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-9850468-B2
Publication Date
2017-12-26
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.
Core Innovation
The invention relates to the discovery and characterization of a hepatitis E virus (HEV) genotype 3 strain, named Kernow-C1, isolated from a chronically infected patient. This virus exhibits an unusual ability to grow well in various cell cultures, notably human hepatoma cells (HepG2/C3A). The invention provides cell cultures, vectors, infectious cDNA clones, and vaccine compositions derived from such viruses that are adapted to replicate efficiently in vitro.
The problem being addressed is the known limited ability of HEV to replicate in cell culture, which impedes the study of the virus life cycle, host range, and zoonotic transmission, as well as vaccine development. Historically, HEV was mainly considered an acute self-limiting infection restricted to developing countries, but recent findings of chronic HEV infections in immunocompromised patients and evidence of zoonotic transmission (genotype 3 and 4) reveal the importance of having replicative cell culture systems. Thus, there is a need for HEV genotype strains that can replicate in cell cultures for research and vaccine development purposes.
The invention provides the identification of a genotype 3 HEV strain that acquires an insertion of 174 ribonucleotides (encoding 58 amino acids) of human ribosomal protein gene S17 in the hypervariable region (HVR) of ORF1 after adaptation to human hepatoma cells. This insertion, along with additional mutations, confers enhanced replication ability in cell culture. The invention also includes infectious cDNA clones comprising this insert, cell culture systems supporting viral replication, and uses of viruses or polypeptides derived from these clones for vaccine production, diagnostics, antiviral screening, and assessment of viral clearance processes.
Claims Coverage
The patent claims encompass multiple independent claims focusing on infectious HEV cDNA clones with a key insert in ORF1, infectious clones of different genotypes, and methods involving these clones and derived viruses.
Infectious HEV type 3 cDNA clone with S17 or S19 protein insert in ORF1 hypervariable region
An HEV genotype 3 infectious cDNA clone comprising an insert encoding 20 to 100 amino acids with at least 85% identity to human ribosomal S17 or S19 protein positioned in the hypervariable region of ORF1, enhancing replicative capacity.
Enhanced replication due to insert encoding S17 or S19 protein segment
The insert encodes a polypeptide sequence of 20 to 100 amino acids matching human ribosomal S17 or S19 protein, which confers improved replication to the HEV clone.
Infectious HEV cDNA clones with at least 75% sequence identity and ORF1 insert
HEV cDNA clones having at least 75% identity to the reference sequence and containing the ORF1 hypervariable region insert related to ribosomal S17 or S19 proteins, supporting replication.
Infectious HEV genotype 1 cDNA clones with ORF1 insert
HEV genotype 1 infectious cDNA clones comprising an insert in the ORF1 hypervariable region encoding 20 to 100 amino acids with at least 85% identity to human ribosomal S17 or S19 protein, enhancing replication in culture.
Cell culture systems harboring infectious HEV clones with ORF1 insert
Cells comprising infectious cDNA clones containing the hypervariable region insert facilitating replication and propagation of HEV in vitro.
Methods of producing immunogenic compositions using RNA from infectious clones
Introducing RNA transcripts obtained from infectious HEV cDNA clones containing the ORF1 insert into cells to produce virus for vaccine or immunogen production.
Methods assessing viral clearance using HEV produced from infectious clones
Introducing HEV produced from infectious cDNA clones with the ORF1 insert into products such as water, blood, or food, subjecting the product to virus inactivation/removal process, and determining residual virus level to assess efficacy.
The claims cover infectious HEV cDNA clones of genotype 3 and 1 that contain a specific insert in the ORF1 hypervariable region encoding human ribosomal protein sequences, cell culture systems using these clones, methods of producing vaccines from the viruses generated by these clones, and methods for assessing viral clearance in products, emphasizing the significance of the ORF1 insert in enhancing viral replication in culture.
Stated Advantages
The insert of a human ribosomal protein gene sequence in the hypervariable region of ORF1 significantly enhances HEV replication efficiency in diverse cell cultures.
The infectious cDNA clones enable the production of viruses capable of infecting multiple host species' cells, facilitating the study of HEV host range and zoonotic transmission.
The infectious clones serve as platforms for development of vaccines and immunogenic compositions against HEV genotype 3 strains.
The virus produced from infectious clones can be used as indicators to assess the efficacy of viral removal and inactivation processes in products such as water, blood, and food.
Documented Applications
Use of infectious HEV cDNA clones to produce virus in cell cultures for vaccine and immunogenic composition development.
Use of recombinant HEV proteins or viruses produced from the clones as diagnostic agents for detecting HEV infection.
Use of the infectious clones and their viruses for screening candidate antiviral agents in cell culture systems supporting viral replication.
Use of HEV viruses produced from infectious clones as indicators to test the efficacy of viral clearance or inactivation in products such as water, food for animal and human consumption, and blood.
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