Center for Innovative Cancer Research, Ottawa Hospital Research Institute, Canada
Virus-based vaccines are arguably societyís most successful modality for the prevention of deadly human and veterinary diseases. However, some-what ironically, vaccine manufacturing has long resisted progress. For many vaccines, manufacturing processes are still dominated by outdated, slow, and unreliable chicken-egg based or primary cell culture methods.
To improve reliability and deal more effectively with the threat of rapidly emerging viral pandemics, the vaccine industry and organizations such as the World Health Organization (WHO) are increasingly advocating the use of continuous cell lines for vaccine production [2-5]. However, due to stringent regulatory guidelines for both human and veterinary vaccines, available continuous cell lines for vaccine manufacturing are still limited. Prominent examples include Vero African green monkey kidney cells, Madin-Darby canine kidney (MDCK) cells, and BHK-21 cells. In addition to these continuous cell lines, a handful of human diploid cells such as MRC-5, FRhl-2, and WI-38 have been used to make human vaccines.
These cell lines provide well defined, reliable, and in the case of continuous cell lines, easily scalable substrates to grow vaccines. However, in many cases low virus yields are obtained using these substrates. Consequently, manufacturers are constantly looking to improve vaccine production yields from cells, often empirically through process optimization. For companies looking to adopt new and more efficient technologies to this end, the decision comes down to simple economics: does the potential increase in vaccine yield justify the overall cost associated to deviating from the methods already in place? With existing technologies, the answer to this question is unfortunately often the negative, preventing the adoption of modern continuous cell-based methods for many vaccines. This is most notably the case for influenza vaccines, which are still predominantly made using chicken eggs. It is clear that there is a pressing need and an emerging market for innovations that will substantially and economically improve vaccine yields from cultured cells.
Likely because of the direct correlation between cell substrate quantity and viral vaccine output, most attempts at improving vaccine yields from cell cultures have focused on improving cell growth properties. Such strategies include optimizing chemically defined media, novel bioreactor technologies, and the selection or design of continuous cell lines with improved growth characteristics. For example, genetically engineered PER.C6 cells (J&J/Crucell) have been reported to grow at very high densities in suspension without the need for animal serum . As a result of these and other characteristics, PER.C6 cells are of high interest to many biotherapeutic companies. Regardless of the success of PER.C6 cells, the use of designer cells for vaccine manufacturing is only in its infancy and to our knowledge there are still no approved vaccine products made with these or other designer cell lines.
Cellular antiviral defenses: overcoming the first hurdle
The cellular antiviral defense is triggered upon viral-infection of a cell and is a common and primary hurdle for all replicating viruses [9, 10]. As invading parasites, most naturally occurring virus strains express virulence proteins that block the cellular antiviral response and thus facilitate virus propagation [11-15]. Virulence proteins in disease-causing viruses are evolutionarily adapted to hijack the antiviral defenses of the specific species, tissues, and cells in which they cause disease (eg. Human airway epithelial cells for human influenza virus). The ability of virulence proteins to overcome antiviral defenses can be compromised in cells that are not the virusí natural target. Similarly, mutations within these virulence proteins can lead to viral attenuation and/or restricted host cell range [13, 16-18]. While reducing virulence is generally desirable to make safe vaccines and other virus-based therapeutics, this also creates viruses that can grow poorly when forced upon non-host manufacturing cell substrates.
To get around this problem, some groups have adapted vaccine strains to improve their growth characteristics in selected manufacturing cells through directed evolution [16, 19, 20]. While this approach can be effective, it necessarily leads to genetic changes in the vaccine, desirable or not, due to its adaptation to the new host cells. Such genetic adaptations on behalf of the virus can be expected to provide a growth advantage by optimizing the virusí ability to co-opt cellular resources in a new context while also effectively retaliating against the new host cellís antiviral defenses. However, this may also impact the vaccineís activity and/or safety.
Another potential approach to improve the growth of vaccines from cell lines is to effectively disable the cellular antiviral defenses of manufacturing cells to fully permit growth of even severely attenuated vaccines. While gene silencing and genetic engineering approaches can and have been considered to this end , they are likely to be too costly and/or insufficiently comprehensive in light of the complexity and redundancy of antiviral defenses. In addition, the genetic engineering strategy is inherently time/resource consuming given the heavy regulatory burdens placed upon new cell line derivatives for vaccine production.
Viral sensitizer technology: a welcome boost from cancer
Surprisingly, one novel approach that can be used to circumvent genetic manipulation of viruses and continuous cell lines comes to us from the field of cancer therapeutics. In the last decade or so, the use of attenuated and tumor-specific oncolytic viruses for the treatment of cancer has become a real possibility. Indeed, Asia can certainly pride itself as being the first to adopt this technology as evidenced by Chinaís approval of Shanghai-based Sunway Biotechís H101 oncolytic adenovirus for the treatment of head and neck cancer, well in advance of the looming approval of similar agents in North America and Europe.
One well recognized issue in the field of oncolytic virotherapy is that tumors often resist infection by these therapeutic agents due to the cellular antiviral response . To overcome this problem, we have recently discovered a group of compounds that robustly enhance virus growth in cells. As a whole, what we have termed viral sensitizer technology (VST) encompasses a collection of compounds that work by broadly and effectively disrupting cellular antiviral defenses.
While we initially discovered this technology in the context of oncolytic virus therapy using a vesicular stomatitis virus , where over 1000-fold increases in viral titers were obtained in some cases, we have since found that our VST can be used to increase the production of vaccine strains in manufacturing cell lines. For example, using VST in continuous BHK-21 cells we have been able to observe increased yields of modified vaccinia ankara by 10-fold, reaching approximately 45-times higher yields than what can be produced in parallel using industry-standard primary/non-continuous chicken embryo cells. Likely owing to the universality of antiviral defenses as a hurdle for efficient viral growth, we have found that such increases in viral yields can be observed for several DNA and RNA viruses and in many different types of continuous cell lines as well as in human diploid cells. It is easy to imagine that such improvements in vaccine yields could have a drastic impact on both the cost and the manufacturing time per vaccine unit.
So how safe is this approach? While we can expect that VST will be held to the same regulatory scrutiny as designer cells and other related technologies, our lead VST compounds for vaccine applications have so far not shown signs of mutagenicity and are below the limit of detection in crudely purified viral preparations. To date, we have also not observed any direct impact of VST on the viruses produced when investigating basic viral characteristics (virus protein expression/size, ability to replicate etc.).
Altogether with VSTís very low cost in relation to other approaches and associated high vaccine yield, VST is an ideal solution to improve existing virus-based vaccine manufacturing processes and for implementation in future vaccine production methods. Because of the obligatory regulatory approval process for all new vaccines/processes, VST is expected to be most beneficial for new vaccine candidates, particularly those that do not grow to sufficiently high titers using available substrates. As alluded to earlier, this is often the case for live attenuated vaccines. It is also foreseeable that VST can be used in addition to technologies that aim to improve the growth characteristic of cells such as chemically-defined media, advanced bioreactor technologies, and designer cell lines. We believe that the Asia-Pacific biotechnology community is a rich source for strategic partnerships to evaluate VST given that market growth for the vaccine sector in the region is expected to grow tremendously in the next few years.
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