Production of viral vaccines on an avian cell line
A virus vaccine and immortalized cell line technology, applied in the field of virus vaccine production on avian cell lines, can solve problems such as inability to carry out
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Embodiment 1
[0231] Example 1. Use Cell lines were used for the replication of wild-type strains of subgroup A1 (C-85473 and CAN99-81), subgroup B1 (CAN97-82) and subgroup B2 (CAN98-75).
[0232] Will Cells were cultured at 37°C in final medium of OptiPro + 4mM L-glutamine in 50ml TubeSpin in a Kühner shaker at 175rpm in 5% CO2 and 85% humidity (d'hygrométrie), And the cells were diluted to 1x10 in 10ml medium 6 cells / ml. Wild virus strains of subgroup A1 (C-85473 and CAN99-81), subgroup B1 (CAN97-82) and subgroup B2 (CAN98-75) were used in the presence of 0.5 μg / mL trypsin (T6763Sigma) ( non-GFP) infected the cells at a multiplicity of infection (MOI) of 0.01.
[0233] Cells were counted in trypan blue every 2 to 3 days to assess cell growth as well as cell death during virus infection. The result is as Figure 1a shown.
[0234] The number of infected particles per ml of culture medium (measured as TCID 50 / ml) to measure virus production. The result is as Figure 1b shown.
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Embodiment 2
[0238] Example 2. Using Cell lines were used for replication of the wild strain C-85473WT, expressing green fluorescent protein (GFP) due to recombination, and the recombinant strains ΔSH-C-85473(GFP) and ΔG-C-85473(GFP).
[0239] The virus strain used in this example has the following genome sequence:
[0240] - the sequence C-85473WT-GFP is represented by SEQ ID NO:4;
[0241] - the sequence ΔSH-C-85473-GFP is represented by SEQ ID NO:5;
[0242] - The sequence ΔG-C-85473-GFP is represented by SEQ ID NO:6.
[0243] Will Cells were maintained in final media of OptiPro + 4 mM L-glutamine in 50 ml TubeSpin in a Kühner shaker at 37° C., 5% CO 2 and 85% humidity at 175 rpm.
[0244] Before infection, dilute cells to 1x10 in 10 ml of culture medium 6 cells / ml, and then in the presence of 0.5 μg / ml of trypsin (T6763 Sigma), the cells were infected by a multiplicity of infection (MOI) of 0.01 using: wild-type recombinant hMPV (C-85473WT), or deletion of the encoding SH protei...
Embodiment 3
[0253] Example 3. Characterization of Virus Particles Obtained According to the Culture Method of Example 2
[0254] Compared with the recombinant virus C-85473WT, this embodiment relates to the measurement of Replication ability of recombinant viruses ΔSH-C-85473 and ΔG-C-85473 produced on cells:
[0255] (i) in monkey kidney epithelial cells LLC-MK2 (ATCC-CCL7), and
[0256] (ii) 3D reconstructed human lung epithelial model (MucilAir TM , Epithelix) and cultured at the air-liquid interface.
[0257] Infect LLC-MK2 cells and MucilAir using TM Cell plateau of healthy reconstituted human respiratory epithelium:
[0258] - Recombinant hMPVΔSH-C-85473 at MOI (multiplicity of infection) of 0.01 and 0.1, respectively;
[0259] - Recombinant hMPVΔG-C-85473 at MOI (multiplicity of infection) of 0.01 and 0.65, respectively.
[0260] Photographs of infected cells taken at 3, 5, 7, 12 and 17 days post-infection are shown in Figure 3a (ΔSH-C-85473) and Figure 3b (ΔG-C-85473). ...
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