Immortalized avian cell lines for virus production
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Publication Number
US-8940534-B2
Publication Date
2015-01-27
Expiration Date
Abstract
The present invention relates to immortalized avian cell lines suitable for production of biologicals or viruses for vaccination. In particular, the cell lines are derived from primary cells which are transformed with at least two viral or cellular genes, one of which causes cell cycle progression whereas the other interferes with innate protective mechanisms of the cell induced by dysregulated replication. The invention moreover relates to the production of said immortalized cell lines and their use for producing biologicals or viruses for vaccination.
Core Innovation
The invention provides immortal duck cell lines for viral vaccine virus production by designer transformation of primary avian cells. The designer transformation introduces a first viral gene and a second viral gene into duck cells so that both are integrated and stably expressed in the resulting immortal duck cell line.
The first viral gene is a Mastadenovirus early region 1A (E1A) gene, with the E1A gene sequence defined as bp 1193 to 2309 of SEQ ID NO: 7 or the sequence complementary to bp 4230 to 3113 of SEQ ID NO: 9. E1A expression is driven by a human phosphoglycerate kinase (PGK) promoter.
The second viral gene codes for a Mastadenovirus early region 1B 55K (E1B 55K) protein, with the E1B gene sequence defined as bp 1145 to 3007 of SEQ ID NO: 8 or the sequence complementary to bp 2345 to 550 of SEQ ID NO: 9. E1B expression is driven by a mouse cytomegalovirus (CMV) immediate early promoter or a human herpesvirus thymidine kinase (tk) promoter.
The document also addresses safety and risk minimization by defining pathways and separating expression units, including minimizing transformation risk and limiting endogenous avian retrovirus and reverse transcriptase activity. The immortalized duck cell lines are described as suitable for cGMP conditions and are characterized by testing for reverse transcriptase activity, including Q-PERT showing duck cells lack RT activity compared with controls.
Claims Coverage
The independent claim clm-00001 defines an immortal duck cell line using two stably integrated and expressed Mastadenovirus early region genes, E1A and E1B 55K, with specified sequence ranges and promoter control, comprising three principal inventive features. Dependent claims further refine the cell sourcing and cell-line/culture properties and narrow related use cases.
Immortal duck cell line with stably integrated Mastadenovirus E1A and E1B 55K genes
A duck cell line comprising a first viral gene comprising a Mastadenovirus early region 1A (E1A) gene and a second viral gene coding for a Mastadenovirus early region 1B 55K (E1B 55K) protein, wherein the first and second viral genes are integrated and stably expressed in the duck cell line.
E1A promoter control via human PGK promoter with specified E1A sequence ranges
The E1A gene is driven by a human phosphoglycerate kinase (PGK) promoter, wherein the E1A gene has the sequence of bp 1193 to 2309 of SEQ ID NO: 7 or the sequence complementary to bp 4230 to 3113 of SEQ ID NO: 9.
E1B 55K promoter control with specified E1B sequence ranges
The E1B gene codes for a Mastadenovirus early region 1B 55K (E1B 55K) protein and is driven by a mouse cytomegalovirus (CMV) immediate early promoter or a human herpesvirus thymidine kinase (tk) promoter, wherein the E1B gene has the sequence of bp 1145 to 3007 of SEQ ID NO: 8 or the sequence complementary to bp 2345 to 550 of SEQ ID NO: 9.
Overall, the claim coverage centers on an immortal duck cell line in which stably integrated Mastadenovirus E1A and E1B 55K genes with explicitly defined sequence ranges are expressed under promoter control, forming the functional basis for the described immortalization while additional dependent features refine sourcing and performance-related properties.
Stated Advantages
Supports viral vaccine virus production using immortalized duck cell lines.
Safety and risk minimization by defining pathways and separating expression units.
Minimizes transformation risk and endogenous avian retrovirus/reverse transcriptase-related concerns.
Duck cells are described as lacking reverse transcriptase activity based on Q-PERT.
Described suitability for cGMP conditions.
Enhanced MVA amplification in duck retina and somite-derived cells, with quantified improvements in burst sizes and titers described as part of the examples.
Documented Applications
Viral vaccine virus production using the immortal duck cell lines.
MVA (modified vaccinia Ankara) amplification in duck retina and duck somite-derived cells, with described increased titers/burst sizes and stability upon serial passaging.
Contacting with pox virus or a pox virus strain MVA as part of the defined use with the immortal duck cell lines derived from duck somites or duck retina.
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