Infectious hepatitis C viruses of genotype 3A and 4A and uses thereof
Inventors
Gottwein, Judith M. • Scheel, Troels Kasper Hoyer • Purcell, Robert • Bukh, Jens
Assignees
Hvidovre Hospital • US Department of Health and Human Services
Publication Number
US-10100346-B2
Publication Date
2018-10-16
Expiration Date
2030-09-16
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Abstract
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.
Core Innovation
The invention provides nucleic acid sequences that comprise the genomes of infectious hepatitis C viruses of genotype 3a (strain S52) and genotype 4a (strain ED43). It includes isolated nucleic acid molecules encoding these viruses, polypeptides encoded by these sequences, and the use of these sequences and polypeptides in producing vaccines, diagnostic assays, and screening assays for antiviral agents against hepatitis C virus (HCV). The invention also relates to viral particles derived from laboratory animals infected with S52 and ED43 viruses and methods for producing HCV by transfecting host cells with RNA transcripts of these nucleic acid sequences.
The problem addressed stems from the extensive genetic heterogeneity of HCV and the lack of infectious clones for all major genotypes, which has hindered the development of universally effective therapies and vaccines. Furthermore, unlike genotype 2a strain JFH1, other HCV isolates of genotypes 3a and 4a did not grow in cell culture, limiting the study of viral replication and pathogenesis and impeding the development of efficient cell culture and animal models. The invention thus aims to provide infectious molecular clones of genotypes 3a and 4a, allowing functional studies, vaccine development, antiviral screening, and the use of chimeric viruses to explore viral gene functions.
Claims Coverage
The claims include one independent claim directed to an isolated nucleic acid molecule comprising a mutated HCV genotype 4a genome, and related constructs, cells, methods of virus production, antiviral agent screening, and cell susceptibility testing. The main inventive features cover the structure of the mutated nucleic acid molecule and methods using it.
Isolated mutated nucleic acid molecule of HCV genotype 4a
An isolated nucleic acid molecule comprising a mutated HCV genotype 4a genome according to SEQ ID NO:4 wherein sequences encoding E1, E2, P7, and NS2 genes are deleted, a part of the Core gene is deleted, and a heterologous reporter gene is inserted. The undeleted HCV genomic portions retain at least 98% sequence identity to a reference sequence with corresponding deletions.
High sequence identity of mutated molecule to reference sequences
The molecule encodes an amino acid sequence with at least 98% identity to a reference amino acid sequence with deletions in E1, E2, P7, and NS2 proteins and part of Core. Also, a version with at least 99% nucleic acid identity to a corresponding reference sequence with these deletions is claimed.
DNA construct and RNA transcript encoding mutated virus
A DNA construct comprising the mutated nucleic acid molecule and its RNA transcript encoding the mutated HCV genotype 4a genome.
Transfected cells containing mutated nucleic acid
Cells transfected with the DNA construct or the RNA transcript encoding the mutated HCV genome.
Method for producing mutated hepatitis C virus genome
A method of producing the mutated hepatitis C virus by transfecting a host cell with the RNA transcript encoding the mutated genome.
Method for assaying candidate antiviral agents
A method comprising exposing a cell containing the mutated hepatitis C virus genome to a candidate antiviral agent and measuring virus replication or correlates thereof in the cell, including by RT-PCR, Western blot, immunofluorescence, reporter gene activity, or immuno-staining.
Method for determining susceptibility of cells to HCV replication
A method comprising growing animal cells in vitro, transfecting them with the mutated nucleic acid, and determining whether they show indicators of HCV replication, e.g., by RT-PCR, Western blot, immunofluorescence, or reporter gene activity.
The claims cover an engineered nucleic acid molecule of HCV genotype 4a with deletions in structural genes and insertion of a reporter gene, related recombinant constructs, and biological materials. They also cover methods for producing virus, screening antiviral agents, and evaluating cell susceptibility, focusing on the mutated genome's use for these purposes.
Stated Advantages
The invention enables production of infectious HCV genomes of genotypes 3a and 4a, which were previously unavailable as infectious clones.
It facilitates the development of vaccines and diagnostic assays specific for these genotypes.
It provides tools for screening antiviral agents using molecular clones and viral proteins, thereby accelerating antiviral drug discovery.
The system allows in vivo infectivity and study of HCV pathogenesis using animal models.
Documented Applications
Development and production of vaccines to prevent hepatitis C in mammals, especially humans, by administering nucleic acid sequences, polypeptides, or viruses of genotype 3a and 4a.
Use in diagnostic assays for detecting hepatitis C virus in biological samples through antibodies generated from the disclosed polypeptides and viruses.
Screening assays for identification of antiviral agents against HCV using cells or animals infected or transfected with the nucleic acid sequences or viral enzymes.
Use in research to identify cell lines capable of supporting replication of HCV in vitro and to study virus replication, virulence, and immune responses.
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