Method for rescuing and producing a virus in avian cells
Inventors
Batard, Luc • Freslon-Evain, Caroline • Perugi, Fabien • Schwamborn, Klaus
Assignees
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Publication Number
US-12053518-B2
Publication Date
2024-08-06
Expiration Date
Abstract
The present invention relates to methods of rescue and/or propagation of paramyxovirus species, particularly wherein both rescue and propagation are carried out in the same cell type; i.e., without the use of helper cells for viral rescue. The paramyxoviruses produced by the disclosed methods may encompass wild-type viruses, chimeric viruses, recombinant viruses or engineered viral products such as virus like particles (VLP). Viruses and/or viral products produced in the method according to the current invention are suitable for medical or veterinary use in such applications as treating or preventing infectious diseases, particularly avian paramyxovirus and human respiratory virus infections, and cancer treatment.
Core Innovation
The invention relates to a method of producing infectious paramyxovirus particles by transfecting a duck cell line with a set of vectors configured for T7-driven expression and then culturing the transfected duck cell line to produce infectious particles. The vector carrying the paramyxovirus nucleic acid sequence is under T7 control and includes a T7 promotor sequence defined by SEQ ID NO: 6 and a T7 terminator sequence defined by SEQ ID NO: 7. A T7 RNA polymerase expression vector is provided in the same transfection to enable transcription of the T7-controlled paramyxovirus nucleic acid.
The core framework additionally uses three further expression vectors encoding paramyxovirus phosphoprotein (P), nucleoprotein (N), and polymerase (L) coding sequences. This arrangement supports production of infectious paramyxovirus particles from T7-controlled nucleic acid together with the required paramyxovirus protein components. In the disclosed embodiments, the approach is applied to Newcastle Disease Virus, including NDV LaSota, NDV Hitchner 131, and NDV AE2240 nucleic acids, and can further encode wild-type genomes, chimeric genomes, recombinant genomes, or virus-like particles containing NDV elements.
Genetic engineering features are described for modifying the paramyxovirus nucleic acid sequence using restriction sites to allow insertion of heterologous coding sequences between paramyxovirus protein coding sequences, and the nucleic acid constructs may include elements such as restriction sites for inserting foreign genes. The document also describes heterologous antigen expression examples, including foreign antigen coding sequences such as hMPV F and hMPV M and sets of antigens including RSV and other listed antigen examples, with sequence identifiers provided in the disclosures.
Claims Coverage
The document includes one independent claim that defines a T7-based reverse-genetics production method in a duck cell line using a T7-controlled paramyxovirus nucleic acid vector plus a T7 RNA polymerase expression vector together with separate expression vectors for P, N, and L; dependent claims narrow the paramyxovirus nucleic acid to specific NDV strain inputs and to specific genome/particle types, and add genetic-design features and sequence-selection constraints.
T7-controlled paramyxovirus nucleic acid vector with defined T7 promotor and terminator
A vector comprising a paramyxovirus nucleic acid sequence under T7 control additionally comprises a T7 promotor sequence as defined by SEQ ID NO: 6 and a T7 terminator sequence as defined by SEQ ID NO: 7.
T7 RNA polymerase supplied by a T7 RNA polymerase expression vector
A T7 RNA polymerase expression vector is transfected together with the T7-controlled paramyxovirus nucleic acid vector.
Separate expression vectors for paramyxovirus P, N, and L proteins
Three further expression vectors comprise, respectively, paramyxovirus phosphoprotein (P), nucleoprotein (N), and polymerase (L) coding sequences.
Duck cell line transfection followed by culturing for infectious particle production
The method comprises transfecting a duck cell line and then culturing the transfected duck cell line under conditions for production of infectious paramyxovirus particles.
Newcastle Disease Virus strain nucleic acid selection
The method specifies that the paramyxovirus nucleic acid is from Newcastle Disease Virus (NDV), including NDV LaSota, NDV Hitchner 131, or NDV AE2240 strain nucleic acid.
Paramyxovirus genome and particle type selection for encoded material
The method specifies that the paramyxovirus nucleic acid encodes either a wild-type Newcastle Disease Virus (NDV) genome, a chimeric NDV genome, a recombinant NDV genome, or a virus-like particle containing NDV elements.
Recombinant NDV genome tied to a specific sequence identifier
The method specifies a recombinant NDV genome defined by SEQ ID NO: 16.
Restriction-site modification to insert heterologous coding sequences
The method includes modifying a paramyxovirus nucleic acid sequence by adding restriction enzyme sites so that a heterologous coding sequence can be inserted between the paramyxovirus protein coding sequences.
Immunogenic hMPV F selection with sequence-identity constraints
The method specifies that the hMPV F protein is selected from the group consisting of SEQ ID NOs: 17-20 and immunogenic variants having at least 95% sequence identity to any one of SEQ. ID NOs: 17-20.
Overall, claim coverage centers on a T7-driven paramyxovirus particle production method in a duck cell line using a T7 promotor/terminator flanked paramyxovirus nucleic acid vector, a T7 RNA polymerase expression vector, and separate P, N, and L expression vectors, with dependent claims narrowing the paramyxovirus inputs to defined NDV strains and genome/particle types, adding restriction-site-based heterologous insertion, and specifying sequence-identity constraints for selected antigen proteins such as hMPV F.
Stated Advantages
Enables production of infectious paramyxovirus particles using the disclosed T7-controlled reverse-genetics arrangement in a duck cell line.
Supports use of different paramyxovirus nucleic-acid formats, including wild-type genomes, chimeric genomes, recombinant genomes, and virus-like particles containing NDV elements.
Allows incorporation of heterologous coding sequences between paramyxovirus protein coding sequences using added restriction enzyme sites.
Provides selection constraints for immunogenic antigen protein variants, including hMPV F proteins with at least 95% sequence identity to defined SEQ ID NOs.
Documented Applications
Production of infectious paramyxovirus particles, including infectious NDV-based particles derived from NDV LaSota, NDV Hitchner 131, or NDV AE2240 nucleic acids.
Expression of heterologous antigens within the disclosed paramyxovirus nucleic-acid constructs, including examples involving hMPV F and hMPV M and referenced antigen sets (e.g., RSV-associated examples).
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