Hvidovre Hospital

The United States of America, as Represented by the Secretary, Department of Health and Human Services

Kobenhavns Universitet

Københavns Universitet

Judith Margarete Gottwein

Jens Bukh

Troels Kasper Hoyer Scheel

Tanja Bertelsen Jensen

Troels Kasper Høyer Scheel

Yiping Li

Robert H Purcell

Jesper Eugen-Olsen

Santseharay Ramirez Almeida

Jannick Prento

Jacob Bo Lademann

Yi-Ping Li

Jannie Pedersen

Santseharay Ramirez Almelda

Long Van Pham

Maria Lisa Knudsen

Daryl Grant Humes

Christian Kjærulff Mathiesen

Anne Finne Pihl

Keven Lothert

Michael Wolff

Anna Offersgaard

The present invention relates to nucleic acid sequences that encode hepatitis C viruses (HCV) of genotype 6a that are useful in the fundamental research of HCV as well as in the search of antivirals and vaccines against HCV. In particular, the present invention relates to nucleic acid sequences that comprises HCVs, which are capable of expressing said virus when transfected into cells and are capable of replication or infectivity in cultured cells.

Efficient cell culture system for hepatitis C virus genotype 6A

The present invention relates to methods for obtaining a whole virus vaccine candidate stock. The present invention also relates to an inactivated whole virus vaccine candidate stock that can be used for vaccination purposes as well as development of novel high titer virus, which is the preferred virus for this technique.

Development of methods for production of a whole virus vaccine candidate stock and novel adaptive mutations in hepatitis C virus

The present invention relates to hepatitis C virus (HCV) culture systems of genotypes 1a, 3a, 4a, 5a, and 6a that directly contribute to HCV drug and vaccine development, to HCV basic research and better-individualized treatment of HCV infected patients.

HCV culture systems and direct-acting antiviral sensitivity

The present invention relates to nucleic acid sequences that encode hepatitis C viruses (HCV) of genotype 2b that are useful in the fundamental research of HCV as well as in the search of a vaccine against HCV. In particular the present invention relates to nucleic acid sequences that comprises HCVs, which are capable of expressing said virus when transfected into cells and are capable of infectivity in vivo.

High-titer HCV full-length genotype 2B infectious cell culture systems and applications thereof

The present invention relates to molecular approaches to the production of nucleic acid sequences, which comprises the genome of infectious hepatitis C virus. In particular, the invention provides nucleic acid sequences which comprise the genomes of infectious hepatitis C viruses of either genotype 3a (strain S52) or genotype 4a (strain ED43). The invention therefore relates to the use of the nucleic acid sequences and polypeptides encoded by all or part of the sequences in the development of vaccines and diagnostic assays for HCV and in the development of screening assays for the identification of antiviral agents for HCV. The invention therefore also relates to the use of viral particles derived from laboratory animals infected with S52 and ED43 viruses.

Infectious hepatitis C viruses of genotype 3A and 4A and uses thereof

The present inventors developed hepatitis C virus recombinants expressing NS5A from genotype 1a, 1b, 2a, 3a, 4a, 5a, 6a or 7a in the context of a genotype 2a backbone. Additional recombinants express NS5A and the structural proteins (Core, E1 and E2), p7 and NS2 from genotype 1a, 1b, 3a, 4a, 5a, 6a or 7a in the genotype 2a backbone. Sequence analysis of the recombinants recovered after viral passage in Huh7.5 cells revealed adaptive mutations in NS5A and/or NS3. The importance of these mutations for improved growth kinetics was shown in reverse genetic studies.

JFH-1 based HCV cell culture systems for NS5A of genotypes 1-7

The present invention relates to nucleic acid sequences that encode hepatitis C viruses (HCV) that are useful in the fundamental research of HCV as well as in the search of a vaccine against HCV. In particular the present invention relates to nucleic acid sequences that comprises HCVs which are capable of expressing said virus when transfected into cells and are capable of infectivity in vivo.

HCV full-length infectious cell culture systems and applications thereof

A robust and genetically stable cell culture system for Hepatitis C Virus (HCV) genotype 3a is provided. A genotype 3a/2a (S52/JFH1) recombinant containing the structural genes (Core, E1, E2), p7 and NS2 of strain S52 was constructed and characterized in Huh7.5 cells. S52/JFH1 and J6/JFH viruses passaged in cell culture had comparable growth kinetics and yielded similar peak HCV RNA titers and infectivity titers. Direct genome sequencing of cell culture derived S52/JFH1 viruses identified putative adaptive mutations in Core, E2, p7, NS3, and NS5A; clonal analysis revealed that all genomes analyzed exhibited different combinations of these mutations. Finally, viruses resulting from transfection with RNA transcripts of five S52/JFH1 recombinants containing these combinations of putative adaptive mutations performed as efficiently as J6/JFH viruses in Huh7.5 cells and were all genetically stable after viral passage.

Cell culture system of a hepatitis C genotype 3a and 2a chimera

The present invention relates to molecular approaches to the production of nucleic acid sequences, which comprises the genome of infectious hepatitis C virus. In particular, invention provides nucleic acid sequences which comprise the genomes of infectious hepatitis C viruses of either genotype 3a (strain S52) or genotype 4a (strain ED43). The invention therefore relates to the use of the nucleic acid sequences and polypeptides encoded by all or part of the sequences in the development of vaccines and diagnostic assays for HCV and in the development of screening assays for the identification of antiviral agents for HCV. The invention therefore also relates to the use of viral particles derived from laboratory animals infected with S52 and ED43 viruses.

The present inventors used the previously developed H77/JFH 1(1a/2a), J4/JFH(1b/2a), J6/JFH(2a/2a), J8/JFH 1(2b/2a), S52/JFH 1(3a/2a), SA13/JFH 1(5a/2a) and HK6a/JFH 1T(6a/2a) constructs for the deletion of Hypervariable Region 1 (HVR1) to construct viable, JFH 1 (genotype 2a) based, genomes. The present inventors serially passaged the viruses in cell culture obtaining relatively high HCV RNA titers and infectivity titers. Sequence analysis of the viruses identified mutations adapting H77/JFH 1(1a/2a), J8/JFH(2b/2a), S52/JFH 1(3a/2a) and J4/JFH 1(1b/2a) to the HVR1 deletion.

Infectious genotype 1a, 1b, 2a, 2b, 3a, 5a, 6a and 7a hepatitis C virus lacking the hypervariable region 1 (HVR1)

Hepatitis C reporter viruses containing Core through NS2 of prototype isolates of all major HCV genotypes and the remaining genes of isolate JFH1, by insertion of reporter genes in domain III of HCV NS5A were developed. A deletion upstream of the inserted reporter gene sequence conferred favorable growth kinetics in Huh7.5 cells to these viruses. These reporter viruses can be used for high throughput analysis of drug and vaccine candidates as well as patient samples. JFH1-based intergenotypic recombinants with genotype specific homotypic 5'UTR, or heterotypic 5&#x2032;UTR (either of genotype 1a (strain H77) or of genotype 3a (strain S52)) were also developed. The present inventors additionally developed J6/JFH1 recombinants with the 5&#x2032;UTR of genotypes 1-6. These recombinants with different 5&#x2032;UTRs are a useful to study the function of the 5&#x2032;UTR in a genotype specific manner.

Hepatitis C virus expressing reporter tagged NS5A protein

The present inventors developed hepatitis C virus 2b/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and the complete NS2 were replaced by the corresponding genes of the genotype 2b reference strain J8. Sequence analysis of recovered 2b/2a recombinants from 2 transfection experiments revealed that 2b/2a was genetically stable. Conclusion: The developed 2b/2a viruses provide a robust in vitro tool for research in HCV genotype 2b, including vaccine studies and functional analysis.

Efficient cell culture system for hepatitis C virus genotype 2B

The present inventors developed 5a/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and all of or part of NS2 were replaced by the corresponding genes of the genotype 5a reference strain SA13. Compared to the J6/JFH control virus, after transfection of in vitro transcripts in Huh7.5 cells, production of infectious viruses was delayed. However, in subsequent viral passages efficient spread of infection and HCV RNA titers as high as for J6/JFH were obtained. Infectivity titers were at all time points analyzed comparable to J6/JFH control virus. Sequence analysis of recovered 5a/2a recombinants from 2 serial passages and subsequent reverse genetic studies revealed adaptive mutations in p7, NS2 and/or NS3. Infectivity of the 5a/2a viruses was CD81 and SR-BI dependant, and the recombinant viruses could be neutralized by chronic phase sera from patients infected with genotype 5a. Conclusion: The developed 5a/2a viruses provide a robust in vitro tool for research in HCV genotype 5, including vaccine studies and functional analyses of an increasingly important genotype in South Africa and Europe.

Efficient cell culture system for hepatitis C virus genotype 5A

The present inventors developed hepatitis C virus 6a/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and the complete NS2 were replaced by the corresponding genes of the genotype 6a reference strain HK6a. Sequence analysis of recovered 6a/2a recombinants from 2 transfection experiments and subsequent reverse genetic studies revealed adaptive mutations in E1 and E2. Conclusion: The developed 6a/2a viruses provide a robust in vitro tool for research in HCV genotype 6, including vaccine studies and functional analysis.

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Frederiksberg C, Denmark

Judith Margarete Gottwein