Hvidovre Hospital

Kobenhavns Universitet

Tanja Bertelsen Jensen

Jens Bukh

Judith Margarete Gottwein

Troels Kasper Hoyer Scheel

Jannick Prento

Troels Kasper Høyer Scheel

Jesper Eugen-Olsen

Yiping Li

Christian Kjærulff Mathiesen

Jacob Bo Lademann

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 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 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 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.

Efficient cell culture system for hepatitis C virus genotype 6A

The present inventors developed hepatitis C virus 1a/2a and 1b/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and NS2 were replaced by the corresponding genes of the genotype Ia reference strain H77C or TN or the corresponding genes of the genotype Ib reference strain J4. Sequence analysis of recovered 1a/2a and 1b/2a recombinants from 2 serial passages and subsequent reverse genetic studies revealed adaptive mutations in e.g. p7, NS2 and/or NS3. In addition, the inventors demonstrate the possibility of using adaptive mutations identified for one HCV isolate in generating efficient cell culture systems for other isolates by transfer of mutations across isolates, subtypes or major genotypes. Furthermore neutralization studies showed that viruses of e.g. genotype 1 were efficiently neutralized by genotype Ia, 4a and 5a serum, an effect that could be utilized e.g. in vaccine development and immunological prophylaxis. The inventors in addition demonstrate the use of the developed systems for screening of antiviral substances in vitro and functional studies of the virus, e.g. identification of receptors required for HCV entry.

Efficient cell culture system for hepatitis C virus genotype 1A and 1B

Genotype 7a has been identified recently, thus not much is known about the biology of this new, major HCV genotype. The present inventors developed hepatitis C virus 7a/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 7a strain QC69 and characterized them in Huh7.5 cells. Sequence analysis of 7a/JFH1 recombinants recovered after viral passage in Huh7.5 cells following 4 independent transfection experiments revealed adaptive mutations in Core, E2, NS2, NS5A and NS5B. In reverse genetic studies the importance of these mutations for improved growth kinetics was shown. Adapted 7a/JFH1 viruses showed growth kinetics, infectivity and RNA titers comparable to a previously developed 3a/JFH1 reference virus. Conclusion: The developed 7a/JFH1 viruses provide a robust in vitro tool for research in HCV genotype 7, including vaccine studies and functional analyses.

Growing community of inventors

Frederiksberg C, Denmark

Tanja Bertelsen Jensen