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Cell-based systems for producing influenza vaccines

a cell-based system and vaccine technology, applied in viruses/bacteriophages, transferases, antibody medical ingredients, etc., can solve the problems of 2004 worldwide influenza vaccine shortage, difficult automation of process, significant risk of contamination, etc., and achieve the effect of preventing or minimizing the infection of influenza virus in a human cell

Inactive Publication Date: 2011-06-23
FLUGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is a method for producing influenza viruses using a CHO cell line that has been genetically modified to express a 2,6-sialyltransferase gene (ST6GAL1). This method involves infecting the CHO cells with an influenza virus and isolating the viruses produced from the cells. The CHO cells that express ST6GAL1 have an increased susceptibility to influenza virus infection compared to CHO cells that do not express the gene. The method also results in a higher yield of influenza viruses per cell compared to non-modified CHO cells. The isolated influenza viruses can be formulated into a vaccine. The invention provides a stable cell line that can be used for detection, analysis, and preparation of seed virus in addition to vaccine production. The generation time for producing the influenza vaccine is about 12 to 18 weeks.

Problems solved by technology

The process is difficult to automate, labor-intensive, costly and creates significant risk of contamination.
For instance, the 2004 worldwide influenza vaccine shortage was the result of contamination at a flu vaccine manufacturing facility.
Another significant drawback to current vaccine manufacturing is poor virus yield.
Vaccine production includes a significant fixed cost component, reaching 90% of the total early stage manufacturing costs.
Once many of the fixed costs have been recovered, however, substantial long-term variable costs still remain.
It is estimated that with current egg-based production methods variable costs can run as high as 37% of the price per vaccine dose depending on the volume produced.
Hence, even a two-fold increase in virus yield could have substantial impact on the cost of manufacturing and the availability of supply.
However, MDCK cells are inherently tumorigenic, while Vero and PER-C6® cells have low virus yields and can have problematic side effects.

Method used

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  • Cell-based systems for producing influenza vaccines
  • Cell-based systems for producing influenza vaccines
  • Cell-based systems for producing influenza vaccines

Examples

Experimental program
Comparison scheme
Effect test

example 1

ST6 CHO Cells with Increased Levels of 2,6-Linked Sialic Acids and Which Produce Increased Yields of Influenza Virus

[0084]CHO cells were incubated with digoxigenin labeled S. nigra agglutinin (SNA), specific for 2,6-linked sialic acids followed by the anti-digoxigenin-fluorescein conjugated antibody, and then analyzed by flow cytometry. The mean fluorescence intensity of WT CHO indicates that WT CHO do not contain 2,6-linked sialic acids on the cell surface. ST6 CHO cells expressing the human ST6Gal 1 gene however contain increased levels of 2,6-linked sialic acids as shown by the curve shift to the right (increased mean fluorescence intensity). See FIG. 4.

[0085]Next, TC-24 cells were seeded with 2×105 CHO or ST6CHO cells. The confluent CHO or ST6CHO monolayers were infected with A / PR / 8 / 34 influenza virus at an MOI=1 the following day. Aliquots were taken out at 48 hours post infection and stored at −80° C. Viral titers were determined on MDCK cells by plaque assay. Briefly, TC-6 ...

example 2

Generate Stable CHO and Vero Cells Expressing Increased Numbers of Human Influenza Virus Specific Receptors

[0088]This example concerns the stable transfection of the ST6Gal I gene, which encodes for 2,6-sialyltransferase I, into the CHO cell genome. This modification is expected to facilitate the production of vaccine virus in CHO cells.

[0089]Cell lines: CHO-S (Cat #11619012, Invitrogen, San Diego, Calif.); Vero (ATCC CCL-81, Manassas, Va.); MDCK (ATCC CCL-34); Plasmids: ST6Gal I (ATCC, Manassas, Va.), pcDNA3.1 (Invitrogen, San Diego, Calif.); Viruses: A / Puerto Rico / 8 / 34 (H1N1), A / Nanchang / 933 / 95 (H3N2), A / Texas / 36 / 91 (H1N1), B / Florida / 4 / 06) (BEI, Manassas, Va.).

[0090]A. Construct Plasmids Expressing the ST6Gal I Gene

[0091]Human (α2,6) ST6Gal I cDNA in the pSPORT vector (Invitrogen) is available from the ATCC (cat #10436251). The ST6Gal I gene was amplified by polymerase chain reaction (PCR) techniques with primers 5′-AAGCTTGCCGCCACCATGATTCACACCAAC-3′ (SEQ ID NO. 2) and 5′-CGGCGCC...

example 3

Quantitate Seasonal and Pandemic Influenza Virus Yields Following Infection of CHO 2,6

[0101]This example will test whether higher expression levels of α2,6-linked sialic acid on the cell surface of the modified cells (CHO-2,6 and Vero-2,6 cells) generated in the preceding Example increases the cell's susceptibility to human influenza virus infection. The α2,6-linked sialic acid is the cell receptor used by the HA glycoprotein on the influenza virus that permits viral attachment to the host cell and leads to infection. The parental and modified CHO and Vero cell lines will be infected with seasonal and pandemic human influenza virus isolates and evaluated for their ability to yield high titers of virus. Populations of all 4 cell lines will be infected with representative influenza viruses (H1N1, H3N2, type B, and H5N1) and growth curves will be generated for each strain. The H1N1, H3N2, and type B influenza viruses represent the different subtypes included in the seasonal influenza ...

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Abstract

The present invention relates to a cell-based method for producing influenza virus vaccines by enriching the population of surface-bound α2,6-sialic acid receptors on a cell surface, such as on a Chinese Hamster Ovary (CHO) cell surface. The host cell therefore presents numerous binding sites to which an influenza virus can bind via its hemagglutinin spike protein and infect the host cell. In contrast to wild-type CHO cells, the surface of the mutated CHO cells of the present invention contains an enriched population of α2,6-sialic acid receptors which makes the inventive CHO cells highly susceptible to viral infection, and therefore safe, effective, and highly efficient cells for rapidly producing influenza vaccines.

Description

[0001]The present application claims priority to U.S. Provisional Application No. 61 / 169,548, filed Apr. 15, 2009, and U.S. Provisional Application No. 61 / 060,653, filed on Jun. 11, 2008, both of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION[0002]The present invention relates to a cell-based method for producing influenza virus vaccines by enriching the population of surface-bound α2,6-sialic acid receptors on a cell surface, such as on a Chinese Hamster Ovary cell surface.BACKGROUND[0003]Influenza vaccines have been manufactured for over 70 years using a process that involves infecting embryonated chicken eggs with influenza virus. The process is difficult to automate, labor-intensive, costly and creates significant risk of contamination. The entire production process requires detailed planning that begins up to 8 months prior to vaccine delivery and leaves little room for error. For instance, the 2004 worldwide influenza vaccine shortage was t...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/145C12N7/00C12N5/10A61P31/16
CPCA61K39/145C12N2760/16234C12N2760/16134C12N2760/16034A61K39/12A61P31/16C12N7/00C12N2760/16051C12N2760/16151C12N2760/16251C12Y204/99007
Inventor BILSEL, PAMUKKAWAOKA, YOSHIHIRONEUMANN, GABRIELE
Owner FLUGEN
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