Phosphatidylinositol 4-Kinase III Alpha Governs Cytoskeletal Organization for Invasiveness of Liver Cancer Cells.

BACKGROUND & AIMS: High expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma. In addition, hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to hepatocellular carcinoma progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process. METHODS: Several hepatic cell culture and mouse models were used to study the functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing, and PI4KIIIα-specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein. RESULTS: High PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased phosphorylation of paxillin and cofilin. This led to morphologic alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver-specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane phosphatidylinositol 3,4-bisphosphate-enriched, invadopodia-like structures that regulate cytoskeletal reorganization by promoting Akt2 phosphorylation. CONCLUSIONS: PI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to the progression of liver cancer and identify promising targets for therapeutic intervention.

Distinct roles of vaccine-induced SARS-CoV-2-specific neutralizing antibodies and T cells in protection and disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific neutralizing antibodies (NAbs) lack cross-reactivity between SARS-CoV species and variants and fail to mediate long-term protection against infection. The maintained protection against severe disease and death by vaccination suggests a role for cross-reactive T cells. We generated vaccines containing sequences from the spike or receptor binding domain, the membrane and/or nucleoprotein that induced only T cells, or T cells and NAbs, to understand their individual roles. In three models with homologous or heterologous challenge, high levels of vaccine-induced SARS-CoV-2 NAbs protected against neither infection nor mild histological disease but conferred rapid viral control limiting the histological damage. With no or low levels of NAbs, vaccine-primed T cells, in mice mainly CD8+ T cells, partially controlled viral replication and promoted NAb recall responses. T cells failed to protect against histological damage, presumably because of viral spread and subsequent T cell-mediated killing. Neither vaccine- nor infection-induced NAbs seem to provide long-lasting protective immunity against SARS-CoV-2. Thus, a more realistic approach for universal SARS-CoV-2 vaccines should be to aim for broadly cross-reactive NAbs in combination with long-lasting highly cross-reactive T cells. Long-lived cross-reactive T cells are likely key to prevent severe disease and fatalities during current and future pandemics.

Memory profiles distinguish cross-reactive and virus-specific T cell immunity to mpox.

Mpox represents a persistent health concern with varying disease severity. Reinfections with mpox virus (MPXV) are rare, possibly indicating effective memory responses to MPXV or related poxviruses, notably vaccinia virus (VACV) from smallpox vaccination. We assessed cross-reactive and virus-specific CD4+ and CD8+ T cells in healthy individuals and mpox convalescent donors. Cross-reactive T cells were most frequently observed in healthy donors over 45 years. Notably, long-lived memory CD8+ T cells targeting conserved VACV/MPXV epitopes were identified in older individuals more than four decades after VACV exposure and exhibited stem-like characteristics, defined by T cell factor-1 (TCF-1) expression. In mpox convalescent donors, MPXV-reactive CD4+ and CD8+ T cells were more prevalent than in controls, demonstrating enhanced functionality and skewing toward effector phenotypes, which correlated with milder disease. Collectively, we report robust effector memory MPXV-specific T cell responses in mild mpox and long-lived TCF-1+ VACV/MPXV-specific CD8+ T cells decades after smallpox vaccination.

Novel prime-boost immune-based therapy inhibiting both hepatitis B and D virus infections.

OBJECTIVE: Chronic HBV/HDV infections are a major cause of liver cancer. Current treatments can only rarely eliminate HBV and HDV. Our previously developed preS1-HDAg immunotherapy could induce neutralising antibodies to HBV in vivo and raise HBV/HDV-specific T-cells. Here, we further investigate if a heterologous prime-boost strategy can circumvent T-cell tolerance and preclude HDV superinfection in vivo. DESIGN: A DNA prime-protein boost strategy was evaluated for immunogenicity in mice and rabbits. Its ability to circumvent T-cell tolerance was assessed in immunocompetent hepatitis B surface antigen (HBsAg)-transgenic mice. Neutralisation of HBV and HDV was evaluated both in vitro and in immunodeficient human-liver chimeric mice upon adoptive transfer. RESULTS: The prime-boost strategy elicits robust HBV/HDV-specific T-cells and preS1-antibodies that can effectively prevent HBV and HDV (co-)infection in vitro and in vivo. In a mouse model representing the chronic HBsAg carrier state, active immunisation primes high levels of preS1-antibodies and HDAg-specific T-cells. Moreover, transfer of vaccine-induced antibodies completely protects HBV-infected human-liver chimeric mice from HDV superinfection. CONCLUSION: The herein described preS1-HDAg immunotherapy is shown to be immunogenic and vaccine-induced antibodies are highly effective at preventing HBV and HDV (super)infection both in vitro and in vivo. Our vaccine can complement current and future therapies for the control of chronic HBV and HDV infection.

Accelerated DNA vaccine regimen provides protection against Crimean-Congo hemorrhagic fever virus challenge in a macaque model.

Crimean-Congo hemorrhagic fever virus (CCHFV) is widely distributed throughout Africa, the Middle East, Southern Asia, and Southern and Eastern Europe. Spread by Hyalomma ticks or by contact with infected animals, CCHF begins non-specifically but can rapidly progress to severe, sometimes fatal, disease. Due to the non-specific early symptoms and often unrecognized infections, patients often present to healthcare systems exhibiting later stages of disease, when treatment is limited to supportive care. Consequently, simple vaccines are critically needed to protect populations at risk of CCHFV infection. Currently, there are no widely approved vaccines for CCHFV. We have previously reported significant efficacy of a three-dose DNA-based vaccination regimen for CCHFV in cynomolgus macaques (Macaca fasicularis). Here, we show that in cynomolgus macaques, plasmid-expressed CCHFV nucleoprotein (NP) and glycoprotein precursor (GPC) antigens elicit primarily humoral and cellular immunity, respectively. We found that a two-dose vaccination regimen with plasmids expressing the NP and GPC provides significant protection against CCHFV infection. Studies investigating vaccinations with either antigen alone showed that plasmid-expressed NPs could also confer protection. Cumulatively, our data show that this vaccine confers robust protection against CCHFV and suggest that both humoral and cellular immunity contribute to optimal vaccine-mediated protection.

A universal SARS-CoV DNA vaccine inducing highly cross-reactive neutralizing antibodies and T cells.

New variants in the SARS-CoV-2 pandemic are more contagious (Alpha/Delta), evade neutralizing antibodies (Beta), or both (Omicron). This poses a challenge in vaccine development according to WHO. We designed a more universal SARS-CoV-2 DNA vaccine containing receptor-binding domain loops from the huCoV-19/WH01, the Alpha, and the Beta variants, combined with the membrane and nucleoproteins. The vaccine induced spike antibodies crossreactive between huCoV-19/WH01, Beta, and Delta spike proteins that neutralized huCoV-19/WH01, Beta, Delta, and Omicron virus in vitro. The vaccine primed nucleoprotein-specific T cells, unlike spike-specific T cells, recognized Bat-CoV sequences. The vaccine protected mice carrying the human ACE2 receptor against lethal infection with the SARS-CoV-2 Beta variant. Interestingly, priming of cross-reactive nucleoprotein-specific T cells alone was 60% protective, verifying observations from humans that T cells protect against lethal disease. This SARS-CoV vaccine induces a uniquely broad and functional immunity that adds to currently used vaccines.

Process Development for Adoptive Cell Therapy in Academia: A Pipeline for Clinical-Scale Manufacturing of Multiple TCR-T Cell Products.

Cellular immunotherapies based on T cell receptor (TCR) transfer are promising approaches for the treatment of cancer and chronic viral infections. The discovery of novel receptors is expanding considerably; however, the clinical development of TCR-T cell therapies still lags. Here we provide a pipeline for process development and clinical-scale manufacturing of TCR-T cells in academia. We utilized two TCRs specific for hepatitis C virus (HCV) as models because of their marked differences in avidity and functional profile in TCR-redirected cells. With our clinical-scale pipeline, we reproduced the functional profile associated with each TCR. Moreover, the two TCR-T cell products demonstrated similar yield, purity, transduction efficiency as well as phenotype. The TCR-T cell products had a highly reproducible yield of over 1.4 × 109 cells, with an average viability of 93%; 97.8-99% of cells were CD3+, of which 47.66 ± 2.02% were CD8+ T cells; the phenotype was markedly associated with central memory (CD62L+CD45RO+) for CD4+ (93.70 ± 5.23%) and CD8+ (94.26 ± 4.04%). The functional assessments in 2D and 3D cell culture assays showed that TCR-T cells mounted a polyfunctional response to the cognate HCV peptide target in tumor cell lines, including killing. Collectively, we report a solid strategy for the efficient large-scale manufacturing of TCR-T cells.

Nucleoside-Modified mRNA Vaccines Protect IFNAR-/- Mice against Crimean-Congo Hemorrhagic Fever Virus Infection.

Crimean-Congo hemorrhagic fever (CCHF), caused by Crimean-Congo hemorrhagic fever virus (CCHFV), is on the World Health Organizations' list of prioritized diseases and pathogens. With global distribution, high fatality rate, and no approved vaccine or effective treatment, CCHF constitutes a threat against global health. In the current study, we demonstrate that vaccination with nucleoside-modified mRNA-lipid nanoparticles (mRNA-LNP), encoding for the CCHFV nucleoprotein (N) or glycoproteins (GcGn) protect IFNAR-/- mice against lethal CCHFV infection. In addition, we found that both mRNA-LNP induced strong humoral and cellular immune responses in IFNAR-/- and immunocompetent mice and that neutralizing antibodies are not necessary for protection. When evaluating immune responses induced by immunization including CCHFV Gc and Gn antigens, we found the Gc protein to be more immunogenic compared with the Gn protein. Hepatic injury is prevalent in CCHF and contributes to the severity and mortality of the disease in humans. Thus, to understand the immune response in the liver after infection and the potential effect of the vaccine, we performed a proteomic analysis on liver samples from vaccinated and control mice after CCHFV infection. Similar to observations in humans, vaccination affected the metabolic pathways. In conclusion, this study shows that a CCHFV mRNA-LNP vaccine, based on viral nucleo- or glycoproteins, mediate protection against CCHFV induced disease. Consequently, genetic immunization is an attractive approach to prevent disease caused by CCHFV and we believe we have necessary evidence to bring this vaccine platform to the next step in the development of a vaccine against CCHFV infection. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a zoonotic pathogen causing Crimean-Congo hemorrhagic fever (CCHF), a severe fever disease. CCHFV has a wide distribution and is endemic in several areas around the world. Cases of CCHF are also being reported in new areas, indicating an expansion of the disease, which is of high concern. Dispersion of the disease, high fatality rate, and no approved vaccine makes CCHF a threat to global health. The development of a vaccine is thus of great importance. Here we show 100% protection against lethal CCHFV infection in mice immunized with mRNA-LNP encoding for different CCHFV proteins. The vaccination showed both robust humoral and cellular immunity. mRNA-LNP vaccines combine the ability to induce an effective immune response, the safety of a transient carrier, and the flexibility of genetic vaccines. This and our results from the current study support the development of a mRNA-LNP based vaccine against CCHFV.

Metabolic Perturbation Associated With COVID-19 Disease Severity and SARS-CoV-2 Replication.

Viruses hijack host metabolic pathways for their replicative advantage. In this study, using patient-derived multiomics data and in vitro infection assays, we aimed to understand the role of key metabolic pathways that can regulate severe acute respiratory syndrome coronavirus-2 reproduction and their association with disease severity. We used multiomics platforms (targeted and untargeted proteomics and untargeted metabolomics) on patient samples and cell-line models along with immune phenotyping of metabolite transporters in patient blood cells to understand viral-induced metabolic modulations. We also modulated key metabolic pathways that were identified using multiomics data to regulate the viral reproduction in vitro. Coronavirus disease 2019 disease severity was characterized by increased plasma glucose and mannose levels. Immune phenotyping identified altered expression patterns of carbohydrate transporter, glucose transporter 1, in CD8+ T cells, intermediate and nonclassical monocytes, and amino acid transporter, xCT, in classical, intermediate, and nonclassical monocytes. In in vitro lung epithelial cell (Calu-3) infection model, we found that glycolysis and glutaminolysis are essential for virus replication, and blocking these metabolic pathways caused significant reduction in virus production. Taken together, we therefore hypothesized that severe acute respiratory syndrome coronavirus-2 utilizes and rewires pathways governing central carbon metabolism leading to the efflux of toxic metabolites and associated with disease severity. Thus, the host metabolic perturbation could be an attractive strategy to limit the viral replication and disease severity.

SARS-CoV-2-specific humoral and cellular immunity persists through 9 months irrespective of COVID-19 severity at hospitalisation.

OBJECTIVES: Humoral and cellular immunity to SARS-CoV-2 following COVID-19 will likely contribute to protection from reinfection or severe disease. It is therefore important to characterise the initiation and persistence of adaptive immunity to SARS-CoV-2 amidst the ongoing pandemic. METHODS: Here, we conducted a longitudinal study on hospitalised moderate and severe COVID-19 patients from the acute phase of disease into convalescence at 5 and 9 months post-symptom onset. Utilising flow cytometry, serological assays as well as B cell and T cell FluoroSpot assays, we assessed the magnitude and specificity of humoral and cellular immune responses during and after human SARS-CoV-2 infection. RESULTS: During acute COVID-19, we observed an increase in germinal centre activity, a substantial expansion of antibody-secreting cells and the generation of SARS-CoV-2-neutralising antibodies. Despite gradually decreasing antibody levels, we show persistent, neutralising antibody titres as well as robust specific memory B cell responses and polyfunctional T cell responses at 5 and 9 months after symptom onset in both moderate and severe COVID-19 patients. CONCLUSION: Our findings describe the initiation and, importantly, persistence of cellular and humoral SARS-CoV-2-specific immunological memory in hospitalised COVID-19 patients long after recovery, likely contributing towards protection against reinfection.

Blocking Entry of Hepatitis B and D Viruses to Hepatocytes as a Novel Immunotherapy for Treating Chronic Infections.

BACKGROUND: Chronic hepatitis B and D virus (HBV/HDV) infections can cause cancer. Current HBV therapy using nucleoside analogues (NAs) is life-long and reduces but does not eliminate the risk of cancer. A hallmark of chronic hepatitis B is a dysfunctional HBV-specific T-cell response. We therefore designed an immunotherapy driven by naive healthy T cells specific for the HDV antigen (HDAg) to bypass the need for HBV-specific T cells in order to prime PreS1-specific T cells and PreS1 antibodies blocking HBV entry. METHODS: Ten combinations of PreS1 and/or HDAg sequences were evaluated for induction of PreS1 antibodies and HBV- and HDV-specific T cells in vitro and in vivo. Neutralization of HBV by PreS1-specific murine and rabbit antibodies was evaluated in cell culture, and rabbit anti-PreS1 were tested for neutralization of HBV in mice repopulated with human hepatocytes. RESULTS: The best vaccine candidate induced T cells to PreS1 and HDAg, and PreS1 antibodies blocking HBV entry in vitro. Importantly, adoptive transfer of PreS1 antibodies prevented, or modulated, HBV infection after a subsequent challenge in humanized mice. CONCLUSIONS: We here describe a novel immunotherapy for chronic HBV/HDV that targets viral entry to complement NAs and coming therapies inhibiting viral maturation.

A DNA-based vaccine protects against Crimean-Congo haemorrhagic fever virus disease in a Cynomolgus macaque model.

There is currently no specific prophylaxis or vaccine against Crimean-Congo haemorrhagic fever virus (CCHFV). Crimean-Congo haemorrhagic fever (CCHF) is a severe febrile illness transmitted by Hyalomma ticks in endemic areas, handling of infected livestock or care of infected patients. We report here the successful protection against CCHFV-mediated disease in a non-human primate disease model. Cynomolgus macaques were vaccinated with a DNA-based vaccine using in vivo electroporation-assisted delivery. The vaccine contained two plasmids encoding the glycoprotein precursor (GPC) and the nucleoprotein (NP) of CCHFV. Animals received three vaccinations and we recorded potent antibody and T cell responses after vaccination. While all sham-vaccinated animals developed viraemia, high tissue viral loads and CCHF-induced disease, the NP + GPC vaccinated animals were significantly protected. In conclusion, this is evidence of a vaccine that can protect against CCHFV-induced disease in a non-human primate model. This supports clinical development of the vaccine to protect groups at risk for contracting the infection.

Sick leave and disability pension in patients with chronic hepatitis C compared with a matched general population: a nationwide register study.

OBJECTIVE: The objective of this study was to evaluate sick leave and disability pension in patients with chronic hepatitis C virus (HCV) infection as compared with a matched general population cohort. DESIGN: Retrospective register study. SETTING: Nationwide in Sweden. PARTICIPANTS: This register-based study used the Swedish National Patient Register to identify working-age patients with HCV in 2012 (n=32 021) who were diagnosed between 1999 and 2007 (n=19 362). Sick leave and disability pension data were retrieved from Statistics Sweden (1994-2012), with up to five matched individuals from the general population. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome was workdays lost due to sick leave episodes (>14 days) and disability pension overall. The secondary outcome was workdays lost per subgroup of patients with chronic HCV. RESULTS: In 2012, 14% of the HCV patients had ≥1 registered sick leave episode compared with 10% in the matched comparator cohort. For disability pension benefits, results were 30% versus 8%, respectively. Overall, in 2012, 57% of patients with HCV did not have any registered workdays lost, whereas 30% were absent ≥360 days compared with 83% and 9% in the matched cohort, respectively. The mean total number of annual workdays lost in 2012 was 126 days in the HCV patient cohort compared with 40 days in the matched general population comparator cohort. Annual days lost increased from a mean of 86 days 5 years before diagnosis to 136 days during the year of diagnosis. CONCLUSIONS: These results show that Swedish HCV patients used more sick days and have a higher frequency of disability pension compared with a comparator cohort from the general Swedish population. Whether earlier diagnosis of HCV and treatment might impact work absence in Sweden warrants further investigation.

Expansion of SARS-CoV-2-Specific Antibody-Secreting Cells and Generation of Neutralizing Antibodies in Hospitalized COVID-19 Patients.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 and has since become a global pandemic. Pathogen-specific Abs are typically a major predictor of protective immunity, yet human B cell and Ab responses during COVID-19 are not fully understood. In this study, we analyzed Ab-secreting cell and Ab responses in 20 hospitalized COVID-19 patients. The patients exhibited typical symptoms of COVID-19 and presented with reduced lymphocyte numbers and increased T cell and B cell activation. Importantly, we detected an expansion of SARS-CoV-2 nucleocapsid protein-specific Ab-secreting cells in all 20 COVID-19 patients using a multicolor FluoroSpot Assay. Out of the 20 patients, 16 had developed SARS-CoV-2-neutralizing Abs by the time of inclusion in the study. SARS-CoV-2-specific IgA, IgG, and IgM Ab levels positively correlated with SARS-CoV-2-neutralizing Ab titers, suggesting that SARS-CoV-2-specific Ab levels may reflect the titers of neutralizing Abs in COVID-19 patients during the acute phase of infection. Last, we showed that IL-6 and C-reactive protein serum concentrations were higher in patients who were hospitalized for longer, supporting the recent observations that IL-6 and C-reactive protein could be used as markers for COVID-19 severity. Altogether, this study constitutes a detailed description of clinical and immunological parameters in 20 COVID-19 patients, with a focus on B cell and Ab responses, and describes tools to study immune responses to SARS-CoV-2 infection and vaccination.

Evidence for B cell maturation but not trained immunity in uninfected infants exposed to hepatitis C virus.

OBJECTIVES: Vertical transmission of hepatitis C virus (HCV) is rare compared with other chronic viral infections, despite that newborns have an immature, and possibly more susceptible, immune system. It further remains unclear to what extent prenatal and perinatal exposure to HCV affects immune system development in neonates. DESIGN: To address this, we studied B cells, innate immune cells and soluble factors in a cohort of 62 children that were either unexposed, exposed uninfected or infected with HCV. Forty of these infants were followed longitudinally from birth up until 18 months of age. RESULTS: As expected, evidence for B cell maturation was observed with increased age in children, whereas few age-related changes were noticed among innate immune cells. HCV-infected children had a high frequency of HCV-specific IgG-secreting B cells. Such a response was also detected in some exposed but uninfected children but not in uninfected controls. Consistent with this, both HCV-exposed uninfected and HCV-infected infants had evidence of early B cell immune maturation with an increased proportion of IgA-positive plasma cells and upregulated CD40 expression. In contrast, actual HCV viraemia, but not mere exposure, led to alterations within myeloid immune cell populations, natural killer (NK) cells and a distinct soluble factor profile with increased levels of inflammatory cytokines and chemokines. CONCLUSION: Our data reveal that exposure to, and infection with, HCV causes disparate effects on adaptive B cells and innate immune cell such as myeloid cells and NK cells in infants.

Liver, Tumor and Viral Hepatitis: Key Players in the Complex Balance Between Tolerance and Immune Activation.

Liver cancer is the third most common cause of cancer related death in the World. From an epidemiological point of view the risk factors associated to primary liver cancer are mainly viral hepatitis infection and alcohol consumption. Even though there is a clear correlation between liver inflammation, cirrhosis and cancer, other emerging liver diseases (like fatty liver) could also lead to liver cancer. Moreover, the liver is the major site of metastasis from colon, breast, ovarian and other cancers. In this review we will address the peculiar status of the liver as organ that has to balance between tolerance and immune activation. We will focus on macrophages and other key cellular components of the liver microenvironment that play a central role during tumor progression. We will also discuss how current and future therapies may affect the balance toward immune activation.

Lower risk of multiple sclerosis in patients with chronic hepatitis C: a nationwide population-based registry study.

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated neurological disease that causes demyelination. The etiology is unknown, but patients with a previous viral infection, such as Epstein-Barr virus, have been shown to be at a higher risk of developing MS. In contrast, people living with HIV have a lower risk of developing MS. Hepatitis C virus (HCV) mainly infects the liver, but patients with HCV can experience several extrahepatic manifestations and studies have shown an association with several autoimmune conditions such as neuropathy and myelitis. The present study aimed to investigate the risk of MS in patients with chronic HCV infection compared with matched comparators. METHODS: Patients were identified using the nationwide Swedish inpatient (2001-2013) and outpatient care registers (2001-2013) for HCV (B18.2) and MS (G35) according to the International Classification of Diseases-10. Up to five comparators (matched on age/sex/place of residency) were drawn from the general population for each HCV patient. Follow-up started at the first HCV visit from 2001 and the patients' accrued person-time until death, emigration or 31 December 2013. Risk of MS diagnosis was calculated as standardized incidence ratio (SIR) with 95% confidence intervals (CIs). RESULTS: HCV patients were at lower risk of MS diagnosis (SIR 0.37; 95% CI 0.26-0.50). The incidence of MS during the study in the HCV cohort was 0.087% compared with 0.27% in the matched comparator cohort. CONCLUSION: Surprisingly, these data suggest HCV patients to have a lower risk of MS diagnosis.

Long-term follow-up after cure from chronic hepatitis C virus infection shows occult hepatitis and a risk of hepatocellular carcinoma in noncirrhotic patients.

OBJECTIVES: Curing of hepatitis C virus (HCV) infection primarily aims to prevent severe liver complications. Our objectives were to investigate the long-term presence and impact of occult HCV infection (OCI) and to study the outcomes in terms of liver disease after virological cure. PATIENTS AND METHODS: A total of 97 patients with achieved sustained virological response (SVR) during 1990-2005 were followed either by a clinical follow-up (FU) visit with blood sampling and liver elastography (n=54) or through national registries for outcomes (n=43). To diagnose OCI among patients with SVR, a highly sensitive method was used to detect HCV-RNA traces in whole blood. The FU duration was a median of 10.5 years, with samples up to 21.5 years after the end of treatment (EOT). RESULTS: The majority of patients [52 (96%)] were HCV-RNA negative at FU, and regression of fibrosis was statistically significant. OCI was found in two (4%) of them at 8 and 9 years after EOT. These patients had F1 and F2 fibrosis before treatment and F2 at FU, but no other abnormal findings. Three previously noncirrhotic men were diagnosed with hepatocellular carcinoma 8-11 years after EOT. CONCLUSION: Occult infection could be detected many years after the achievement of SVR but was not associated with serious liver disease. The majority had persistent viral eradication and regression of fibrosis after SVR. However, an increased risk of hepatocellular carcinoma may persist in the long term after SVR even in noncirrhotic patients. Further studies with FU after direct-acting antiviral therapy and on the long-term impact after cure are needed.