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 • Government of the United States of America
Publication Number
US-8946398-B2
Publication Date
2015-02-03
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, 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 comprising the genomes of infectious hepatitis C viruses of genotype 3a (strain S52) and genotype 4a (strain ED43). It relates to methods for producing hepatitis C virus by transfecting a host cell with RNA transcripts of these nucleic acids, as well as polypeptides encoded by these sequences. The invention extends to the use of these nucleic acid sequences and encoded polypeptides in the development of vaccines, diagnostic assays, and screening assays for antiviral agents against hepatitis C virus.
The background identifies a significant problem with hepatitis C virus (HCV) infection: its genetic heterogeneity, limited cell culture systems for reproducible viral growth, and lack of convenient small animal models hinder progress in understanding HCV replication and development of effective therapies and vaccines. While infectious clones have been developed for other genotypes, the absence of clones for major genotypes like 3a and 4a is problematic, especially given genotype-dependent differences in disease severity and response to therapy. The invention addresses this gap by providing infectious clones of genotype 3a and 4a, which enable molecular analysis, study of viral pathogenesis, and support development of vaccines and antiviral agents.
Claims Coverage
The patent includes one independent claim focused on an isolated nucleic acid molecule of hepatitis C virus genotype 3a and several dependent claims related to constructs, transfections, viruses produced, methods for antiviral screening and cell susceptibility, and compositions.
Isolated nucleic acid molecule encoding infectious hepatitis C virus genotype 3a
An isolated nucleic acid molecule comprising a consensus sequence encoding human hepatitis C virus genotype 3a, capable of infectivity in vivo, encoding the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence with at least 98% identity to SEQ ID NO: 1 over its entire length.
Recombinant DNA construct comprising the nucleic acid molecule
A recombinant DNA construct containing the nucleic acid molecule encoding the infectious hepatitis C virus genotype 3a as described.
RNA transcript of the DNA construct
An RNA transcript derived from the recombinant DNA construct encoding hepatitis C virus genotype 3a.
Cells transfected with DNA construct or RNA transcript
Cells transfected with either the recombinant DNA construct or its RNA transcript capable of producing hepatitis C virus genotype 3a.
Hepatitis C virus produced by transfected cells
Production of hepatitis C virus genotype 3a by cells transfected with the recombinant DNA construct or RNA transcript.
Hepatitis C virus genome comprising the nucleic acid molecule
Hepatitis C virus whose genome includes the nucleic acid molecule encoding genotype 3a virus.
Method for producing hepatitis C virus via transfection
A method for producing hepatitis C virus comprising transfection of a host cell with an RNA transcript of the nucleic acid molecule encoding genotype 3a virus.
Method for assaying candidate antiviral agents
A method for assaying candidate antiviral agents by exposing a cell containing hepatitis C virus genotype 3a to a candidate antiviral agent and measuring presence or absence of viral replication or correlates thereof.
Method for determining cell susceptibility to HCV infection
A method for determining in vitro susceptibility of animal cells to support HCV infection by transfecting cells with the nucleic acid molecule and detecting indicators of viral replication.
Pharmaceutical composition comprising the nucleic acid molecule
A composition comprising the nucleic acid molecule encoding hepatitis C virus genotype 3a suspended in a pharmaceutically acceptable diluent or excipient.
Nucleic acid molecule encoding genotype 3a with sequence identity or exact sequence
Nucleic acid molecule encoding genotype 3a virus comprising nucleic acid sequence with at least 98% identity to SEQ ID NO: 3 or the exact sequence of SEQ ID NO: 3.
The claims cover the isolated infectious nucleic acid molecule of hepatitis C virus genotype 3a, recombinant constructs, their RNA transcripts, their use to produce infectious virus in vitro and in vivo, methods for antiviral screening and susceptibility testing, and pharmaceutical compositions comprising the nucleic acids.
Stated Advantages
Provides infectious clones of hepatitis C virus genotypes 3a and 4a, facilitating study of viral replication and pathogenesis.
Enables development of vaccines and diagnostic assays specific for diverse HCV genotypes.
Supports screening assays for identification of antiviral agents targeting hepatitis C virus.
Allows production of attenuated viruses and chimeric constructs for vaccine development and functional studies.
Documented Applications
Development of vaccines to prevent hepatitis C infection in mammals, especially humans, using nucleic acid sequences, viruses, or polypeptides derived from genotypes 3a and 4a.
Diagnostic assays for detecting hepatitis C virus infection using antibodies produced against polypeptides or viruses encoded by the nucleic acid sequences.
Screening assays for antiviral agents by testing candidate compounds against hepatitis C virus replication in cell cultures or animal models transfected or infected with genotype 3a or 4a nucleic acid sequences or viruses.
Identification of cell lines susceptible to HCV infection by transfection with nucleic acid sequences.
Production of antibodies for therapeutic and prophylactic treatment of hepatitis C virus infection.
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