Hepatitis B virus-specific CD4 T cell responses differentiate functional cure from chronic surface antigen+ infection.

BACKGROUND & AIMS: With or without antiviral treatment, few individuals achieve sustained functional cure of chronic hepatitis B virus (HBV) infection. A better definition of what mediates functional cure is essential for improving immunotherapeutic strategies. We aimed to compare HBV-specific T cell responses in patients with different degrees of viral control. METHODS: We obtained blood from 124 HBV-infected individuals, including those with acute self-limiting HBV infection, chronic infection, and chronic infection with functional cure. We screened for HBV-specific T cell specificities by ELISpot, assessed the function of HBV-specific T cells using intracellular cytokine staining, and characterized HBV-specific CD4 T cells using human leukocyte antigen (HLA) class II tetramer staining, all directly ex vivo. RESULTS: ELISpot screening readily identified HBV-specific CD4 and CD8 T cell responses in acute resolving infection compared with more limited reactivity in chronic infection. Applying more sensitive assays revealed higher frequencies of functional HBV-specific CD4 T cells, but not CD8 T cells, in functional cure compared to chronic infection. Function independent analysis using HLA multimers also identified more HBV-specific CD4 T cell responses in functional cure compared to chronic infection, with the emergence of CD4 T cell memory both after acute and chronic infection. CONCLUSIONS: Functional cure is associated with higher frequencies of functional HBV-specific CD4 memory T cell responses. Thus, immunotherapeutic approaches designed to induce HBV functional cure should also aim to improve CD4 T cell responses. LAY SUMMARY: Immunotherapy is a form of treatment that relies on harnessing the power of an individual's immune system to target a specific disease or pathogen. Such approaches are being developed for patients with chronic HBV infection, in an attempt to mimic the immune response in patients who control HBV infection spontaneously, achieving a so-called functional cure. However, what exactly defines protective immune responses remains unclear. Herein, we show that functional cure is associated with robust responses by HBV-specific CD4 T cells (a type of immune cell).

Increase in Hepatitis A Cases Linked to Imported Strains to Rio de Janeiro, Brazil: A Cross-Sectional Study.

This study aims to evaluate the epidemiological and molecular features associated with HAV transmission in adults in Rio de Janeiro during a period of increased registered cases of HAV (2017-2018). Socio-epidemiological data and serum samples from anti-HAV IgM+ individuals were obtained. HAV RNA was RT-PCR amplified and sequenced for further phylogenetic and phylogeographic analyses. From fifty-two HAV IgM+ individuals, most were men (78.85%; p = 0.024), aged 20-30 years old (84.61%; p < 0.001), resided in the Rio de Janeiro north zone (31/52; 59.62%; p = 0.001), and are men who have sex with men (MSM) (57.69%; p = 0.002). Sexual practices were more frequent (96%) than others risk factors (food-borne (44%), water-borne (42.31%), and parenteral (34.62%)). Individuals who traveled to endemic regions had a 7.19-fold (1.93-36.04; p < 0.01) increased risk of HAV. Phylogenetic analysis revealed four distinct clades of subgenotype IA, three of them comprised sequences from European/Asian MSM outbreaks and one from Brazilian endemic strains. Bayesian Inference showed that the imported strains were introduced to Brazil during large mass sportive events. Sexual orientation and sexual practices may play a role in acquiring HAV infection. Public policies targeting key populations must be implemented to prevent further dissemination of HAV and other STIs.

The Inflammatory Cytokine Profile Associated With Liver Damage Is Broader and Stronger in Patients With Chronic Hepatitis B Compared to Patients With Acute Hepatitis B.

Liver damage in hepatitis B is immune driven and correlates with inflammatory markers in patient serum. There is no comparison of these markers to determine if inflammatory profiles are distinct to different types of liver damage across patients at different stages of disease. We measured 25 inflammatory markers in patients with acute hepatitis B and chronic hepatitis B with hepatitis B e antigen seroconversion and chronic patients stopping nucleoside analogue therapy. Myeloid markers dominated the inflammatory profile in all stages of hepatitis B. More inflammatory markers were detectable in chronic patients, including elevated concentrations of cytotoxic effectors Fas ligand, TRAIL, and TNF-α.

A Systematic Evaluation of IgM and IgG Antibody Assay Accuracy in Diagnosing Acute Zika Virus Infection in Brazil: Lessons Relevant to Emerging Infections.

Accurate diagnostics underpin effective public health responses to emerging viruses. For viruses, such as Zika virus (ZIKV), where the viremia clears quickly, antibody-based (IgM or IgG) diagnostics are recommended for patients who present 7 days after symptom onset. However, cross-reactive antibody responses can complicate test interpretation among populations where closely related viruses circulate. We examined the accuracy (proportion of samples correctly categorized as Zika positive or negative) for antibody-based diagnostics among Brazilian residents (Rio de Janeiro) during the ZIKV outbreak. Four ZIKV enzyme-linked immunosorbent assays (ELISAs; IgM and IgG Euroimmun, IgM Novagnost, and CDC MAC), two dengue ELISAs (IgM and IgG Panbio), and the ZIKV plaque reduction neutralization test (PRNT) were evaluated. Positive samples were ZIKV PCR confirmed clinical cases collected in 2015-2016 (n = 169); negative samples (n = 236) were collected before ZIKV was present in Brazil (≤2013). Among serum samples collected ≥7 days from symptom onset, PRNT exhibited the highest accuracy (93.7%), followed by the Euroimmun IgG ELISA (77.9%). All IgM assays exhibited lower accuracy (<75%). IgG was detected more consistently than IgM among ZIKV cases using Euroimmun ELISAs (68% versus 22%). Anti-dengue virus IgM ELISA was positive in 41.1% of confirmed ZIKV samples tested. The Euroimmun IgG assay, although misdiagnosing 22% of samples, provided the most accurate ELISA. Anti-ZIKV IgG was detected more reliably than IgM among ZIKV patients, suggesting a secondary antibody response to assay antigens following ZIKV infection. Antibody ELISAs need careful evaluation in their target population to optimize use and minimize misdiagnosis, prior to widespread deployment, particularly where related viruses cocirculate.

Differentiation of exhausted CD8+ T cells after termination of chronic antigen stimulation stops short of achieving functional T cell memory.

T cell exhaustion is associated with failure to clear chronic infections and malignant cells. Defining the molecular mechanisms of T cell exhaustion and reinvigoration is essential to improving immunotherapeutic modalities. Here we confirmed pervasive phenotypic, functional and transcriptional differences between memory and exhausted antigen-specific CD8+ T cells in human hepatitis C virus (HCV) infection before and after treatment. After viral cure, phenotypic changes in clonally stable exhausted T cell populations suggested differentiation toward a memory-like profile. However, functionally, the cells showed little improvement, and critical transcriptional regulators remained in the exhaustion state. Notably, T cells from chronic HCV infection that were exposed to antigen for less time because of viral escape mutations were functionally and transcriptionally more similar to memory T cells from spontaneously resolved HCV infection. Thus, the duration of T cell stimulation impacts exhaustion recovery, with antigen removal after long-term exhaustion being insufficient for the development of functional T cell memory.

Differentiation of exhausted CD8 T cells after termination of chronic antigen stimulation stops short of achieving functional T cell memory

T cell exhaustion is associated with failure to clear chronic infections and malignant cells. Defining the molecular mechanisms of T cell exhaustion and reinvigoration is essential to improving immunotherapeutic modalities. Analysis of antigen-specific CD8+ T cells before and after antigen removal in human hepatitis C virus (HCV) infection confirmed pervasive phenotypic, functional, and transcriptional differences between exhausted and memory CD8+ T cells. After viral cure, we observed broad phenotypic and transcriptional changes in clonally stable exhausted T-cell populations suggesting differentiation towards a memory-like profile. However, functionally, the cells showed little improvement and critical transcriptional regulators remained in the exhaustion state. Notably, T cells from chronic HCV infection that were exposed to antigen for shorter periods of time because of viral escape mutations were functionally and transcriptionally more similar to memory T cells from spontaneously resolved acute HCV infection. Thus, duration of T cell stimulation impacts the ability to recover from exhaustion, as antigen removal after long-term T cell exhaustion is insufficient for the development of key T cell memory characteristics.

Evolution of the innate and adaptive immune response in women with acute Zika virus infection.

Zika virus (ZIKV) is a flavivirus that is closely related to other human pathogens, such as dengue virus (DENV)1. Primary transmission usually involves Aedes aegypti, which has expanded its distribution range considerably2, although rarer infection routes, including mother-to-fetus transmission, sexual contact and blood transfusion, have also been observed3-7. Primary ZIKV infection is usually asymptomatic or mild in adults, with quickly resolved blood viraemia, but ZIKV might persist for months in saliva, urine, semen, breast milk and the central nervous system8-12. During a recent ZIKV outbreak in South America, substantial numbers of neurological complications, such as Guillain-Barré syndrome, were reported13,14 together with cases of microcephaly and associated developmental problems in infants born to women infected with ZIKV during pregnancy15-20, highlighting the clinical importance of this infection. Analyses of the human immune response to ZIKV are lacking21-28, but the recent outbreak has provided an opportunity to assess ZIKV immunity using current immunological methods. Here, we comprehensively assess the acute innate and adaptive immune response to ZIKV infection in ten women who were recruited during early infection and followed through reconvalescence. We define a cascade of events that lead to immunological control of ZIKV, with previous exposure to DENV impacting some, but not all, mediators of antiviral immunity.

Genetic Diversity of the Hepatitis B Virus Subgenotypes in Brazil.

Hepatitis B virus (HBV) subgenotypes may be related to clinical outcomes and response to antiviral therapy. Most Brazilian studies on HBV subgenotypes are restricted to some regions and to specific population groups. Here, we provide an insight about genetic diversity of HBV subgenotypes in 321 serum samples from all five geographical regions, providing a representative overview of their circulation among chronic carriers. Overall, HBV/A1 was the most prevalent subgenotype, being found as the major one in all regions except in South Brazil. Among HBV/D samples, subgenotype D3 was the most prevalent, found in 51.5%, followed by D2 (27.3%) and D4 (21.2%). D2 and D3 were the most prevalent subgenotypes in South region, with high similarity with European strains. D4 was found in North and Northeast region and clustered with strains from Cape Verde and India. For HBV/F, the most frequent subgenotype was F2 (84.1%), followed by F4 (10.1%) and F1 (5.8%), closely related with strains from Venezuela, Argentina and Chile, respectively. Phylogeographic analyses were performed using an HBV full-length genome obtained from samples infected with genotypes rarely found in Brazil (B, C, and E). According to Bayesian inference, HBV/B2 and HBV/C2 were probably introduced in Brazil through China, and HBV/E from Guinea, all of them mostly linked to recent events of human migration. In conclusion, this study provided a comprehensive overview of the current circulation of HBV subgenotypes in Brazil. Our findings might contribute to a better understand of the dynamics of viral variants, to establish a permanent molecular surveillance on the introduction and dispersion patterns of new strains and, thus, to support public policies to control HBV dissemination in Brazil.

Hepatitis A Strain Linked to the European Outbreaks During Gay Events between 2016 and 2017, Identified in a Brazilian Homosexual Couple in 2017.

Hepatitis A virus (HAV) outbreaks among men who have sex with men (MSM) have been reported worldwide and associated primarily with sexual transmission through oral-anal sex. Here, we provide the molecular and evolutionary description of a European strain, linked to HAV outbreaks among MSM, detected in a Brazilian homosexual couple. Bayesian analysis provided evidence that the viral isolates were introduced in Brazil from Spain between the end of 2016 and the beginning of 2017.

Accuracy of rapid test for diagnosis of hepatitis A with different infection rate settings and with predictive modeling.

AIM: We evaluated the accuracy of a commercial rapid immunochromatographic test (rapid test [RT]) for hepatitis A (HA) diagnosis and epidemiological studies. MATERIALS & METHODS: The accuracy of a RT was evaluated in laboratory and in field conditions. Predictive modeling estimated the test performance in a hypothetical population. RESULTS: The RT showed sensitivities of 66-86%, and specificities of 21-100%, depending on the antibody isotype (IgM or IgG) analyzed and prevalence of infection. CONCLUSION: The RT is a good alternative for diagnostic in HA outbreaks. The predictive model indicates that it should not be used alone for HA diagnosis in low prevalence populations. These data can be used in the future to strengthen decision-making during the implementation of rapid diagnostic methods in health services.

Phenotype and function of HBV-specific T cells is determined by the targeted epitope in addition to the stage of infection.

OBJECTIVE: Chronic HBV infection affects more than 250 million people worldwide and remains a global healthcare problem in part because we lack curative treatment. Sustained viral control requires HBV-specific T cells, but these become functionally impaired in chronic infection. Clinical evidence indicates that functional cure of HBV infection by the host immune response is feasible. Developing T cell-based therapies able to achieve functional cure will require identification of the requirements for a successful T cell response against HBV and the relative contribution of individual T cell specificities to HBV control. DESIGN: The phenotype and function of HBV-specific T cells were studied directly ex vivo using fluorochrome-labelled multimers. We studied multiple HBV-specific T cell specificities targeting different HBV proteins in individuals with either an acute self-limiting or chronic HBV infection. RESULTS: We detected strong T cell responses targeting multiple HBV viral proteins in acute self-limiting and low-frequency core and polymerase-specific T cells in chronic infection. Expression of the T cell inhibitory receptor PD-1, as well as T cell differentiation, T cell function and T cell regulation differed by stages and outcomes of infection. In addition, these features differed significantly between T cells targeting different HBV specificities. CONCLUSION: HBV-specific T cells with different target specificities are characterised by distinct phenotypical and functional profiles. These results have direct implications for the design of immunological studies in HBV infection, and are potentially relevant for informing immunotherapeutic approaches to induce functional cure.

Hepatitis D infection in Brazil: Prevalence and geographical distribution of anti-Delta antibody.

In Brazil, the Amazon Basin is endemic for hepatitis D virus (HDV) infection; however, studies in other regions of the country are scarce. This study aims to map the seroepidemiological situation of anti-Delta antibodies in chronic hepatitis B carriers in all five Brazilian geographic regions. Serum samples from 1240 HBsAg positive individuals (55.4% men; mean age 43.1 ± 13.4 years) from 24 of 26 Brazilian states were tested for the presence of anti-Delta antibodies using a commercial immunoassay. Anti-Delta antibodies were detected in 40 samples (3.2%; 52.5% female; mean age of 38.1 ± 13.8 years). Age less than 20 years was significantly associated with anti-HDV positivity (P < 0.001). The distribution of anti-Delta differed markedly in the diverse regions of the country. The highest prevalence of anti-HDV was found in the North (8.5%; P < 0.001), followed by Central West (2.5%), Southeast (1.7%), Northeast (0.8%), and South (0.0%). Anti-Delta antibodies were detected in 12 states, but more than 60% of the positive cases were observed in two states, Amazonas and Acre, located in the western portion of the Amazon region. The overall HDV prevalence of 3.2% emphasizes that HDV is far from being a disease under control in Brazil. Despite the low HDV prevalence in non-endemic regions, this infection persists as a major concern in two states (Acre and Amazonas) in the north of the country, indicating that a continuous epidemiological surveillance program should be implemented in all Brazilian regions.

Early Transcriptional Divergence Marks Virus-Specific Primary Human CD8+ T Cells in Chronic versus Acute Infection.

Distinct molecular pathways govern the differentiation of CD8+ effector T cells into memory or exhausted T cells during acute and chronic viral infection, but these are not well studied in humans. Here, we employed an integrative systems immunology approach to identify transcriptional commonalities and differences between virus-specific CD8+ T cells from patients with persistent and spontaneously resolving hepatitis C virus (HCV) infection during the acute phase. We observed dysregulation of metabolic processes during early persistent infection that was linked to changes in expression of genes related to nucleosomal regulation of transcription, T cell differentiation, and the inflammatory response and correlated with subject age, sex, and the presence of HCV-specific CD4+ T cell populations. These early changes in HCV-specific CD8+ T cell transcription preceded the overt establishment of T cell exhaustion, making this signature a prime target in the search for the regulatory origins of T cell dysfunction in chronic viral infection.

Hypovitaminosis D and its relation to demographic and laboratory data among hepatitis C patients.

BACKGROUND: The relationship between 25-hydroxyvitamin D [25(OH)D] serum levels and response to antiviral therapy and laboratory data in HCV infection remains unclear. The aim of this study was to determine pre-treatment 25(OH)D serum level among HCV infected individuals and to evaluate the association between vitamin D status, virological response, and laboratory data. MATERIAL AND METHODS: Baseline serum 25(OH)D levels were measured in 237 chronic HCV infected patients (139 female, age 53.7 ± 11.2 years) using chemiluminescence immunoassay. Correlations between serum 25(OH)D levels, virological and laboratory data regarding HCV infection as well as sustained virological response (SVR) to antiviral therapy were evaluated. RESULTS: Mean serum values of 25(OH)D was 26.2 ± 12 ng/mL and prevalence of vitamin D deficiency (< 30 ng/mL) was 66.2%. Advanced age (> 55 years), high mean values of LDL, total cholesterol, HDL and low mean values of alkaline phosphatase and hemoglobin were statistically associated to vitamin D deficiency. Antiviral treatment was underwent by 133 HCV patients and 44.3% of them achieved SVR. Most of individuals that presented SVR also presented 25(OH)D level higher than 30ng/mL (55.9%). SVR was associated to low mean values of LDL, total cholesterol and platelets; high mean values of ALT, AST and low fibrosis grade. CONCLUSIONS: In conclusion, low vitamin D levels were observed among HCV infected patients and was associated to laboratory findings, however baseline 25(OH)D level is not independently associated with SVR.

Detection of mixed populations of wild-type and YMDD hepatitis B variants by pyrosequencing in acutely and chronically infected patients.

BACKGROUND: Lamivudine (LAM) is associated with the highest known rate of resistance mutations among nucleotide analogs used to treat chronic hepatitis B virus (HBV) infection. Despite this, LAM continues in widespread use, especially in combination therapies. The primary LAM resistance mutation (rtM204V/I) occurs in the YMDD motif of HBV polymerase. The aim of this study was to characterize Brazilian HBV isolates from acute and chronic cases by direct sequencing, and to identify HBV quasispecies in the YMDD motif using a pyrosequencing method capable of detecting single-nucleotide polymorphisms. HBV DNA from serum samples of 20 individuals with acute HBV infection and 44 with chronic infection undergoing antiviral therapies containing LAM were analyzed by direct sequencing and pyrosequencing methods. RESULTS: Phylogenic analyses of direct-sequenced isolates showed the expected genotypes (A, D and F) for the Brazilian population in both acute and chronic infections. However, within genotype A isolates, subgenotype A2 was more frequently detected in acute cases than in chronic cases (P = 0.012). As expected, none of the individuals with acute hepatitis B had LAM-resistant isolates as a dominant virus population, whether detected by direct sequencing or pyrosequencing. However, pyrosequencing analyses showed that 45% of isolates (9/20) had minor subpopulations (4-17%) of LAM-resistant isolates. Among chronic patients undergoing LAM treatment, YMDD mutants were frequently found as a dominant virus population. In cases where wild-type virus was the dominant population, subpopulations of YMDD variants were usually found, demonstrating the complexity of HBV quasispecies. CONCLUSIONS: YMDD variants were frequently detected as a minor population in acute HBV infection. The occurrence of pre-existing variants may lead to a high frequency of resistant mutants during antiviral therapy in the chronic phase. In chronic infection, detection of YMDD variants before virological or biochemical breakthrough might contribute to making better therapy choices and thus improving treatment outcome.

Antibody dynamics and spontaneous viral clearance in patients with acute hepatitis C infection in Rio de Janeiro, Brazil.

BACKGROUND: The anti-HCV antibody response has not been well characterized during the early phase of HCV infection and little is known about its relationship to the clinical course during this period. METHODS: We analyzed serial anti-HCV antibodies longitudinally obtained from a prospective cohort of 65 patients with acute HCV infection by using a microparticle enzyme immunoassay AxSYM HCV 3.0 (Abbott Diagnostics) during the first 12 months from HCV acquisition in Rio de Janeiro, Brazil. Spontaneous viral clearance (SVC) was defined as undetectable HCV RNA in serum, in the absence of treatment, for three consecutive HCV PCR tests within 12-months of follow-up. RESULTS: Baseline antibody values were similar among patient groups with self-limiting HCV evolution (n = 34) and persistent viremia (n = 31) [median (interquartile range) signal/cut-off ratio (s/co) 78.7 (60.7-93.8) vs. 93.9 (67.8-111.9), p = 0.26]. During 12-months follow-up, patients with acute spontaneous resolving HCV infection showed significantly lower serial antibody response in comparison to individuals progressing to chronic infection [median (interquartile range) s/co 62.7 (35.2-85.0) vs. 98.4 (70.4-127.4), p < 0.0001]. In addition, patients with self-limiting HCV evolution exhibited an expeditious, sharp decline of serial antibody values after SVC in comparison to those measured before SVC [median (interquartile range) s/co 56.0 (25.4-79.3) vs. 79.4 (66.3-103.0), p < 0.0001]. CONCLUSION: Our findings indicate a rapid short-term decline of antibody values in patients with acute spontaneous resolving HCV infection.

Prospective follow-up of patients with acute hepatitis C virus infection in Brazil.

BACKGROUND: The natural outcome of infection with hepatitis C virus (HCV) varies substantially among individuals. However, little is known about host and viral factors associated with a self-limiting or chronic evolution of HCV infection. METHODS: From 1 January 2001 through 31 December 2008, a consecutive series of 65 patients from Rio de Janeiro, Brazil, with a well-documented diagnosis of acute HCV infection, acquired via various routes, were enrolled in this study. Patients were prospectively followed up for a median of 40 months after the estimated date of HCV infection with serial measurements of serum alanine aminotransferase, HCV RNA, and anti-HCV antibodies. Spontaneous viral clearance (SVC) was defined as undetectable levels of HCV RNA in serum, in the absence of treatment, for 3 consecutive HCV polymerase chain reaction tests within the first 6 months of follow-up. Cox proportional hazards regression was used to identify host and viral predictors of SVC. RESULTS: The cumulative rate of SVC was 44.6% (95% confidence interval, 32.3%-57.5%). Compared with chronic HCV evolution, patients with self-limiting disease had significantly lower peak levels of anti-HCV antibodies (median, 109.0 vs 86.7 optical density-to-cutoff ratio [od/co]; P<.02), experienced disease symptoms more frequently (69.4% vs 100%; P<.001), and had lower viral load at first clinical presentation (median, 4.3 vs 0.0 log copies; P=.01). In multivariate analyses, low peak anti-HCV level (<93.5 od/co) was the only independent predictor for SVC; the hazard ratio compared with high anti-HCV levels (> or =93.5 od/co) was 2.62 (95% confidence interval, 1.11-6.19; P=.03). CONCLUSION: Our data suggest that low levels of anti-HCV antibodies during the acute phase of HCV infection are independently related to spontaneous viral clearance.

Viral sequence evolution in acute hepatitis C virus infection.

CD8(+)-T-cell responses play an important role in the containment and clearance of hepatitis C virus (HCV) infection, and an association between viral persistence and development of viral escape mutations has been postulated. While escape from CD8+ -T-cell responses has been identified as a major driving force for the evolution of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), a broader characterization of this relationship is needed in HCV infection. To determine the extent, kinetics, and driving forces of HCV sequence evolution, we sequenced the entire HCV genome longitudinally in four subjects monitored for up to 30 months after acute infection. For two subjects the transmission sources were also available. Of 53 total non-envelope amino acid substitutions detected, a majority represented forward mutations away from the consensus sequence. In contrast to studies in HIV and SIV, however, only 11% of these were associated with detectable CD8+ T-cell responses. Interestingly, 19% of non-envelope mutations represented changes toward the consensus sequence, suggesting reversion in the absence of immune pressure upon transmission. Notably, the rate of evolution of forward and reverse mutations correlated with the conservation of each residue, which is indicative of structural constraints influencing the kinetics of viral evolution. Finally, the rate of sequence evolution was observed to decline over the course of infection, possibly reflective of diminishing selection pressure by dysfunctional CD8+ T cells. Taken together, these data provide insight into the extent to which HCV is capable of evading early CD8+ T-cell responses and support the hypothesis that dysfunction of CD8+ T cells may be associated with failure to resolve HCV infections.

Immunologic evidence for lack of heterologous protection following resolution of HCV in patients with non-genotype 1 infection.

Chronic hepatitis C virus (HCV) infection is typically characterized by a lack of virus-specific CD4(+) T-cell-proliferative responses, but strong responses have been described in a subset of persons with persistent viremia. One possible explanation for these responses is that they were primed by an earlier resolved infection and do not recognize the current circulating virus. We defined all targeted epitopes using overlapping peptides corresponding to a genotype 1a strain in 44 patients chronically infected with different HCV genotypes (GT). Surprisingly, more HCV-specific CD4(+) T-cell responses were detected in patients with chronic non-GT1 infection compared with patients with chronic GT1 infection (P = .017). Notably, we found serologic evidence of a previous exposure to GT1 in 4 patients with non-GT1 infection, and these persons also demonstrated significantly more responses than non-GT1 patients in whom genotype and HCV serotype were identical (P < .001). Comparison of recognition of GT1-specific peptides to peptides representing autologous virus revealed the absence of cross-recognition of the autologous circulating virus. These data indicate that persistent HCV infection can occur in the presence of an HCV-specific T-cell response primed against a heterologous HCV strain, and suggest that clearance of 1 GT does not necessarily protect against subsequent exposure to a second GT.