Accelerated mass production of influenza virus seed stocks in HEK-293 suspension cell cultures by reverse genetics

Abstract

Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Vaccine Virus (CVV) as reported by the 2015 WHO Informal Consultation report titled "Influenza Vaccine Response during the Start of a Pandemic". In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza strains, and advances in the large-scale transfection of suspension cultured HEK-293 cells. We demonstrate the efficient generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CVV for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Furthermore, the scalability of the transfection technology combined with the HEK-293 platform has been extensively demonstrated at >100L scale for several biologics, including recombinant viruses. Thus, this innovative approach is better suited to rationally engineer and mass produce influenza CVV within significantly shorter timelines to enable an effective global response in pandemic situations.

Personalized vaccinology: A review

“…Egg-based influenza vaccines take >6 months to create. The recent licensure of cell culture-based influenza vaccines demonstrate that rapid, scalable processes can now be implemented in order to create vaccine against emerging influenza strains (e.g., H1N1, H5N1, H7N9, H9N2, H7N8) within weeks [99] and can be safely administered to individuals with egg allergies [100] . The Ebola outbreak in Liberia, Sierra Leone, and Guinea in 2015 provides an example of the need to rapidly develop vaccine candidates [101] .…”

Extended gene expression for Gag VLP production achieved at bioreactor scale

“…This is very important during initial stages of development when several candidates need to be tested on a small scale and when the product of interest varies often and rapidly. This is the case of several viruses such as the influenza virus, where a new vaccine has to be generated on a yearly basis …”

“…This is the case of several viruses such as the influenza virus, where a new vaccine has to be generated on a yearly basis. 22,23 One of the main drawbacks of TGE is that plasmid DNA is not integrated into the cell genome and thus the gene of interest is lost as cells divide. 24,25 Some experimental approaches have been developed to overcome this problem.…”

A pooled genome-wide screening strategy to identify and rank influenza host restriction factors in cell-based vaccine production platforms

“…As a result, egg-based vaccines exhibit a 15–20% decrease in protection rate compared to similarly formulated cell-based vaccines 6 – 8 . Cell-based platforms can also drastically reduce production lead time by accelerating seed stock reassortment via reverse genetics 9 . This, in turn, reduces the chance of major changes in circulating strains occurring between initial strain selection and vaccine release, an issue that rendered the 2014–2015 seasonal vaccine largely ineffective 10 , 11 .…”