Major outbreak throughout the history of human civilizations .
Infectious diseases threatened humankind countless times through history, when knowledge on microorganisms was absent and medical capabilities were limited. Pandemics and outbreaks caused death of millions, brought empires to their knees and even wiped some ancient civilizations. In “modern” days, despite of improved medical application, sanitary precautions and effective medicines, infectious diseases are still cause of more than 54% of total mortality in developing countries. Millions of people are protected from the infectious diseases annually as a result of mass immunization campaigns. Nevertheless, novel diseases as COVID-19, MERS-CoV, avian influenza, Ebola, Zika and possible future infections require dynamic vaccine research and investment. Along with all the advantages of vaccines, there are several limitations regarding cost, biosafety/biosecurity, storage, distribution, degradation topics. Plant-based vaccine production for humans and animals has been under serious consideration to overcome some of these limitations. Nowadays, plant biotechnology brought new insight to vaccines research through gene transfer strategies to plants and improvements in amount, isolation and purification and addition of adjuvant for production of recombinant vaccine antigens in plants. Recombinant vaccines can undeniably offer us new standards and legal regulations to be introduced for the development, approval, authorization, licensing, distribution and marketing of such vaccines. The aim of this chapter is to exploit uses, methods and advantages of recombinant DNA technology and novel plant biotechnology applications for plant-based vaccine research in respect to existing infectious diseases.
- Plant-based vaccine
- recombinant protein
- virus-like particles
- transgenic plant
- molecular farming
- oral vaccine
- chloroplast transformation
- transient expression
- stable nuclear transformation
There are many breakthrough events during ongoing human civilization process. All of these individual events contributed the process in different significance. Nevertheless, agricultural development and animal domestication significantly accelerated all the other developments due to the fact that they saved people from daily boundary of finding nutrition and allowed more spare time for socialization and thinking.
Advantages of settled life style increased population in early cities rapidly. Ancient cities as Rome, Athens, Fayum varied in population between a hundred thousand to a million in various eras [1, 2]. Lacking the knowledge of microorganisms, hygiene and sanitation precautions, underdeveloped sewer systems and living so close to domesticated animals and people resulted to the rise of “civilization pathogens”. It is hypothesized that virulent pathogens were present but not persistent due to the limited population in human communities before agricultural development and urbanization. Most of the animals tend to live together in herds. Even though the herd lifestyle is very suitable for the transmission of pathogens, there were limited contact between humans and animals since hunting was the only viable way. Developments in agriculture and animal domestication lifted that barrier and allowed animal diseases to be transmitted to humans more frequently in higher population densities. Major fatal human diseases as measles, tuberculosis, smallpox, influenza, pertussis, and falciparum malaria are linked to early domesticated animals via phylogenetic analysis . Throughout the history of human civilization there are several outbreaks of pandemic diseases which shaped the world socially, economically and politically (Table 1).
|Common Name||Year(s)||Cause||Estimated Death (Million)|
|Plague of Athens||430 B.C.||0.1|
|Antonine Plague||165–180||Small pox (||5|
|Plague of Justanian||541–542||30–50|
|New World Smallpox||1520 - NA||25–55|
|Great Plague of London||1665||0.075–0.1|
|Russian Flu||1889–1890||A/H3N8, A/H2N2, or coronavirus OC43 (uncertain)||1|
|Yellow Fewer||Late 1800s||RNA virus from ||0.15|
|Spanish Flu||1918–1920||Influenza strains of A/H1N1||50|
|Asian Flu||1957–1958||Influenza A virus subtype H2N2||1|
|Hong Kong Flu||1968–1970||H3N2 strain of the influenza A virus||1|
|HIV-AIDS||1981-ongoing||35 and counting|
|Swine Flu||2009||H1N1 influenza virus||0.2|
|Covid-19||2019-ongoing||Coronavirus (SARS-CoV-2)||2.52 and counting|
In present day, vaccines are the vital part of the preventive healthcare globally. Many of the once deadly diseases are not present for decades, since the mass vaccination campaigns were applied worldwide. Based on their production method and protection mechanisms, vaccines are categorized under several classes including live attenuated, inactive (killed whole organism), toxoid, subunit (purified native or recombinant protein, polysaccharide or peptide), virus-like particle, outer membrane particle, protein-polysaccharide conjugate, viral vectored, nucleic acid, bacterial vectored, antigen-presenting cell vaccines [5, 6]. Despite the various new approaches to the vaccine production, most of the vaccines which are applied in immunization programs are either live attenuated, inactive or subunit vaccines. WHO (World Health Organization) vaccine-preventable diseases: monitoring system  offers important and comparable data on this topic based on all countries. Even so, there are public concerns over the topics as age and schedule of vaccination, common (injection site pain, redness and swelling, fever, malaise, headache) and rare side effects (anaphylaxis, idiopathic thrombocytopenic purpura, narcolepsy, autism), immunodeficiency or antigenic overload issues. As in all daily life matters, misinformation on these topics and issues in social media and search engines greatly challenges vaccine production methods and public acceptance, although the scientific evidences prove the contrary.
As illustrated in Figure 1, one of the greatest challenges in vaccine research is based on logistics and distribution. Along with post-production purification and packaging issues, logistics of traditional vaccines under cold chain conditions in limiting shelf-life durations exhibit certain difficulties . Especially in underdeveloped countries, lack of health infrastructure and required conditions threaten overall process. Moreover, world once again faced the same dilemma in Covid-19 pandemic as 16 of total 256 vaccine candidates passed to phase III trials by February, 2021 . These manufacturers announced their plans to produce 10 billion doses (at least 2 doses are required for immunity in most) until the end of 2021 in their best estimations. Apparent insufficient annual production capacity leads priority groups to be formed for the early products. As in this latest ongoing example, inequity in access to vaccines is always a major issue .
Leading Covid-19 vaccine candidates are mainly developed by private/industry association by 72%. Remaining 28% is consisted of academic, public and non-profit organizations. Also, there are bigger multinational vast vaccine manufacturer companies as Janssen, Sanofi, Pfizer and GlaxoSmithKline which may ensure tolerating lack of large scale vaccine manufacturing inexperience and capacities of these relatively smaller organizations . Even though, commercial viability apparently is not an issue for potential Covid-19 vaccine, it is a real drawback for many diseases. These diseases are mostly have devastating effects on restricted local areas as poor communities. In case of such rare infection diseases, development costs offset potential income. Vaccines against this kind of diseases as Ebola, which multinational manufacturers hesitate to invest due to commercial viability, are called ‘orphan vaccines’ . More profitable vaccine production methods may withdraw hesitation over these diseases which are only producible with government assistance and still have high mortality rates regionally.
Another and probably the most challenging factor in vaccine research is based on immunological issues. Commercially viable vaccine targets for diseases like HIV, gonorrhea (
Following the rapid development of biotechnology and bioinformatics, precise genomic and proteomic target identification methods and instruments are emerged. Therefore, knowledge on structural biology and immunology is mostly available for many infectious diseases. Deciding vaccine production methods is based on delivery, immunogenicity, production capacity and speed, transport requirements and shelf life and economic viability. Along with the methods as viral vectored vaccines, nucleic acid-based vaccines, RNA vaccines, outer membrane vesicles, plant based vaccines present promising contribution to the field. Experimental and commercial applications of plant based vaccines will be evaluated further in this chapter in perspective of future preventive healthcare alternative.
2. Plant-based vaccine production
Especially in the last two decades, the expression studies of vaccine antigens in plants have been accelerated with the developments in the production of recombinant proteins in plants and it has been provided possibility to design effective plant-based vaccines against many diseases. In this process, both developments in transgenic approaches and transformation methods and improvements in various areas such as promoter selection, codon optimization, plastid transformation for increasing yield of recombinant protein have paved the way for the production of vaccine antigens. The significant increase in the world population and the emergence of epidemic and pandemic diseases cause demands that exceed the vaccine production capacity. However, the success of national vaccine programs is marred by both high cost-per-dose of producing vaccines and the limitations in the distribution of vaccine. In addition, there is a risk of exposure to dangerous pathogens caused by injection procedures during vaccination, resulting in diseases like HIV, hepatite C that can be transmitted through blood. Moreover, risk of contamination of other viruses such as SV40 and foamy virus, which can cause disease in humans and animals, is another important factor that should be evaluated in terms of health, although it depends on the nature of the vaccine (attenuated or inactivated), the titer of the contaminant, the degree of inactivation and pathogenicity . When all these disadvantages are examined, it is seen that the use of plant systems for vaccine production has the potential to provide a biotechnological solution, considering that it can provide high-scale production and reduce the cost-per-dose and minimize the problems that may occur during vaccine production and distribution. While plants produce complex proteins similar to other eukaryotic systems, they can fold and modify these proteins post-translationally. However, they contain minor differences in glucosylation patterns compared to mammalian cells .
In general, the technical points taken into consideration for the plant transformation and the production of recombinant protein in plants should also be taken into account in planning for the production of recombinant vaccine antigens in plant systems. Two different systems are used in the production of recombinant proteins in plants known as stable genetic transformation and transient gene expression. Stable nuclear transformation results in stable expression in plant tissues by ensuring the insertion of recombinant DNA into the nuclear genome of the plant cell . In addition, transgenes can stably integrate into the plastid genome other than the nuclear genome. The transfer of recombinant DNA is carried out by using direct and indirect methods and this preference varies according to the target plant species, target genome (nuclear or plastid), gene construct to be introduced. Natural plant pathogens,
2.1 Nuclear transformation
It is ensured that vaccine antigens can be produced in large amounts in the tissues of transgenic plants that are transformed by nuclear transformation, and oral administration of the vaccine becomes possible thanks to the expression in edible plant organs in such as lettuce (
For many years, the nuclear genome has been the main target of plant gene transfer studies, which has enabled the production of recombinant vaccine antigens by nuclear transformation in plants to come to the fore and to be performed relatively easily. Thanks to nuclear transformation in plants, the production of vaccines against many disease factors from enteric bacteria to viruses that threaten human and animal health has been achieved (Table 2).
|Plant||Pathogen/Disease||Antigent||Transfer Method||% of Total Soluble Protein or μg of antigen/g fresh biomass||Adjuvant||Immunization||Target Organism||Immun Response||Ref.|
|Hepatitis B virus||small surface antigen|
|CaMV 35S promoter/||22–63 μg/g FW||With adjuvant (alhydrogel)||Orally-tablet, intramuscularly /total 200 ng||Human||IgA|||
|HIV / AIDS||Gp 120 and Gp 41 attached poly-HIV||PEG-Mediated by Protoplast||3.7 μg/g FW||With adjuvant (Freund’s complete adjuvant and Freund’s incomplete adjuvant)||Subcutaneously/ 34 ng||Human||IgG İnduction|||
|TBAg-ELP Fusion Protein||CaMV 35S promoter/||4% TSB||With adjuvant (Freund’s complete adjuvant and Freund’s incomplete adjuvant)||Intraperitoneal/ for mice 10 ||Human||Induced IgG|||
|Measles virus||Measles virus hemagglutinin (MV-H)||NA||Only intranasal with adjuvant (crude saponins)||Intranasally (40-100 μg-tsb)/ Intraperitonealy/ (400-1000 μg-tsb)/ intramuscularly (50 μg-single dose)||Human|
|Infectious bronchitis virus (IBV)||Spike (S) protein||CaMV 35S promoter/||2.5 μg/g FTW||Witout adjuvant||Orally 35 g/ intramuscularly 35 g extract||Poultry||Induced chIL-2|||
|Norwalk virus (NV)||Recombinant capsid protein rNV||Dual-enhancer 35S promoter /||ST:120 μg/g FW|
LE:150 μg/g FW
|Without adjuvant||Orally/ 5 g dry weight||Human||Induced serum IgG and intestinal IgA|||
|Rabies virus RV||G protein||Maize ubiquitin promoter / transformed by biolistics||25 μg/g FW||Without adjuvant||Orally / 0.5–2 mg x 5 fresh kernels||Human and Dog||NA|||
|The LTBentero protein||CaMV 35S promoter/||5.29 μg/g FW||With complete Freund’s adjuvant / incomplete Freund’s adjuvant||Subcutaneously (10 mg)||Human||Induced IgG and mucosal IgA|||
Every day, 200 million people in the world experience health problems due to gastroenteritis. In developing countries, more than 2 million people die annually from such enteric diseases . It has been reported that a multiepitopic protein from the antigens of enterotoxigenic
In recent years, except advanced monocot and dicot transgenic plants, various moss groups in the plant kingdom have started to be preferred for vaccine production. Env-based HIV (Human Immunodeficiency Virus) multi-epitope protein (poly-HIV) produced in transgenic moss lines has been reported to elicit an immune response in mice as a candidate for subunit vaccine. Moss can be propagated under
Considerable efforts have been made to develop methods that can simplify the purification procedure as the downstream processing of recombinant vaccine antigens will significantly increase production costs. ELPylation as one of these methods, generally increases the accumulation of transgenic proteins in plants as reported in various examples . In addition, ELPylated proteins can be rapidly purified by membrane-based inverse transition cycling (mITC) procedure. Using ELPylation, in which an elastin-like polypeptide (ELP) consisting of a series of pentapeptides is fused to the end of the target protein, an increase in antigen accumulation is observed in both transiently and stably transformed plants . It has also been reported that the immune response increases significantly with the proper folding and trimerization of the antigen. In the study, in which the hemagglutinin of the avian influenza virus (AIV HA) was produced as a monomer and ELPylated trimer form in the tobacco plant (transient in
Virus-like particles that do not contain viral nucleic acids and are formed by viral capsid proteins become prominent as much more reliable candidates when compared with attenuated viral vaccines. Studies in which capsid proteins are expressed in plants by nuclear transformation have been shown to induce specific antibodies comparable to attenuated vaccines when administered orally with an adjuvant . VP2, VP6 and VP7 capsid proteins of group A rotavirus, one of the most common cause of human pathogen, acute infantile and pediatric gastroenteritis worldwide, have been expressed in transgenic tobacco plants. It has been reported to be significantly higher IgG antibodies in the serum of mice of the group VP 2/6/7 (immunized with transgenic tobacco plants) than group VP 2/6 (immunized with transgenic tobacco plants) and the group RV (immunized with orally attenuated RV vaccine) group IgG antibodies are significant. In addition, it was reported that the serum IgG titer in the VP 2/6 group was almost as high as that found in the RV group .
Using the nuclear transformation system, plant-based vaccines can be developed on the basis of low-cost multiepitopic recombinant proteins to contain epitope variants that can induce broad-spectrum antibodies. In addition, sequences containing adjuvant activity can be added to these multiepitopic proteins. Multiepitopic vaccines produced in plants trigger local and systemic immune responses more than their counterparts produced in bacteria. In orally inoculated BALBc mice, lettuce-derived HIV C4 (V3) 6 multiepitopic protein showed a higher immunogenic potential than
Plant systems are also used in the production of vaccines against various parasitic diseases other than bacteria and viruses.
2.2 Chloroplast transformation
This section emphasized on recent advances in creation and development of transplastomic plants to produce plant-derived vaccines and protection against infectious diseases. Plastid transformation has come to be advantageous for the production of vaccines due to the high copy number in a single transformation allowing high levels of transgene expression and the absence of effects leading to gene silencing and the lack of concerns about positional and pleiotropic effects. In addition, as a result of maternal inheritance, the containment of foreign genes in the chloroplast genome and the absence of transgenes in the pollen are other advantages. Moreover, chloroplast transformation ensures expression of multiple genes in prokaryotic-like operon systems. However, this system is limited by the insufficient number of the target plant variety and the trials of very few plant varieties according to nuclear transformation. Another disadvantage is the lack of glucosylation ability of chloroplasts. Therefore, difficulty of expression of eukaryotic human or viral genes in prokaryotic chloroplasts is most important barrier to the use of transplastomic plants. Some major costs associated with the production of recombinant proteins in fermentation-based systems can be reduced by using chloroplasts as bioreactors. Chloroplast-derived therapeutics, especially when administered orally, eliminate expensive purification steps, cold storage, transportation and sterile injection requirements .
Advances in transgenic approaches and the development of gene gun and biolistic (particle bombardment method) technologies have enabled the transgene to be transferred directly to living cells. With these systems where tungsten and gold particles are used as microcarriers, the transformation of plastids can be carried out effectively. As an alternative to this system, plastids are transformed via polyethylene glycol (PEG) [31, 32]. PEG-mediated transformation allows the simultaneous transformation of many samples as a simple and efficient method and enables a large number of transformed cells with a high survival rate. However, it has a lower success rate than biolistic-mediated transformation. Despite its high transformation efficiency, biolistic is not available in many laboratories and standardization of its protocol is very difficult. It has been shown repeatedly that recombinant proteins capable of eliciting protective mammalian immune responses can also be produced in chloroplasts of plants. Thanks to chloroplast transformation, vaccines can be produced against viral diseases such as polio (poliovirus), human immunodeficiency (HIV), human papilloma virus (HPV), as well as numerous contagious and fatal bacterial infections and diseases such as cholera, tuberculosis, plague and anthrax (Table 3).
|Antigens||Disease/Pathogen||% of Total Soluble Protein or μg of antigen/g fresh biomass||Plant||Immunization||Transfer|
|Viral protein 1||Poliovirus||—||Orally (ORV)||Biolistic|||
|gp120 and gp41 multiepitope|
|HIV||16 μg /g fresh weight||Orally (ORV)||Biolistic|||
|gp120 and gp41 multiepitope|
|HIV||16 μg /g fresh weight||Orally (ORV)||Biolistic|||
|7.5% for ESAT-6 and 1.2% for Mtb72F||Orally (ORV)||Biolistic|||
|mmpI with Lymphotoxin-beta (LTB)||—||—||Polyethylene glycol (PEG)|||
|C4V3 polypeptide||HIV||—||Orally (ORV)||Biolistic|||
|L1 capsid protein fused with LTB||Human papillomavirus||2%||—||Biolistic|||
|dengue-3 serotype polyprotein (prM/E)||Dengue virus||—||Orally (ORV)||Biolistic|||
|13.17 and 10.11%||Subcutaneously (SQV) or orally (ORV)||Biolistic|||
|7.3 and 6.1%||Subcutaneously (SQV) or orally (ORV)||Biolistic|||
|VP1||Foot and mouth dis ease virus (FMDV)||51%||Inoculated||Biolistic|||
|HIV-1 Pr55gag polyprotein||HIV||6.75%||—||Biolistic|||
|labile toxin B subunit heat-stable|
|ETEC-induced diarrhoeal disease||2.3%||Orally (ORV)||Biolistic|||
|E7 and E7CP HPV protein||Human papillomavirus||0.1% and 0.5%||—||Biolistic|||
|multi-epitope DPT fusion protein||0.8%||Orally (ORV)||Biolistic|||
|HIV-1 Pr55Gag, p24 and p17/p24||HIV||4 μg/g fresh weight||Intramuscularly|||
|L1 capsid protein||Human papillomavirus||24%||Intraperitoneal (IP)||Biolistic|||
|L1 capsid protein||Human papillomavirus||1.5%||—||Biolistic|||
|Structural protein E2||Swine|
fever virus (CSFV)
|1–2%||Subcutaneous and intragastric||Biolistic|||
|TetC||10%||Oral and İntranasal (IN)||Biolistic|||
|Severe acute respiratory syndrome coronavirus||0.2%||Orally (ORV)||Biolistic|||
|2.7 and 1.7%||—||Biolistic|||
Virus-like particles formed by viral capsid proteins that do not contain viral nucleic acids are frequently preferred within vaccine production in plants due to their ability to elicit protective immune responses. In this context, Lenzi et al.  reported that the self-assembled L1 capsid protein of human papilloma virus (HPV), that is the causative agent of cervical cancer which is one of the most common causes of death for women, can be produced in chloroplasts of the
The level of transgene expression in chloroplasts varies depending on the origin of the coding sequence. Although the amount of transcript depends on the high copy number of the transgenes, it is also closely related to the efficiency of the promoter chosen and the regulatory sequences that affect translation. Most of the transgenes expressed in chloroplasts utilize the psbA promoter. It has also been reported that the 5’UTR sequence of psbA shows higher translation activity compared to many 5’UTR sequences. For this reason, studies connected with improving codon optimization benefited from psbA sequences, and it has been reported that codon optimization significantly increases translation in chloroplasts . It has been indicated that the expression of the gene of the vaccine subunit is increased 50 times in chloroplasts by codon optimization .
In recent years, the production of vaccine antigens in the chloroplasts of edible plants like lettuce and ensuring oral vaccination have come to the fore as an important development in order to eliminate the economically demanding and extremely expensive steps such as fermentation, purification, cold storage, cold chain and transportation. Lyophilized plant cells stored at ambient temperature retain their efficacy and antigen folding/assembly, thus eliminating the need for cold chain . Thus, chloroplast bioreactors have become an important alternative to the production of fermentation-based vaccine antigens. Arlen et al.  have achieved the production of high levels of F1-V antigen, as much as 14.8% of the total soluble protein. In a study conducted by aerosol challenge with
Besides terrestrial plant systems, photosynthetic unicellular alga
Production of vaccine antigens (HIV gag transgene; Pr55gag) by biolistic-mediated chloroplast transformation resulted in significantly greater protein accumulation than
2.3 Plant virus based expression system
Plant viruses are generally described as safe for humans and animals. Therefore, they are preferred for the production of therapeutic molecules. TMV (tobacco mosaic virus), PVX (potato virus X), BaMV (bamboo mosaic virus), CPMV (cowpea mosaic virus) are highly stable to high temperature, pressure and pH conditions and can be purified from host plants in amounts exceeding hundreds of mg/kg plant biomass . Virus-like particles (VLPs) are multi-subunit molecules consist of self-assembly protein structures that are the same or highly similar to the general structure of authentic virus. Due to the fact that VLPs do not contain viral nucleic acids, therefore conversion to infectious viruses is not possible which is an important risk factor for attenuated vaccines. In recent years, the use of both VLPs and plant viruses for vaccine production has increased rapidly (Table 4). There are various recombinant vaccine production strategies that stand out in which different vaccine antigens can be produced by using plant viral structures. Launch vector-based on virus for plant transient expression system, plant virus-based vector systems by the incorporation of 2A peptide, plant virus conjugated with purified antigen from bacterial expression system are some of the systems by which recombinant vaccine antigens can be produced.
|Antigens||Disease/ Pathogen||Recombinant vaccine platforms for production of antigen||Plant||Immunization||Virus||Ref.|
|JEV envelope protein domain III (EDIII)||Japanese encephalitis virus (JEV)||BaMV-based vector system||Intraperitoneally (IP)||Bamboo mosaic virus (BaMV)|||
|OmpA-like protein (OmpA), chaperone protein DnaK and lipoprotein Tul4||Bacterial system (||Intranasally (IN), subcutaneously (SC.)||Tobacco mosaic virus (TMV)|||
|Dengue virus envelope glycoprotein|
(E) domain III
|Dengue virus||TMV-based vector system||Orally (ORV)||Tobacco Mosaic Virus (TMV)|||
|RhoA-derived peptide (Antiviral peptide production/inhibitor)||Respiratory|
syncytial virus (RSV)
|TMV-based vector system||—||Tobacco Mosaic Virus (TMV)|||
|Launch vector-based plant transient expression system||Intramuscularly (IM)||Tobacco mosaic virus (TMV)|||
|Pfs25 VLP||Launch vector-based on Tobacco mosaic virus||Intramuscularly (IM)||Tobacco Mosaic Virus (TMV)|||
|Norwalk virus capsid|
|Norwalk virus||BeYDV-based geminiviral replicon|
|—||Bean yellow dwarf virus (BeYDV)|||
|PCV2 capsid protein||Porcine Circovirus (PCV)||CMV-based expression system||Intraperitoneally (IP)||Cucumber|
mosaic virus (CMV)
|Extracellular domain M2 protein (M2e) fused to hepatitis B core antigen (HBc)||Influenza||Potato X virus-based vector system||Intraperitoneally (IM)||Potato X virus (PXV)|||
|Hemagglutinin (HA) protein||H5N1 influenza virus||Launch vector-based plant transient expression system||Subcutaneously (s.c.)||Tobacco mosaic virus (TMV)|||
|BTV coat proteins||Bluetongue virus (BTV)||Cowpea mosaic virus–based|
HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system
|Subcutaneously (s.c.)||Cowpea mosaic virus|||
Multiple doses of multivalent vaccine can be administered in the immunization of mice without any adverse effects. In addition, multivalent subunit vaccines can be developed against various diseases using an efficient TMV-based delivery platform. A multivalent subunit vaccine consisting of the combination of OmpA, DnaK chaperone and Tul4 protective antigens of the
The genomes of both RNA and DNA viruses can be modified for recombinant protein production. Geminiviral replicon systems are one of the plant-viral based expression systems used to increase the expression of vaccine antigens in plants. In geminiviral derived vectors with a single stranded DNA genome, the viral genes encoding the coat and movement proteins are deleted and the expression cassette for protein of interest is inserted. In these strategies, it has been reported that the viral vector has transient expression only 4 days after it was transferred to
2.4 Transient expression
In addition to stable transgene expression (nuclear or plastid transformation), transient expression is often preferred for the expression of genes encoding vaccine proteins in plant tissues. Since transient expression does not contain chromosomal integration, it is not affected by position effect. In addition, the expression of extrachromosomal transgenes can be detected 3 hours after transfer and can last for about 10 days . The major advantage of transient expression is production of vaccine antigen rapidly at low cost and high yield. In addition, the easy applicability of the system and its ability to allow the production of complex proteins composed of subunits encourages its use against the novel viral diseases that emerge suddenly. Plant-based vaccines developed by transient expression are given in Table 5.
|Plant||Pathogen/ Disease||Antigent||Transfer Method||% of Total Soluble Protein or μg of antigen/g fresh biomass||Adjuvant||Immunization||Target organism||Immun response||Ref.|
|HPV-16/ Cervical cancer||E7 protein fused 16E7SH||0.4–6 g/kg FW||With adjuvant/ (Freund’s incomplete adjuvant)||Subcutaneous/ 5 μg||Human||Tumor size decrease and IgG ınduction|||
|Hepatitis B virus||HBsAg||MagnICON viral|
|0,64 mg/g FW||With adjuvant (alum)||Intraperitoneal 346 mIU/mL at week||Human||anti-HBsAg response|||
|HIV/AIDS||Subtype C Envelope gp140||4.9–6.2 mg/kg FW||With adjuvant (Alhydrogel®)||Intramuscularly/ 50 μg||Human||NA|||
|Poliovirus (PV) type 3||Capsid protein VP1||60 mg/kg FW||—||Intraperitoneal / intramuscular||Human||—|||
|Infectious Bursal Disease Virus (IBDV)||Structural VP2 protein||1% TSB||With adjuvant (Freund’s complete adjuvant and Freund’s incomplete adjuvant)||Intramuscular/ (12 μg of VP2)||Poultry||NA|||
|FMDV||Capsid precursor P1-2A and the protease 3C fusion||3–4 mg/kg FW||With adjuvant (Montanide ISA 50)||Intraperitoneal/ 500 ng||meat-producing animals||NA|||
|Plasmodium Surface Protein Pf38 fused to Red floresan Protein||CaMV 35S promoter/||4–12 μg/g FW||With Gerbu MM Adjuvant||Intraperitoneal (17 μg)||Human||Induced IgG|||
Margolin  achieved
It is imperative to produce vaccines at rates which offset mutation frequency of viral infections such as influenza, for which a new and unique epidemic strain appears within a few years. In recent years, recombinant vaccines become prominent as one of the most important options to solve this problem. New recombinant strategies provided by plant biotechnology and the production of plant-based vaccines are becoming widespread in the struggle with pandemic and epidemic diseases such as Influenza A H1N1, Influenza H5N1, plague, Ebola, Zika, SARS-CoV and SARS-Cov-2 [78, 79, 80, 81]. Plant-based vaccines developed for pandemic and epidemic diseases are given in Table 6.
|Spreading area||Plant||Pathogen/Disease||Antigent||Transfer Method||% of Total Soluble Protein or μg of antigen/g fresh biomass||Adjuvant||Immunization||Immun response||Ref.|
|E||Cholera toxin B-subunit||NA||Witout adjuvant||Orally/150 mg seed||Induced IgG and mucosal IgA|||
|E||SARS-CoV||SARS-CoV nucleocapsid (rN) protein||0.8–1% of the TSP||With complete Freund’s adjuvant/incomplete Freund’s adjuvant||Intraperitoneal/500 mg fresh tobacco leaves||Induced IgG1 and IgG2/Increased IFN-_ and IL-10/not changed IL-2 and IL-4|||
|P||Influenza A H1N1||Soluble protein H1/H1-VLP||NA||-||-||Induced CD4+ and CD8+ T cells|||
|E||Influenza A H5N1||Matrix protein 2 ectodomain (M2e) fused to N-terminal proline-rich domain (Zera®) of the γ-zein protein of maize||125–205 mg/kg FW||Without adjuvant||Intramuscular/4.5 μg||Induced IgG|||
|E||Major capsular protein F1-V antigen fused||NT: 1-4% FW|
LE: 4-10% mg DW
|With adjuvant NT: aluminum hydroxide t/LE: cholera toxin||NT: Subcutaneously (10μg purified)/LE: Orally|
(2 g tomato fruit)
IgG1, IgG2a and mucosal IgA
|E||Flavivirus/Yellow fever (YF)||YF virus envelope protein (YFE) fusion to the bacterial enzyme lichenase (YFE-LicKM)||NA||With Alhydrogel adjuvant||Intramuscularly 5 μg x3/5 μg x 2/30 μg x 3||Induced IgG, İncreased IFNγ|||
|E||Ebola virus (EBOV)||Envelope-associated protein VP40||2.6 μg/g FW||With complete Freund’s adjuvant/incomplete Freund’s adjuvant||Orally (25 ng)/|
Subcutaneously (125 ng)
|Induced IgM, IgG and intestinal IgA|||
|E||Zika virus (ZIKV)||Envelope (E) protein||160 μg/g FW||With aluminium hydroxide gel adjjuvant||Subcutaneously/50 μg x 24||Induced IgG1|
and IgG2, Increased IFN-γ, IL-4 and IL-6
|P||SARS-CoV-2||Spike specific monoclonal antibody (mAb) CR3022||130 μg/g FW||-||-||-|||
Especially the commercial scale production of these vaccines and their examination at Phase I, Phase II and Phase III levels beyond the functional evaluations in animal models indicates that in the future, plant-derived vaccines will be an important part of the struggle against pandemic and epidemic diseases. Plant-based vaccines produced in commercial scale and candidate vaccines are given in Table 7 by companies. For instance, COVID-19 (severe acute respiratory syndrome coronavirus 2/SARS-CoV-2), which has become a major threat to global health, has also significantly impacted the world economy and social mobility. So far, with its high contagiousness, rapid spreading nature and high mortality rate, more than 2.5 million people have died from COVID-19, and more than 116 million people have been infected with COVID-19 worldwide . In order to control the pandemic, the whole world work hard to develop new strategies to be applied in the field of health, to deliver vital medical supplies to those in need, to develop and apply safe and effective vaccines. Especially, the development of vaccines and drugs for this new pathogenic Coronavirus, which emerged suddenly and mutated at certain times, became an inevitable target. Until now, the number of vaccines in preclinical development are 182 and the number of vaccines in clinical development is 74, worldwide . Plant-based vaccines have also proven that they can play an active role in fighting against COVID-19 with their promising results in preclinical and clinical stages. Along with the initiative of commercial companies using plant biotechnology, transient expression of the SARS-CoV-2 antigen in plants was achieved, and a plant-based COVID-19 vaccine candidate was produced with a high-scale production technology . The COVID-19 vaccine developed by Medicago company using a plant-based platform started Phase II clinical trials. In this approach, virus-like particles (SARS-CoV-2 spike protein self-assambles into VLPs) could be produced by transient expression in
Prophylaxis of SARS-CoV-2
spike protein fused lichenase protein /
Prophylaxis of Classical swine fever (CSF)
CSFV E2 glycoprotein /
Prophylaxis of H7N9 influenza / Phase 2 /
Prophylaxis of pertussis, diphtheria, tetanus, poliomyelitis and prophylaxis of Hib infection in infants /
Phase 3 /
Prophylaxis of seasonal influenza /
Prophylaxis of rotavirus gastroenteritis / Phase 1 /
Prophylaxis of SARS-CoV-2/ Phase 2 /
Prophylaxis of H5N1 influenza / Phase 2 /
Prophylaxis of Ebola virus /
Prophylaxis of Norovirus / Phase I /
Prophylaxis of Staphylococcal Enterotoxin B /
|Prophylaxis of H1N1 influenza||Prophylaxis of H1N1 influenza||Prophylaxis of Malaria|
2.5 Evaluation of plant-based vaccines side effect
Vital part of vaccine research is the risk assessment through randomized, double-blind placebo-controlled multicentre trials. Vaccine side effects can be evaluated under two categories as common side effects (high fever, vomiting, dizziness, anxiety and nausea) and rare side effects (risk of hospitalization, death or long-term morbidity). Same evaluation processes is required for plant-based vaccines as all traditional and recombinant vaccines. In literature, there are various studies in emphasis to safety and side effects of plant-based vaccines.
Plant-based vaccines can be evaluated in two different ways: cases in which the plant content is directly applied in pure form and the cases where the vaccine content is isolated and mixed with adjuvant. Phase studies were initiated for many candidate vaccines, where antigen or VLP was produced and then mixed with adjuvant before injection. In reported Phase I vaccine case against influenza A presented common side effects on volunteers as high fever, vomiting, dizziness, anxiety and nausea . Similarly, in another case local effects occurred in the vaccination area and 93% of side effects were mild effects . McCormick et al., stated in their studies that volunteers showed symptoms that were described as severe at a very low rate, but recovered within 1–2 days without the need for medical intervention, and the vaccine candidate was quite safe . Moreover, Pillet et al. tested the vaccines with 300 healthy adults and 450 volunteers over the age of 50 in their phase III study on two different age groups. As a result, a higher rate of fatigue was observed in volunteers over 50 years old . Ward et al. reported that the most common side effect was pain at the injection site in their studies on 22,854 volunteers and mortality rate was slightly higher for inactivated.
vaccine comparing to virus like particles . Chichester et al., (2018) reported that 94% of the volunteers developed at least one of the side effects of high fever, vomiting, dizziness, anxiety and nausea . In all of these studies with both oral and injectable vaccines, the observed effects were evaluated as mild to moderate. In this way, plant-based production has been defined as an effective and safe vaccine production tool . Production of the adjuvants to be used to stimulate mucosal and peripheral immunity in the plant or the selection of plant which produce appropriate secondary metabolites that can act as mucosal adjuvants contributes to the decrease in the incidence of side effects. The number of studies in which plant-based vaccine candidates have passed to phase studies were much less than classical vaccine studies. When these studies are evaluated, there is no significantly increased side effect risk report concerning plant-based vaccines against any other vaccines production options .
2.6 Legal regulations involving plant-made pharmaceuticals
Regulatory processes of the vaccine development, approval, authorization, licensing, distribution, and marketing are as challenging as the production. There are both national and centralized regulatory agencies. These agencies emphasize on scientific evaluation of data, quality of the product, safety for human use, verification of reported efficacy and authenticity of product labels. In USA, centralized regulatory agency is Food and Drug Administration known as FDA. As plant based-vaccines are considered in biological materials apart from chemical entities, plant-made pharmaceutics are regulated under Biologics License Application (BLA) . The European Union (EU) members have both their own national regulation and the centralized regulation under European Medicines Agency (EMA) which acts as the counterpart of FDA in Europe. The Committee on Herbal Medicinal Products (HMPC) is the EMA’s committee responsible for compiling and assessing scientific data on herbal substances, preparations and combinations, to support the harmonization of the European market . However, considering the nature of plant based vaccines, they are under authorization of The Committee for Medical Products for Human Use (CHMP) which plays a vital role in the authorization of medicines in the European Union (EU).
Strictness of the regulation is mostly based on the production method and the host plant. Non-food plants as
Regulatory approval of a PMPs and other biotechnologically derived products may take up to a year depending on the legal response limits of the regulation agencies. However, Covid-19 pandemic triggered a realization on EU and UK for national and global needs. Therefore, product review, conditional approval and deployment timelines are significantly reduced for PMPs .
In conclusion, infectious diseases threatened humanity countless times throughout history. In particular, as pandemic and epidemic diseases killed millions of people, it increases in importance to develop safe and cost-effective vaccines and their storage and rapid distribution. Apart from many diseases such as diphtheria, cholera, typhoid, tuberculosis, which are controlled by vaccine campaigns in developed countries, new vaccine production systems using recombinant DNA technologies are needed for emerging diseases such as COVID-19, MERS-CoV, avian influenza, Ebola, Zika and possible future infections. Plant-based vaccine production for humans and animals stands out as an important alternative that can be used to overcome the disadvantages of existing conventional vaccines. Within the scope of plant biotechnology, it became possible to produce cost-effective, immunogenic and safe vaccines thanks to the development of gene transfer strategies to plants and improvements in amount, isolation and purification and addition of adjuvant for production of recombinant vaccine antigens in plants. It is an undeniable fact that the possibilities that recombinant vaccines can offer us will increase with new standards and legal regulations to be introduced for the development, approval, authorization, licensing, distribution and marketing of such vaccines. In scope of future preventive healthcare, it is hard to assume monopoly of one particular vaccine technology. There will always be some ups and downs in any vaccine production methods. However, plant based vaccines represent considerable strong suits and offer swift and viable solutions over traditional and other recombinant vaccines.