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To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A high-throughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein Bcl-xL, and inhibition of Bcl-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable. Significance: Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of Bcl-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor. This article is highlighted in the In This Issue feature, p. 2355

ispartof: LANCET MICROBE vol:4 issue:3 pages:e132-e133 ispartof: location:England status: published

BACKGROUND: Mpox (formerly monkeypox) is a viral disease caused by the mpox virus (MPXV), endemic in Central and West Africa and currently causing a global outbreak of international concern. Much remains unknown about sample types most suited for mpox laboratory diagnosis. While it is established that high viral loads can be found in active skin lesions (currently the recommended mpox laboratory confirmation specimen type), WHO mpox testing guidelines encourage the use of oropharyngeal swabs as an additional sample type for mpox diagnosis and suggest investigating the value of other specimens like blood samples. OBJECTIVE: In this study, we verified the value of select alternative specimen types for mpox laboratory confirmation. METHODS: We included 25 patients with MPXV-confirmed skin lesions to compare diagnostic sensitivity of MPXV PCR testing on EDTA plasma and two upper respiratory specimens: oropharyngeal swabs and saliva. RESULTS: In our patient cohort with MPXV-confirmed skin lesions, diagnostic sensitivity of MPXV PCR was 80% in EDTA plasma, 64% in oropharyngeal swabs, and 88% in saliva. MPXV viral loads were significantly higher in saliva compared to oropharyngeal swabs and EDTA plasma. DISCUSSION: The WHO recommendation to collect oropharyngeal swabs as an additional specimen for mpox diagnosis might need to be revised to include saliva wherever feasible. We suggest investigating saliva as a diagnostic specimen in the absence of active skin lesions or during the phase preceding skin manifestations. Moreover, the relatively high MPXV DNA content of saliva warrants elucidating its potential role in disease transmission. ispartof: JOURNAL OF CLINICAL VIROLOGY vol:159 ispartof: location:Netherlands status: published

ObjectiveThe available epidemiological and clinical evidence from the currently ongoing monkeypox (MPX) outbreak in non-endemic areas suggests an important factor of sexual transmission. However, limited information on the behaviour and experiences of individuals with an MPX infection has to date been provided. We aimed to describe the initial phase of the MPX outbreak in Belgium, and to provide a more in-depth description of sexual behaviour and transmission contexts.MethodsWe used routine national surveillance data of 139 confirmed MPX cases with date of symptom onset until 19 June 2022, complemented with 12 semistructured interviews conducted with a subsample of these cases.ResultsSexualised environments, including large festivals and cruising venues for gay men, were the suspected exposure setting for the majority of the cases in the early outbreak phase. In-depth narratives of sexual behaviour support the hypothesis of MPX transmission through close physical contact during sex. Despite awareness of the ongoing MPX outbreak, low self-perceived risk of MPX acquisition and confusing initial signs and symptoms for other STIs or skin conditions delayed early detection of an MPX infection. In addition, we describe relevant contextual factors beyond individual behaviour, related to sexual networks, interpersonal interactions and health systems. Some of these factors may complicate early MPX detection and control efforts.ConclusionOur results highlight the role of sexual contact and networks in the transmission of MPX during the early phase of the outbreak in Belgium. Risk communication messages should consistently and transparently state the predominant sexual transmission potential of MPX virus, and prevention and control measures must be adapted to reflect multilevel factors contributing to MPX transmission risk.

The magnitude of the 2022 multi-country monkeypox virus (MPXV) outbreak has surpassed any preceding outbreak. It is unclear whether asymptomatic or otherwise undiagnosed infections are fuelling this epidemic. In this study, we aimed to assess whether undiagnosed infections occurred among men attending a Belgian sexual health clinic in May 2022. We retrospectively screened 224 samples collected for gonorrhea and chlamydia testing using an MPXV PCR assay and identified MPXV-DNA-positive samples from four men. At the time of sampling, one man had a painful rash, and three men had reported no symptoms. Upon clinical examination 21-37 days later, these three men were free of clinical signs, and they reported not having experienced any symptoms. Serology confirmed MPXV exposure in all three men, and MPXV was cultured from two cases. These findings show that certain cases of monkeypox remain undiagnosed and suggest that testing and quarantining of individuals reporting symptoms may not suffice to contain the outbreak.

Adeno-associated virus (AAV) vector serotypes vary in their ability to transduce hepatocytes from different species. Chimeric mouse models harboring human hepatocytes have shown translational promise for liver-directed gene therapies. However, many variables that influence human hepatocyte transduction and transgene expression in such models remain poorly defined. Here, we aimed to test whether three experimental conditions influence AAV transgene expression in immunodeficient, fumaryl-acetoactetate-hydrolase-deficient (Fah−/−) chimeric mice repopulated with primary human hepatocytes. We examined the effects of the murine liver injury cycle, human donor variability, and vector doses on hepatocyte transduction with various AAV serotypes expressing a green fluorescent protein (GFP). We determined that the timing of AAV vector challenge in the liver injury cycle resulted in up to 7-fold differences in the percentage of GFP expressing human hepatocytes. The GFP+ hepatocyte frequency varied 7-fold between human donors without, however, changing the relative transduction efficiency between serotypes for an individual donor. There was also a clear relationship between AAV vector doses and human hepatocyte transduction and transgene expression. We conclude that several experimental variables substantially affect human hepatocyte transduction in the Fah−/− chimera model, attention to which may improve reproducibility between findings from different laboratories., Graphical Abstract, Liver chimeric mice are widely used to model human hepatocyte susceptibility to adeno-associated virus (AAV) gene vector transduction with inconsistent finding between laboratories. Zou et al. quantify to what extent three experimental conditions—namely, mouse liver injury, human donor variability, and AAV doses—affect human hepatocyte transduction in chimeric mice.

Coronavirus disease 2019 (COVID-19) is a viral lower respiratory tract infection caused by the highly transmissible and pathogenic SARS-CoV-2 (severe acute respiratory-syndrome coronavirus-2). Besides respiratory failure, systemic thromboembolic complications are frequent in COVID-19 patients and suggested to be the result of a dysregulation of the hemostatic balance. Although several markers of coagulation and fibrinolysis have been studied extensively, little is known about the effect of SARS-CoV-2 infection on the potent antifibrinolytic enzyme carboxypeptidase U (CPU). Blood was collected longitudinally from 56 hospitalized COVID-19 patients and 32 healthy controls. Procarboxypeptidase U (proCPU) levels and total active and inactivated CPU (CPU+CPUi) antigen levels were measured. At study inclusion (shortly after hospital admission), proCPU levels were significantly lower and CPU+CPUi antigen levels significantly higher in COVID-19 patients compared to controls. Both proCPU and CPU+CPUi antigen levels showed a subsequent progressive increase in these patients. Hereafter, proCPU levels decreased and patients were, at discharge, comparable to the controls. CPU+CPUi antigen levels at discharge were still higher compared to controls. Baseline CPU+CPUi antigen levels (shortly after hospital admission) correlated with disease severity and the duration of hospitalization. In conclusion, CPU generation with concomitant proCPU consumption during early SARS-CoV-2 infection will (at least partly) contribute to the hypofibrinolytic state observed in COVID-19 patients, thus enlarging their risk for thrombosis. Moreover, given the association between CPU+CPUi antigen levels and both disease severity and duration of hospitalization, this parameter may be a potential biomarker with prognostic value in SARS-CoV-2 infection.

Background: COVID-19 patients experience several features of dysregulated immune system observed in sepsis. We previously showed a dysregulation of several proline-selective peptidases such as dipeptidyl peptidase 4 (DPP4), fibroblast activation protein alpha (FAP), prolyl oligopeptidase (PREP) and prolylcarboxypeptidase (PRCP) in sepsis. In this study, we investigated whether these peptidases are similarly dysregulated in hospi- talized COVID-19 patients. Methods: Fifty-six hospitalized COVID-19 patients and 32 healthy controls were included. Enzymatic activities of DPP4, FAP, PREP and PRCP were measured in samples collected shortly after hospital admission and in longi- tudinal follow-up samples. Results: Compared to healthy controls, both DPP4 and FAP activities were significantly lower in COVID-19 pa- tients at hospital admission and FAP activity further decreased significantly in the first week of hospitalization. While PRCP activity remained unchanged, PREP activity was significantly increased in COVID-19 patients at hospitalization and further increased during hospital stay and stayed elevated until the day of discharge. Conclusion: The changes in activities of proline-selective peptidases in plasma are very similar in COVID-19 and septic shock patients. The pronounced decrease in FAP activity deserves further investigation, both from a pathophysiological viewpoint and as its utility as a part of a biomarker panel.

Objectives: Hepatitis E virus (HEV) genotype 3 is an emerging pathogen in developed countries. We evaluated the performance of two new serological assays for the detection of HEV, VIDAS® anti-HEV IgM and IgG. Methods: VIDAS® assays were performed on 77 clinical samples: 68 samples from patients suspected for HEV infection and 9 samples which previously tested positive for HEV IgM, IgG or HEV PCR. All samples were also tested using Wantai HEV assays. Cross-reactivity was assessed. To get a better view on the natural course of HEV infections, three clinical cases are described. Results: The concordance rate between VIDAS® and Wantai assays was good for HEV IgM (0.75,CI 0.52-0.98) and very good for HEV IgG (0.85,CI 0.72-0.98). Four samples tested borderline/positive with Wantai IgM but negative with VIDAS® IgM. All of these samples were HEV RNA negative, HEV IgG was positive in 2/4 samples. Five samples produced conflicting HEV IgG results. These tested positive with VIDAS® but negative with Wantai IgG. All five samples were HEV IgM and RNA negative. We detected no cross-reactivity. The clinical cases illustrate that HEV serology can still be negative in the very beginning of an acute infection. Conclusions: There is a good agreement between VIDAS® and Wantai anti-HEV IgM and IgG assays. Discrepant HEV IgM results probably reflect false positive Wantai IgM results (RNA-/IgG- samples) and longer-lasting positive Wantai IgM (RNA-/IgG+ samples). Discrepant HEV IgG results, could either represent resolved HEV infections (false negative Wantai IgG results) or false positive VIDAS® HEV IgG results.

Despite the general agreement on the importance of T cells during SARS-CoV-2 infection, the clinical impact of specific and cross-reactive T-cell responses remains uncertain, while this knowledge may indicate how to adjust vaccines and maintain robust long-term protection against continuously emerging variants. To characterize CD8+ T-cell response to epitopes unique to SARS-CoV-2 (SC-unique) or shared with other coronaviruses (CoV-common), we trained a large number of TCR-epitope recognition models for MHC-I-presented SARS-CoV-2 epitopes from publicly available data. Applying those models to longitudinal COVID-19 TCR repertoires of critical and non-critical COVID-19 patients, we discovered that notwithstanding comparable CD8+ T-cell depletion and the sizes of putative CoV-common CD8+ TCR repertoires in all symptomatic patients at the initial stage of the disease, the temporal dynamics of putative SC2-unique TCRs differed depending on the disease severity. Only non-critical patients had developed large and diverse SC2-unique CD8+ T-cell response by the second week of the disease. Additionally, only this patient group demonstrated redundancy in CD8+ TCRs putatively recognizing unique and common SARS-CoV-2 epitopes. Our findings thus emphasize the role of thede novoCD8+ T-cell response and support the argument against the clinical benefit of pre-existing cross-reactive CD8+ T cells. Now, the analytical framework of this study can not only be employed to track specific and cross-reactive SARS-CoV-2 CD8+ T cells in any TCR repertoire but also be generalized to more epitopes and be employed for adaptive immune response assessment and monitoring to inform public health decisions.

Users of anatomical donors are at risk of exposure to bloodborne pathogens. This study evaluated screening algorithms for hepatitis B and C virus, human immunodeficiency virus and Treponema pallidum during donor allocation and assessed the impact of postmortem time on hemolysis and how hemolysis affects test results and donor discard rate.From 2011 to 2018, demographic data of anatomical donors, time of postmortem blood sampling, presence of sample hemolysis, serological test results (negative; active infection; false reactive screening test; historic infection; inconclusive; technically impracticable) and the actual donor allocation were collected.Donors (n = 537) had a mean age of 77.53 ± 13.67 (24-103) year. Nine (1.68%) had laboratory test results indicative for active infection for hepatitis B (n = 1) and C virus (n = 2), human immunodeficiency virus (n = 5) and T. pallidum (n = 1). Negative screenings ranged from 74.67 to 97.58%, depending on the pathogen. According to the original screening algorithms, 479 (89.20%) donors should have been accepted. In practice, a donor acceptance rate of 91.20% was found. Analysis of potential donor allocation interpretation obstacles resulted in simplification of the in-house laboratory testing algorithms and addition of a nucleic acid test to increase the reliability for identification of active (acute) human immunodeficiency virus infection. Hemolysis was more common when sampling was performed more than 24 h after death (p0.001). Hemolytic samples more frequently showed a reactive or indeterminate human immunodeficiency virus test result (p0.001). Screening for human immunodeficiency virus and T. pallidum was technically more impracticable when hemolysis was present (p = 0.042 and p = 0.003, respectively). Donors with hemolytic blood samples were more often discarded (46.88%) compared to bodies with non-hemolytic samples (6.32%) (p0.001).Despite the implementation of donor screening algorithms, a significant number of bodies have an inconsistent allocation. New algorithms, to be evaluated in future research, were suggested. Early postmortem blood sampling is key as hemolysis can influence certain test results and donor allocation.

Background & Aims Hepatitis C virus (HCV) is a leading cause of chronic liver diseases and the most common indication for liver transplantation in the United States. HCV particles in the blood of infected patients are characterized by heterogeneous buoyant densities, likely owing to HCV association with lipoproteins. However, clinical isolates are not infectious in vitro and the relative infectivity of the particles with respect to their buoyant density therefore cannot be determined, pointing to the need for better in vivo model systems. Methods To analyze the evolution of the buoyant density of in vivo–derived infectious HCV particles over time, we infected immunodeficient human liver chimeric fumaryl acetoacetate hydrolase-/- mice with J6/JFH1 and performed ultracentrifugation of infectious mouse sera on isopicnic iodixanol gradients. We also evaluated the impact of a high sucrose diet, which has been shown to increase very-low-density lipoprotein secretion by the liver in rodents, on lipoprotein and HCV particle characteristics. Results Similar to the severe combined immunodeficiency disease/Albumin-urokinase plasminogen activator human liver chimeric mouse model, density fractionation of infectious mouse serum showed higher infectivity in the low-density fractions early after infection. However, over the course of the infection, viral particle heterogeneity increased and the overall in vitro infectivity diminished without loss of the human liver graft over time. In mice provided with a sucrose-rich diet we observed a minor shift in HCV infectivity toward lower density that correlated with a redistribution of triglycerides and cholesterol among lipoproteins. Conclusions Our work indicates that the heterogeneity in buoyant density of infectious HCV particles evolves over the course of infection and can be influenced by diet., Graphical abstract

Significance The ability to study human liver disease is limited by available hepatocyte models. Primary human hepatocytes (PHH) and xenograft models suffer from limited availability, donor-to-donor variability, and high cost. Here we report two transformative advances. First, the alkaloid retrorsine improves humanization of the murine liver, which allows routine production of highly humanized mice and high-quality mouse-passaged PHH. Second, the ability to genetically modify PHH cultures and retransplant to create highly humanized mice with genetically altered grafts. When combined, these two advances open new frontiers for creating disease-specific PHH models and for performing genetic and other screens in PHH., Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum. mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.

To evaluate the potential of dried blood spots (DBS) as a congenital cytomegalovirus (cCMV) testing specimen, the laboratory diagnostic accuracy of polymerase chain reaction (PCR) on DBS was compared to viral urine cultures from neonates suspected for cCMV. Two different extraction methods (EasyMAG, bioMerieux versus Qiagen) and 2 real-time PCR protocols (in-house versus Argene) were compared. We were able to collect both DBS and urine samples in 6 Belgian neonatal units from 276 neonates suspected for cCMV registered in CMVREG (an online neonatal registry system). Forty-eight neonates (17.4%) were positive by viral culture in urine. Laboratory diagnostic accuracy parameters of DBS-PCR were both extraction method and PCR protocol dependent. Not all DBS-CMV-PCR methods successfully detected urine-culture-positive neonates born after first-trimester seroconversions. Interestingly, however, all urine-culture-positive neonates having clinical signs of cCMV did consistently score positive. Keywords: Cytomegalovirus Congenital Dried blood spot testing (C) 2020 Elsevier Inc. All rights reserved.

Introduction: Complement functional analyses provide insight into the integrity of the entire complement reaction cascade. These tests are suitable for investigating suspected complement deficiencies. Falsely reduced test outcomes may result from preanalytical instabilities of individual complement components. To generate rationale for this or potential alternative practices, this study aimed to extend the knowledge on the preanalytical stability of widely used tests to screen the complement system. We assessed the influence of time, temperature and EDTA on classical (CH50) and alternative pathway (AP50) functional assay test results. Materials and methods: We used nephelometric (C3d) and immunofixation (C3c) techniques to support the investigation of the preanalytical phase of basic complement system activity tests. Quantitative determination of classical and alternative pathway function was performed with a haemolytic activity assay and a C5b-9 neo-epitope ELISA-based assay respectively. Blood of five healthy volunteers was sampled and complement components allowed to degrade under different conditions. Results: CH50 and AP50 remain stable for approximately one week in serum samples incubated on ice. CH50 activity decreased almost twice as fast in EDTA plasma compared to serum at room temperature. AP50 activity contrastingly, decreased twice as slow in EDTA plasma compared to serum at room temperature. Conclusion: Serum on ice remains the preferred specimen for functional complement analyses. In the absence of serum transported on ice, serum kept at room temperature (not exceeding 24h) is suitable for classical and alternative pathway analyses. For alternative pathway analyses specifically, the C3-stabilising effect of EDTA allows for the extended use of EDTA plasma (not over 4 days). In these conditions, at least 85% of baseline complement activity remains.

Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in “humanized” mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (~3-times, ~8-times, and ~80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.

ABSTRACT Hepatitis E virus (HEV) infections appear to be an emerging problem in Europe. Infections are mainly caused by viruses of genotype 3. Pigs and wild boar are the main reservoirs of HEV in Europe and most autochthonous infections are probably caused by the consumption of uncooked or undercooked infected meat. Nevertheless, transfusion-associated transmission has been described in different European countries but the efficiency of this route of transmission need to be further investigated. Most acute infections are asymptomatic or the induced symptoms are rather nonspecific. Although people that are otherwise completely healthy can spontaneously clear an HEV infection, people with underlying liver disease and/or suffering from immune deficiencies may require treatment to avoid chronicity and exacerbation of liver disease. In this review, we give an epidemiological overview of HEV in Europe and the potential complications.

Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer. Cell entry of HCV and other pathogens is mediated by tight junction (TJ) proteins, but successful therapeutic targeting of TJ proteins has not been reported yet. Using a human liver-chimeric mouse model, we show that a monoclonal antibody specific for the TJ protein claudin-1 (ref. 7) eliminates chronic HCV infection without detectable toxicity. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Antibody treatment reduces the number of HCV-infected hepatocytes in vivo, highlighting the need for de novo infection by means of host entry factors to maintain chronic infection. In summary, we demonstrate that an antibody targeting a virus receptor can cure chronic viral infection and uncover TJ proteins as targets for antiviral therapy. INTERREG-IV-Rhin Supérieur-FEDER-Hepato-Regio-Net 2009 and 2012

Hepatitis C virus (HCV)-induced endstage liver disease is currently a major indication for liver transplantation. After transplantation the donor liver inevitably becomes infected with the circulating virus. Monoclonal antibodies (mAbs) against the HCV coreceptor scavenger receptor class B type I (SR-BI) inhibit HCV infection of different genotypes, both in cell culture and in humanized mice. Anti-SR-BI mAb therapy is successful even when initiated several days after HCV exposure, supporting its potential applicability to prevent HCV reinfection of liver allografts. However, HCV variants with reduced SR-BI dependency have been described in the literature, which could potentially limit the use of SR-BI targeting therapy. In this study we show, both in a preventative and postexposure setting, that humanized mice infected with HCV variants exhibiting increased in vitro resistance to SR-BI-targeting molecules remain responsive to anti-SR-BI mAb therapy in vivo. A 2-week antibody therapy readily cleared HCV RNA from the circulation of infected humanized mice. We found no evidence supporting increased SR-BI-receptor dependency of viral particles isolated from humanized mice compared to cell culture-produced virus. However, we observed that, unlike wild-type virus, the in vitro infectivity of the resistant variants was inhibited by both human high density lipoprotein (HDL) and very low density lipoprotein (VLDL). The combination of mAb1671 with these lipoproteins further increased the antiviral effect. Conclusion: HCV variants that are less dependent on SR-BI in vitro can still be efficiently blocked by an anti-SR-BI mAb in humanized mice. Since these variants are also more susceptible to neutralization by anti-HCV envelope antibodies, their chance of emerging during anti-SR-BI therapy is severely reduced. Our data indicate that anti-SR-BI receptor therapy could be an effective way to prevent HCV infection in a liver transplant setting. (Hepatology 2014;60:1508–1518)

Type III interferons (IFN-λs) signal through a heterodimeric receptor complex composed of the IFN-λR1 subunit, specific for IFN-λs, and interleukin-10Rβ (IL-10Rβ), which is shared by multiple cytokines in the IL-10 superfamily. Low affinity of IL-10Rβ for cytokines has impeded efforts aimed at crystallizing cytokine-receptor complexes. We used yeast surface display to engineer a higher-affinity IFN-λ variant, H11, which enabled crystallization of the ternary complex. The structure revealed that IL-10Rβ uses a network of tyrosine residues as hydrophobic anchor points to engage IL-10 family cytokines that present complementary hydrophobic binding patches, explaining its role as both a cross-reactive but cytokine-specific receptor. H11 elicited increased anti-proliferative and antiviral activities in vitro and in vivo. In contrast, engineered higher-affinity type I IFNs did not increase antiviral potency over wild-type type I IFNs. Our findings provide insight into cytokine recognition by the IL-10R family and highlight the plasticity of type III interferon signaling and its therapeutic potential.

PMC5292934; Due to the highly restricted species-tropism of Hepatitis C virus (HCV) a limited number of animal models exist for pre-clinical evaluation of vaccines and antiviral compounds. The human-liver chimeric mouse model allows heterologous challenge with clinically relevant strains derived from patients. However, to date, the transmission and longitudinal evolution of founder viral populations in this model have not been characterized in-depth using state-of-the-art sequencing technologies. Focusing on NS3 protease encoding region of the viral genome, mutant spectra in a donor inoculum and individual recipient mice were determined via Illumina sequencing and compared, to determine the effects of transmission on founder viral population complexity. In all transmissions, a genetic bottleneck was observed, although diverse viral populations were transmitted in each case. A low frequency cloud of mutations ( 1% restricted to a subset of nucleotides. The population of SNVs >1% was reduced upon transmission while the low frequency SNV cloud remained stable. Fixation of multiple identical synonymous substitutions was apparent in independent transmissions, and no evidence for reversion of T-cell epitopes was observed. In addition, susceptibility of founder populations to antiviral therapy was assessed. Animals were treated with protease inhibitor (PI) monotherapy to track resistance associated substitution (RAS) emergence. Longitudinal analyses revealed a decline in population diversity under therapy, with no detectable RAS >1% prior to therapy commencement. Despite inoculation from a common source and identical therapeutic regimens, unique RAS emergence profiles were identified in different hosts prior to and during therapeutic failure, with complex mutational signatures at protease residues 155, 156 and 168 detected. Together these analyses track viral population complexity at high-resolution in the human-liver chimeric mouse model post-transmission and under therapeutic intervention, revealing novel insights into the evolutionary processes which shape viral protease population composition at various critical stages of the viral life-cycle.

Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization levels observed for mAb 2A5 were generally high and mostly superior to those obtained with AP33, a well-characterized HCV-neutralizing monoclonal antibody. Using humanized mice, complete protection was observed after genotype 1a and 4a HCV challenge, while only partial protection was achieved using gt1b and 6a isolates. Epitope mapping revealed that mAb 2A5 binding is conformation-dependent and identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain. CONCLUSION: mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence represent a valuable candidate to prevent HCV recurrence in LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines. PMC5785094 NIHMS935524

Background & Aims Human liver chimeric mice are useful models of human hepatitis virus infection, including hepatitis B and C virus infections. Independently, immunodeficient mice reconstituted with CD34 + hematopoietic stem cells (HSC) derived from fetal liver reliably develop human T and B lymphocytes. Combining these systems has long been hampered by inefficient liver reconstitution of human fetal hepatoblasts. Our study aimed to enhance hepatoblast engraftment in order to create a mouse model with syngeneic human liver and immune cells. Methods The effects of human oncostatin-M administration on fetal hepatoblast engraftment into immunodeficient fah −/− mice was tested. Mice were then transplanted with syngeneic human hepatoblasts and HSC after which human leukocyte chimerism and functionality were analyzed by flow cytometry, and mice were challenged with HBV. Results Addition of human oncostatin-M enhanced human hepatoblast engraftment in immunodeficient fah −/− mice by 5–100 fold. In contrast to mice singly engrafted with HSC, which predominantly developed human T and B lymphocytes, mice co-transplanted with syngeneic hepatoblasts also contained physiological levels of human monocytes and natural killer cells. Upon infection with HBV, these mice displayed rapid and sustained viremia. Conclusions Our study provides a new mouse model with improved human fetal hepatoblast engraftment and an expanded human immune cell repertoire. With further improvements, this model may become useful for studying human immunity against viral hepatitis. Lay summary Important human pathogens such as hepatitis B virus, hepatitis C virus and human immunodeficiency virus only infect human cells which complicates the development of mouse models for the study of these pathogens. One way to make mice permissive for human pathogens is the transplantation of human cells into immune-compromised mice. For instance, the transplantation of human liver cells will allow the infection of these so-called "liver chimeric mice" with hepatitis B virus and hepatitis C virus. The co-transplantation of human immune cells into liver chimeric mice will further allow the study of human immune responses to hepatitis B virus or hepatitis C virus. However, for immunological studies it will be crucial that the transplanted human liver and immune cells are derived from the same human donor. In our study we describe the efficient engraftment of human fetal liver cells and immune cells derived from the same donor into mice. We show that liver co-engraftment resulted in an expanded human immune cell repertoire, including monocytes and natural killer cells in the liver. We further demonstrate that these mice could be infected with hepatitis B virus, which lead to an expansion of natural killer cells. In conclusion we have developed a new mouse model that could be useful to study human immune responses to human liver pathogens.

Infections with HCV represent a major global health problem. End-stage liver disease caused by chronic HCV infection is the most common indication for liver transplantation. Limited efficacy and severity of side effects hamper the use of pegylated interferon combined with ribavirin in a liver transplant setting. Therefore, new therapeutic options should be made available. Viral entry, the first step of the viral life cycle, represents an interesting target for therapeutic intervention. Understanding the mechanisms of viral entry is necessary to define the viral and cellular factors involved. In this review, we summarize these factors, highlight their potential as therapeutic targets and review the current (pre)clinical development of molecules that interfere with HCV entry.

An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8(+) and class II CD4(+) T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm(-3). The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8(+) T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8(+) T-cell responses and will have important implications for the development of a preventative vaccine for HCV.

To explore mechanisms of hepatitis C viral (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action. Conclusion: These observations reveal novel details about HCV membrane fusion; moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as a cost-effective component of HCV combination therapies. (Hepatology 2016;63:49–62)

Objective: Direct-acting antivirals (DAAs) inhibit hepatitis C virus (HCV) infection by targeting viral proteins that play essential roles in the replication process. However, selection of resistance-associated variants (RAVs) during DAA therapy has been a cause of therapeutic failure. In this study, we wished to address whether such RAVs could be controlled by the co-administration of host-targeting entry inhibitors that prevent intrahepatic viral spread. Design: We investigated the effect of adding an entry inhibitor (the anti-scavenger receptor class B type I mAb1671) to a DAA monotherapy (the protease inhibitor ciluprevir) in human-liver mice chronically infected with HCV of genotype 1b. Clinically relevant non-laboratory strains were used to achieve viraemia consisting of a cloud of related viral variants (quasispecies) and the emergence of RAVs was monitored at high resolution using next-generation sequencing. Results: HCV-infected human-liver mice receiving DAA monotherapy rapidly experienced on-therapy viral breakthrough. Deep sequencing of the HCV protease domain confirmed the manifestation of drug-resistant mutants upon viral rebound. In contrast, none of the mice treated with a combination of the DAA and the entry inhibitor experienced on-therapy viral breakthrough, despite detection of RAV emergence in some animals. Conclusions: This study provides preclinical in vivo evidence that addition of an entry inhibitor to an anti-HCV DAA regimen restricts the breakthrough of DAA-resistant viruses. Our approach is an excellent strategy to prevent therapeutic failure caused by on-therapy rebound of DAA-RAVs. Inclusion of an entry inhibitor to the newest DAA combination therapies may further increase response rates, especially in difficult-to-treat patient populations.

The development and evaluation of effective therapies and vaccines for the hepatitis C virus (HCV) and the study of its interactions with the mammalian host have been hindered for a long time by the absence of suitable small animal models. Immune compromised mouse models that recapitulate the complete HCV life cycle have been useful to investigate many aspects of the HCV life cycle including antiviral interventions. However, HCV has a high propensity to establish persistence and associated histopathological manifestations such as steatosis, fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Better understanding of these processes requires the development of a permissive and fully immunocompetent small animal model. In this review we summarize the in vivo models that are available for the study of HCV.

Hepatitis E virus (HEV) is yearly responsible for approximately 20 million infections worldwide. Although most infections occur in developing countries, HEV appears to be an emerging problem in several industrialized countries, where it is mostly associated with either traveling to an HEV endemic area or contact with pigs, which represent a major reservoir of HEV. The major risk groups for HEV infection and its ensuing complications are elderly men, pregnant women, young children, immunocompromised patients, patients with preexisting liver disease, and workers that come into close contact with HEV-infected animals. Whereas HEV mainly causes acute self-limiting infections, chronic infections may occur among immunocompromised patients (e.g., transplant recipients and human immunodeficiency virus [HIV]-infected patients). Accordingly, HEV-HIV coinfection leads to accelerated liver cirrhosis and increased mortality rates compared to HEV infection alone, which is, except during pregnancy, usually associated with only low mortality. In the Western world, the most common genotype (gt) causing HEV infection is gt 3. Ribavirin (RBV) and interferon have been used successfully for treatment of HEV, but this treatment is contraindicated in certain patient groups. Therefore, novel antiviral compounds are highly needed, especially given that viral isolates with RBV resistance have been recently identified. Moreover, eradication of HEV is hampered by long-term environmental persistence of the virus, which represents a continuous source of the virus. In 2011, the first prophylactic HEV vaccine, Hecolin, was approved in China, but it is not yet globally available. In this review, we will discuss the molecular virology of HEV, mode of transmission in industrialized countries, and potential implications for different specific patient populations.

Background & Aims Efforts to develop an effective vaccine against hepatitis C virus (HCV) have been hindered by the propensity of the virus to evade host immune responses. HCV particles in serum and in cell culture associate with lipoproteins, which contribute to viral entry. Lipoprotein association has also been proposed to mediate viral evasion of the humoral immune response, though the mechanisms are poorly defined. Methods We used small interfering RNAs to reduce levels of apolipoprotein E (apoE) in cell culture−derived HCV−producing Huh7.5-derived hepatoma cells and confirmed its depletion by immunoblot analyses of purified viral particles. Before infection of naive hepatoma cells, we exposed cell culture−derived HCV strains of different genotypes, subtypes, and variants to serum and polyclonal and monoclonal antibodies isolated from patients with chronic HCV infection. We analyzed the interaction of apoE with viral envelope glycoprotein E2 and HCV virions by immunoprecipitation. Results Through loss-of-function studies on patient-derived HCV variants of several genotypes and subtypes, we found that the HCV particle apoE allows the virus to avoid neutralization by patient-derived antibodies. Functional studies with human monoclonal antiviral antibodies showed that conformational epitopes of envelope glycoprotein E2 domains B and C were exposed after depletion of apoE. The level and conformation of virion-associated apoE affected the ability of the virus to escape neutralization by antibodies. Conclusions In cell-infection studies, we found that HCV-associated apoE helps the virus avoid neutralization by antibodies against HCV isolated from chronically infected patients. This method of immune evasion poses a challenge for the development of HCV vaccines.

Tyrosine phosphorylation is a hallmark for activation of STAT proteins, but their transcriptional activity also depends on other secondary modifications. Type I IFNs can activate both the ISGF3 (STAT1:STAT2:IRF9) complex and STAT3, but with cell-specific, selective triggering of only the ISGF3 transcriptional program. Following a genome-wide RNAi screen, we identified the SIN3 transcription regulator homolog A (Sin3a) as an important mediator of this STAT3-targeted transcriptional repression. Sin3a directly interacts with STAT3 and promotes its deacetylation. SIN3A silencing results in a prolonged nuclear retention of activated STAT3 and enhances its recruitment to the SOCS3 promoter, concomitant with histone hyperacetylation and enhanced STAT3-dependent transcription. Conversely, Sin3a is required for ISGF3-dependent gene transcription and for an efficient IFN-mediated antiviral protection against influenza A and hepatitis C viruses. The Sin3a complex therefore acts as a context-dependent ISGF3/STAT3 transcriptional switch.

BACKGROUND & AIMS: Hepatitis C virus (HCV)-induced end-stage liver disease is currently the major indication for liver transplantation in the Western world. After transplantation, the donor liver almost inevitably becomes infected by the circulating virus and disease progression is accelerated in immune suppressed transplant patients. The current standard therapy, based on pegylated interferon and ribavirin, induces severe side effects and is often ineffective in this population. Therefore, new strategies to prevent graft re-infection are urgently needed. We have previously shown that monoclonal antibodies (mAbs) against the HCV co-receptor scavenger receptor class B type I (SR-BI/Cla1) inhibit infection by different HCV genotypes in cell culture. METHODS: Using phage display libraries, we have generated a large set of novel human mAbs against SR-BI and evaluated their effectiveness in preventing HCV infection and direct cell-to-cell spread in vitro and in vivo using uPA-SCID mice with a humanized liver. RESULTS: Eleven human monoclonal antibodies were generated that specifically recognize SR-BI. Two antibodies, mAb8 and mAb151, displayed the highest binding and inhibitory properties and also interfered with direct cell-to-cell spread in vitro. Studies in humanized mice showed that both antibodies were capable of preventing HCV infection and could block intrahepatic spread and virus amplification when administered 3 days after infection. Interestingly, anti-SR-BI therapy was effective against an HCV variant that escaped the control of the adaptive immune response in a liver transplant patient. CONCLUSIONS: The anti-SR-BI mAbs generated in this study may represent novel therapeutic tools to prevent HCV re-infection of liver allografts.

Hepatitis C virus (HCV)-infected patients undergoing liver transplantation universally experience rapid reinfection of their new liver graft. Current treatment protocols do not prevent graft reinfection and, in addition, an accelerated disease progression is observed. In the present study, we have evaluated a novel strategy to prevent HCV infection using a lectin, griffithsin (GRFT) that specifically binds N-linked high-mannose oligosaccharides that are present on the viral envelope. The antiviral effect of GRFT was evaluated in vitro using the HCV pseudoparticle (HCVpp) and HCV cell culture (HCVcc) systems. We show here that preincubation of HCVpp and HCVcc with GRFT prevents infection of Huh-7 hepatoma cells. Furthermore, GRFT interferes with direct cell-to-cell transmission of HCV. GRFT acts at an early phase of the viral life cycle by interfering in a genotype-independent fashion with the interaction between the viral envelope proteins and the viral receptor CD81. The capacity of GRFT to prevent infection in vivo was evaluated using uPA +/+ -SCID mice (uPA stands for urokinase-type plasminogen activator) that harbor human primary hepatocytes in their liver (chimeric mice). In this proof-of-concept trial, we demonstrated that GRFT can mitigate HCV infection of chimeric mice. Treated animals that did become infected demonstrated a considerable delay in the kinetics of the viral infection. Our data demonstrate that GRFT can prevent HCV infection in vitro and mitigate HCV infection in vivo . GRFT treatment of chronically infected HCV patients undergoing liver transplantation may be a suitable strategy to prevent infection of the liver allograft.