Generation of helper virus-free adeno-associated viral vector packaging/producer cell lines based on a human suspension cell line

The emerging number of clinical trials in the gene therapy field poses the challenge to the industry to produce viral vectors in a scalable, reproducible and cost-efficient manner. To address this issue, our CAP-GT platform comprises high density, serum free suspension cell lines that enable reproducible, scalable transfection and high titer production of viral vectors. An adeno-associated virus (AAV) based vector was the first approved gene therapy product in clinical applications. Attractive features of AAV as a gene therapy vector are e.g. its lack of pathogenicity and its ability to transduce dividing and non-dividing cells. Moving away from mainly targeting ultra-rare diseases and taking more common indications into focus will need to see significant improvements concerning productivity and consistent quality of AAV vector production in order to ensure supply. For this purpose, we are developing a helper virus-free packaging cell line that can easily be turned into a producer cell line by only one additional step of cell line development. Base of this packing cell line is the generation of a cell line with stable Tet-inducible expression of Rep proteins. Extensive screening of Rep expressing single cell clones resulted in clonal cell lines which produced high AAV titers upon induction and transfection of the missing components. In a next step, the adenoviral helper functions E2A and E4orf6 are introduced, due to their toxicity also under the control of a Tet-inducible promoter. In addition, the VA RNA is encoded by the same construct. Finally, a Tet-inducible capsid function and GFP as transgene flanked by the ITRs combined on one construct will be stably integrated resulting in a proof of principle producer cell line. This approach should enable a consistent quality production of AAV vectors that abolishes the drawbacks of transient transfection concerning reproducibility, consistency and high costs for GMP-grade DNA. Process optimization in regard to process duration, feeding strategy etc. is currently ongoing for further improving the vector yields

Analysis of Serine Codon Conservation Reveals Diverse Phenotypic Constraints on Hepatitis C Virus Glycoprotein Evolution

Serine is encoded by two divergent codon types, UCN and AGY, which are not interchangeable by a single nucleotide substitution. Switching between codon types therefore occurs via intermediates (threonine or cysteine) or via simultaneous tandem substitutions. Hepatitis C virus (HCV) chronically infects 2 to 3% of the global population. The highly variable glycoproteins E1 and E2 decorate the surface of the viral envelope, facilitate cellular entry, and are targets for host immunity. Comparative sequence analysis of globally sampled E1E2 genes, coupled with phylogenetic analysis, reveals the signatures of multiple archaic codonswitching events at seven highly conserved serine residues. Limited detection of intermediate phenotypes indicates that associated fitness costs restrict their fixation in divergent HCV lineages. Mutational pathways underlying codon switching were probed via reverse genetics, assessing glycoprotein functionality using multiple in vitro systems. These data demonstrate selection against intermediate phenotypes can act at the structural/functional level, with some intermediates displaying impaired virion assembly and/or decreased capacity for target cell entry. These effects act in residue/isolate-specific manner. Selection against intermediates is also provided by humoral targeting, with some intermediates exhibiting increased epitope exposure and enhanced neutralization sensitivity, despite maintaining a capacity for target cell entry. Thus, purifying selection against intermediates limits their frequencies in globally sampled strains, with divergent functional constraints at the protein level restricting the fixation of deleterious mutations. Overall our study provides an experimental framework for identification of barriers limiting viral substitutional evolution and indicates that serine codon-switching represents a genomic "fossil record" of historical purifying selection against E1E2 intermediate phenotypes

Supercritical fluid extraction of heather (Calluna vulgaris) and evaluation of anti-hepatitis C virus activity of the extracts

Previous studies using lipid extracts of heather (Calluna vulgaris) leaves showed the presence of high concentrations of ursolic and oleanolic acid. These two compounds have been reported to present antiviral activity against hepatitis C virus (HCV). In this work, the supercritical fluid extraction of heather was studied with the aim of assessing a potential anti-HCV activity of the extracts owing to their triterpenic acid content. Supercritical extraction assays were carried out exploring the pressure range of 20-50. MPa, temperatures of 40-70. °C and 0-15% of ethanol cosolvent. The content of oleanolic and ursolic acid in the extracts were determined, and different samples were screened for cellular cytotoxicity and virus inhibition using a HCV cell culture infection system. Antiviral activity was observed in most extracts. In general, superior anti-HCV activity was observed for higher contents of oleanolic and ursolic acids in the extracts.This work has been supported by project ALIBIRD-S2009/AGR-1469 grant number, P2013/ABI-2728 from Comunidad Autónoma de Madrid.Peer Reviewe

Mechanisms of methods for hepatitis C virus inactivation.

Virus inactivation by chemical disinfectants is an important instrument for infection control in medical settings, but the mechanisms involved are poorly understood. In this study, we systematically investigated the effects of several antiviral treatments on hepatitis C virus (HCV) particles as model for enveloped viruses. Studies were performed with authentic cell culture-derived viruses, and the influence of chemical disinfectants, heat, and UV treatment on HCV was analyzed by the determination of infectious particles in a limiting-dilution assay, by quantitative reverse transcription-PCR, by core enzyme-linked immunosorbent assay, and by proteolytic protection assay. All different inactivation methods resulted in a loss of HCV infectivity by targeting different parts of the virus particle. Alcohols such as ethanol and 2-propanol did not affect the viral RNA genome integrity but disrupted the viral envelope membrane in a capsid protection assay. Heat and UV treatment of HCV particles resulted in direct damage of the viral genome since transfection of viral particle-associated RNA into permissive cells did not initiate RNA replication. In addition, heat incubation at 80°C disrupted the HCV envelope, rendering the viral capsid susceptible to proteolytic digest. This study demonstrated the molecular processes of viral inactivation of an enveloped virus and should facilitate the development of effective disinfection strategies in infection control not only against HCV but also against other enveloped viruses

Hepatitis C Virus Stimulates Murine CD8α-Like Dendritic Cells to Produce Type I Interferon in a TRIF-Dependent Manner.

Hepatitis C virus (HCV) induces interferon (IFN) stimulated genes in the liver despite of distinct innate immune evasion mechanisms, suggesting that beyond HCV infected cells other cell types contribute to innate immune activation. Upon coculture with HCV replicating cells, human CD141+ myeloid dendritic cells (DC) produce type III IFN, whereas plasmacytoid dendritic cells (pDC) mount type I IFN responses. Due to limitations in the genetic manipulation of primary human DCs, we explored HCV mediated stimulation of murine DC subsets. Coculture of HCV RNA transfected human or murine hepatoma cells with murine bone marrow-derived DC cultures revealed that only Flt3-L DC cultures, but not GM-CSF DC cultures responded with IFN production. Cells transfected with full length or subgenomic viral RNA stimulated IFN release indicating that infectious virus particle formation is not essential in this process. Use of differentiated DC from mice with genetic lesions in innate immune signalling showed that IFN secretion by HCV-stimulated murine DC was independent of MyD88 and CARDIF, but dependent on TRIF and IFNAR signalling. Separating Flt3-L DC cultures into pDC and conventional CD11b-like and CD8α-like DC revealed that the CD8α-like DC, homologous to the human CD141+ DC, release interferon upon stimulation by HCV replicating cells. In contrast, the other cell types and in particular the pDC did not. Injection of human HCV subgenomic replicon cells into IFN-β reporter mice confirmed the interferon induction upon HCV replication in vivo. These results indicate that HCV-replicating cells stimulate IFN secretion from murine CD8α-like DC independent of infectious virus production. Thus, this work defines basic principles of viral recognition by murine DC populations. Moreover, this model should be useful to explore the interaction between dendritic cells during HCV replication and to define how viral signatures are delivered to and recognized by immune cells to trigger IFN release

Environmental Stability and Infectivity of Hepatitis C Virus (HCV) in Different Human Body Fluids.

Hepatitis C virus (HCV) is a hepatotropic, blood-borne virus, but in up to one-third of infections of the transmission route remained unidentified. Viral genome copies of HCV have been identified in several body fluids, however, non-parental transmission upon exposure to contaminated body fluids seems to be rare. Several body fluids, e.g., tears and saliva, are renowned for their antimicrobial and antiviral properties, nevertheless, HCV stability has never been systematically analyzed in those fluids. We used state of the art infectious HCV cell culture techniques to investigate the stability of HCV in different body fluids to estimate the potential risk of transmission via patient body fluid material. In addition, we mimicked a potential contamination of HCV in tear fluid and analyzed which impact commercially available contact lens solutions might have in such a scenario. We could demonstrate that HCV remains infectious over several days in body fluids like tears, saliva, semen, and cerebrospinal fluid. Only hydrogen-peroxide contact lens solutions were able to efficiently inactivate HCV in a suspension test. These results indicate that HCV, once it is present in various body fluids of infected patients, remains infective and could potentially contribute to transmission upon direct contact

Type I IFN production is dependent on IFNAR and TRIF signaling.

<p>Flt3-L DC were generated from (A) C57BL/6, (B) IFNAR^-/-, (C) IL28R^-/-, (D) CARDIF^-/-, (E) MyD88^-/- and (F) TRIF^-/- mice and cocultured with mock or HCV transfected cells or stimulated with VSV-M2 at a MOI 1. After 18 h, IFN-α was quantified in cell-free supernatant by ELISA (n = 3–8). Dashed line indicates the lowest value of the standard of the respective ELISA assay, n.d. not detected. (****, p≤ 0.0001, ***, p≤ 0.001; **, P≤0.01; *, P≤0.05; Mann-Whitney test, means + SD; n.s. not significant).</p

Type I IFN production by Flt3-L DC cultures is dependent on HCV RNA replication and independent of cell-to-cell contact.

<p>(A) Huh7.5 cells were mock transfected or transfected with SGR, Jc1 or Jc1ΔGDD (ΔGDD) RNA, co-cultivated with Flt3-L derived DC cultures and the amount of IFN-α in the supernatant was determined (n = 3). (B) Mock or HCV RNA transfected hepatoma cells were treated with 0.5 μg/mL RNAse or 1 unit DNAse before Flt3-L DC were added in a coculture. After 18 h, IFN-α was detected in the cell-free supernatants (n = 3). Flt3-L derived DC were seeded and stimulated with Jc1 (C) or 5 μL concentrated SN from Mock or HCV SGR transfected cells (D) (n = 3). (E) Extracellular vesicles were isolated from concentrated SN from Mock, pUCΔGDD (ΔGDD) or HCV SGR transfected cells. 5 μL of isolated vesicles were used to stimulate Flt3-L DC for 18 h and IFN-α was quantified in the cell-free supernatant by ELISA (n = 6). (F) Protein content of isolated extracellular vesicles was analyzed using antibodies against polypeptides typically enriched in exosomes (Hsp70, AnxII, CD81, CD63 and actin). Dashed line indicates the lowest value of the standard of the respective ELISA assay, n.d. not detected. (****, p≤ 0.0001, ***, p≤ 0.001; **, P≤0.01; *, P≤0.05; Mann-Whitney test and 2-way ANOVA, means + SD; n.s. not significant).</p