Abstract
BackgroundInfluenza viruses cause hundreds of thousands of respiratory diseases worldwide each year, and vaccination is considered the most effective approach for preventing influenza annual epidemics or pandemics. Since 1950, chicken embryonated eggs have been used as the main method for producing seasonal influenza vaccines. However, this platform has the main drawback of a lack of scale-up flexibility, and thus, egg-based vaccine manufacturers cannot supply sufficient doses within a short period for use for pandemic prevention. As a result, strategies for reducing the manufacturing time and increasing production capacity are urgently needed. Non-virion vaccine methods have been considered an alternative strategy against an influenza pandemic, and the purpose of maintaining an immunogenic capsule structure with infectious properties appears to be met by the virus-like particle (VLP) platform.ResultsAn influenza H7N9-TW VLP production platform using insect cells, which included the expression of hemagglutinin (HA), NA, and M1 proteins, was established. To scale up H7N9-TW VLP production, several culture conditions were optimized to obtain a higher production yield. A high level of dissolved oxygen (DO) could be critical to H7N9-TW VLP production. If the DO was maintained at a high level, the HA titer obtained in the spinner flask system with ventilation was similar to that obtained in a shake flask. In this study, the HA titer in a 5-L bioreactor with a well-controlled DO level was substantially improved by 128-fold (from 4 HA units (HAU)/50 μL to 512 HAU/50 μL).ConclusionsIn this study, a multigene expression platform and an effective upstream process were developed. Notably, a high H7N9-TW VLP yield was achieved using a two-step production strategy while a high DO level was maintained. The upstream process, which resulted in high VLP titers, could be further used for large-scale influenza VLP vaccine production.
Highlights
Influenza viruses cause hundreds of thousands of respiratory diseases worldwide each year, and vaccination is considered the most effective approach for preventing influenza annual epidemics or pandemics
Establishment of the H7N9-TW virus-like particle (VLP) expression system The HA, NA, and M1 genes from the Influenza A/ Taiwan/1/2013 (H7N9) strain were cloned into the pFastBac DUAL vector (Invitrogen, USA) (Fig. 1)
The HA titer of the H7N9-TW VLPs produced using Sf-21 cells did not exceed 64 (HA units (HAU)/50 μL), whereas the High FiveTM cells produced H7N9-TW VLPs with an HA titer of 512 (HAU/50 μL) (Table 2), which demonstrated that H7N9-TW VLPs are more produced by High FiveTM rather than Sf-21 cells
Summary
Influenza viruses cause hundreds of thousands of respiratory diseases worldwide each year, and vaccination is considered the most effective approach for preventing influenza annual epidemics or pandemics. Previous studies [14,15,16] have shown that a number of factors can influence protein expression, and these include the production media, the level of dissolved oxygen (DO), the virus multiplicity of infection (MOI) and the time point after infection used for harvesting. These established insect cell lines show variable abilities to amplify baculovirus and express soluble protein.
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