HERACLIS-HDV cohort for the factors of underdiagnosis and prevalence of hepatitis D virus infection in HBsAg-positive patients.

BACKGROUND AND AIMS: Hepatitis D virus (HDV) underdiagnosis remains common. We assessed the HDV screening and prevalence rates in HBsAg-positive patients seen at tertiary liver centres throughout Greece as well as factors affecting HDV diagnosis. METHODS: All adult HBsAg-positive patients seen within the last 5 years were included. Non-screened patients who visited or could be recalled to the clinics over a 6-month period were prospectively tested for anti-HDV. RESULTS: Of 5079 HBsAg-positive patients, 53% had anti-HDV screening (41% before and 12% after study initiation). Pre-study (8%-88%) and total screening rates (14%-100%) varied widely among centres. Screening rates were associated with older age, known risk group, elevated ALT, centre location and size and period of first visit. Anti-HDV prevalence was 5.8% without significant difference in patients screened before (6.1%) or after study initiation (4.7%, p = 0.240). Anti-HDV positivity was associated with younger age, parenteral drug use, born abroad, advanced liver disease and centre location. Overall, HDV RNA detectability rate was 71.6% being more frequent in anti-HDV-positive patients with elevated ALT, advanced liver disease and hepatitis B therapy. CONCLUSIONS: Anti-HDV screening rates and recall capabilities vary widely among Greek liver clinics being higher in HBsAg-positive patients of known risk group with active/advanced liver disease seen at smaller centres, while non-medical factors are also important. Anti-HDV prevalence varies throughout Greece being higher in patients born abroad with younger age, parenteral drug use and advanced liver disease. Viremia is more frequently but not exclusively detected in anti-HDV-positive patients with elevated ALT and advanced liver disease.

Frequency, Private Specificity, and Cross-Reactivity of Preexisting Hepatitis C Virus (HCV)-Specific CD8+ T Cells in HCV-Seronegative Individuals: Implications for Vaccine Responses.

UNLABELLED: T cell responses play a critical role in controlling or clearing viruses. Therefore, strategies to prevent or treat infections include boosting T cell responses. T cells specific for various pathogens have been reported in unexposed individuals and an influence of such cells on the response toward vaccines is conceivable. However, little is known about their frequency, repertoire, and impact on vaccination. We performed a detailed characterization of CD8(+) T cells specific to a hepatitis C virus (HCV) epitope (NS3-1073) in 121 HCV-seronegative individuals. We show that in vitro HCV NS3-1073-specific CD8(+) T cell responses were rather abundantly detectable in one-third of HCV-seronegative individuals irrespective of risk factors for HCV exposure. Ex vivo, these NS3-1073-specific CD8(+) T cells were found to be both naive and memory cells. Importantly, recognition of various peptides derived from unrelated viruses by NS3-1073-specific CD8(+) T cells showed a considerable degree of T cell cross-reactivity, suggesting that they might in part originate from previous heterologous infections. Finally, we further provide evidence that preexisting NS3-1073-specific CD8(+) T cells can impact the T cell response toward peptide vaccination. Healthy, vaccinated individuals who showed an in vitro response toward NS3-1073 already before vaccination displayed a more vigorous and earlier response toward the vaccine. IMPORTANCE: Preventive and therapeutic vaccines are being developed for many viral infections and often aim on inducing T cell responses. Despite effective antiviral drugs against HCV, there is still a need for a preventive vaccine. However, the responses to vaccines can be highly variable among different individuals. Preexisting T cells in unexposed individuals could be one reason that helps to explain the variable T cell responses to vaccines. Based on our findings, we suggest that HCV CD8(+) T cells are abundant in HCV-seronegative individuals but that their repertoire is highly diverse due to the involvement of both naive precursors and cross-reactive memory cells of different specificities, which can influence the response to vaccines. The data may emphasize the need to personalize immune-based therapies based on the individual's T cell repertoire that is present before the immune intervention.

Comprehensive phenotyping of regulatory T cells after liver transplantation.

Regulatory T cells (Tregs) play an important role in controlling alloreactivity after solid organ transplantation, but they may also impair antiviral immunity. We hypothesized that the Treg frequency and the Treg phenotype are altered in hepatitis C virus (HCV)-infected recipients of liver transplantation (LT) with possible prognostic implications. Tregs from 141 individuals, including healthy individuals, LT recipients with or without persistent HCV infections, and nontransplant patients with chronic HCV, were studied. A comprehensive phenotypic analysis was performed with multicolor flow cytometry, which included standard Treg markers [CD4(+), CD25(hi), CD127(-), and FoxP3(+) in addition to HLA DR, CCR7, CD45RA, CD62L, CD49d, CD39, ICOS and LAP-TGFß stainings. Healthy individuals and LT patients displayed similar Treg frequencies and largely comparable Treg phenotypes, which were stable over time after transplantation. In contrast, Tregs with a CD45RA(-) CCR7(-) effector phenotype were enriched in LT recipients with chronic HCV versus HCV-negative transplant patients. HCV infection, rather than LT, altered the expression of functional markers on Tregs. A principal component analysis revealed distinct Treg phenotypes in HCV-infected LT recipients with rejection and patients with recurrent graft HCV. In conclusion, Treg phenotypes are altered in HCV-infected LT patients. An investigation of Tregs may possibly help to distinguish recurrent HCV from graft rejection. Further functional studies are needed to define the role of Tregs in determining the balance between antiviral and allogenic immunity.

Hepatitis E virus (HEV)-specific T-cell responses are associated with control of HEV infection.

Hepatitis E virus (HEV) infection is usually self-limited but may lead to acute hepatitis and rarely to fulminant hepatic failure. Persistent HEV infections have recently been described in organ transplant recipients receiving immunosuppressive medications, suggesting that HEV is controlled by adaptive immune responses. However, only few studies have investigated HEV-specific T-cell responses and immune correlates for the failure to clear HEV infection have not been established so far. We investigated T-cell responses against HEV in 38 subjects including anti-HEV-positive (exposed, n = 9) and anti-HEV-negative (n = 10) healthy controls, 12 anti-HEV-positive but HEV RNA-negative organ transplant recipients, and seven transplant recipients with chronic hepatitis E. Proliferation as well as cytokine production of CD4+ and CD8+ T cells was studied after stimulation with overlapping peptides spanning all proteins encoded by HEV-open reading frame (ORF)2 and HEV-ORF3. We show that (1) strong and multispecific HEV-specific T-cell responses are present in exposed healthy controls, and to a lesser extent also in recovered patients after transplantation; (2) that these responses are absent in patients with chronic hepatitis E but become detectable after viral clearance; and (3) that HEV-specific T-cell responses can be restored in vitro by blocking the PD-1 or CTLA-4 pathways. However, a combination of PD-1 and CTLA-4 blockade had no synergistic effects. We conclude that chronic hepatitis E is associated with impaired HEV-specific T-cell responses and suggest that enhancing adaptive cellular immunity against HEV might prevent persistent HEV infections.

Interferon-alpha-induced TRAIL on natural killer cells is associated with control of hepatitis C virus infection.

BACKGROUND & AIMS: Pegylated interferon-alpha (PEG-IFNalpha), in combination with ribavirin, controls hepatitis C virus (HCV) infection in approximately 50% of patients by mechanisms that are not completely understood. Beside a direct antiviral effect, different immunomodulatory effects have been discussed. Natural killer (NK) cells might be associated with control of HCV infection. We examined the effects of IFNalpha on human NK cells and its relevance to HCV infection. METHODS: We performed gene expression profiling studies of NK cells following stimulation of peripheral blood mononuclear cells with IFNalpha. We evaluated IFNalpha-induced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression using flow cytometry analyses of NK cells isolated from patients with acute or chronic hepatitis C that had received PEG-IFNalpha therapy. RESULTS: TRAIL was among the most up-regulated genes after IFNalpha stimulation of NK cells from healthy controls. After in vitro stimulation with IFNalpha, CD56(dim) NK cells from patients who had responded to PEG-IFNalpha therapy expressed higher levels of TRAIL than cells from patients with chronic hepatitis C. TRAIL expression, ex vivo, was inversely correlated with HCV-RNA levels during the early phase of PEG-IFNalpha therapy. In patients with acute hepatitis C, TRAIL expression on CD56(bright) NK cells increased significantly compared with cells from controls. In in vitro studies, IFNalpha-stimulated NK cells eliminated HCV-replicating hepatoma cells by a TRAIL-mediated mechanism. CONCLUSIONS: IFNalpha-induced expression of TRAIL on NK cells is associated with control of HCV infection; these observations might account for the second-phase decline in HCV-RNA levels during PEG-IFNalpha therapy.

Intrahepatic long-term persistence of parvovirus B19 and its role in chronic viral hepatitis.

Parvovirus B19 (B19V) has been detected in the liver of Asian patients infected with HBV and may contribute to acute and chronic liver disease. This study aimed to investigate the impact of B19V infection in European patients with viral hepatitis. B19V DNA was detected in 1/91 and 0/50 serum samples from patients with chronic hepatitis C and B, respectively. In contrast, B19V DNA was amplified frequently from explanted end-stage liver tissues (37/50, 74%) and from routine biopsy samples (14/32, 44%) (P < 0.05). However, there was no significant difference in B19V copy number per cell between these two groups. B19V-specific CD4(+) T-cell responses to two dominant MHC-class-restricted epitopes were detected in a similar frequency in healthy anti-B19V-positive individuals (3/19; 16%) and patients with chronic hepatitis C (3/13; 23%). These results indicate that B19V can persist in the liver. However, there is no evidence that B19V is a "hepatitis virus" worsening liver disease in European patients with chronic hepatitis C.

Effect of peptide pools on effector functions of antigen-specific CD8+ T cells.

Peptide pools are routinely used to study antigen specific T cell responses, both in epitope discovery as well as immune monitoring. However, optimal assay conditions such as concentration of peptides or the best possible number of peptides per pool have not been defined. Thus, we examined whether different peptide concentrations or varying number of peptides per pool influence effector functions of antigen-specific human T-cells. PBMC isolated from HLA-A2-positive individuals with known responses to frequently recognised dominant CD8+ T cell epitopes derived from four different viruses (influenza virus, CMV, EBV, or HCV) were studied. PBMC were cultured with one of these HLA-A2 restricted peptides and varying concentrations of overlapping peptide pools derived from unrelated viruses specific for the hepatitis D and E viruses, the subjects have not been exposed to. Importantly, unrelated peptide pools inhibited the proliferation of IV-M1(58), CMVpp65(495-503), EBV-BMLF(1259-267) and HCV NS3(1073)-specific CD8 T-cells in a dose dependent manner. Similarly, an increase in the number of peptides per pool also impaired antigen specific CD8+ T cell proliferation. In contrast, secretion of cytokines such as IL-2, IL-10, IFN-gamma, TNF-alpha or IP-10 as well as cytotoxicity was not affected by these unrelated peptide pools. The inhibition of proliferation could be restored by blocking PD-1/PDL-1 interaction and was not dependent on DMSO when DMSO concentration was

Persistence of occult hepatitis B after removal of the hepatitis B virus-infected liver.

Occult hepatitis B is defined as the persistence of hepatitis B virus (HBV) DNA in persons without HBV surface antigen (HBsAg). The primary site for HBV persistence in persons with occult hepatitis B is considered to be the liver. We provide virological and immunological evidence for long-term persistence of HBV, even after removal of the infected liver, in 25 consecutive, randomly selected liver transplant recipients who tested positive for anti-HBV core antigen (anti-HBcAg) and negative for HBsAg at the time of transplantation. Furthermore, in a cohort of 101 anti-HBcAg-positive/HBsAg-negative patients, 2 showed clinical HBV reactivation after transplantation. Thus, these data indicate that a long-term extrahepatic HBV reservoir exists, which is relevant not only for liver transplantation but also for other types of transplantations, including bone marrow grafting.