Suspended cell lines for inactivated virus vaccine production

ABSTRACT

Introduction

Inactivated virus vaccines are the most widely used tool to prevent disease. To meet vaccine production demands, increasing attention has been placed on identifying methods to improve vaccine production efficiency. The use of suspended cells can greatly increase vaccine production. Suspension acclimation is a traditional method to convert adherent cells to suspension strains. Furthermore, as genetic engineering technology has developed, increasing attention has focused on the development of suspension cell lines using targeted genetic engineering techniques.

Areas covered

This review systematically summarizes and analyzes the development and research progress of various inactivated viral vaccine production suspension cell lines and provides protocols and candidate target genes for the engineered establishment of additional suspension cell lines for vaccine production.

Expert opinion

The use of suspended cells can significantly improve the production efficiency of inactivated virus vaccines and other biological products. Presently, cell suspension culture is the key component to improve many vaccine production processes.

KEYWORDS:

• Inactivated virus vaccine
• Suspension cell
• Suspension domestication
• Target gene
• Genetic engineering methods
• Siat7e
• Cell adhesion

1. Introduction

Inactivated virus vaccines can produce a wide range of immune responses against multiple targets and have numerous benefits, including safety, relatively low production cost, heat resistance, and long-term storage capacity. Therefore, inactivated virus vaccines are presently the most widely used disease prevention tool [1] for a broad range of diseases, such as influenza [2], poliovirus [3], enterovirus 71 [4], hantavirus [5], and rabies virus [6], and they occupy a large proportion of the global market.

For instance, in 2021, over 80 million domestic influenza vaccines will be distributed, of which approximately 55 million – or more than 65%—would be quadrivalent inactivated influenza vaccines. It is expected that the number of quadrivalent inactivated influenza vaccine issued will increase year by year, accounting for a larger proportion of the total number of all influenza vaccines issued. Currently, the use of cells to produce viral vectors and vaccines is receiving increasing attention. There are two main types of cell culture: adherent culture and suspension culture. In addition to blood cells, most vertebrate-derived cell lines require adherent culture, which is limited by the surface area of the culture medium and therefore difficult to expand the production scale. Compared to adherent culture, firstly, suspension culture can greatly increase the cell density in medium, significantly increase the vaccine yield, and reduce the production cost. For example, the growth density of suspended cells can generally reach 1 × 10⁶ cells/mL. Secondly, serum-free suspension cell cultures are more suitable for bioreactor-based production processes, which enable large-scale cell culture and a more stable and controlled vaccine production process. In particular, the use of bioreactors can meet the global market demand for vaccines when they are urgently needed during a pandemic. It also significantly simplifies the processes of cell culture and process scale-up, making the vaccine production process more stable and controllable, and improving the process stability of the vaccine. With the development of modern biotechnology, the industry trend toward the mainstream production of biological products using cell suspension culture technology is inevitable. For example, from 2014 to 2018, approximately 84% of approved monoclonal antibodies were derived from industrially produced CHO cells. Cell suspension culture technology has developed rapidly, and many previously adherent cell lines have been converted to suspension culture [7]. Moreover, the cells in suspension culture still maintain their original susceptibility and biological properties to viruses, so the advantages of using cellular matrix for the production of virus-based vaccines are more obvious and have been applied in the production of various virus vaccines such as COVI-VAC [8], Newcastle disease [9], poliovirus [10], rabies [11], and Foot-and-Mouth disease virus (FMDV) [12]. Presently, suspension acclimation and gene editing are the main methods to obtain suspension cell lines. The suspension acclimation of adherent cells, which is currently the primary method to obtain suspension cell lines for vaccine production, is mainly achieved by gradually reducing the serum content in the culture medium. This acclimation process allows cells to adapt to low serum or serum-free culture and eventually lose their adhesion ability and therefore grow in suspension. Serum-free suspension culture is gaining more and more attention as it can further reduce the production cost, simplify the production process, exclude the influence of serum, shorten the production time, improve the production efficiency, and facilitate the expansion of culture. Therefore, it is currently the main way to obtain suspension cell lines for vaccine production. Microcarrier suspension cell culture is an additional method for converting adherent cells to suspension culture and easily promotes the scale-up of production in large-scale industrial bioreactors. With this method, microcarriers [13–15] are added to provide sufficient surface area required by the adherent cells in suspension culture, thereby promoting the scale-up of production. For example, Sanofi Pasteur uses microcarriers in a 1000 L reactor to culture Vero cells to produce rabies and poliomyelitis vaccines for human use. Additionally, the continuous development of biotechnology and genetic engineering in recent years has resulted in the successful application of gene editing technology to transform adherent cells into suspended cell lines, allowing these cells to proliferate easily. This highly targeted transformation of cells at the gene level makes the cells more suitable for vaccine production.

2. Domestication of suspension cell lines

Suspension culture technology can be divided into whole suspension cell culture and microcarrier suspension culture according to cell adhesion. Suspended cells, such as Chinese hamster ovary (CHO) and baby hamster kidney-21 (BHK-21) cells, can be directly expanded in bioreactors for vaccine production. Their benefits include free cell growth, uniform culture environment, simple sampling methods, simple and controllable culture operation, convenient amplification, and low contamination rate and cost. The microcarrier suspension culture system established by Van Wezel has made the large-scale culture of adherent cells a reality [16]. Although this microcarrier culture method is sufficient to obtain high virus yields [14,15], relative to serum-free total suspension cell cultures, it is a cumbersome process with many shortcomings and uncertainties related to vaccine production, such as limited cell culture area, difficult separation of cells from carriers, and expensive microcarriers. Therefore, continual efforts are ongoing to find a simple and convenient large-scale culture technology.

The strategy of low serum adaptation to serum-free suspension culture is the simplest and most direct method to domesticate adherent cells into suspension cells. The serum composition in mammalian cell culture is highly variable, causing substantial differences between different batches of serum containing maintenance solution. Additionally, the serum is difficult to obtain and preserve, which increases production costs. Furthermore, it is often easy to introduce foreign pollutants from animal sources, such as mycoplasma and bovine virus, which is especially problematic for virus-based processes, such as the production of the influenza vaccine [13]. The quality of vaccine products may also be uncontrollable when serum is used [17,18]. Therefore, in order to meet the growing demand for vaccine production safety, it is necessary to continually research the industrial development of serum-free and protein-free culture media. The conversion from serum-containing complete medium (SCM) to serum-free medium (SFM) has been the key factor in suspension cell culture research for many years. Suspension cells can be adapted from CM to SFM by gradually decreasing the serum concentration while gradually increasing SFM [19]. Since the 1960s, this technology has developed rapidly; presently, more than 80 kinds of cells, including Madin-Darby Canine Kidney (MDCK) [20], CHO [21], human embryonic kidney (HEK) 293 [22], BHK-21 [23], and Vero cells [24], have been successfully domesticated and used in the industrial large-scale production of many biological products such as animal vaccines, monoclonal antibodies, and pharmaceutical proteins. Based on current research, we systematically collated the suspension cell lines obtained through suspension domestication that have potential uses for vaccine production (Table 1).

Table 1. Existing cell lines acclimated to suspension for use in vaccine production.

Cell line	Domestication method	Cell suitable for virus growth or recombinant expression	Related vaccines type	Licensed	Manufacturer	References
MDCK	Serum-free suspension acclimation	Influenza vaccine	Inactivated influenza vaccine	Yes	Seqirus UK Ltd	[25–30]
			Inactivated subunit vaccine	No	Novartis Vaccines
			Subunit vaccine	No	SKChemicals、Seqirus*
Vero	Microcarrier suspension	Rabies	Inactivated rabies vaccine	No	SANOFI PASTEUR S.A. r	[31–34]
		SARS-CoV 2	Inactivated COVID-19 vaccine	Yes	SinoPharm
		Adenovirus type 5	Adenovirus vectored vaccine	N/A	N/A
		NDV	N/A	N/A	N/A
		Poliovirus	Inactivated poliovirus vaccine	N/A	N/A	[35,36]
		Hantaan virus	Inactivated Hantaan virus vaccine	N/A	N/A	[37]
		Ev71	Inactivated enterovirus type 71 vaccine	N/A	N/A	[38]
CHO	Serum-free suspension acclimation	Influenza virus/Plasmid	AIV（H7N9) vaccine	No	N/A	[39]
BHK-21	Serum-free suspension acclimation	FMDV	N/A	No	N/A	[23,40,41]
		Zika	N/A	No
HEK 293	Serum-free suspension acclimation	Recombinant vesicular stomatitis viruses (rVSV)	Ebola virus vaccine (rVSV-ZEBOV)	No	NRC Canada	[42]
	Low-serum suspension culture	SARS-CoV-2 vaccine	Adenovirus-vectored-SARS-CoV-2 vaccine	Yes	the University of Oxford in partnership with AstraZeneca	[43]
MDBK	Serum-free suspension acclimation	BoHV-1	Inactivated BoHV-1 vaccine	No	IDT Biologka	[44]
Hi5	Serum-free suspension acclimation	Recombinant protein	HIV-1 virus-like particle (VLP)	No	N/A	[45]
Marc −145	Serum-free suspension acclimation	PRRSV	N/A	No	N/A	[46]
PK-15	Serum-free suspension acclimation	Recombinant expression	PCV2 vaccine	No	N/A	[47]
PBG.PK2.1	CD-U5 medium adapted to suspension growth	Influenza virus	N/A	No	N/A	[48]
ST	Microcarrier suspension	Recombinant SPV	CSFV vaccine	No	N/A	[49]
EB66	Serum-free suspension acclimation	Influenza virus	Inactivated H5N1 vaccine	No	GSK	[50–54]
		MVA	MVA vectored vaccine	No	N/A
		YFV、ZIKV	N/A	No	N/A
		DTMUV	Inactivated　DTMUV　vaccine	No	N/A
UMNSAH	Microcarrier suspension	MDV	Living vaccine	No	N/A	[55]

Note: N/A　in the Related vaccines means the cell line suitable for virus growth, but no related vaccine production.

2.1. MDCK suspension cells

MDCK cells are adherent culture-type cell lines established by Madin and Darby in 1958 from the kidney tissue of American Cocker Spaniel dogs [56]. Because of its high influenza virus infection efficiency, rapid proliferation, and resistance to mutation, it is recognized as one of the cell lines suitable for influenza virus vaccine production [57]. MDCK cells were one of the first continuous cell lines recommended by the World Health Organization (WHO) in the mid-1990s to replace eggs in seasonal influenza vaccine production [58]. Its potential for influenza vaccine preparation was evaluated and validated by several academic and industrial laboratories and culminated with the market approval of Flucelvax_® (Seqirus) and Optaflu_® (Novartis), the first cell culture-derived influenza vaccine [25]. MDCK cells were initially cultured on microcarriers as adherent cells [14]. However, in order to meet increasing demand, and with the development of cell lines and media, MDCK suspension cell lines were produced. In 2011, Huang et al. [59] acclimated and obtained a cell line suitable for single cell suspension culture in SFM. Currently, high cell density (HCD) perfusion culture technology has also been applied to suspension culture of MDCK cells, which can effectively improve virus production [60].

2.2. Vero suspension cells

Vero cell lines were established from African green monkey kidney cells in 1962 [61] and are adherent-dependent transformation cells. Vero cells are considered by the WHO as the most widely accepted subculture cell line for the production of virus vaccines for human use [62], because they can be subcultured indefinitely, allow a wide range of cell characterization, and can establish a large cell library. Collectively, these features provide a valuable advantage over primary cell lines with limited subculture capacity, such as chicken embryo fibroblasts [63]. Many vaccines can be produced using Vero cells, including vaccines against SARS-CoV-2 (Inactivated COVID-19 Vaccine (Vero Cell), Sinopharm/Beijing Institute of Biological Products Co., Ltd. (BIBP); CoronaVac, Sinovac) [31], poliovirus (PEDIARIX_®, Glaxo Smith Kline; IPOL_®, Sanofi Pasteur), rabies (VERORAB_®, Sanofi Pasteur), Japanese encephalitis virus (IXIARO_®, Intercell Biomedical), enterovirus type 71 vaccine [38], smallpox (ACAM2000_®, Sanofi Pasteur), and rotavirus gastroenteritis (Rotarix_®, Glaxo Smith Kline; RotaTeq_®, Merck) [64]. Vero cells are adherent cells that can only proliferate when a suitable surface is provided [65]. Given that many vaccines are currently produced using Vero cells as substrates and that the production efficiency of these vaccines needs to be significantly increased to cope with the outbreak of infectious diseases, the suspension of Vero cells has become the focus of the vaccine industry. In 2009, Paillet et al. [24] generated a Vero cell line (sVero) suitable for suspension growth in SFM through suspension domestication. Acclimated suspended Vero cell cultures can be used to produce H1N1 in SFM [66]. In 2018, Logan [67] used OptiPro SFM medium to adapt Vero cells to serum-free conditions and then gradually further adapted the cells to various media formulations, in order to achieve an optimized suspension culture medium formulation to support the growth of Vero cells in suspension. CF Shen [65] reported that adherent Vero cells successfully adapted to suspension growth in an internally developed serum-free and animal-free medium (IHM03), which was applied to the production of RVSV-GFP, resulting in higher cell density and greatly increased virus productivity.

2.3. CHO suspension cells

CHO cells were first isolated by Puck laboratory in 1957 [68] through enzymatic digestion of 0.1 g of ovarian tissue from a female Chinese hamster obtained from Dr. George Yerganian’s laboratory at the Cancer Research Foundation in Boston. It is the most representative mammalian expression vector used in genetically engineered vaccine research and is the most successful cell type used to express foreign proteins. In the past decade, the CHO cell line has been the most frequently reported expression system [68]. CHO cells are naturally adherent and preferentially grow in suspension growth culture to obtain higher cell density and productivity [69]. Many vaccines have been developed using CHO cells, including vaccines against H5N1 avian influenza [70], H7N9 avian influenza [39], Chikungunya [71], HIV [72], and hepatitis B [73], therefore serving an important role in vaccine production and quality of life. In 1973, Thompson [74] isolated a strain of CHO cells that could be used for suspension culture and named this strain CHO-S. In 2002, after domesticating CHO-K1 from ECACC to suspension serum-free culture, Lonza Company established the CHO-K1SV cell line [75], which was widely used in its glutamine synthetase (GS) expression platform. In 2016, Lee et al. [76] acclimated CHO-K1 cells from adherent cells to suspension cells and used RNA sequencing and gene editing techniques to identify two genes necessary for cell adaptation to suspension culture, namely, IGFBP44 and AQP1. However, by only using the same single cell line to effectively modify the cell function, the results were affected by a large false positive rate, therefore limiting the popularization of their technique. In 2006, Wu Jieheng et al. [77] obtained CHO-S suspension cells by acclimating CHO cells in the logarithmic growth phase to SFM from low serum media.

2.4. BHK-21 suspension cells

The BHK-21 cell line is a continuous cell line for vaccine production. BHK-21 cells are anchor-dependent but can be grown rapidly in bioreactors after suspension culture for large-scale viral propagation, and for cost-effective vaccine production using SFM [12]. The BHK-21 cell line can be used for the proliferation and purification of a variety of viruses, such as FMDV, Encephalomyocarditis virus (EMCV), Reovirus, Vesicular stomatitis virus (VSV), and Zika virus (ZIKV) [40]. Presently, BHK-21 suspension cells are the first choice for the industrial production of FMDV vaccine antigens [78]. In 1967, suspension culture used microcarriers for carrying adherent cells, thereby maximizing the use of bioreactor volume [16].

2.5. HEK293 suspension cells

The HEK293 cell line is a commonly used human cell line for the expression of a variety of recombinant proteins. This cell line derives from the kidney of aborted human female embryos and was originally immortalized in 1973 by integrating a 4 kb adenovirus 5 (Ad5) genome fragment containing the E1A and E1B genes on chromosome 19 [79]. Two industrially relevant suspension cell lines are 293-F and 293-H, both of which can grow rapidly in SFM with high transfection rates. 293-H cells were originally derived from the more adherent HEK293 cell clone strain, which showed strong adhesion in the assay. Adherent cells have traditionally been widely used to produce viruses, such as adenoviruses (AAV) and lentiviruses, for clinical research. Although certain experimental procedures are more effective with adherent cells, such as chemical transfection and viral infection, large-scale production is more effective with suspended cells due to their ability to not form cell clumps in the medium. At present, the HEK293 cell line has been used in an influenza A virus vaccine, as a candidate for a rabies vaccine [80], SARS‐CoV‐2 vaccines [43], and is currently in advanced phase III clinical trials for the Ebola virus vaccine (rVSV-ZEBOV) [42].

2.6. MDBK suspension cells

The Madin-Darby bovine kidney (MDBK) cell line is an immortal cell line derived from bovine kidney, which is naturally adherent in culture medium containing FBS [56]. MDBK cells are susceptible to a variety of viruses and are widely used for virus proliferation and infection. However, due to the limitation of its growth conditions, it is usually cultured on roller bottles or microcarriers for industrial large-scale vaccine production [81,82]. Meanwhile, studies have shown that production of bovine adenovirus (BAdV) in suspended MDBK cells can achieve higher virus production than in adherent cells [83]. Bovine herpesvirus 1 (BOHV-1) was used as an example to study the adaptability of MDBK cell suspension culture and its effect on BOHV-1 production. It was found that increasing cell density could significantly increase the production of BOHV-1 in suspension culture, which has obvious advantages over adherent cell culture. More importantly, BOHV-1 produced using the suspension culture method has superior quality and immunogenicity. In conclusion, compared with adherent cell culture, suspension culture is space-saving and can support cell growth at a higher concentration; therefore, it is more suitable for industrial production.

2.7. Marc-145 suspension cells

Marc-145 derives from the rhesus monkey kidney cell line and is commonly used to isolate porcine reproductive and respiratory syndrome virus (PRRSV) for diagnostic and research purposes and vaccine production [46]. Currently, it is the most important host cell for PRRSV proliferation in vitro and the most suitable cell type for PRRSV vaccine production [84].

3. Genetic engineering methods

Although many suspension cell lines have been obtained by suspension acclimation techniques, there remain some shortcomings with these methods, namely, lengthy suspension acclimation cycles, high number of cell passages, and the potential risk of reduced cell proliferation activity and virus sensitivity. With the discovery of gene editing technology, attention has increasingly been given to functional gene expression modifications in cells to change cell traits. For adherent cells, there are a variety of adhesion molecules between the cells and the extracellular matrix (ECM), which enable cells to have the function of adherent growth. If the expression and function of these adhesion molecules are inhibited, the cell adhesion process will theoretically be effectively inhibited, allowing the cell to grow easily in suspension.

3.1. Existing genetically engineered suspension cells

The siat7e gene, also known as ST6GalNac-V, is a member of the α2–6 sialic acid transferase family. The ST6GalNAc family consists of six GalNAc α2,6-sialic acid transferases-three of which communicate with glycolipids (ST6GalNAc-III, V, and VI), and three that communicate with the N-acetylgalactoamine (GalNAc) residues of O-glycosylproteins (ST6GalNAc- I, II, and IV) – that promote the introduction of Neu5Ac [85], which is considered to be one of the genes that controls cell adhesion. Previous results show that when siat7e content is high, the cells maintain a low degree of adhesion; conversely, adhesion is enhanced after using siRNA to inhibit siat7e transcription [86]. Additionally, ST6GalNAc-V has been shown to mediate brain metastasis in breast cancer [87]. It was proven that MDCK cells could be transformed into non-anchor cells after introduction of the human siat7e gene; the successful incorporation and transcription of this human gene in the MDCK cells dramatically changed the cell phenotype. It was shown that the net surface charge of siat7e-expressing cells changed. Furthermore, the increased negative charge on the cell surface may help to reduce the adhesion between cells and the surface and lead to electrostatic repulsion between cells, thus allowing cells to grow in suspension. Siat7e-expressing cells were not only able to grow in suspension and produce the same virus as those produced in embryos, but also secreted approximately 20 times more hemagglutinin (HA) than the anchored parent cells. Established MDCK cell lines expressing siat7e have the potential to significantly increase the efficiency of influenza vaccine production, and are therefore likely to help reduce vaccine costs and provide a wider range of vaccines to a larger number of recipients worldwide [88].

3.2. Potential target genes for promoting suspension cell growth

The cell membrane surface of adherent culture cells contains a large number of adhesion factors. These adhesion factors regulate the activity of adherent culture cells and promote cell growth and proliferation by mediating cell-ECM or cell-cell contact and activating different signaling pathways. Theoretically, using gene editing methods to inhibit the expression or function of cell adhesion molecules, the cell-cell and cell-ECM adhesion contacts could be altered to allow cells to grow easily in suspension. ECM is one of the factors that determines the adhesion characteristics of cells in suspension culture. For example, the ECM-protein adhesion and cell aggregation abilities of suspension-adapted BHK-21 cells were significantly reduced relative to wild-type adherent BHK-21 cells [12]. Therefore, we can hypothesize that during suspension adaptation, cell survival and proliferation in serum-free conditions can reduce cell adhesion through the rearrangement of cell membrane integrins and the actin cytoskeleton [89,90]. Presently, genetically engineered suspended cells are relatively limited. Therefore, we screened eleven key cell adhesion genes as potential targets that have been shown by previous research to have significant effects on cell adhesion – through knockdown or overexpression studies – but have little effect on virus proliferation (Table 2). Furthermore, the cellular action pathways of these potential genes are shown in Figure 1.

Figure 1. Molecular model of potential target gene interactions in genetically engineered suspension cells. The cell membrane surface contains a large number of adhesion factors, which regulate the activity of adherent cultured cells and promote cell growth and proliferation by mediating the contact between cells and ECM or between cells to activate different signaling pathways. Integrin is an important adhesion factor and activates focal adhesion kinase (FAK) by mediating the contact between cells and ECM. FAK inhibits endogenous apoptosis by activating the PI3K/AKT and ERK pathways, thereby regulating cell growth. Therefore, genes that affect α6β4 integrin expression, namely, Itgb1, Talin-1, Kindlin-2, and HDs, and genes that affect adhesive spots, namely Src-1 and Paxillin, can be used as targets for genetic engineering modifications. The PINCH-1 gene regulates and maintains cell adhesion stability through ILK, parvin, and actin regulatory proteins related to the LIM2-LIM5 domain. Talin-1/kindlin-2/Src-1 and Paxillin/ILK/parvin/PINCH-1 form the adhesive plaque complex. Bridgemen mediates cell-cell adhesion. Plakoglobin is in an important class of signaling and adhesion molecules; it binds to classical cadherin and desmosomal cadherin and regulates the stability and strength of its connection. E-cadherin and α-Catenin, and β-Catenin and P120 form cadherin catenin adhesion complex and affect cell adhesion. E-cadherin plays an adhesive role through the Wnt pathway. The introduction of α-Catenin restores the cell response to JNK inhibition and leads to cell-cell adhesion; therefore, knocking out α-Catenin prevents cells from forming adhesive connections. The red font in the figure indicates the 11 key genes that act between cells or between cells and ECM, which can significantly affect cell adhesion through knockout or overexpression. Siat7e plays a negative regulatory role, while the other genes are positive regulators.

Table 2. Potential genetically engineered cell lines for vaccine production.

Gene targets	Edit mode	Regulation mode	Reported cell line	Signal pathway	References
Itgb1	Knock out	Positive control	Itgb1/^− -^iPSCs，keratinocytes–Itgb1，epidermal stem cell–Itgb1	PI3K/AKT signaling pathway	[91–93]
HDs (Itgb4)	Knock out	Positive control	Keratinocytes–Itgb4	PI3K/AKT signaling pathway	[94,95]
Talin-1	Knock out	Positive control	Fibroblasts–T1^Ko,MHCC-97L–Talin-1	PI3K/AKT signaling pathway	[96,97]
Kindlin-2	Knock out	Positive control	Fibroblasts–K2^Ko,HUVEC–kindlin-2,Mesenchymal Cancer Cell–Mig-2	-	[96,98,99]
SRC-1	Knock out	Positive control	Breast Cancer Cell–SRC-1^KO	FAK/PI3K/Akt signaling pathway	[100,101]
Paxillin	Knock out	Positive control	Fibroblasts–Paxillin，MEF–PXN^KO	AKT/PKB signaling pathway	[102,103]
PINCH-1	Knock out	Positive control	Deficient keratinocytes –PINCH-1	PI3K/AKT signaling pathway	[104,105]
E-cadherin	Knock out	Positive control	Highly undifferentiated carcinoma classified as grade IV ，	Rho-Rock pathway	[106–108]
α-catenin	Knock out	Positive control	Keratinocytes，F9 cells,the poorly differentiated cell lines，Cardiomyocyte	Ras-MAPK pathway	[109,110]
Plakoglobin	Knock out	Positive control	Keratinocyte	-	[111,112]
Siat 7e	Overexpression	Negative control	HeLa -Siat7e，Vero-Siat7e	PI3K/AKT signaling pathway	[63,86]

Integrins are heterodimers composed of an α subunit noncovalently associated with a β subunit with 18 α subunits and 9 β subunits currently identified. The β1-containing integrin (Itgb1) mainly mediates adhesion between cells and ECM components. It has been experimentally demonstrated that Itgb1 knockdown reduces iPSC-ECM adhesion and simultaneously increases ECM cross-migration in vitro [91]. Additionally, it plays a role in reducing cell adhesion between epidermal cells and ECM and keratinocytes [92,93]. Hemidesmosomes (HDs), with α6β4-integrin as the core, act as complex adhesive junctions linking the basement membrane to the intracellular keratin cytoskeleton and mediate stable anchoring to the ECM. α6β4 is a receptor for laminin, which mainly binds to laminin 5. Thus, disruption of HDs by depleting α6- or β4-integrin expression promotes collective cell migration and modulates migration activity. Experiments have shown that the destruction of HDs can affect the molecular diffusion rate of focal adhesion kinase (FAK), thereby reducing the adhesion of cell adhesins [94]. Additionally, some experiments have demonstrated that its deletion will reduce proliferation, survival, and differentiation of keratinocytes [95].

Talin is the most characteristic binding protein linking integrins to actin and is a major component of focal adhesions (FAs). Talin has two subtypes, namely talin-1 and talin-2. Talin-1 gene knockout cell lines were established in fibroblasts, and it was found that they could not activate their integrins nor bind to the fibrin in ECM, thus leading to a significant decline in cell adhesion [96]. Other studies have confirmed the role of talin-1 in carcinogenesis and provided a new therapeutic target for the treatment of hepatocellular carcinoma (HCC). Furthermore, talin-1 may promote cell adhesion by regulating the epithelial-mesenchymal transition (EMT) process [97]. Studies have also shown that T cells and T cell exocrines lacking talin-2 show reduced binding with integrin ligands ICAM-1 and MAdCAM-1 [113], demonstrating that knockdown of the talin-2 gene can reduce cell adhesion. Kindlins, which contain three members (Kindlin-1, Kindlin-2, and Kindlin-3), are key cell-ECM adhesion proteins and key activators of integrins. Kindlin-2, also known as mig-2, can directly bind to the β1- and β3-integrin tail [98]. Studies have shown that knockdown of the Kindlin-2 gene can prevent the activation of integrins, thus significantly affecting the adhesion ability of cells [99]. Additionally, deficiencies in Kindlin-1 and Kindlin-3 can lead to diseases. For example, Kindlin-1 deficiency causes skin weakness and blistering (called Kindler syndrome), and Kindlin-3 deficiency causes hemorrhagic disease and immune deficiency.

The Src family, also known as the P160 steroid receptor coactivator family, contains three members: Src-1, Src-2, and Src-3. The lack of Src-1 (also known as NCOA1) in human breast cancer cells has been shown to significantly prolong the time of adhesion plaque disassembly and reassembly and reduce the cell adhesion and migration ability on fibronectin [100]. In MCF-7 breast cancer cells, Src-2 knockdown reduced estrogen-induced cell proliferation and target gene expression [114]. Paxillin is a plaque-associated protein involved in regulating integrin signaling and organizing the actin cytoskeleton. Paxillin is associated with many signaling molecules, including adaptor molecules (p130Cas and CRK), kinases (FAK, Pyk2, PAK, and Src), tyrosine phosphatases (PTP – PEST), ARF – GAP proteins (p95pkl and PAG3), and papillomavirus E6 oncoproteins [115]. Paxillin knockout cells have defects in adhesion remodeling [102]; furthermore, loss of Paxillin leads to impaired activation of its downstream target FAK, which is an outward signaling marker of integrin [103].

PINCH has two PINCH proteins (PINCH-1 and PINCH-2, also known as LIMS1 and LIMS2, respectively); each protein has five LIM domains followed by a C-terminal nuclear localization signal. PINCH-1 forms a ternary complex downstream of the integrin with integrin-linked kinase (ILK) and parvin. The first LIM domains of PINCH-1 and PINCH-2 bind to ILK. PINCH-1 has been shown to regulate and maintain cell adhesion stability through ILK, parvin, and actin regulatory proteins associated with the LIM2-LIM5 domain. When the PINCH-1 gene is knocked out, keratinocytes showed significantly decreased adhesion, diffusion, and migration [104]; therefore, it could be used as a potential candidate gene for genetic modification of adherent cells in suspension culture.

The calcium dependent cell adhesins (cadherin) family is one of the earliest adhesion molecules that Takeichi discovered to mediate cell aggregation. In the presence of Ca²⁺, it can resist protease hydrolysis. The connection between the cytoskeleton transmembrane protein E-cadherin (also known as uvomorulin, L-CAM, cell CAM 120/80, or ARC-1) and actin filament is the key to cell-cell adhesion. Cells lacking E-cadherin expression do not aggregate or adhere to each other because the production of cadherin-catenin adhesion complexes is affected [106]. α-Catenin is a key subunit of the cadherin catenin adhesion complex, which affects cell-cell adhesion. In the absence of stable α-Catenin expression in poorly differentiated cell lines, the addition of α- Catenin cDNA will increase Ca²⁺-dependent cell-cell aggregation, indicating that α-Catenin directly leads to the loss of cell-cell adhesion in some cell lines [109]. In α-Catenin-ablated keratinocytes, cell-cell connection defects could be observed using immunofluorescence microscopy along with excessive cell proliferation [110].

Plakoglobin (PG) is a member of the adhesion/signaling protein family, which can bind to classical cadherin and desmosomal cadherin. PG connects the cytoplasmic domain of cadherin to the catenin/vinculin in the cytoskeletal connexin adhesion connection and the plakin family member desmoplakin (DP) in the desmosome; moreover, it regulates the stability and strength of these connections. Some studies have shown that the loss of PG leads to the reduction of the number and structural changes of desmosomes in the epidermis of mice [116]. In addition, the keratinocyte culture established from PG knockout mice showed decreased adhesion and increased motility in the transwell migration test [111].

4. Conclusion

In order to meet the increasing demand for vaccines, cell suspension culture is particularly important. Suspension culture of cells can maximize the use of large bioreactors for cost-effective and large-scale vaccine production; therefore, it is critical to adapt adherent cells to suspension culture. The suspension adaptation process of adherent cells primarily uses artificial interventions to resist apoptosis [117]. The process of suspension adaptation can generally be divided into three steps: serum-free adaptation culture, suspension adaptation culture, and bioreactor high-density adaptation culture; these steps are straightforward and frequently used in the suspension adaptation process of various mammalian cells. In large-scale industrial production, suspension culture of adherent cells in the reactor can provide the surface area required by microcarrier-dependent cells [13,14], thus expanding cell production. However, relative to serum-free suspension cell culture technology, carrier suspension culture technology has many disadvantages and uncertainties in the process of vaccine production, such as limited cell culture area, difficult separation of cells from carriers, and expensive microcarriers. Therefore, the adaptation of low serum to serum-free suspension culture is the most practical method to acclimate adherent cells to suspension cells.

There are three key points in the process of cell suspension acclimation. First, the correct low serum culture medium should be selected, while taking into account the growth and adaptation period of the cells. Second, the general serum concentration should be reduced in stages from 10% to 5–8% to 1–3%, and the subculture should be continued until there is no significant difference between the growth and proliferation of cells cultured with low serum and the adherent rate of cells cultured with high serum, at which point cell acclimation is considered complete. Third, the cell culture conditions should be maintained at 37°C and 5% CO₂, with a rotating speed generally ranging between 100–110 rpm, depending on the specific cells and media. For example, although both cells were cultured in suspension at 37°C and 5% CO₂, HEK293SF cells were cultured with a rotating speed of 110 rpm [42] while CHO-K1 cells were stirred in a rotating flask at 100 rpm [118] (Figure 2).

Figure 2. Illustration of cell suspension culture methodology.

In addition to the traditional method of engineering the domestication of cells, the use of genetic engineering to reduce or eliminate the expression of adhesion molecules is more likely to facilitate cell suspension. The genetically engineered methods to achieve suspension are more targeted and can effectively avoid the problem of excessive generations that occur during suspension domestication. Additionally, multigene editing offers several advantages, namely, easily achieved suspension, lack of tumorigenesis, and high virus production, which can collectively improve vaccine production efficiency and biological safety. Therefore, as potential targets of genetic engineering, we selected several key cell adhesion genes that have been proven to significantly affect cell adhesion through knockout or overexpression. The aim is to further improve vaccine production strategy by integrating the methods of genetically engineered cell line improvement, upstream optimization, and downstream processing, and to provide a basis for large-scale culture of other viral vaccines. However, we need to consider potential problems that could result from genetic engineering methods: First, adhesion is crucial to the normal growth and proliferation of cells, and the expression of adhesion molecules often involves multiple gene families. For example, talin and Kindlins cooperate to activate integrins, leading to FN binding and adhesion [113]. Therefore, if single gene editing has little effect, multigene editing can be attempted. Secondly, some target genes are multifunctional, and knockdown may cause multiple downstream effects that could result in death or substantial changes in cell or virus proliferation. Furthermore, the function of some genes may vary with cell type. For example, knockdown of Itgb1 in induced pluripotent stem cells promoted the migration of extracellular stromal cells but had no significant effect on cell apoptosis and proliferation [93]. However, in keratinocytes, Itgb1 knockdown reduced cell proliferation [94]. Therefore, gene function can create difficulties with gene editing technology and should be considered when selecting gene editing methods. Finally, since vaccine production requires the establishment of a long-term safe and stable cell line, the genetic stability and tumorigenicity of cell lines are important items checked by regulatory agencies. Currently, regulatory agencies mainly focus on genetic stability using qPCR, dPCR, and HTS tests. To determine the genetic stability of cell lines, the most widely used verified molecular approach is qPCR. It is simple to validate, conforms with Good Manufacturing Practice (GMP) standards, and is approved by regulatory authorities [119]. According to WHO test requirements, the tumorigenicity of cells is evaluated, and typically athymic nude mice are used to assess the tumorigenicity of mammalian cells [120]. Therefore, when obtaining suspension cells by genetic engineering, it is necessary to take into account the genetic stability and tumorigenicity of the cell lines.

These two methods of obtaining suspended cell lines have their own advantages and drawbacks. The method of suspension domestication is simple and controllable, with low pollution rate and cost. However, the experimental period is long, the number of cell passages required is too high, and there may be a risk of decreased cell proliferation activity and virus sensitivity. The genetic engineering method is more targeted and has enormous potential because of its fast design and construction speed. However, this method has a complicated operation and high cost (Table 3).Therefore, the combination of genetic engineering and suspension acclimation should be considered for the development of suspended cell lines; this combined approach may create an optimized method that can enhance benefits while minimizing weaknesses of the two individual approaches, resulting in an effective method more conducive to achieving high quality suspended cell lines.

Table 3. The advantages and disadvantages of the suspension acclimation methods.

	Serum-free suspension acclimation	Microcarrier suspension	Genetic engineering suspension
advantages	Free cell growth and convenient amplification	Provides a great culture surface area/volume ratio	Design and build fast
	Uniform culture environment	High domestication quality	Wide range of cells
	Simple and controllable culture operation	Sufficient to obtain high virus yields	More targeted
	Simple sampling methods	Less space for cultivation	Enormous potential
	Low contamination rate and low cost	Low labor demand	-
disadvantages	Long domestication period	Limited cell culture area	Complex operation
	The number of cell passages required is too high	Difficult separation of cells from carriers	High cost
	Cell proliferation activity decreased	Microcarriers are expensive	Off-target risk
	Decreased virus sensitivity	Process is not easy to scale up	-

This review systematically summarizes and analyzes the development and research progress of all suspended cell lines for inactivated viral vaccine production. The relevant research will contribute to a more comprehensive understanding of the molecular mechanism of cell adhesion and provide protocols and candidate target genes for the artificial establishment of suspended cell lines for vaccine production.

5. Expert opinion

Inactivated viral vaccines are currently the main form of vaccination for most viruses worldwide and are characterized by their high safety profile, stability, and inability to replicate in the body, making them available to immunodeficient individuals. Because it has more complete information about the antigenic epitopes, it can activate a more comprehensive humoral immune response, and its effectiveness can be tested over a long period of time. The total value of the global inactivated vaccine market reached $37.6 billion in 2019, accounting for one-sixth of the global vaccine market value, and is expected to grow to $47.5 billion in 2026, at a CAGR of 3.3%. For instance, in 2021, over 80 million domestic influenza vaccines will be distributed, of which approximately 55 million – or more than 65%—would be quadrivalent inactivated influenza vaccines. It is expected that the number of quadrivalent inactivated influenza vaccine issued will increase year by year, accounting for a larger proportion of the total number of all influenza vaccines issued.

Animal cells are good substrates for different viral expansions and are widely used in the production process of various vaccines. The cells are classified into two types of growth: adherent growth and suspension growth. Suspension cells can get rid of the limitation of the surface area of the culture vector to achieve high density culture and significantly increase the vaccine yield. Serum-free suspension culture can further reduce production costs, simplify the production process, exclude the influence of serum, make the production process more controllable, and facilitate the expansion of culture scale. Currently, suspension culture has been achieved by MDCK cells, CHO cells, BHK-21 cells, HEK293 cells, and others, but the majority of them still have not. As a result, scientists are still investigating and attempting to get cells to switch from wall growth to suspension growth.

Through systematic analysis of existing studies and applications, we found that there are two main ways of obtaining suspension cells, mainly the stepwise descending serum suspension domestication method and the genetic engineering modification method. Currently, some suspension cells obtained through domestication have been used in the production of human or veterinary vaccines. Examples include the use of MDCK suspension cells in the production of influenza vaccine and the use of EB66 suspension cells in the production of diphasic inactivated vaccines for chicken Newcastle disease and avian influenza (subtype H9). However, there are still a lot of adherent cells that have not been domesticated successfully, and there may be a series of problems in the traditional suspension domestication process, such as high cell generation, reduced virus susceptibility and cell activity during domestication, and not easy to grow in a serum-free environment, which eventually lead to domestication failure, then using gene editing technology to target cell adhesion genes may become a new solution, for example, overexpression of siat7e gene can reduce the adherent performance of MDCK cells and thus easy to obtain MDCK suspension cells.

Cell adhesion processes exhibit exceptionally high tissue and cell specificity, and our understanding of cell adhesion mechanisms for vaccine production is still very limited. In this paper, we systematically compiled the major existing cell adhesion gene pathways and predicted 11 potential target genes that promote cell suspension to provide a new idea for establishing suspension cells for vaccine production. While modern vaccines are diverse – such as recombinant protein vaccines, adenovirus vector vaccines and mRNA vaccines each have advantages and are equally promising – inactivated vaccines remain the most widespread form of vaccine and the mainstay of global disease prevention because of their safety, stability and long-term storage capacity. With the progressive understanding of these cell lines and cell adhesion mechanisms, it is expected that in the next 5–10 years, targeted cell modification using new technologies of genetic engineering will lead to the development of high quality suspension cell lines with a variety of advantageous traits such as high yield and virus susceptibility, and it is expected that single cell suspension bioreactor production processes, such as the use of VERO suspension cells Rabies vaccine and polio vaccine, etc. It can effectively increase the vaccine yield and improve the biosafety, making it better for the production of different inactivated virus vaccines.

Article highlights

• Inactivated virus vaccines are the most widely used tool to prevent disease.

• serum-free suspension cell cultures are more suitable for bioreactor-based production processes, which enable large-scale cell culture and a more stable and controlled vaccine production process and improve the process stability of the vaccine.

• The strategy of low serum adaptation to serum-free suspension culture is the simplest and most direct method to domesticate adherent cells into suspension cells.

• Suspension cells can greatly improve the vaccine production efficiency, and many cell lines have been adapted to suspension culture and applied to the production of various virus vaccines, such as COVI-VAC, Newcastle disease, poliovirus, rabies, Foot-and-Mouth disease virus.

• With the discovery of gene editing technology, attention has increasingly been given to functional gene expression modifications in cells to change cell traits.

• For adherent cells, there are a variety of adhesion molecules between the cells and the extracellular matrix (ECM), which enable cells to have the function of adherent growth. If the expression and function of these adhesion molecules are inhibited, the cell adhesion process will theoretically be effectively inhibited, allowing the cell to grow easily in suspension.

• To systematically organize cell adhesion-related studies and propose 11 potential target genes for cell suspension for the first time, which can provide a new idea for the establishment of suspension cells for vaccine production.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Author contributions

All authors contributed to the article and approved the submitted version. All authors have read and agreed to the published version of the manuscript.

Additional information

Funding

This research was funded and supported by the National Natural Science Foundation of China (32160164) and National major scientific and technological special project for Significant New Drugs Development (2015ZX09102016).