Infectious hepatitis E virus genotype 3 recombinants
Inventors
Emerson, Suzanne U. • Shukla, Priyanka • Nguyen, Hanh T. • Purcell, Robert H. • Dalton, Harry R. • Bendall, Richard
Assignees
Royal Cornwall Hospital Trust • US Department of Health and Human Services
Publication Number
US-9181530-B2
Publication Date
2015-11-10
Expiration Date
2032-01-10
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Abstract
The invention relates to the discovery of an HEV strain from a chronically infected patient. The virus grow unusually well in numerous cell cultures. Thus, the invention provides cell cultures, vectors, and vaccine compositions based on the virus. The invention relates, in part, on the identification of a new strain of HEV genotype 3 virus. Strain Kernow-C1 (genotype 3) of HEV, which was isolated from a chronically infected patient, was used to identify human, pig and deer cell lines permissive for infection. Adaptation of the Kernow-C 1 strain to growth in human hepatoma cells selected for a rare virus recombinant that contained an insertion of 174 ribonucleotides (58 amino acids) of a human ribosomal protein gene and additional mutations.
Core Innovation
The invention relates to the discovery and characterization of a new strain of hepatitis E virus (HEV) genotype 3, designated Kernow-C1, isolated from a chronically infected patient. This virus strain grows unusually well in numerous cell cultures, including human, pig, and deer cell lines. Adaptation of the Kernow-C1 strain to growth in human hepatoma cells selected for a rare virus recombinant containing an insertion of 174 ribonucleotides (encoding 58 amino acids) derived from a human ribosomal protein gene, along with additional mutations.
The problem being solved addresses the difficulties in culturing HEV, particularly genotype 3 strains, which replicate to low titers in vivo and have been exceedingly difficult to grow in cell culture. HEV infection was historically considered an acute, self-limiting disease not progressing to chronicity, but recently chronic HEV infections have been identified, especially genotype 3 infections in immunocompromised individuals. The epidemiology, host range, and zoonotic transmissions of HEV are not fully understood, and there is a need for HEV genotype strains capable of replicating efficiently in cell culture to facilitate research, vaccine development, and the study of cross-species infection.
The invention includes infectious cDNA clones of replicative genotype 3 HEV with an insertion in the hypervariable region of ORF1, which enhances viral replication and cell culture adaptation. The invention also provides cell culture systems comprising such HEV variants that allow studying zoonotic spread and enable the production of vaccines and immunogenic compositions. In particular, the recombinant viruses with the insertion derived from the human ribosomal protein S17 confer enhanced replication abilities in cell culture, which was demonstrated through sequential passaging, cloning, and mutagenesis studies.
Claims Coverage
The patent claims cover several inventive features relating to infectious hepatitis E virus cDNA clones, including insertions in the ORF1 hypervariable region that enhance replication, their sequences, and methods for producing viruses and using them in cell culture systems.
Insertion improving replication in the hypervariable region of ORF1 in HEV genotype 3 cDNA clones
An infectious HEV genotype 3 cDNA clone comprising an insert in the hypervariable region of ORF1 that encodes an in-frame polypeptide sequence of 35 to 65 amino acids having at least 85% identity to SEQ ID NO:9, with the insert positioned such that the first amino acid starts at position 750 relative to SEQ ID NO:6.
Infectious HEV cDNA clones with specified sequence identity and insert length
HEV cDNA clones having at least 75% identity to SEQ ID NO:1 and comprising an insert in the hypervariable region of ORF1 encoding an in-frame polypeptide sequence of 35 to 65 amino acids with at least 85% identity to SEQ ID NO:9, optionally possessing at least 85% or 95% identity to SEQ ID NO:1 and encoding the polypeptide sequence of SEQ ID NO:9.
Infectious genotype 1 or genotype 3 HEV cDNA clones with an insert in ORF1
Genotype 1 or genotype 3 infectious HEV cDNA clones that include an insert in the hypervariable region of ORF1 encoding an in-frame polypeptide sequence of 35 to 65 amino acids with at least 85% identity to SEQ ID NO:9.
Cell culture systems comprising cells harboring infectious HEV cDNA clones
Cell culture systems containing cells comprising the infectious HEV cDNA clones with the described ORF1 insert, supporting virus replication in culture.
Methods of producing viral compositions from infectious HEV cDNA clones
Methods involving introducing RNA transcribed from the infectious HEV cDNA clones into cells to obtain virus, including versions incapable of producing ORF3 protein.
Use of infectious HEV cDNA clones encoding ORF1 inserts to produce viruses lacking ORF3
Methods of producing viruses from infectious cDNA clones that include the ORF1 hypervariable region insert but are incapable of producing ORF3 protein, with virus production in cell culture.
Method of assessing virus status of treated products using HEV from infectious clones
A method of assessing virus removal or inactivation efficacy in products by spiking with HEV produced from infectious cDNA clones containing the ORF1 insert, subjecting to virus treatment, and quantifying remaining virus in the product.
The claims cover infectious hepatitis E virus cDNA clones comprising specific insertions in the hypervariable region of ORF1, which enhance replication, methods for producing viruses from these clones including ORF3-deficient variants, cell culture systems supporting replication, and applications in vaccine production and viral clearance assays.
Stated Advantages
The virus strain grows unusually well in numerous cell cultures, facilitating research and vaccine development.
The insertion in the hypervariable region of ORF1 enhances replication efficiency and cell culture adaptation of HEV genotype 3.
The infectious cDNA clones enable production of high titer infectious virus capable of cross-species infection in cell culture systems.
The model system permits detailed virological studies including zoonotic spread and immune response evaluation.
The ability to assess virus removal or inactivation from blood, food, or water improves safety testing for viral contaminants.
Documented Applications
Use of HEV cDNA clones and viruses in developing vaccines for HEV, including live or inactivated vaccine compositions.
Utilization of cell culture systems comprising human, pig, and deer cells permissive for viral infection studies.
Methods for producing viral compositions from infectious cDNA clones for research and vaccine use.
Application in screening assays for antiviral agents targeting HEV replication.
Use in assessing virus removal or inactivation efficacy in products such as water, food, and blood by spiking with HEV virus produced from infectious clones.
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