Plant Species and Expression Systems on Vaccines Production: Comparison
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Please note this is a comparison between Version 3 by Abdullah Makhzoum and Version 2 by Camila Xu.

Use of plant cells as vaccine production hosts is appropriate for the expression of complex antigens in a cost-efficient manner. Recombinant proteins can be produced using various expression systems, each of which has its own benefits and disadvantages pertaining to the yield, time required for expression and the costs incurred in this process.

 

  • vaccine
  • transgenic plant
  • plant-made vaccine
  • plant molecular pharming

1. Introduction

Plant-based vaccines have gained importance over the last 20 years because traditional technologies for vaccine productions are crucial in terms of vaccine efficacy and safety [1][2]. Plant-derived vaccines can overcome such shortcomings and allow the expression of recombinant proteins on a large scale. Also, the application of transgenic plants for vaccine production dispenses with issues arising from contamination factors associated with mammalian-derived vaccines [3][4][5].

The recent pandemic caused by SARS-CoV-2 has underlined the significance of vaccines particularly against viral diseases and necessitates viable solutions for prevention and spread of viral pathogens. There is now great demand for high-quality recombinant vaccines generated using novel genetic engineering technologies and this has provided new platforms for production of recombinant proteins using various expression hosts. Importantly, plant molecular pharming (PMP) has evolved tremendously due to the invention of transient protein expression technologies yielding vaccine / therapeutic proteins on a large scale within short timeframes.

Presently, bacteria, yeast, insect cell systems and mammalian cell cultures are being used primarily, as they are well-characterized [3][6]. Amongst these systems, yeast-based expression provides the capability for post-translational modifications while bacterial expression platforms generate elevated yields of recombinant proteins in brief time periods. Nevertheless, a majority of currently approved biopharmaceuticals are based on mammalian cell culture expression technologies mainly using viral vectors [7][8].

2. Host Plant Species Used during Recombinant Protein Production

The plant molecular pharming technology has enabled the expression of pharmaceutical proteins in plants. Transgenic plants provide the capability to generate recombinant proteins essential for preclusion, diagnosis and therapy of various diseases. Amongst plant systems, the appropriate choice of plant species as hosts for vaccine production is highly significant. Popularly, tobacco has been used as a host plant for generation of therapeutic polypeptides such as antibodies, vaccines and cytokines [9][10]. Tobacco-based expression affords a facile and advantageous tool for expression of abundant amounts of recombinant proteins due to its large foliage [11][12] in addition to having high content of soluble proteins [12]. Transgenic tobacco plants have demonstrated high-level expression of transgenes encoding therapeutic proteins [13].

Among the transgenic plant species used for recombinant protein production, Arabidopsis thaliana plants allow increased ease of Agrobacterium-mediated transformation using the floral dip technique [14]. Also, Arabidopsis plants possess small, genetically adaptable genomes allowing easy transformation using genetic engineering technologies while being capable of generating numerous seeds. These plants have been considered as model protein-expression organisms for the above reasons. Nevertheless, A. thaliana is not appropriate for plant molecular pharming due to the small size of its foliage that yields low amounts of biomass in addition to being difficult to cultivate on a large-scale basis [14].

Crops such as maize, rice, barley and wheat are increasingly used as popular expression platforms for recombinant protein expression. When compared to maize, the annual yield of rice grains is slightly lower and rice seeds contain lesser protein content (about 8%) than that of maize seeds [14]. Despite this, rice forms an ideal model among cereal species because its genome has been completely sequenced [15][16]. Nevertheless, the major constraint of using rice as a host plant rather than maize is that the former is expensive to acquire, which may dissuade developing countries from employing rice for the expression of recombinant proteins although rice serves as a staple food in Asia and Africa.

On the other hand, maize is more favorable than other cereal crops as it has a higher grain yield and elevated seed protein content (about 10%) in addition to being the most extensively cultivated crop in North America [8] which makes it cheaper to procure when compared to rice. Also, maize is amenable to easy in vitro transformation and can be grown quickly in fields. ProdiGene Inc., has produced two recombinant proteins using maize plants [17][18].

Among tubers, potato contains very high yield of annual tuber biomass of about 125 tonnes/ha and therefore can be used to produce recombinant proteins in large amounts as tubers of potatoes are tractable to harbor proteins in large quantities [14]. However, the major drawback of using potatoes for edible vaccine delivery is that potatoes are used as cooked food prior to consumption and the heat applied during cooking could denature the expressed proteins thereby hindering their capability to induce immune response. Also, potatoes inherently contain starch in elevated amounts that could hinder the downstream processing of the expressed pharmaceutical proteins.

Whereas tomatoes are amenable to be cultivated in greenhouses allowing for their production on a large scale as well as containment. Tomato plants are also advantageous due to their elevated yield of annual fruit biomass of about 60 tonnes/ha and have been employed to produce several vaccine candidates [19]. Another host for the production of recombinant proteins is algae that can be grown rapidly enabling scale up within brief time periods, thus making them ideal platforms for expression of recombinant proteins. Algae are inexpensive to grow as their culture media incur low costs and can be reused to grow algae in a continuous cycle [20].

Yet another tractable system for recombinant protein production is the rapidly grown lettuce which generates diminished levels of secondary metabolites such as alkaloids and phenolics [21], thereby simplifying the purification process of the recombinant proteins in addition to reducing production costs. However, despite having an annual biomass of 30 tonnes/ha, lettuce plants incur high costs to produce and purchase [14]. Also, lettuce has elevated water content (98%), which negatively impacts protein stability and therefore yield [14]. Table 1 enlists some of the uses of different host species in the production of recombinant proteins. Table 1 summarizes some of the applications of these plant host species that have been reported over time.

Table 1. Applications of different plant species in recombinant protein production.

Host Plant Species

Applications

Tobacco

- Cancer antibodies [22][23] and vaccine [24] production

- HIV antibodies [25] and vaccines [26][27] production

Arabidopsis thaliana

- α-creatine kinase MAK33 mAb (Fab) expression with up to 6% protein accumulation [28]

- Helicobacter pylori TonB protein production for the immunization against Helicobacter infections [29].

Rice (Oryza sativa)

- Recombinant hGM-CSF expression with high yield of about 129 mg/L [30]

- Recombinant human lysozyme protein production [31]

Maize (Zea Mays)

- β-glucuronidase (GUS) expression with accumulation levels of up to 0.7% of water-soluble protein extracted from dry seeds

- Protease inhibitor aprotinin production [32]

Potato (Solanum tuberosum)

- Production of pharmaceutical proteins such as human interleukins [33][34] and human interferons [35]

- Vaccines production against different enteric diseases [36]

Tomato (Lycopersicon esculentum)

- Different pharmaceutical proteins expression such as the scFv recognizing carcinoembryonic antigen [37].

- Vaccines expression such as the respiratory syncytial virus-F protein [38]

Alga (Chlamydomonas reinhardtii)

- Monoclonal antibodies production [20]

Lettuce (Lactuca sativa)

- Recombinant proteins production such as the hepatitis B surface antigen [39][40]

- Antibodies production against colorectal cancer, rabies, and anthrax [41].
3. Different Expression Systems Used during Recombinant Protein Production

The advent of recombinant protein technologies in the 1970s and the resultant discovery that eukaryotic genes can be expressed in E. coli [42] were major milestones in the progress of molecular biology. Bacterial cells such as E. coli and mammalian cells were typically used as protein expression systems for a long time. Nevertheless, on account of their ease of cultivation and cost-efficacy, plants have become popular for expressing recombinant proteins particularly for the generation of vaccine candidates against various diseases, some of which have successfully undergone clinical trials [43][44][45][46][47][48]. The selection of an ideal recombinant protein expression system is a crucial step. The most commonly used systems are derived from bacterial cells (primarily E. coli) [49], yeasts (Saccharomyces cerevisiae and Pichia pastoris) [50], insect cells, mammalian cells and plants.

3.1. Plants

Plants as recombinant expression systems

Protein-based biologicals are among the largest and rapidly emerging category of pharmaceutical products [51]. Transgenic plant systems afford a low-cost, safe and easily scalable platform for production of recombinant proteins. The successful launch of ZMapp [52], the anti-Ebola virus drug is one example of the significant advancement of plant-made pharmaceuticals (PMP). Nevertheless, small differences in the protein N-glycosylation patterns do exist between human and plant cells which are of major concern as they may induce plant-glycan-specific antibodies that could lower the therapeutic efficacy of the plant-made protein candidate and could therefore lead to adverse outcomes. Glycoengineering of plants knocks out genes encoding plant-specific glycosylation while recruiting mammalian glycosylation genes thus resulting in the production of plant hosts capable of expressing mAbs harboring authentic human N-glycans. Additionally, these plant-made mAbs possess a degree of glycan homogeneity that cannot be accomplished by mammalian cells or in vitro expression platforms. This is beneficial at the stage of regulatory ratification of the respective plant-based product. Thus, the on-demand availability of plant lines expressing biomolecules that harbor tailor-made mammalian N-glycans enables the development of vaccines and therapeutics with more potent efficiency and safety compared to those expressed using other recombinant production platforms [51]. Plants dispense with the caveat of microbial contamination typically found with other expression systems such as mammalian cells and bacteria [51].

Agrobacterium tumefaciens has been recognized as a plant pathogen for a long time. However, its capability to introduce foreign DNA into plants has only been explored in recent times [53]. This bacterium causes crown gall disease in a broad range of plant hosts in which it transfers and integrates a portion of its own DNA, known as the T-DNA into the genome of the concerned plant [54]. Agrobacterium gains entry into plants via cuts or wounds occurring in the roots, leaves or stem of the plants followed by the insertion of its T-DNA which induces the development of swollen galls in the plant. This enables interkingdom transfer of genetic material which makes Agrobacterium a propitious vector for generation of foreign proteins in transgenic plants. Additionally, the virulence genes of Agrobacteria used for generating transgenic plants are removed, in other terms, the bacterium is ‘disarmed’ by deletion of most of its T-DNA excepting the left and right border sequences, through which the respective foreign gene is integrated into the plant genome. Figure 1 below depicts the various steps in the bioengineering of vaccines and their expression in transgenic plants.

Even though plant-based vaccine production systems seem highly promising, there are concerns of regulatory approval that need to be addressed for commercial, large-scale expression of recombinant biopharmaceuticals in plants. Since the generation of recombinant proteins in mammalian and microbial cell cultures is labor and capital intensive, the emergence of plant-based expression platforms is a welcome advancement.

3.2. Mammalian Cells

The application of mammalian cells calls for the judicious consideration of the cell line of choice. The decision to use a given mammalian cell line is determined by factors such as the post-translational modifications required, whether transient or stable protein expression is needed and consideration of the production scale of the recombinant protein [55]. Human embryonic kidney 293 cells (HEK-293) and Chinese hamster ovary cells (CHO) are the most popularly used mammalian cells for recombinant protein expression [56] since these cells can be used both as suspension and adherent cultures.

Mammalian cells are the singular platform that enable the expression of recombinant proteins as similar as possible to their wild type equivalents in genetic terms. This is especially true for antibodies generated in mammalian cells that are nearly the same as their human-expressed counterparts [57]. By virtue of their capability to carry out complex post-translational modifications, mammalian cells are typically employed for the synthesis of complex recombinants [56]. However, the drawback in the use of mammalian cells for this purpose is that they require high-cost culture media and require specially designated production facilities in addition to the problem of contamination inherent to all animal cell-based systems [51].

3.3. Insect Cells

Aside from mammalian cells, insect cells are popularly used to express proteins having complex post-translational modifications and are comparatively more facile to culture and maintain in vitro in addition to enabling both stable ad transient expression [58]. Insect cells display greater tolerance to osmolality and express higher levels of the recombinant protein candidates [59]. The most popular insect cell line is that of the Drosophila melanogaster. Similar to mammalian cells, the choice of a given insect cell line for recombinant protein expression is dependent upon whether stable or transient expression is desired [60]. Importantly, insect cells are increasingly being used along with the baculovirus expression vector system as recombinant expression platforms [59]. The exclusion of serum from media used for culturing insect cells is being seriously considered which would be advantageous in discounting any xenogeneic contamination issues.

3.4. Bacterial Systems

Bacteria are facile systems to manipulate for the purposes of genetic engineering. E. coli, in particular is malleable in vitro and is genetically as well as phenotypically diverse, making it the most widely used bacterial host for producing recombinant proteins. E. coli exhibits low degree of conservation between its various strains and is therefore one of the most diverse bacterial species [61]. Currently active research is ongoing towards augmenting the efficiency of E. coli as a host for recombinant protein expression. For this purpose, E. coli has been manipulated by deleting some of the genes from its genome to remove damaged genes, transposons and insertion sequences [62]. These bacteria require short time periods to culture and have obtained regulatory approval for use in recombinant protein expression.

3.5. Yeasts

Yeasts are amenable to easy genetic modification, thus making them quintessential for recombinant protein expression. Aside from the widely used Saccharomyces cerevisiae for this purpose, other yeast species such as Kluyveromyces lactis, Komagataella sp. and Yarrowia lipolytica have emerged as efficient choices [50]. Perhaps the most appealing characteristic of yeasts is their capability to incorporate post-translational modifications which enhances the stability of the expressed proteins [63]. Also, yeasts are able to withstand low pH conditions and therefore can be used for large-scale production of the therapeutic / vaccine candidates. Table 2 enlists some the benefits and disadvantages of the various protein expression platforms.

Table 2. Advantages and drawbacks of the different expression systems used for recombinant protein expression [64][65][66].

Expression System

Advantages

Disadvantages

Plant

- Low cost and high scalability

- Ability to produce complex proteins

- Growth protocols are optimized

- No pathogenic contamination risks

- Insufficient or incomplete glycosylation

- Lacks regulatory approval

- Inconsistent quality product

- Difficult downstream purification

Mammalian cells

- High protein yield

- Extensive industrial funding

- Accurate post-translational modifications

- Regulatory approval exists

- Native protein

- High production process

- Time consuming to culture and poor scalability

- High risks of human pathogen contamination

- Costly to culture

- Heterologous product

Insect cells

- Ability to produce complex proteins

- High protein expression

- Accurate post-translational modifications

- Native protein

- Regulatory approval exists

- High scalability

- Costly to culture

- Extended production time

- Undesirable post-translational modifications

Bacteria

- Regulatory approval exists

- High protein expression

- High scalability

- Docile

- Cell lines well characterized

- Short production time

- Cheap

- No post-translational modifications

- Accumulation of endotoxins

- Inactive proteins

Yeast

- High growth rate and scalability

- Cheap

- Regulatory approval exists

- Native protein

- Docile

- Cell lines well characterized

- Accurate post-translational modifications

- Excessive protein glycosylation

- Thick cell walls make cell disruption difficult

- Low capacity for glycosylation

From the above, it is evident that plants are increasingly gaining ground as quintessential systems for the production of recombinant proteins. Nevertheless, commercialization of these plant systems is still impending due to the requirement of protracted downstream processing procedures, low yields and inconsistent product quality. In particular, the yield of the recombinant protein indicates the host plants’ intrinsic productivity. The major targets of recombinant expression systems are primarily high quality, high yield and low cost [6]. Initially, the low-cost and easy scalability of plant-based expression systems were expected decrease manufacturing costs. Notwithstanding, this has not been found to be true due to the low yields, poor product recovery and high purification costs.

Even as cell-specific productivity (qP) that indicates the daily production capacity of a given expression system has been demonstrated for other platforms such as the CHO mammalian cells [67], equivalent findings are not available for plant-based systems. Also, plant cells are notably larger when compared to other cell types such as bacterial and mammalian cells [6], due to which it is difficult to augment productivity in suspension cultures simply by increasing cell numbers. Although this can be circumvented by increasing production volumes, this would necessitate scale-up of plant cell suspension cultures which increases the costs and is therefore commercially inviable [68]. This is distinct from whole plants which can generate high biomass at low costs. Yet another intriguing plant system that has been used successfully for recombinant protein expression is the hairy roots system which provides more genetic stability over prolonged time periods [69][70][71][72].

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