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-9683269-B2
Publication Date
2017-06-20
Expiration Date
2030-09-16
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
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 isolated nucleic acid sequences comprising the genomes of infectious hepatitis C viruses (HCV) of genotypes 3a (strain S52) and 4a (strain ED43). These nucleic acid molecules encode the viral polyproteins, including structural and nonstructural proteins necessary for HCV propagation. The invention further encompasses methods for producing infectious HCV by transfecting host cells with RNA transcripts derived from these nucleic acid sequences, as well as polypeptides encoded by these sequences.
The problem addressed is the absence of infectious clones for all major HCV genotypes, which has hampered the development of effective therapies, vaccines, and laboratory models. Prior to this invention, infectious clones existed primarily for genotypes 1a, 1b, and 2a, with genotype 2a's JFH1 strain uniquely capable of robust in vitro culture. The extensive genetic heterogeneity among HCV genotypes necessitates infectious clones representing genotypes 3a and 4a to study viral replication, pathogenesis, and to develop genotype-specific antiviral agents and vaccines.
The invention also relates to the construction of chimeric nucleic acid sequences combining genomic regions from different HCV genotypes or subtypes. These chimeric clones facilitate studies of viral function, growth, and virulence, and support the development of multivalent vaccines. Additionally, the nucleic acid sequences, derived viruses, and encoded polypeptides can be employed in diagnostic assays, vaccine compositions, development of antiviral screening assays, and antibody production. The invention further covers methods for assessing antiviral compounds using cells or animal models infected with viruses derived from these nucleic acid sequences.
Claims Coverage
The patent includes a set of independent claims primarily focused on a mutated nucleic acid molecule of genotype 3a HCV and related biological and assay methods. The main inventive features involve the specific genome modifications, transfection methods, and antiviral agent assays.
Nucleic acid molecule with genotype 3a HCV genome deletions and reporter gene insertion
An isolated nucleic acid molecule comprising a mutated HCV genotype 3a genome based on SEQ ID NO:3, where all sequences encoding E1, E2, P7, and NS2 genes are deleted, a portion of the Core gene is deleted, and a heterologous reporter gene sequence is inserted. The deleted genome portions maintain at least 98% sequence identity to a reference sequence with these deletions.
Nucleic acid molecule encoding deleted genotype 3a polyprotein with high sequence identity
The molecule encodes an amino acid sequence having at least 98% identity to a reference SEQ ID NO:1 sequence with deleted E1, E2, P7, NS2 amino acid sequences, and part of the Core sequence deleted.
High identity nucleic acid molecule encoding mutated genotype 3a HCV genome
The nucleic acid molecule encodes genotype 3a HCV with at least 99% identity to the reference SEQ ID NO:3 with the specified gene deletions.
DNA construct comprising the mutated nucleic acid molecule
A DNA construct comprising the isolated nucleic acid molecule described above encoding the mutated genotype 3a HCV genome.
RNA transcript encoding the mutated hepatitis C virus genome
An RNA transcript of the DNA construct that encodes the mutated HCV genotype 3a genome with defined gene deletions and reporter gene insertion.
Cell transfected with DNA construct
A cell transfected with the DNA construct comprising the mutated hepatitis C virus genome nucleic acid molecule.
Cell transfected with RNA transcript
A cell transfected with the RNA transcript derived from the DNA construct encoding the mutated HCV genome.
Method of producing mutated hepatitis C virus genome
A method for producing the mutated HCV genome by transfecting a host cell with the RNA transcript of the mutated nucleic acid molecule.
Method for assaying antiviral agents using cells with mutated HCV genome
A method for assessing candidate antiviral agents by exposing a cell containing the mutated hepatitis C virus genome to the agent and measuring viral replication or correlates thereof.
Assay measuring HCV replication by multiple detection methods
Measuring replication through negative strand RT-PCR, quantitative RT-PCR, Western blot, immunofluorescence, reporter gene activity, or non-fluorescent immuno-staining.
Method determining susceptibility of cells to support HCV replication
Growing animal cells in vitro, transfecting with the mutated nucleic acid, and determining if cells show signs of HCV replication.
Use of human cells for in vitro susceptibility testing
The method for determining susceptibility of cells is specifically applicable to human cells.
Measurement of replication by molecular and immunological assays
Replication measurement by RT-PCR, Western blot, immunofluorescence, or reporter gene activity in susceptibility assays.
The claims cover an isolated mutated genotype 3a hepatitis C virus nucleic acid molecule with specific gene deletions and reporter insertion, DNA and RNA constructs thereof, transfected cells, methods for virus production and antiviral testing, and assays to determine cell susceptibility to HCV replication. The inventive features focus on the specific genome modifications, utility in producing mutated virus genomes, and application in antiviral agent screening.
Stated Advantages
Provides infectious nucleic acid sequences for HCV genotypes 3a and 4a, facilitating development of genotype-specific therapies and vaccines.
Enables production of authentic viral particles and proteins for vaccine, diagnostic, and antiviral screening applications.
Allows creation of chimeric HCV sequences to study viral determinants of virulence and adaptation.
Enables identification of cell lines supporting HCV replication to overcome previous culture system limitations.
Facilitates screening and identification of antiviral agents targeting various viral proteins using the nucleic acid sequences and encoded enzymes.
Documented Applications
Development of vaccines and pharmaceutical compositions against hepatitis C virus using nucleic acid sequences, viruses, or polypeptides of genotypes 3a and 4a.
Use in diagnostic assays for detecting HCV antigens or antibodies in biological samples.
Screening assays to identify candidate antiviral agents active against HCV by measuring viral replication or protease activity.
Research tools for studying HCV replication, pathogenesis, and viral protein functions via chimeric viruses and mutated sequences.
Production of antibodies against HCV for prophylaxis, therapy, or diagnostic use.
Interested in licensing this patent?