Influenza virus is a major health concern that has huge impacts on the human society. Historically responsible for millions of deaths in pandemics, the virus also causes seasonal outbreaks during colder months in temperate regions which annually result in up to 500,000 deaths worldwide . Although antiviral drugs for acute treatment are available in some countries, vaccination remains as one of the most effective ways to mitigate this disease.
Both inactivated vaccine and the live attenuated Influenza vaccines are commercially available. Although the live attenuated virus vaccine has been used in Russia since the 1960s , concerns regarding safety and possible virus shedding have precluded it from use in the rest of the world until recently: In 2003, a cold adapted, egg grown, live attenuated influenza virus vaccine by MedImmune was licensed for use in the US [3, 4]. Live attenuated virus vaccines have the added advantage of being more cross-reactive than traditional inactivated vaccines [5–7]. This type of vaccine is also easier to administer, since it is delivered in the form of nasal sprays, compared to injections for the traditional inactivated influenza vaccines.
One promising live attenuated Influenza that has completed Phase I clinical trial is deltaFLU, a deletion mutant lacking the viral Nonstructural Protein 1 (NS1) gene developed by Avir Green Hills Biotechnology [8–12]. As NS1 is an interferon antagonist , the NS1 deletion virus is replication defective in interferon competent host systems, enabling its use as a live attenuated vaccine [9–11]. Another consequence of this gene deletion is that this virus vaccine can only propagate effectively in cells with a deficiency in the interferon-mediated antiviral response . Vero (African Green Monkey kidney) is one such cell line as the gene locus encoding the main Type I interferons, Interferon α and β, are missing from its genomic DNA [14, 15]. Consequently, it has been previously demonstrated that the NS1 deletion Influenza virus grows efficiently in Vero cells, but not in MDCK or mice [8, 16, 17]. This NS1 deletion virus is also interesting because it may find applications in cancer therapy [18, 19] and other prophylactics .
Regardless of vaccine type (inactivated or live attenuated), virus vaccine production requires the initial step of propagating the Influenza viruses carrying the haemaglutinin and neuraminidase antigens of the strains that the vaccine is providing prophylaxis for. These viruses are traditionally propagated in embryonated hen eggs. Two important limitations of this process are the inflexible supply of high quality specific pathogen free (SPF) eggs and possible low titres of emerging viruses, such as the highly pathogenic Influenza A (H5N1) strain. To provide an alternative to egg-based vaccine production, mammalian cell culture based production has been developed in recent years . This provides a flexible and scalable platform that can make use of existing biopharmaceutical infrastructure for Influenza vaccine production.
Three cell lines commonly used for Influenza virus production are the PER.C6 cells, MDCK (Madin-Darby Canine Kidney) and Vero (African Green Monkey Kidney). All three cell lines can be grown in serum-free media. While PER.C6 and MDCK can be cultured in suspension [22, 23], microcarriers are commonly used for culturing MDCK [22, 24–30] and Vero cells [31–34] because these cell lines are typically anchorage dependent. The seasonal and pandemic Influenza vaccine produced in MDCK cells by Novartis has gained various regulatory approvals in 2007 and 2009 respectively, while those produced in Vero cells by Baxter has also gained approvals in 2010 and 2009 respectively.
Although bioreactor production of Influenza virus has been developed, serum-free production processes described in literature commonly use proprietary in-house cell culture media [24, 25, 29, 30, 32]. To our knowledge, there are a few reports describing Influenza virus production using MDCK cells in commercially available serum-free medium [21, 26–28], while that using Vero cells is described in only one recent report  although the medium used contains animal components. Related literature described serum-free media for Vero cells [36, 37] and microcarrier bioreactor processes for the production of other viruses using Vero cells [37–46]. It is important to bridge this gap to provide a scalable animal-component free, serum-free platform for researchers and academics to produce different Influenza viruses using Vero cells.
In this report, we describe for the first time, a scalable bioreactor process for the production of Influenza A virus lacking NS1 in Vero cells using commercially available animal-component free, serum-free media. We chose to use Cytodex 1 microcarriers for our bioreactor cell culture, since this microcarrier has been previously reported for Vero cells [31, 33, 34, 39–43, 45, 46]. We evaluated five commercially available animal-component free, serum-free media for Vero cells by comparing the cell yield in these media. The medium giving the highest cell densities was then used to develop the bioreactor process for Influenza virus production. This involved studies of parameters that will affect the virus production process, namely trypsin concentration, time-point of infection (TOI), and multiplicity of infection (MOI). These parameters were validated in classical stirred tank bioreactor processes. Finally, we also compared the production of the NS1 truncated Influenza A virus with that of the corresponding wild type Influenza A virus.