Hepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide.

RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer3. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.

Inactivated genotype 1a, 2a and 3a HCV vaccine candidates induced broadly neutralising antibodies in mice.

OBJECTIVE: A prophylactic vaccine is needed to control the HCV epidemic, with genotypes 1-3 causing >80% of worldwide infections. Vaccine development is hampered by HCV heterogeneity, viral escape including protection of conserved neutralising epitopes and suboptimal efficacy of HCV cell culture systems. We developed cell culture-based inactivated genotype 1-3 HCV vaccine candidates to present natively folded envelope proteins to elicit neutralising antibodies. DESIGN: High-yield genotype 1a, 2a and 3a HCV were developed by serial passage of TNcc, J6cc and DBN3acc in Huh7.5 cells and engineering of acquired mutations detected by next-generation sequencing. Neutralising epitope exposure was determined in cell-based neutralisation assays using human monoclonal antibodies AR3A and AR4A, and polyclonal antibody C211. BALB/c mice were immunised with processed and inactivated genotype 1a, 2a or 3a viruses using AddaVax, a homologue of the licenced adjuvant MF-59. Purified mouse and patient serum IgG were assayed for neutralisation capacity; mouse IgG and immune-sera were assayed for E1/E2 binding. RESULTS: Compared with the original viruses, high-yield viruses had up to ~1000 fold increased infectivity titres (peak titres: 6-7 log10 focus-forming units (FFU)/mL) and up to ~2470 fold increased exposure of conserved neutralising epitopes. Vaccine-induced IgG broadly neutralised genotype 1-6 HCV (EC50: 30-193 µg/mL; mean 71 µg/mL), compared favourably with IgG from chronically infected patients, and bound genotype 1-3 E1/E2; immune-sera endpoint titres reached up to 32 000. CONCLUSION: High-yield genotype 1-3 HCV could be developed as basis for inactivated vaccine candidates inducing broadly neutralising antibodies in mice supporting further preclinical development.

Identification of novel neutralizing determinants for protection against HCV.

BACKGROUND AND AIMS: HCV evasion of neutralizing antibodies (nAb) results in viral persistence and poses challenges to the development of an urgently needed vaccine. N-linked glycosylation of viral envelope proteins is a key mechanism for such evasion. To facilitate rational vaccine design, we aimed to identify determinants of protection of conserved neutralizing epitopes. APPROACH AND RESULTS: Using a reverse evolutionary approach, we passaged genotype 1a, 1b, 2a, 3a, and 4a HCV with envelope proteins (E1 and E2) derived from chronically infected patients without selective pressure by nAb in cell culture. Compared with the original viruses, HCV recombinants, engineered to harbor substitutions identified in polyclonal cell culture-passaged viruses, showed highly increased fitness and exposure of conserved neutralizing epitopes in antigenic regions 3 and 4, associated with protection from chronic infection. Further reverse genetic studies of acquired E1/E2 substitutions identified positions 418 and 532 in the N1 and N6 glycosylation motifs, localizing to adjacent E2 areas, as key regulators of changes of the E1/E2 conformational state, which governed viral sensitivity to nAb. These effects were independent of predicted glycan occupancy. CONCLUSIONS: We show how N-linked glycosylation motifs can trigger dramatic changes in HCV sensitivity to nAb, independent of glycan occupancy. These findings aid in the understanding of HCV nAb evasion and rational vaccine design, as they can be exploited to stabilize the structurally flexible envelope proteins in an open conformation, exposing important neutralizing epitopes. Finally, this work resulted in a panel of highly fit cell culture infectious HCV recombinants.

High-Titer Hepatitis C Virus Production in a Scalable Single-Use High Cell Density Bioreactor.

Hepatitis C virus (HCV) infections pose a major public health burden due to high chronicity rates and associated morbidity and mortality. A vaccine protecting against chronic infection is not available but would be important for global control of HCV infections. In this study, cell culture-based HCV production was established in a packed-bed bioreactor (CelCradle™) aiming to further the development of an inactivated whole virus vaccine and to facilitate virological and immunological studies requiring large quantities of virus particles. HCV was produced in human hepatoma-derived Huh7.5 cells maintained in serum-free medium on days of virus harvesting. Highest virus yields were obtained when the culture was maintained with two medium exchanges per day. However, increasing the total number of cells in the culture vessel negatively impacted infectivity titers. Peak infectivity titers of up to 7.2 log10 focus forming units (FFU)/mL, accumulated virus yields of up to 5.9 × 1010 FFU, and a cell specific virus yield of up to 41 FFU/cell were obtained from one CelCradle™. CelCradle™-derived and T flask-derived virus had similar characteristics regarding neutralization sensitivity and buoyant density. This packed-bed tide-motion system is available with larger vessels and may thus be a promising platform for large-scale HCV production.

Inactivated whole hepatitis C virus vaccine employing a licensed adjuvant elicits cross-genotype neutralizing antibodies in mice.

BACKGROUND & AIMS: A prophylactic vaccine is required to eliminate HCV as a global public health threat. We developed whole virus inactivated HCV vaccine candidates employing a licensed adjuvant. Further, we investigated the effects of HCV envelope protein modifications (to increase neutralization epitope exposure) on immunogenicity. METHODS: Whole virus vaccine antigen was produced in Huh7.5 hepatoma cells, processed using a multistep protocol and formulated with adjuvant (MF-59 analogue AddaVax or aluminium hydroxide). We investigated the capacity of IgG purified from the serum of immunized BALB/c mice to neutralize genotype 1-6 HCV (by virus neutralization assays) and to bind homologous envelope proteins (by ELISA). Viruses used for immunizations were (i) HCV5aHi with strain SA13 envelope proteins and modification of an O-linked glycosylation site in E2 (T385P), (ii) HCV5aHi(T385) with reversion of T385P to T385, featuring the original E2 sequence determined in vivo and (iii) HCV5aHi(ΔHVR1) with deletion of HVR1. For these viruses, epitope exposure was investigated using human monoclonal (AR3A and AR4A) and polyclonal (C211 and H06) antibodies in neutralization assays. RESULTS: Processed HCV5aHi formulated with AddaVax induced antibodies that efficiently bound homologous envelope proteins and broadly neutralized cultured genotype 1-6 HCV, with half maximal inhibitory concentrations of between 14 and 192 µg/ml (mean of 36 µg/ml against the homologous virus). Vaccination with aluminium hydroxide was less immunogenic. Compared to HCV5aHi(T385) with the original E2 sequence, HCV5aHi with a modified glycosylation site and HCV5aHi(ΔHVR1) without HVR1 showed increased neutralization epitope exposure but similar immunogenicity. CONCLUSION: Using an adjuvant suitable for human use, we developed inactivated whole HCV vaccine candidates that induced broadly neutralizing antibodies, which warrant investigation in further pre-clinical studies. LAY SUMMARY: A vaccine against hepatitis C virus (HCV) is needed to prevent the estimated 2 million new infections and 400,000 deaths caused by this virus each year. We developed inactivated whole HCV vaccine candidates using adjuvants licensed for human use, which, following immunization of mice, induced antibodies that efficiently neutralized all HCV genotypes with recognized epidemiological importance. HCV variants with modified envelope proteins exhibited similar immunogenicity as the virus with the original envelope proteins.

Efficacy of Ion-Channel Inhibitors Amantadine, Memantine and Rimantadine for the Treatment of SARS-CoV-2 In Vitro.

We report the in vitro efficacy of ion-channel inhibitors amantadine, memantine and rimantadine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In VeroE6 cells, rimantadine was most potent followed by memantine and amantadine (50% effective concentrations: 36, 80 and 116 µM, respectively). Rimantadine also showed the highest selectivity index, followed by amantadine and memantine (17.3, 12.2 and 7.6, respectively). Similar results were observed in human hepatoma Huh7.5 and lung carcinoma A549-hACE2 cells. Inhibitors interacted in a similar antagonistic manner with remdesivir and had a similar barrier to viral escape. Rimantadine acted mainly at the viral post-entry level and partially at the viral entry level. Based on these results, rimantadine showed the most promise for treatment of SARS-CoV-2.

In vitro efficacy of artemisinin-based treatments against SARS-CoV-2.

Effective and affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are needed. We report in vitro efficacy of Artemisia annua extracts as well as artemisinin, artesunate, and artemether against SARS-CoV-2. The latter two are approved active pharmaceutical ingredients of anti-malarial drugs. Concentration-response antiviral treatment assays, based on immunostaining of SARS-CoV-2 spike glycoprotein, revealed that treatment with all studied extracts and compounds inhibited SARS-CoV-2 infection of VeroE6 cells, human hepatoma Huh7.5 cells and human lung cancer A549-hACE2 cells, without obvious influence of the cell type on antiviral efficacy. In treatment assays, artesunate proved most potent (range of 50% effective concentrations (EC50) in different cell types: 7-12 µg/mL), followed by artemether (53-98 µg/mL), A. annua extracts (83-260 µg/mL) and artemisinin (151 to at least 208 µg/mL). The selectivity indices (SI), calculated based on treatment and cell viability assays, were mostly below 10 (range 2 to 54), suggesting a small therapeutic window. Time-of-addition experiments in A549-hACE2 cells revealed that artesunate targeted SARS-CoV-2 at the post-entry level. Peak plasma concentrations of artesunate exceeding EC50 values can be achieved. Clinical studies are required to further evaluate the utility of these compounds as COVID-19 treatment.

Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 In Vitro.

Antivirals targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could improve treatment of COVID-19. We evaluated the efficacy of clinically relevant hepatitis C virus (HCV) NS3 protease inhibitors (PIs) against SARS-CoV-2 and their interactions with remdesivir, the only direct-acting antiviral approved for COVID-19 treatment. HCV PIs showed differential potency in short-term treatment assays based on the detection of SARS-CoV-2 spike protein in Vero E6 cells. Linear PIs boceprevir, telaprevir, and narlaprevir had 50% effective concentrations (EC50) of ∼40 µM. Among the macrocyclic PIs, simeprevir had the highest (EC50, 15 µM) and glecaprevir the lowest (EC50, >178 µM) potency, with paritaprevir, grazoprevir, voxilaprevir, vaniprevir, danoprevir, and deldeprevir in between. Acyclic PIs asunaprevir and faldaprevir had EC50s of 72 and 23 µM, respectively. ACH-806, inhibiting the HCV NS4A protease cofactor, had an EC50 of 46 µM. Similar and slightly increased PI potencies were found in human hepatoma Huh7.5 cells and human lung carcinoma A549-hACE2 cells, respectively. Selectivity indexes based on antiviral and cell viability assays were highest for linear PIs. In short-term treatments, combination of macrocyclic but not linear PIs with remdesivir showed synergism in Vero E6 and A549-hACE2 cells. Longer-term treatment of infected Vero E6 and A549-hACE2 cells with 1-fold EC50 PI revealed minor differences in the barrier to SARS-CoV-2 escape. Viral suppression was achieved with 3- to 8-fold EC50 boceprevir or 1-fold EC50 simeprevir or grazoprevir, but not boceprevir, in combination with 0.4- to 0.8-fold EC50 remdesivir; these concentrations did not lead to viral suppression in single treatments. This study could inform the development and application of protease inhibitors for optimized antiviral treatments of COVID-19.

High density Huh7.5 cell hollow fiber bioreactor culture for high-yield production of hepatitis C virus and studies of antivirals.

Chronic hepatitis C virus (HCV) infection poses a serious global public health burden. Despite the recent development of effective treatments there is a large unmet need for a prophylactic vaccine. Further, antiviral resistance might compromise treatment efficiency in the future. HCV cell culture systems are typically based on Huh7 and derived hepatoma cell lines cultured in monolayers. However, efficient high cell density culture systems for high-yield HCV production and studies of antivirals are lacking. We established a system based on Huh7.5 cells cultured in a hollow fiber bioreactor in the presence or absence of bovine serum. Using an adapted chimeric genotype 5a virus, we achieved peak HCV infectivity and RNA titers of 7.6 log10 FFU/mL and 10.4 log10 IU/mL, respectively. Bioreactor derived HCV showed high genetic stability, as well as buoyant density, sensitivity to neutralizing antibodies AR3A and AR4A, and dependency on HCV co-receptors CD81 and SR-BI comparable to that of HCV produced in monolayer cell cultures. Using the bioreactor platform, treatment with the NS5A inhibitor daclatasvir resulted in HCV escape mediated by the NS5A resistance substitution Y93H. In conclusion, we established an efficient high cell density HCV culture system with implications for studies of antivirals and vaccine development.