Posted at 11.28.2018
Importance of the field: Cell culture-based method for influenza vaccines development offers various advantages over egg-based processing method during influenza pandemics.
Areas covered in this review: Great things about cell culture-based method for influenza vaccines production have been mentioned, including: Increasing vaccines development potential during pandemics, stopping the development of mutations and providing better processing control. Besides that, recent obstacles of using cell culture instead of egg-based method have been also covered, such as low yield of some influenza disease strains in cell culture and capability of the selected virus pressure to grow on specific cell culture line.
What the audience will gain: The reader will gain an overview of the benefits of using cell culture-based method alternatively way to the existing egg-based manufacturing way for creation of influenza vaccines. Also, the reader will come to learn the recent issues facing the new cell culture founded technology.
Take home concept: Influenza vaccines made by cell culture method promote safe and innovative vaccine regimens that may significantly broaden and lengthen protection against influenza.
Keywords: influenza, cell culture, vaccines, egg-based method
Influenza "Flu" is a one of the most crucial viral diseases that remains a risk to humans worldwide because of its great health insurance and economic impacts. The disease is highly contagious and impacting breathing disease [1-3]. The key feature of influenza trojan is due to its ability to cause sporadic pandemics in human population with following persistence which threats the public health constantly . The genome of influenza virus includes eight, single-stranded RNAs . Influenza infections are grouped into three types: A, B and C predicated on the virus antigens (nucleoprotein and matrix protein). Type A is the main one since it affect individual as well as dog too. While types B and C are influencing individual only. Influenza infections type A are typed in line with the expression of two surface proteins mounted on the envelop of the disease: the hemagglutinin (HA) and neuraminidase (NA). For example, influenza A subtype H3N2 is refer to an expression of hemagglutinin 3 and neuraminidase 2. Influenza virus continuously changes its antigenicity. Therefore, in each season new influenza virus strain comes out and results in an accumulation of many disease strains. However, H1N1 and H3N2 influenza pathogen strains are the most prevalent real human influenza viruses . Two key features, antigenic drift and antigenic shift, are the reasons behind the continuous changing of the antigenicity of influenza viruses, and this renews the web host susceptibility to influenza viruses and cause consistent changes in the viral components of seasonal influenza vaccines [5, 6]. Antigenic drift is a replication mistake, which results in hereditary mutations mainly in HA . While antigenic change is the capability to shift the genetic materials, because its genome is segmented, between two different influenza viruses in the same infected number cell (co-infection), and bring about reassortment (transfer) of these genetic materials. Improve individuals welfare by protecting against influenza may be accomplished widely by using vaccines . Vaccination against influenza is the primary procedure in the elimination and control programs of the disease . Two main types of influenza vaccines have been developed for pandemic influenza on the bases of existing seasonal individual influenza, the inactivated and the live attenuated disease vaccines [1, 4, 5]. Consequently, to achieve the maximum effect of influenza vaccines, the vaccine viruses strains and the individuals isolated strains need to be antigenically matched . The typical method for production of influenza vaccines is using the fertilized rooster eggs to expand the disease. However, this method has many constraints including: difficulties to create large numbers of influenza vaccines in relatively small amount of time because it is limited by quantity of embryonated eggs available. Furthermore, long time necessary for planning and creation could complicate the creation process. Furthermore, the utilization of egg-based method is associated with era of high progress reassortants (HGR) and difficult production control like contamination that could be happen during vaccine producing steps [3, 9]. The constraints of currently available egg-based method demanded more effective, rapid, effective, and reliable vaccine production technology in the event of a pandemic [1, 4]. Consequently, researchers tried to evaluate cell culture vaccine creation methods to replace egg-based production method [10-12], and different types of cell lines have been developed and used effectively to produce influenza vaccines, for example, the continuous mammalian cell lines from monkey kidney skin cells named Vero cells which is abbreviation of "Verda Reno", Madin Darby canine kidney skin cells (MDCK)  and human-derived PER. C6 cells [14, 15]. Huge progresses have been made and many released articles present data that support the benefits of replacing egg-based development by cell culture method. However, up to day, this issue still controversial and many troubles still lie in advance . This review centered on benefits and issues of using cell culture method as an alternative to the existing egg-based solution to produce influenza vaccines.
Figure 1 Vaccine development by traditional egg-based technology and novel cell-culture processes: Timeline contrast and features of cell culture-based making methods .
The first use of mammalian cell cultures was in the early 20th century for cell physiology studies. However, the animal cell culture at an commercial level is not used until the 50's . Since last 2 decades, huge funds and resources have been spent to build up and improve cell cultures to replace hen embryonated eggs for influenza vaccines creation. Shifting vaccine development from eggs to cell culture can provide benefits including, boost the ability to create enough vaccines when pandemics appear, prevent the opportunity of mutations that may happen in the virus surface glycoproteins especially the HA and offer most effective control of vaccine creation and processing steps by using shut systems [18, 19].
2. 1. Increasing vaccines creation capability during pandemics
Cell culture-based vaccine development method offers time saving rather egg-based creation methods and considerably shorten making timelines. Among the main limitations of egg-based vaccine development is frustrating process and limited egg resources could be available during the time of a pandemic (Shape 1). Therefore, continuous supply of eggs is needed to keep up with the vaccine development [16, 20]. Another limitation is the power of influenza infections to grow on egg. Not absolutely all influenza trojan strains can be grow during most important isolation and sometimes adaptation you need to performed to increase the likelihood of the disease to grow in egg [3, 18, 21, 22]. Alternatively, Donis, et al.  were successfully able to isolate influenza disease (H3N2) strains using MDCK cell lines, which have been difficult to isolate straight in eggs. Le Ru, et al.  demonstrated that cell culture could scale-up the creation process with high titers of computer virus particle. They used serum free suspension of HEK-293 human cell series for replication of different subtypes and variations of influenza disease, including A/H1, A/H3, to create up to 109 IVP/mL (infectious pathogen particles) in three liters bioreactor. Aggarwal, et al.  were efficiently able to create a system process for making influenza vaccine and reduce the production time and energy to approximately three weeks. Finally, cell culture method can be cryopreserved, reconstituted, and scaled-up anytime which means it can significantly save time and initiatives. These results obviously proven that influenza viruses isolated in authorized cell culture may give a good option to lessen the steps needed to produce influenza vaccine. Hence, it can significantly save enough time required for production of the vaccine especially in case of pandemic [26, 27].
2. 2. Preventing the development of mutations
Production of seasonal influenza vaccines are usually performed using viruses adapted to expand in eggs. These vaccines have been became safe and effective. However, the use of egg-based method is associated with regular increasing of mutations that impact the antigenicity of the circulating influenza infections. This will likely lead to era of high expansion reassortants, and finally mismatching between the circulating pressure and vaccine pressure would happen which cause low efficiency of the vaccine [28-30]. Influenza vaccines derived from cell culture would allow influenza virus vaccine strain to be produced nearer to the circulating strain, and finally slow up the probability of mismatches that may be occurred between them . Additionally, selecting antigenic variants, which is essential for version and progress of the trojan in the eggs, could be omitted by using cell culture to isolate and develop the vaccine trojan strains. Based on the prior facts, it is clear that the cell culture-derived vaccines would be probably more effective than egg-derived vaccines. Hence it might offer more security against the circulating pathogen strains .
2. 3. Providing better developing control
Production in cell culture offer greater production control factors over egg-based method [3, 16]. The problem variables like changing press and adding supplements can be easily performed. Furthermore, monitoring and modification of oxygen contents and pH can be controlled according to the growth period [11, 31]. On the other hand, the production process based on the infection of eggs is more complicated than the cell cultivation using fermentation procedures as each egg signify a separate unit of vaccine creation . Another control factor relates to the safeness of the produced vaccines. The well characterized mammalian cell ethnicities can match the increasing protection needs by regulatory specialists. The growth of validated skin cells which are analyzed for purity, identity in line with the relevant rules and guidelines can truly add superior benefit of using cell culture rather than eggs for vaccine development [2, 33]. Although influenza vaccines creation mainly depends on egg-based method, concerns about the chance of contamination anticipated to hen flora was significantly increased. The possibility of contaminants of the provided egg by avian pathogens or microbial impurities that might occur during vaccine control, can add more challenges for the egg-based method [4, 31]. Cell culture offer gets rid of the opportunity of contaminants, and using shut manufacturing systems can greatly minimize the risk of contamination during manufacture process [3, 34].
As commented above, cell culture has many advantages over egg-based way for development of influenza virus. However, many complex problems are still unsolved which stand as an road blocks and prevents the replacement unit of egg-based method. Small, et al.  pointed out, "Although cell culture has been launched successfully in developing influenza vaccines, different issues about the type of cell line, research disease derivation and even the regulatory process, have to be assessed, and whether or not they have an adverse effect on cell culture manufacturing processes". On this review two limits will be discussed, the low yield and the ability of the chosen virus pressure to expand on specific cell culture series. Low produce of some influenza virus strains in cell culture is considered as one of the constraints that still challenging this technique. Factors impacting on both of system control and cell culturing conditions need to be controlled and fine-tuned in the manner that can offer suitable environment and provide maximum virus production. For instance, the carrier/reactor process with microcarrier, adherent cells, is currently the preferred method to cultivate the skin cells needed for influenza viral replication due to its high surface to size ratio and better control of the cultivation parameters. However, issues facing the application of this technology at commercial level are still persist. Cell culture using microcarriers requires further steps including skin cells distribution on a surface for subsequent progress, maintenance of the suspension system in the fermenter, and lastly products parting from the microcarriers [34, 35]. Another restriction that facing cell culture is the power of the selected virus pressure to increase on specific cell culture collection. It is obvious that some crazy type reference point strains could give low produce of antigen using the egg control method which brings about produce HGR. At the same time, sufficient growing of any wild type computer virus in cell culture can reduce the HGR. However, there is a significant risk a virus able to grow using one type of cell culture might not exactly have the ability to expand on others. The MDCK cells are revealed to be much better than eggs for influenza disease isolation. However, variant between different MDCK cell lines used for vaccine creation could be task as a few of influenza strains are not able to increase properly and Vero or PER. C6 may be needed to achieve proper isolation and succeeding steps for vaccine production [36, 37]. This consider as a substantial problem which requires development of different prospect vaccine viruses each year appropriate with the culture substrate .
The benefits of cell-culture technology for development of influenza vaccines offers benefits over egg-based vaccine creation method, with a high potential to better face future influenza-related challenges. More specialized and scientific improvements efforts need to be done to achieve both ease of manipulation and sponsor compatibility targets.