic makeup of the virus in 22 chronically infected women to the original strain that had infected them more than 20 years before. The women were among hundreds accidentally infected in 1977 by a blood product tainted with hepatitis C, providing the researchers with unique access to the source of the infection, which came from a single donor unaware of having the illness.
Using computer analysis techniques developed at Hopkins, the scientists mapped these changes against the genetic makeup of the women's immune response. The researchers found that when viral mutations were clustered in epitopes specific to each woman's immune system, the changes were directed away from the consensus sequence, suggesting immune escape. However, when mutations were clustered in epitopes that were not specific, the mutations were reversions back to the consensus sequence.
When the individual genome changes in each woman were mapped on a grid, each woman formed a unique cluster indicating individual, evolutionary selection. However, some of the changes were shared, suggesting convergence, which would not have occurred had the virus simply mutated at random.
"Our results raise the possibility that a hepatitis-C consensus sequence could be the best practical option for a vaccine," says infectious disease specialist David Thomas, M.D., a professor of medicine at Hopkins who served as senior author of the study of Irish women. "If we can focus vaccine development on the common genetic element in chronically infected patients, then we may be able to make a more effective vaccine."
Funding for these studies, which took place from January 2002 to January 2005, was provided by the National Institutes of Health, including the National Institute for Allergy and Infectious Disease, and the National Institute on Drug Abuse.
Other Hopkins investigators in this research were Timothy Mosbruger; Qing Mao, M.D., Ph.D.; Zhi Liu, M.D.; Xiao-Hong Wang, M.D.; Hung-Chih Yang; Xiao-Hang Wang, Ph.D., Dale Netski, Ph.D.; and Drew Pardoll, M.D. Co-investigators in the first study also included John Sidney, Ph.D., and Alessandro Sette, Ph.D., from the La Jolla Institute for Allergy and Immunology, in San Diego, Calif. Collaborators in the second study, conducted at Hopkins and at Cork University Hospital in Ireland, were Liam Fanning, Ph.D., and Kelizaeth Kenny-Walsh, M.D.
Hepatitis C is the leading cause of liver disease in the United States, causing an estimated 10,000 to 12,000 deaths each year. Hepatitis C is transmitted when blood and possibly other body fluids of an infected person enter another person, primarily through injection drug use, exposures in health care settings, from an infected mother to her baby during birth, and occasionally through sexual exposure. Symptoms of hepatitis C may not appear for years after infection, and diagnosis must be confirmed by blood tests. However, in addition to liver inflammation and tumors, earlier signs of infection are persistent flu-like symptoms, including any combination of body aches, headaches, night sweats, loss of appetite, diarrhea, fatigue, rash, nausea and mild abdominal pain. Current treatments for hepatitis C involve weekly injections of pegylated interferon for one year, plus twice daily doses of oral ribavirin. While some patients recover on their own, with their immune system attacking the virus and clearing it from the body, most do not. Scientists have not yet determined why this happens in some patients and not in others.
Journal of experimental Medicine
Published 6 June 2005
COMMENTARY (Editorial)
Mutational escape from CD8+ T cell immunity : HCV evolution, from chimpanzees to man
David G. Bowena,b and Christopher M. Walkera,b
a D.G.B. and C.M.W. are at Center for Vaccines and Immunity, Columbus Children's Research Institute, Columbus, OH 43205.
b C.M.W. is at College of Medicine and Public Health, Ohio State University, Columbus, OH 43210.
Are CTL escape mutations in HCV important for viral persistence?
Although it is clear that CTL escape mutations occur in HCV genomes, the relevance of this mechanism to viral persistence is an open question. Mutations usually occur within the first 3-4 months of infection (3, 4), consistent with the delayed generation of CD8+ T cell responses that is an unusual feature of acute HCV infections (1). Such observations are compatible with release from early immune selection pressure as viral escape is established, and perhaps suggest a role for CTL escape mutations in the genesis of chronic infection. However, as observed in SIV infection (9), the rate at which escape mutations occur during HCV infection can be highly variable, occurring between 10 weeks to 2 years postinfection in chimpanzees (3). It is notable that once established, escape mutations in HCV tend to remain fixed within the circulating quasispecies of an individual (3, 15), perhaps driven by persisting CD8+ T cell responses, which are often concentrated within the liver (1). The cause of this variability in the timing of viral escape remains to be determined and may well be governed largely by the stochastic nature of viral evolution and a requirement for the development of compensatory mutations for viruses containing certain escape mutations to remain viable. However, differences in the kinetics of the immune response might also be involved in the variable dynamics of viral evolution. Indeed, future detailed prospective study of factors such as the temporal relationship between the emergence of escape mutations and breadth and persistence of the HCV-specific CD4+ T cell response, already demonstrated as important in disease resolution and containment of mutational escape (1, 22), might not only further elucidate a role for these cells in mutational escape, but also the role of mutational escape in viral persistence.
Concluding remarks
Although recent data confirms the generation of escape mutations in HCV, it is clear that this field is in its infancy. As is becoming increasingly apparent from the immunodeficiency virus literature, studies comparing circulating viral genomes to consensus sequences are extremely difficult to interpret. Further detailed prospective studies of acute and evolving HCV infection are required to delineate the role of escape mutations in HCV persistence, as well as to define both the immunological and virological factors governing viral evolution. In particular, careful dissection of these factors will be critical to the design of any effective CTL-based vaccine.
The mechanisms by which the hepatitis C virus (HCV) establishes persistence are not yet fully understood. Previous chimpanzee and now human studies suggest that mutations within MHC class I-restricted HCV epitopes might contribute to viral escape from cytotoxic T lymphocyte (CTL) responses. However, there are several outstanding questions regarding the role of escape mutations in viral persistence and their fate in the absence of immune selection pressure.
The hepatitis C virus is a small positive-stranded RNA virus within the Flaviviridae family that persists in 70% of infected individuals, considerably increasing their risk of developing cirrhosis and hepatocellular carcinoma. With an estimated 170 million infected individuals worldwide, HCV presents a major global health challenge (1). Studies of chimpanzees, the only nonhuman HCV host, have indicated that escape mutations in MHC class I-restricted epitopes may play a role in evasion of the antiviral CTL response (2, 3). In this issue, three reports demonstrate that CTL escape mutations also occur in human HCV infection (4-6) and reinforce the general relevance of this immune evasion mechanism to persistence of RNA viruses in humans.
Escape mutations in RNA viruses: precedents for HCV infection
The majority of RNA viruses produce RNA polymerases that lack proofreading activity, and thus encode viral genomes containing random base mutations. In the presence of immune selection pressure exerted by CTLs against wild-type virus, this genomic diversity could facilitate preferential expansion of mutant progeny encoding altered epitopes that evade recognition by effector T cells. The principle of "escape mutations" in MHC class I-restricted epitopes was first demonstrated in the model of murine lymphocytic choriomeningitis viral infection (7). However, this finding remained controversial, as escape was forced by the presence of relatively high affinity T cell receptor (TCR) transgenic T cells, and resultant mutations did not enable immune evasion from a broader epitope-specific response by nontransgenic CTLs. The relevance of this mechanism was ultimately established in studies of HIV and simian immunodeficiency virus (SIV) infections, where immune selection pressure by CD8+ T cells results in development of escape mutations (8, 9), particularly during the acute phase of infection (10, 11). The importance of escape mutations in immune evasion by these viruses has been confirmed in studies demonstrating that mutations in immunodominant epitopes are associated with progression to AIDS (12), with escape and subsequent disease progression after transfer of an autologous CTL clone (13), and with escape from partial control afforded by prior vaccination (14).
CTL escape in the chimpanzee model of HCV infection
Virus-specific CD8+ T cells are unquestionably important in the outcome of HCV infection because the onset of this response is temporally correlated with control of acute phase viremia, and antibody-mediated depletion of the CD8+ T cell subset prolongs viral replication in chimpanzees (1). HCV replication is directed by the error-prone RNA-dependent RNA polymerase encoded by the viral NS5b gene, which due to its propensity to introduce mutations into the viral genome might provide the same selective advantage enjoyed by HIV and SIV. This mechanism of immune escape may contribute to the remarkable ability of HCV to persist in infected individuals.
Initial investigations of HCV CTL mutational escape were performed in the chimpanzee. The major advantage of this model is that the sequence of the infecting inoculum is known, allowing for accurate analysis of viral evolution. The initial description of CTL escape in a single epitope was made relatively early after the discovery of HCV (2), but statistical proof that CD8+ T cells select for HCV escape mutations has only recently become available. In a study of three chimpanzees that developed persistent infection, mutations abrogating CTL function were described in multiple regions of the viral genome encoding known epitopes, and were largely confined to animals expressing the appropriate restricting MHC class I allele or a closely related subtype (3). The ratio of nonsynonymous base substitution (which changed the amino acid encoded) to synonymous base substitution (which left encoded amino acids unchanged) was higher in regions encoding these epitopes than in flanking sequences, consistent with Darwinian selection pressure (3). Thus, the limited data available from this sole animal model has supported the supposition that CTL escape mutations occur during the course of HCV infection.
CTL escape mutation in human HCV infection
Until very recently, investigation of escape mutation in human HCV infections has been limited by practical considerations. As acute hepatitis C is often cryptic, patients usually do not present until late in infection. Study of viral evolution throughout the course of infection has hence proved extremely difficult. Furthermore, as the genomic sequence of the infecting inoculum is rarely known, interpretation of sequences obtained from circulating quasispecies in later phases of infection is problematic. Comparison to published prototypical HCV sequences partially circumvents these limitations, and this approach has provided preliminary indications that CTL escape mutations occur during human HCV infection (15).
Proof of the escape hypothesis would ideally require comparisons between the evolving viruses and the infectious inoculum, and not external reference sequences of published viruses. Much effort has therefore recently been directed to the surveillance of populations at high risk of HCV infection, including intravenous drug users, health care workers suffering needle stick injuries, and patients undergoing medical procedures. This allows prospective monitoring of HCV evolution, analysis that has to date been unavailable. In addition, cohorts of individuals infected by a single source of HCV of known sequence have also been studied. Such analyses allow definitive assessment of changes within the viral genome, which are critical to determining the role of immune selection pressure in viral evolution. Thus, in a recent publication in the Journal of Experimental Medicine, Timm et al. studied the evolution of an immunodominant HLA-B*08-restricted NS3 epitope during acute HCV infection in two HLA-B*08-positive patients, one infected by a needle stick injury, and the other by an undetermined route (16). CTL-mediated responses to this epitope were followed by the development of escape mutations in both individuals (16).
In this issue, three additional studies complement and extend this observation (4-6). These manuscripts in toto constitute a critical mass of evidence for CTL escape mutations in human HCV infection. Tester et al. followed two individuals acutely infected from a single source; in the recipient who did not spontaneously resolve infection, escape mutation in an immunodominant epitope was observed (6). Cox et al. analyzed the evolution of HCV by partial genome sequencing in eight acutely infected individuals, defining escape mutations in multiple CTL epitopes (4). In the third study, Ray et al. used the unique approach of comparing the sequences of viruses from 22 humans with chronic hepatitis C with the sequence of the single common virus that initiated these infections 20 year earlier (5). The expression of HLA-B*07, HLA-B*35, or HLA-B*37 alleles were found to be linked to the presence of mutations in epitopes presented by these alleles, indicating a likely role for CTL-mediated pressure in driving viral evolution (5).
Mechanisms of CTL escape
Thus, as with other highly mutable RNA viruses, escape mutations in MHC class I-restricted epitopes are a feature of HCV infection and can diminish CTL responses via several mechanisms. Most recently it has been demonstrated that amino acid substitutions within or adjacent to CTL epitopes can alter proteosomal processing causing epitope destruction before transport to the endoplasmic reticulum for MHC binding (16-18). A loss of epitope phenotype can also occur when amino acid anchor residues required for MHC binding are changed (3, 15). CTL recognition of epitopes may also be diminished despite apparent conservation of peptide MHC binding (15), perhaps due to reduced TCR recognition of the neo-epitope-MHC complex. Such mutated peptide-MHC complexes may alternatively antagonize responses to the wild-type epitope (15, 19, 20).
It should be noted that CTL escape has not been observed in all studied epitopes (3, 5, 21). It is unclear why HCV-specific CTLs directed against these unaltered epitopes do not mediate viral clearance in persistently infected individuals, but recent studies have indicated that their antiviral functions may be impaired in chronic infection (1).
The factors constraining the emergence of escape mutations are incompletely understood. Even in chronically HCV-infected individuals, CD8+ T cell responses can sometimes be directed against a wide range of MHC class I-restricted epitopes (1). Indeed, one major criticism of the escape hypothesis has been the assumption that viral persistence would require almost simultaneous mutations within multiple epitopes targeted by CTLs. However, it is possible that the spontaneous loss of HCV-specific CD4+ T cells that is consistently observed in subjects who develop chronic HCV infection (1) could reduce the effectiveness of CD8+ T cells to a point where they select for viral escape variants, rather than containing infection. Direct support for this concept was obtained by antibody-mediated CD4+ T cell depletion of immune chimpanzees that had resolved prior HCV infections (22). Rechallenge of animals lacking CD4+ T cells with the same HCV strain resulted in viral persistence. Importantly, the frequency and breadth of the memory CD8+ T cell response was reduced in this setting, and persistent viruses developed escape mutations in multiple MHC class I-restricted epitopes (22). Consistent with the importance of CD4+ T cell responses in containing viral escape, in the study of Tester et al., mutational escape was associated with undetectable HCV-specific CD4+ T cell responses, whereas in the subject who resolved infection without mutational escape, broad sustained CD4+ T cell responses were demonstrated (6). In addition, a study of HCV-specific CD8+ T cell responses in HCV-infected chimpanzees indicated that the diversity of clonal TCR usage might also be a factor in the development of escape mutations (23). In a majority of cases, escape mutation was associated with reduced CTL clonal TCR diversity in comparison to epitopes in which escape mutations were not observed or those analyzed in chimpanzees that resolved infection (23). Similar analysis in SIV infection has also indicated a link between diverse clonotypic TCR repertoires and lack of mutation in the targeted epitope, whereas a more highly conserved clonotypic TCR repertoire was associated with viral escape (24).
Viral reversion and the meaning of consensus sequences
It is likely that the emergence of escape mutations is at least in part governed by the "fitness cost" imposed on HCV replication. However, although impairment of propagation may constrain the emergence of escape mutations in some structurally critical residues (25), evidence from studies in HIV and SIV indicates that compensatory viral mutations can occur, both within epitopes and in other regions, which allow for the generation of escape mutations in highly constrained sites (26-28). Consistent with mutational escape exacting fitness cost, recent studies of a limited number of HIV and SIV epitopes have indicated that reversion of epitopes to wild-type sequence can occur after transmission of mutated virus into a host that does not express the restricting MHC class I allele (29-31).
Although fitness cost is likely an important factor shaping HCV evolution, how individual amino acid substitutions alter replication is not known. In the acute infection cohort of Cox et al., sequences from the subjects' HCV genomes external to known CTL epitopes were compared with a corresponding HCV consensus sequence, and a tendency to mutate toward viral consensus was found in these regions (4). Similarly, in the study by Ray et al., mutation of the HCV genomes toward consensus was noted in regions outside known CTL epitopes. Additionally, in HLA-A*01- and B*08-negative individuals, absence of these alleles was associated with evolution toward consensus within epitopes restricted by these MHC molecules (5). These results have been interpreted as indicating that such mutations occur due to viral reversion to a more fit ancestral state (4, 5). Although the available data supports this model, conclusions are tempered by the absence of MHC haplotype information for the donor of the HCV inocula that would provide additional insight into the selective forces acting on the HCV genomes before and after transmission.
The concept that consensus sequences represent genomes that are the most antigenic as well as replicatively fit is being challenged by studies of HIV evolution and CTL responses. Recent population-based studies have indicated that genetic polymorphisms within certain HIV CTL epitopes are significantly associated with expression of the restricting MHC class I alleles (32), indicating that common HLA alleles may leave a "footprint" on this virus, with loss of epitopes restricted by highly prevalent HLA class I alleles (33). Analysis of a limited set of HIV and SIV CTL escape mutations has indicated that for some mutations maintenance of a variant epitope sequence may be largely confined to subjects expressing the appropriate restricting MHC molecule (29-31). On the other hand, recent data indicates that at least some HIV CTL escape mutations persist within the viral genome in individuals who do not express the restricting MHC class I molecule (29, 34). Indeed, such escape mutations may even become prevalent enough to enter the consensus sequence (34), indicating that for these epitopes, marked fitness cost is not exacted by viral escape, and reversion to a more immunogenic ancestral state is not automatic upon passage to a host in which immune selection pressure is absent. Although similar evidence is not available for HCV infection, it is interesting to note that a mutation reported in an HLA-A*02-restricted epitope in which CTL escape was mediated by altered proteosomal processing was not observed at a statistically significant higher frequency among the HLA-A*02-positive population in comparison to the control HLA-A*02-negative population studied (17). It is tempting to speculate that this phenomenon might be due to low fitness cost associated with this particular mutation, thus allowing persistence of the variant sequence in the absence of immune selection pressure.
Thus, due to likely interplay between the opposing forces of immune selection pressure and viral fitness cost, a variety of outcomes are possible upon initial infection with HCV, or after subsequent transmission of the virus to a recipient in whom the initial MHC class I alleles are not expressed.
Excerpts from the two main research articles
Divergent and convergent evolution after a common-source outbreak of hepatitis C virus
Stuart C. Ray1, Liam Fanning2, Xiao-Hong Wang1,3, Dale M. Netski1, Elizabeth Kenny-Walsh2, and David L. Thomas1
1 Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21231
2 Hepatitis C Unit, Department of Medicine, Cork University Hospital, National University of Ireland, Cork, UK
3 Southwest Hospital, Third Military Medical University, Chongqinq, Peoples Republic of China 400038
The genomic sequences of viruses that are highly mutable and cause chronic infection tend to diverge over time. We report that these changes represent both immune-driven selection and, in the absence of immune pressure, reversion toward an ancestral consensus. Sequence changes in hepatitis C virus (HCV) structural and nonstructural genes were studied in a cohort of women accidentally infected with HCV in a rare common-source outbreak. We compared sequences present in serum obtained 18-22 yr after infection to sequences present in the shared inoculum and found that HCV evolved along a distinct path in each woman. Amino acid substitutions in known epitopes were directed away from consensus in persons having the HLA allele associated with that epitope (immune selection), and toward consensus in those lacking the allele (reversion). These data suggest that vaccines for genetically diverse viruses may be more effective if they represent consensus sequence, rather than a human isolate.
A virus capable of genetic variation and of causing chronic infection will evolve to optimize its fitness in each host, a process which is the net sum of immune recognition (positive selection) and functional constraint on replication (negative selection). Because an estimated 1012 virions are produced each day through an error-prone, nonproofreading NS5B RNA polymerase, hepatitis C virus (HCV) is especially capable of viral evolution (1, 2). However, we previously showed that evolution is not driven by replication alone. In the acute phase of infection before adaptive immune responses (but after weeks of replication supporting a viral RNA level of more than 105 IU/ml), the same major viral variant was detected in each of a serial passage of eight chimpanzees (3). In contrast, the sequence of envelope genes, particularly HVR1, changes in virtually all humans who have been persistently infected (including the source of the inoculum passaged through this chimpanzee lineage [4]), a notable exception being persons with attenuated humoral immune responses (agammaglobulinemia), who have been shown to have reduced variability in HVR1 (5). Longitudinal studies of chimpanzees experimentally infected with HCV have revealed that amino acid replacements in immunodominant CD8+ T cell epitopes presented on MHC class I in an allele-restricted manner contribute to viral persistence (6). Thus, we hypothesized that the net evolution of HCV would demonstrate both functional constraint (reversion of sequences toward consensus) as well as positive pressure (and thus reveal immunodominant epitopes).
Although it required that persons be infected with the same inoculum, it was possible to test this hypothesis because between May 1977 and November 1978 over 500 women were inadvertently infected with HCV from a single acutely infected source, as a result of treatment with contaminated anti-D immune globulin (7). In a single amplicon, a 5.2-kb cDNA spanning 5'UTR through the NS3-NS4A junction was cloned from serum collected from 22 women 18-22 yr after infection, as well as in two specimens of frozen plasma from the inoculum donor.
Indirectly, these results suggest that immune escape is costly to the virus. Fitness is conventionally measured as a competition among genetic variants, and when the viral population size is large the most successful variants present at any one time in a host are by definition the most fit under the overall selection pressures. We assume that over very long periods of time (relative to the viral life cycle) the residues in the viral genome have the opportunity to vary substantially, and independently to a first approximation (this is a fundamental assumption of phylogenetic analysis). Therefore, although there may be some covariation, analytically the positions in the sequence can be considered independent variables. Finding a strong positive correlation between the presence of HLA alleles and substitutions in allele-associated epitopes suggests that those changes increase viral fitness in the presence of the associated immune response. Likewise, a strong association between the absence of alleles and substitutions in allele-associated epitopes, particularly when found to represent reversion to consensus, suggests that positive selection in a previous host carried a fitness cost in terms of viral replicative capacity.
Prior studies have demonstrated reversion of CTL escape-variant sequences in macaques experimentally infected with SIV (13), reversion of one epitope each of HIV-1 and HCV in humans (14, 15), and evidence of HIV-1 adaptation to common HLA alleles (16). This is the first report of viral adaptation to multiple HLA alleles across multiple genes in HCV, and provides additional support for the suggestion, based on minimizing differences between vaccine and circulating strains, that vaccine effectiveness may be enhanced by using a consensus (17) or ancestral (18) sequence.
The ability of viruses to restrict adaptive immune responses and evade those that are formed contributes to persistence and is a major barrier to vaccine development. These data suggest that escape variants have greater fitness in the presence of an individual host's immune response, and that immune evasion contributes to the sequence divergence observed in a each persistently infected host. Nonetheless, this divergence may actually reduce the fitness of the virus in the population (that is, in other hosts). From an evolutionary perspective, these forces maintain the virus as a distinct pathogen. However, the data also suggest that immune responses to consensus sequences (rather than a product based on the sequence in a given host) may establish the highest barrier to viral escape and consequently the most effective protection against chronic infection.
Cellular immune selection with hepatitis C virus persistence in humans
Andrea L. Cox1,2, Timothy Mosbruger1, Qing Mao1, Zhi Liu1, Xiao-Hong Wang1, Hung-Chih Yang1, John Sidney6, Alessandro Sette6, Drew Pardoll1,2,3,4, David L. Thomas1,5, and Stuart C. Ray1
1 Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD 21231
2 Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD 21231
3 Department of Molecular Biology and Genetics, Johns Hopkins Medical Institutions, Baltimore, MD 21231
4 Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD 21231
5 Department of Epidemiology, Johns Hopkins Medical Institutions, Baltimore, MD 21231
6 La Jolla Institute for Allergy and Immunology, San Diego, CA 92121
ABSTRACT
Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific cellular immune responses. To determine if immunologically driven sequence variation occurs with HCV persistence, we coordinately analyzed sequence evolution and CD8+ T cell responses to epitopes covering the entire HCV polyprotein in subjects who were followed prospectively from before infection to beyond the first year. There were no substitutions in T cell epitopes for a year after infection in a subject who cleared viremia. In contrast, in subjects with persistent viremia and detectable T cell responses, we observed substitutions in 69% of T cell epitopes, and every subject had a substitution in at least one epitope. In addition, amino acid substitutions occurred 13-fold more often within than outside T cell epitopes (P < 0.001, range 5-38). T lymphocyte recognition of 8 of 10 mutant peptides was markedly reduced compared with the initial sequence, indicating viral escape. Of 16 nonenvelope substitutions that occurred outside of known T cell epitopes, 8 represented conversion to consensus (P = 0.015). These findings reveal two distinct mechanisms of sequence evolution involved in HCV persistence: viral escape from CD8+ T cell responses and optimization of replicative capacity.
The World Health Organization estimates there are 170 million persons with hepatitis C virus (HCV) infection worldwide, and an estimated 4 million persons are infected with HCV in the United States (1, 2). In most countries, HCV infection is found in 1-2% of the general population and may cause cirrhosis or hepatocellular cancer, but only when infection persists (3-7).
Patients in the acute phase of HCV infection are much more likely to respond to therapy that is designed to eradicate the virus than are patients after progression to chronicity (8-10). The features unique to acute infection that allow increased responsiveness to interferon therapy remain unknown. Spontaneous clearance of HCV infection occurs in 20% of acutely infected individuals and is associated with a broadly specific and vigorous cellular immune response (11-14). Although the cellular immune response is often less vigorous and more narrowly directed in those who fail to clear the infection, nonetheless, a cellular immune response often is present in early infection and may persist into chronic infection. Why the early response fails to control viremia in those who progress to chronic infection is not clear, but responses generated in acute infection have been shown to decline in some subjects who remained persistently infected, and chronic infection is characterized by low frequencies of CD8+ T cells in peripheral blood (13, 15-20). Although the liver has the potential to delete antigen-specific T cell responses, HCV-specific CD8+ CTL lines have been generated from the liver of chronically infected humans and chimpanzees; this suggests that elimination of HCV-specific lymphocytes from the liver is neither universal nor necessary for HCV persistence (21-24). Survival of HCV, despite virus-specific CD8+ CTL, might be explained by impaired cellular effector functions (proliferation, cytokine secretion, cytolytic activity), T cell exhaustion, or dendritic cell dysfunction (16, 25-27). A final possibility is that persistence is facilitated by viral evolution over the course of infection, enabling escape by mutation of key epitopes targeted by T lymphocytes. Mutational escape from T cell responses has been noted in HIV, which uses an error-prone RNA polymerase similar to HCV (28).
Mathematical models of viral kinetics suggest that up to 1012 virions are produced each day in a human with chronic hepatitis C (29). This rate exceeds comparable estimates of the production of HIV by more than an order of magnitude, and, coupled with the absence of proofreading by the HCV NS5B RNA polymerase, results in frequent mutations within the HCV genome. Mutation of class I or II MHC restricted T cell epitopes could alter the outcome of infection by preventing or delaying clearance of infected hepatocytes (30). In the face of a vigorous multispecific CTL response, mutation of several epitopes, perhaps simultaneously, would be required for survival of the virus. In HCV infection, a strong association between viral persistence and the development of escape mutations has been demonstrated in the chimpanzee model (31), and one group examined viral evolution in a single HLA-B8-restricted NS3 epitope (32); however, evidence of evasion of a multispecific CTL response in humans is lacking. Although mutations in class I MHC-restricted HCV epitopes have been observed in humans with chronic HCV infection, it is uncertain that these mutations result from CD8+ T cell selection pressure or that they occur during the acute phase of infection, when clearance or persistence is determined (33-35).
Studies of the cellular immune response to acute HCV infection have been challenging because acute hepatitis C usually is clinically silent; this makes early virus isolates and CTL difficult to obtain. In addition, no consistent pattern of HCV epitope dominance has emerged in humans so large numbers of PBMCs are needed for the broad screening that is required to identify CTL responses. We have overcome these challenges to test the hypothesis that CTL-driven sequence variation occurs with progression to persistent HCV in humans. We prospectively studied HCV antibody-negative injection drug users at risk for HCV infection and compared the viral sequences at initial viremia to sequences that were obtained at multiple time points in acute HCV infection. In parallel, a genome-wide analysis of T cell responsiveness was performed. As evidence of immune selection pressure, we determined the percentage of T cell epitopes that underwent substitution, assessed the likelihood of amino acid changing substitutions to occur within versus outside T cell epitopes, and examined the effects of observed amino acid sequence changes in epitopes on T cell recognition and MHC class I binding. For amino acid substitutions outside T cell epitopes, we investigated explanations other than T cell pressure. Our results provide strong evidence in humans that immune and fitness selection occur during the acute phase of HCV infection.
Participants
The Risk Evaluation Assessment of Community Health prospective study of young injection drug users in Baltimore examined the incidence and risk factors for HCV infection, as described previously (49). Participants who were eligible for the study were anti-HCV antibody negative, between 15 and 30 years of age, and acknowledged use of injection drugs. Participants were invited to co-enroll in a substudy of acute hepatitis C; those who consented had blood drawn for isolation of serum, plasma, and PBMCs in a protocol that was designed for monthly follow-up. At each visit, participants were provided counseling to reduce the risks of drug use. The Risk Evaluation Assessment of Community Health protocol and the HCV substudy protocols were approved by the institutional review boards of the Johns Hopkins Schools of Medicine and Hygiene and Public Health.
AUTHOR DISCUSSION
This study suggests a mechanistic linkage between viral sequence variation and progression to chronicity. The arrested development of new T cell responses, despite ongoing viremia with sequence evolution, distinguishes the acute and chronic phases of HCV infection. Enhanced understanding of cellular immune failure that leads to chronic HCV infection could accelerate development of vaccines to prevent viral persistence, as was suggested elsewhere (39).
In this investigation of sequence variation in T cell epitopes as a potential mechanism for viral persistence, we show that amino acid substitutions during acute HCV infection are nonrandom and may be explained, in part, by escape from CD8+ T cell recognition and convergence, possibly because of replicatively unfit substitutions that were selected in a previous host. Significantly, there is early fixation of the T cell repertoire because we observed no instances of de novo development of T cells that recognized mutant t6 epitopes better than the original t0 epitope using lines or PBMCs.
Using the chimpanzee model of HCV infection, mutation of multiple class I MHC-restricted epitopes early in the course of chronic HCV infection was demonstrated (31). The role of CD8+ CTL in control of HCV replication was reinforced further by a statistically significant increase in the number of mutations that resulted in amino acid changes in class I MHC-restricted epitopes, but not unrestricted epitopes or flanking sequences of the viral genome. These data indicate that in chimpanzees, amino acid substitutions in class I MHC-restricted epitopes are selected and possibly maintained by HCV-specific CD8+ CTL populations that exert positive Darwinian selection pressure.
We also observed a statistically significant increase in the number of mutations that resulted in amino acid changes in class I MHC restricted epitopes versus portions of the viral genome outside T cell epitopes, and that amino acid substitutions in class I MHC-restricted epitopes resulted in escape from CD8+ T cell recognition in acutely HCV-infected humans who progressed to chronic infection. New T cells specific for the sequences that were detected 6 mo after initial viremia were not detected, despite follow-up for as long as 3 yr after infection. Thus, selection of HCV variants that evade CD8+ T cell recognition may represent a mechanism for persistence of HCV infection in humans. Supporting this, we observed no substitutions within recognized CD8+ T cell epitopes in the subject who cleared infection, and substitution in 60-75% of CD8+ T cell epitopes in subjects with persistent infection. The number of CTL epitopes with substitutions was shown previously to correlate with control of HIV viremia (28); however, a relationship between HCV control and maintenance of T cell epitopes had not been shown previously.
Although the observed substitutions were disproportionately contained within the portion of the HCV polyprotein in T cell epitopes, many were found outside of detectable T cell epitopes. It is possible that we missed CD8+ T cell epitopes and that some of the substitutions that were observed outside of T cell epitopes actually represented substitutions within T cell epitopes. We took several steps to minimize the chances of this occurring. Where there were substitutions and the H77 sequence that was used to make overlapping peptides as potential antigens differed from that of the subject, we tested for recognition of overlapping peptides representing autologous sequence. Where the subject's sequence matched H77 in areas of amino acid substitution, but there no responses were detected, we tested for recognition of additional overlapping peptides with different termini to minimize the possibility that we cut within a region that contained an epitope. We detected no additional epitopes via ELISPOT analysis with either method of antigen modification (unpublished data).
Because there is no clinical indication for liver biopsy in acute infection, we could only assess responses in the periphery. Therefore, it is possible that some of the substitutions may represent pressure for T cell responses present in the liver but not detectable in the periphery. We do not favor this explanation because previous studies have shown that the majority of T cell responses in the liver also are detectable in the peripheral blood (40, 41).
There are several possible alternative mechanisms for selection of these substitutions occurring outside of observed CD8+ T cell epitopes. The first is that they represent substitutions in CD4+ T cell epitopes. Although we detect a few CD4+ T cell epitopes using our ELISPOT assay, the conditions of the assay preferentially detect CD8+ T cell epitopes and we may fail to detect all of the possible CD4+ T cell epitopes. The second possible explanation is that they represent substitutions in B cell epitopes. Mutations in dominant antibody epitopes that are located in the HVR-1 of envelope glycoprotein 2 (E2) have been linked to persistence of HCV infection (42). B cell epitopes are predicted to occur within the envelope regions of the polyprotein and the majority of mutations outside of T cell epitopes were found in the envelope proteins for most subjects. Another possible explanation is that the mutations outside of T cell epitopes are selected because they confer a viral replication advantage. Some mutations may represent epitopes that are recognized by the previous host that revert to a more stable sequence when the new host fails to mount similar immune pressure. This may occur when the new host lacks the MHC allele that is required to present that epitope. Loss of escape mutations upon passage of simian immunodeficiency virus to new animals (43) and HIV to humans (44), that do not exert immune pressure on that region was described recently, with the inference that escape from CTL responses may reduce viral fitness.
The relevance of those studies to human infection with HCV is supported by a recent study of one epitope in an acutely HCV infected patient (32), and our accompanying study of chronically infected individuals (39). The former study supports the nonrandom nature of reversion at a site of previous immune escape. The latter study shows that HCV amino acid sequence tends to revert to consensus in areas outside of T cell epitopes in subjects who have persistent infection. The consensus sequence likely represents a more replicatively fit state than the initial infecting strains, which presumably have adapted to the immune response of the previous host. Taken together, these results reveal at least two types of sequence variation that occur simultaneously in progression of acute HCV to persistence: immune pressure that selects T cell escape variants, and reversion to consensus sequence that is likely to result in enhanced replicative fitness.
Despite viral mutation that results in the production of new potential antigens, no new T cell responses developed in response to mutant peptides that escaped initial recognition over months, and in some cases years, after the appearance of the mutation. This phenomenon also has been observed frequently with HIV sequence evolution where the failure to prime new responses may be due to impaired CD4+ T cell function. Although overall CD4+ T cell function is intact in HCV infection, chronic HCV infection has been linked to loss of HCV-specific CD4+ T cell responses (45). In addition, HCV has been linked to impaired DC function, decreased IFN regulatory factor 3 signaling, and protein kinase RNA-activated inhibition, which may inhibit priming of an immune response to the mutated peptides (26, 27, 46, 47). However, the failure to prime responses to the newly generated sequences is observed even in HIV infected individuals with relatively high CD4 counts, and there is no evidence in those with HCV infection of impaired priming of immune responses to other antigens, as would be evident by global immunosuppression. An alternative explanation is that original antigenic sin (the higher threshold required for stimulation of an immune response to an epitope resembling a previously recognized epitope [48]) may be responsible for the lack of response to mutant sequences seen in HIV and HCV, although this phenomenon has not been demonstrated in humans. Lastly, because the selective pressures of the immune system favor the emergence of a viral sequence that fails to elicit a productive response, we may be observing sequences that cannot be processed effectively for presentation or that resemble self-antigens, and therefore, are incapable of stimulating an immune response.
Journal of Experimental Medicine
Published 6 June 2005
Immune evasion versus recovery after acute hepatitis C virus infection from a shared source
Ian Tester1,2, Susan Smyk-Pearson1,2, Ping Wang3, Anne Wertheimer2, Ermei Yao3, David M. Lewinsohn1,2, John E. Tavis3,4, and Hugo R. Rosen1,2
1 Department of Medicine, Portland Veterans Administration Medical Center
2 Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland OR 97239
3 Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine
4 Saint Louis University Liver Center, St. Louis, MO 63103
ABSTRACT
Acute infection with hepatitis C virus (HCV) rarely is identified, and hence, the determinants of spontaneous resolution versus chronicity remain incompletely understood. In particular, because of the retrospective nature and unknown source of infection in most human studies, direct evidence for emergence of escape mutations in immunodominant major histocompatibility complex class I-restricted epitopes leading to immune evasion is extremely limited. In two patients infected accidentally with an identical HCV strain but who developed divergent outcomes, the total lack of HCV-specific CD4+ T cells in conjunction with vigorous CD8+ T cells that targeted a single epitope in one patient was associated with mutational escape and viral persistence. Statistical evidence for positive Darwinian selective pressure against an immunodominant epitope is presented. Wild-type cytotoxic T lymphocytes persisted even after the cognate antigen was no longer present.
Chronic hepatitis C virus (HCV) infection is common and affects 3% of the world's population; however, the diagnosis of acute HCV rarely is made and usually is inferred based on the history of a known exposure (1, 2). Emerging data suggest that immunologic and virologic events in the early stages of infection determine the eventual outcome (3). Spontaneous resolution of acute HCV infection has been associated with robust T cell responses in chimpanzees (4, 5) and humans (6-8), and it has been suggested that a threshold frequency of CTL is required for clearance of HCV (9). Although emergence of escape mutations in class I MHC-restricted epitopes was shown to lead to immune evasion in HCV-infected chimpanzees (10, 11), direct evidence for this mechanism in humans is limited. Tsai et al. (12), by focusing on a single HLA A2-restricted HCV E1 epitope in the hypervariable region, observed variant epitope sequences with CTL antagonist activity 3 mo from onset of acute hepatitis in two patients who developed persistence. Timm et al. (13) recently described the development of CTL responses against an HLA-B8-restricted epitope (including one patient after antiviral therapy); however, mutations within this immunodominant epitope did not impact HLA class I binding or TCR recognition.
We comprehensively studied two patients who developed homologous acute HCV infection after patellar tendon transplantation from a deceased donor who was in the "window" phase of acute HCV (i.e., negative for HCV antibody but positive for HCV RNA). Antigenic and viral genetic mapping revealed striking differences in these two individuals who shared several HLA alleles but had divergent outcomes. To assess the total and specific CD4+ and CD8+ T cell response against all potential HCV epitopes in an unbiased manner, we used 750 overlapping 15-mer peptides that spanned the entire HCV polyprotein. The lack of HCV-specific CD4+ T cells, in conjunction with vigorous CD8+ T cells that targeted a single immunodominant epitope, was associated with mutational escape and viral persistence. In contrast, the patient who demonstrated vigorous and multispecific CD4+ and CD8+ T cell responses spontaneously eradicated HCV infection.
EXCERPTS FROM RESULTS & DISCUSSION
Divergent outcomes after exposure to identical HCV-infected donor tissue
Two subjects with no previous history of exposure to HCV underwent elective patellar ligament (with bone) transplantation for knee reconstructive surgery in April 2002. Within 2 mo, both patients developed symptoms of fatigue and elevation in liver function tests. The first patient (PD101, a 49-yr-old white man) had elevation in his liver function tests and persistent viremia, and was treated with pegylated interferon and ribavirin. The second patient (PD102, a 51-yr-old white woman) became jaundiced with a peak total serum bilirubin of 8.1 mg/dl and spontaneously cleared HCV RNA from serum within 3 mo. Remarkably, both patients shared several HLA alleles, including HLA A2.
Reports of individuals who were exposed to a single source HCV infection have been limited to subjects who acquired the infection in the remote past (18), and consequently, immunologic and virologic analyses were performed long after the outcome of infection was determined. In this study, we prospectively tracked two patients early after they acquired HCV infection from the same acutely infected, "window period" donor. Comprehensive mapping of CD4+ and CD8+ T cell responses with overlapping 15 mer-peptides spanning the entire HCV polyprotein revealed statistically significant differences in the breadth and vigor of T cell responses between both individuals. These results are even more compelling when one considers that the autologous, infecting viral sequences and the peptides that were used to screen responses were nearly identical (presumably facilitating immune recognition), and that the subjects shared several HLA alleles, including the highly prevalent HLA A2 (also present in the donor).
Our results are consistent with the concept that the variant HCV carrying a substitution within an immunodominant CD8+ T cell epitope was selected by the monospecific antiviral CTL response, and the variant epitope (perhaps because of lack of HCV-specific CD4+ T cell help [11]) failed to generate new variant-specific CTL populations. The only peptide region that elicited IFN- response in PD101 was the class I-restricted epitope region where a nonsynonymous mutation arose and was maintained. In contrast, the likelihood of CTL escape is low when the CTL response is directed against multiple viral epitopes simultaneously, as in the case of PD102. We acknowledge the possibility that T cell responses were broader at time points earlier than the 6 mo when samples first became available.
One of the most striking findings in our study was that the CTL effector response (IFN- production and proliferative capacity) in PD101 was maintained even when the cognate antigen that it recognizes was no longer present (after viral escape and after therapeutic viral eradication). This apparent paradox was described previously in a human study of HBV infection (19) and in HCV-infected chimpanzees (9); it could be related to recurrent stimulation from an undefined reservoir of wild-type virus (20), as suggested by the recent demonstration of low-level HCV replication in PBMCs of patients with therapy-induced or spontaneous resolution of HCV infection (20). Alternatively, mutant epitopes could stimulate expansion of the wild-type-specific CTL—a phenomenon known as "original antigenic sin"—that initially was described for antibodies, but also extended to class I HLA-restricted responses (9, 21). Direct enumeration of the frequency of mutant-specific CTL with tetramers was not possible because attempts to synthesize them failed on two occasions, possibly a reflection of the fact the mutant peptide bound to the HLA-A2 molecule with lower affinity than the wild-type peptide (which also might explain the lack of antagonistic activity of the mutant peptide).
In summary, our findings—derived by combining viral sequence data and unbiased, functional T cell analyses that examine every potential HCV epitope after homologous challenge—indicate that the absence of CD4+ T cell help, coupled with strong selective pressure exerted by wild-type-specific CTLs, favor the emergence of immune escape (11) and indicate a patient profile associated with chronicity that might benefit from early antiviral therapy. Elucidation of the mechanisms which underlie the early failure to develop HCV-specific CD4+ T cells and their instructive signals during memory CD8+ T cell differentiation has implications for vaccine strategies that are designed to induce protective immunity to this common disease (22).
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