Edward Berger (NIAID)
Miriam Triyatni (NIAID)
- Proof of concept.
- Preliminary tools and techniques for screening strategies.
HCV has infected an estimated 3% of the world population in whom viral infection persists for more than two third of the cases, often resulting in life-threatening complications. The standard of care (pegylated interferon alpha-2 plus ribavirin) is efficient in only 50% of treated patients, costly and has numerous side effects. In addition, viral resistance to newly developed drugs -- targeting viral protease or RNA polymerase -- has been described, but no vaccine is yet available. The difficulty in developing HCV vaccines is largely due to the broad sequence-diversity displayed by HCV, the frequent occurrence of viral mutations within immunogenic epitopes in vivo, and the lack of proper standard/definition for viral neutralization.
One alternative strategy in HCV-vaccine or drug development comprises measuring viral entry, the first step in viral infection. Such measurements are limited by the available screening systems, in that, HCV pseudo-typed retroviral particles have a different envelope conformation and contain foreign components that are likely to interfere with the measured HCV entry. Moreover, HCV lab strain requires intensive replication for its in vitro production, resulting in numerous mutations that impede development of convenient screening tools.
The inventors have developed a system for generating infectious HCV particles and HCV-like particles (HCV-LP) suitable for a qualitative single-cycle entry assay, completely independent of HCV replication. To adapt this system as a single assay to study HCV-LP entry, HCV non-structural genes were replaced with a heterologous gene that upon viral-entry triggers firefly luciferase and EGFP expressions in target as well as non-permissive cells. The pretreatment of HCV-replication permissive HuH-7.5 cells with siRNA targeting HCV candidate receptors inhibited viral entry. These new systems enable production of authentic HCV infectious particles as well as HCV-LPs suitable for single-cycle entry assays adaptable to high throughput screening.
- Screening a library expressed in non-permissive cells for identifying new HCV candidate receptor(s) or entry molecule(s).
- Testing drugs or compounds inhibiting HCV particle entry or viral genome uncoating, or neutralizing antibodies in target cells.
- Testing drugs or compounds that inhibit virus assembly, maturation and/or egress, or genome packaging, in producer cells.
- Incorporating a 'tag' in the genome of various HCV genotypes to more conveniently study virus spreading and dissemination in an organ, tissue and/or small animal model.
- Enhancing immune response in patients: one way to trigger high level anti-HCV immunity is by isolating antigen-presenting cells from patients and incubating them with HCV particles produced with this system using replication-defective viral genome (with or without an immunogenic tag and/or in combination with other viral epitopes) and eventually re-inject their primed cells to the patients.
- These systems do not use pseudo-typed HCV particles, i.e. no foreign proteins present in the virus particles.
- Particle production in the producing cells is independent of HCV RNA replication, hence avoids the occurrence of adaptive mutations that could be detrimental for virus particle's infectivity or could alter tags or nucleotide sequences incorporated in the viral genome.
- These systems are not specifically dedicated to HCV of a particular genotype, i.e. they can be used to generate HCV particles of various genotypes without requiring the use of chimeras.