**Table 6.**

*Plant-based vaccines for pandemics and epidemics infectious diseases.*

## *Next Generation of Transgenic Plants: From Farming to Pharming DOI: http://dx.doi.org/10.5772/intechopen.102004*

cVLP vaccine. CPG adjuvant vaccine was administered in healthy adult subjects in two age groups, Part A (18–49 years) and Part B (50–85 years). The company also produces seasonal influenza vaccines through *Nicotiana benthamiana* plants [86]. BaiyaPharming™ produced a subunit-based plant-derived vaccine against SARS-CoV-2 in *N. benthamiana*. Baiya SARS-CoV-2 Vax 1 was chosen between six candidates which showed better immunogenicity in mice and monkeys. Baiya Phytopharm expects to initiate human trials by September 2021 [87]. Plant-derived vaccines for humans and animals stand out as a viable alternative that can be used to overcome the barriers of conventional vaccines. Within the scope of transgenic plants, it is possible to produce cost-effective, immunogenic and safer vaccines in plants with an enhanced amount, effective isolation and purification methods. Correct use of adjuvants along with the production of recombinant vaccine antigens also seems equally crucial for the future of this technology.

#### **3.2 Replacement human proteins**

Plants are usually referred to as molecular factories to provide humans with many useful molecules for many purposes. In the last decades, it has also become available to produce specific heterologous proteins as a replacement in humans. In this manner, the first plant-derived pharmaceutical was human growth hormone, which was expressed in transgenic tobacco as a fusion with *Agrobacterium* nopaline synthase enzyme in 1986 [88]. Today, recombinant human proteins are a considerable part of FDA-approved biotechnological drugs and recombinant plant-derived proteins are extended in many categories as industrial enzymes, research intended technical proteins, nutritional supplements and polymers as well as antibodies and vaccines which were mentioned in the previous section. Replacement human proteins include products as growth hormone, HSA, α-interferon, erythropoietin (EPO), human secreted alkaline phosphatase, aprotinin, collagen, α1-antitrypsin and more (**Table 7**).

Human growth hormone (hGH) has various biological functions on protein synthesis, cell proliferation and metabolism. After the first successful plantderived production, many different strategies were achieved. In a recent approach, a synthetic hGH gene (shGH) has been synthesized in a plant expression vector under the control of the rice amylase 3D (Ramy3D) promoter. The plant expression vector was introduced into rice calli (*O. sativa* L.) via the particle bombardment transformation method. The shGH protein expression was verified and quantified as 57 mg/L in the transgenic rice cell suspension medium. Biological activities of the shGH were found similar to the conventional *E. coli*-derived recombinant hGH. Likewise, many different plant tissues and expression systems are suggested as effective hGH production replacements [101].

Human serum albumin (HSA) is the most abundant protein in human blood plasma. HSA is a soluble, globular, unglycosylated, monomeric multidomain protein. The single polypeptide of HSA consists of 585 amino acids with a range of structural configurations that fold into three helical domains [104]. It is also the first full-size native human protein expressed in plants and there is more than 500 tons annual demand. The transient expression level of the HSA gene in different genotypes was achieved in many plants. Recently, the A. tumefaciens strains LB4404 and GV3101 containing pBI121-HSA binary vector were infiltered in *Nicotiana benthamiana* and *N. tabacum* varieties. The bioactive HAS expression in tobacco leaves through the expression of the HSA gene in the plant system is suggested as the first transient expression success in literature [105].

α-interferon is the first human pharmaceutical protein produced in rice. The plasmid pIG3031 containing human α-interferon cDNA and the neomycin


#### *Next Generation of Transgenic Plants: From Farming to Pharming DOI: http://dx.doi.org/10.5772/intechopen.102004*


**Table 7.**

*Replacement human proteins produced from transgenic plants.*

*Genetically Modified Plants and Beyond*

**70**

phosphotransferase II coding sequence was introduced to Indica rice protoplasts via lipofection-mediated transformation. Transgenic plants were regenerated from transformed calli. Extracts of transgenic cell cultures and plants presented apparent interferon activity proven by the resistance of human amniotic cell lines to viral infection in the presence of plant extracts. This production encouraged many other strategies as human α-interferon cDNA was correctly expressed in rice cells [106].

Erythropoietin (EPO) is the first human replacement protein produced in tobacco suspension cells. EPO is a cytokine that regulates and maintains the physiological level of circulating erythrocytes. The survival of erythroid precursor cells is also achieved through EPO. It stimulates the proliferation and differentiation of the precursor cells by plasma membrane EPO-receptor interactions. The first transgenic approach introduced human Epo cDNA via *A. tumefaciens*-mediated gene transfer to tobacco BY2 cells (*N. tabacum* L. cv. Bright Yellow 2). EPO is a heavily glycosylated protein, therefore glycosylation of tobacco-derived EPO by smaller oligosaccharides led the molecule to remain attached to the cell wall. However, it induced the differentiation and proliferation of erythroid cells in in vitro biological activity trials [107]. Recently, mammalian cell-derived recombinant human erythropoietin (rhuEPOM) is a multimodal neuroprotectant in experimental stroke models. However, the rhuEPOM clinical trials were terminated due to the increased risk of thrombosis, largely ascribed to its erythropoietic function. A rhuEPO derivative without sialic acid residues was produced in a plant-based expression system which is lacking sialylation capacity to produce asialo-rhuEPO<sup>P</sup> . Repeated intravenous injection (44 μg/kg bw) in mice presented no increase in hemoglobin levels and red blood cells. Hence, Asialo-rhuEPO<sup>P</sup> that lacks erythropoietic activity and immunogenicity suggested as a great multimodal neuroprotectant for stroke treatment [108].

Pathogen contamination risk of animal-derived collagen initiated the need for safe recombinant production of this complex molecule. Collagen is the first human structural-protein polymer produced in a plant-derived system. The use of the tobacco plant as a novel expression system for the production of human homotrimeric collagen I was achieved in 2000. cDNA encoding the human proalpha1(I) chain was introduced to tobacco. Expressed recombinant procollagen has been folded to stable homotrimeric triple helix-shaped collagen as in animal cells [109]. *A. tumefaciens*-mediated transient expression of the recombinant hydroxylated homotrimeric collagen in tobacco plants that are co-transformed with a human type I collagen and a chimeric proline-4-hydroxylase (P4H) improved the quality of collagen by enhancing thermostability to 37°C [110].

Human 1-antitrypsin (AAT) is a 394-amino-acid glycoprotein that inhibits the activity of the serine protease neutrophil elastase. Healthy individuals control elastase activity by producing sufficient quantities of AAT into the bloodstream. When the circulating concentration drops below 15%, various diseases as emphysema, hepatitis and skin disorders occur. Various expression systems, cell types and tissues of plants were used for molecular pharming earlier. However, rice suspension cells were used for the molecular farming of recombinant human 1-antitrypsin (rAAT) in biologically active form for the first time in 1999. Transformation of rice callus tissues with a p3D-AAT expression vector containing the cDNA for mature human AAT protein was achieved. The promoter, signal peptide and terminator of a rice-amylase gene Amy3D, which tightly controls simple sugars such as sucrose, regulated expression and secretion of rAAT. Expression of the rAAT was initiated by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. For that time being, the rice cell culture system clearly contributed to the molecular pharming field.

As a concluding summary, plants have various upsides against traditional microbial and animal cell culture systems in respect to molecular pharming. Some of these include cost efficiency, easier up-scale production, absence of human pathogens and accurate maturation (folding and assembly) of proteins. The potential of molecular pharming in plants attracts more and more entrepreneurs following the numerous successful products and companies. Achieving secretory properties and targeting of plant products also surpass many other production systems. The use of plants as bioreactors is well known, and its applications are increasing for both recombinant protein expression and recombinant pharmaceutical production. This chapter showcases the various plant biotechnology application and strategies as applied in the production of recombinant antibodies, antigens, therapeutic proteins and enzymes, that are used in the treatment of various diseases. Now with the COVID-19 pandemic, more than ever this approach is taking center stage. This is so important as it will reveal the hidden treasure that transgenic plants already offered but mired in the genetically modified organism debate and therefore rejected before these other applications beyond food could be readily realized. Indeed, different systems exist within the system utilizing many different parts and tissues of the plant to produce products. Furthermore, this system is more advantageous in a eukaryotic system that performs post-translational modification as would animal and yeast cells thus yielding the final desired therapeutic product comparable to what is already produced by humans, for example.
