|Year : 2021 | Volume
| Issue : 4 | Page : 366-373
Plant-based vaccines: Potentiality against severe acute respiratory syndrome coronavirus 2
Pramita Sharma1, Himel Mondal2, Shaikat Mondal3, Rabindranath Majumder4
1 Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Howrah; Department of Zoology, Hooghly Mohsin College (Affiliated to University of Burdwan), Chinsurah, West Bengal, India
2 Department of Physiology, Santiniketan Medical College, Bolpur, West Bengal, India
3 Department of Physiology, Raiganj Government Medical College, Raiganj, West Bengal, India
4 Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Howrah, West Bengal, India
|Date of Submission||14-Aug-2021|
|Date of Acceptance||17-Oct-2021|
|Date of Web Publication||14-Dec-2021|
Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Shibpur, Howrah - 711 103, West Bengal
Source of Support: None, Conflict of Interest: None
The pandemic of novel coronavirus disease-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has stimulated scientists from different backgrounds to gear up on developing vaccines against the virus. Several antigenic epitopes of the virus have the potential to induce an immunogenic response, among which viral spike protein (“S” protein) is considered to be the most suitable vaccine candidate. In this review, the latest progress in the field of plant molecular pharming (PMF), its application, limitations, and commercial initiatives toward the production of the SARS-CoV-2 vaccine have been discussed. Vaccine production by PMF has gained considerable attention these days and can be used for large-scale commercial production of antigenic proteins, antibodies, and other biopharmaceuticals. New age plant breeding techniques facilitated by CRISPR-Cas-based genome editing technology and next-generation sequencing methods also help to achieve greater precision and rapidity. Several unique advantages are offered by plant-based vaccine production techniques over that of the microbial or mammalian cell culture system. Virus-like particles and Agrobacterium-mediated transient somatic expression systems have a high potential for the large scale, cost-effective, and robust production of plant-derived vaccines against SARS-CoV-2.
Keywords: COVID-19 vaccines, molecular pharming, plant vaccine, recombinant proteins, SARS-CoV-2 spike glycoprotein
|How to cite this article:|
Sharma P, Mondal H, Mondal S, Majumder R. Plant-based vaccines: Potentiality against severe acute respiratory syndrome coronavirus 2. Biomed Biotechnol Res J 2021;5:366-73
|How to cite this URL:|
Sharma P, Mondal H, Mondal S, Majumder R. Plant-based vaccines: Potentiality against severe acute respiratory syndrome coronavirus 2. Biomed Biotechnol Res J [serial online] 2021 [cited 2022 Jan 23];5:366-73. Available from: https://www.bmbtrj.org/text.asp?2021/5/4/366/332455
| Introduction|| |
The magnitude of damage, unpredictability, and terrifying features of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people globally. The worrisome characteristic of the virus is its ability to mutate rapidly, its fast transmissibility, and to create major setbacks to the elderly and persons with comorbidity., To prevent the viral spread, the discovery of a vaccine that is safe, effective, and can be easily administered to provide immunity by the production of neutralizing antibodies is imminent., The vaccine development methods witnessed significant changes over the decades from the direct use of pathogens (live/attenuated) to the application of recombinant subunit vaccines based on desired antigens. In general, the expression of recombinant antigenic proteins is evaluated in either prokaryotic or eukaryotic cells in a laboratory. Now, with the advanced plant-based molecular pharming techniques, the recombinant antigenic proteins can be harvested using the specific plant systems.,, Among several antigenic epitopes of the SARS-CoV-2, spike (S) glycoprotein might serve as a suitable candidate for designing a recombinant vaccine against the coronavirus; however, either the expression of total S1 protein or only the receptor-binding domain (RBD) portion can induce sufficient innate and humoral immunity or not, is yet to be solved. To meet the demand for large-scale vaccine production, plants may be a good platform that is aided by the plant molecular pharming technique (PMF). In this review, different methods of PMF with special reference to Agrobacterium-mediated transient and rapid protein expression, method of construction of virus-like particles (VLPs), and its amplification in plant-based platforms for the production of potential drug or vaccine candidates for SARS-CoV-2 have been critically analyzed.
| The General Concept of Plant-Based Vaccine Production|| |
Plants are used for the expression and production of high-value recombinant proteins used for diagnostic, therapeutics, monoclonal antibodies, and vaccines. In the 1990s, plant-based edible subunit vaccines gained considerable interest among researchers. The plant vaccine development involves several steps including the selection of antigenic epitope, choosing a suitable plant platform, selecting a method of insertion of a gene into plasmids or virus-derived vectors, achieving a stable or transient expression of recombinant protein, and optimum amplification in the plants. After harvesting plant material bearing a recombinant protein, it undergoes downstream processing, purification, and subsequent preclinical and clinical evaluation. The general sequence of plant vaccine production is depicted in [Figure 1].
|Figure 1: A flow diagram showing sequential steps of plant vaccine formulation.|
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| Advantages of Plant Vaccines|| |
The plants are the preferred platform mainly because they do not harbor any human pathogen neither do they transmit any pathogenic load in the process, their cultivation requires no sterile environment or expensive media like mammalian cell culture, the process is cost-effective and the yield is satisfactory. Plant-based vaccines may be formulated for oral delivery which can stimulate the first line (mucosal) of defense in the mouth, nose and thus prevents the entry of virus-like SARS-CoV-2 at their entry through these checkpoints inside the body. The plant can yield greater antigenic proteins than cell culture does. The recombinant protein can be harvested from the target plants almost after 8 weeks of the vector mediated insertion of deoxyribonucleic Acid (DNA). The proteins can undergo post-translational modification just like in mammalian cells and can increase the pharmacological activities of products. The different features of vaccines from the plant and the other conventional methods are compared in [Table 1]. Earlier, S-glycoprotein of SARS-CoV was stably expressed in Nicotiana benthamiana and Lactuca sativa. Lycopersicon esculentum and Nicotiana tabacum for SARS virus were selected as platforms for vaccine production. They have the potential to be used as edible vaccine.
| The Possible Road Map for Production of Plant Vaccine against SARS-CoV-2|| |
Selection of gene
There are four structural proteins encoded by SARS-CoV-2, namely spike (S), envelope (E), membrane (M), and nucleocapsid (N), of which the spike (S) protein is the most efficient antigen of choice to induce immune response through CD4+/CD8+ T-cells., S protein is a major transmembrane protein of the virus and exhibits amino acid sequence diversities among coronaviruses. The convalescent patients' sera showed an immunoreactivity reaction to the immunoreactive epitopes on the S1 region. The N protein is conserved for all coronavirus strains and thus cannot provide specific protective immunity; while M and E are reported to have a weak immune response. Thus, surface spike protein is considered as a good candidate for specific vaccine production against SARS-CoV-2. The RBD of the SARS-CoV-2 spike protein S1 binds to the human angiotensin-converting enzyme 2 (ACE2) which is present on the host cell surface and provides viral access. This subunit is reported to elicit particular cellular immunity. The subunit vaccine can also be functionally improved by complementing adjuvant or VLP for greater immunogenic reactivity.
Methods of insertion of a gene into the plant and its amplification
Stable nuclear genome transformation
The sequence of the gene of interest eliciting antigenic responses is integrated into the nuclear genome of a plant through a plant transformation vector such as Agrobacterium tumefaciens or microprojectile bombardment methods. The integration of the transgene into the chromosome of plants creates permanent genetic changes in recipient cells'. Thus, a transgenic line for inheritable antigen production is established and seed banks may be created. However, improper insertion of the transgene and longer time required to establish accurate recombinant proteins are its limitations. The production of SARS-CoV-1 spike protein in tomato and low nicotine content tobacco plants elicited immunoglobulin A (IgA) and IgG in mice.
Agroinfection-mediated transient expression
This strategy involves the Agrobacterium tumefaciens-mediated infiltration of the desired microbial DNA encoding for the epitope transiently or temporarily in the host cells without being integrated into the genome of plants. Therefore, the antigen production is not heritable; seed banks cannot be generated and requires purification cost. This expression system is used for multiple epitope vaccine productions; and the transgenic proteins are expressed on a large scale in a shorter period. Coronavirus N protein was transiently expressed in N. benthamiana which induced the production of IgG1 and IgG2 in mice and upregulated interferon (IFN)-γ, and interleukin-10 (IL-10) in splenocytes.
It includes the site-directed insertion of foreign DNA into the chloroplast genome by homologous recombination. A high yield of antigenic protein due to large copies of transgenes, no gene silencing effect, and the possibility of multiepitope vaccine formulations are the advantages of this process. However, the lack of posttranslational modifications and longer duration for the generation of transplastomic lines reduce the efficiency of the process.
VLPs are self-assembled structures derived from viral antigens that mimic the native architecture of viruses but lack the viral genome hence are noninfective. VLPs have the potential to be used as a universal vaccine platform due to their safety, immunogenicity, and easier manufacturing process. Multiple foreign antigenic epitopes may be displayed as viral coat proteins., A plant virus is designed to form a chimeric gene for the viral coat protein and this modified virus would deliver the transgene into the plants followed by expressing antigen transiently in plants. The recombinant virus thus expresses the desired protein or peptide during viral replication in the plants. To meet the challenge of large scale production of vaccines, plants are used conveniently as a suitable platform for multiplication of VLPs. However, this production method also causes the death of the host plants after infection. Thus, the re-infection procedure has to be done repeatedly for continuous vaccine production. The SARS-CoV-1 VLP emulsified with Freund's adjuvant accelerated production of IFN-γ, IL-4, cytokine, IL-6, IFN-α, upon subcutaneous administration in mice. These expression systems are depicted in [Figure 2].
|Figure 2: An overview of different expression systems and down-stream processing for plant-derived vaccine production|
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| Harvesting of the Vaccine and Evaluation|| |
The plant harboring the recombinant gene produces the antigenic proteins and their respective antibodies in its various plant parts. These edible parts can be processed for the production of orally administered vaccines unlike previously when these edible parts were consumed directly. Based on the diversity and complexity of antigen types and the platforms used for vaccine production, a wide range of vaccine harvesting strategies are adopted. Traditional purification schemes typically involve multiple steps including extraction and concentration of vaccines, precipitation using calcium chloride, ammonium sulfate, polyethylene glycol, etc., followed by filtration or centrifugation and different chromatographic techniques. Often some of these techniques have certain limitations for commercial production. Affinity chromatography (AC) relies on highly specific interactions between the target and the immobilized ligand. Immuno AC, lectin mediated or metal affinity-based chromatography have earlier been used for vaccine production. Lately some scaffolds, molecularly imprinted polymers, and peptidomimetics were also tried at laboratory scale for purification of vaccine proteins. The recombinant protein may be fused with elastin-like polypeptides (ELP) which allows the desired protein precipitation by altering the temperature., The antigens Ag85B and ESAT6 from Mycobacterium tuberculosis, hemagglutinin from influenza was fused with ELP and injected subcutaneously to mice which evoked humoral immune responses and showed long-lasting results. The vaccine developed by VLPs (chimeric or native) and immune-complexes requires simple purification.
| Preclinical Vaccine Evaluation|| |
Preclinical evaluation of the immunization process (antigen inoculated to model) and immunotherapy (antibodies respective to an antigen are charged for passive immunotherapy) is essential. The selection of proper animal models is highly challenging due to their inability to mimic the clinical manifestation and record pathogenesis of the disease., The smaller animal models are more convenient than larger ones due to easy handling, lower cost, availability, and ability to manipulate at the genetic level. Some of the models for determining SARS vaccine were inbred mouse species (129S, BALB/c),, golden Syrian hamster (strain LVG, Charles River Laboratories), ferrets (Mustela furo) showed successful evaluation efficacy. More research should be conducted to determine the models by specifying the receptor compatibility of SARS-CoV-2 followed by the manifestation of the disease, viral pathogenesis, recording the immune response in the concerned animal models for successful antiviral drugs and vaccines.
| Initiatives to Develop Plant Vaccine against SARS COV-2|| |
Virus-like particle for the production of antigenic epitope
Coronavirus-VLPs are reported to express S, E, M, N of SARS-CoV in cell cultures. The VLP generation in plants has been initiated by a biopharmaceutical company, Medicago Inc. in Canada. For successful posttranslational modification and glycosylation, the VLP-based nuclear expression should be manipulated for the secretion in the trans-Golgi route. In a comparative study for the routes of vaccine administration of SARS-CoV-1 VLP, where S protein fused with M1 from influenza protein and expressed in baculovirus showed greater
levels of IgG during intraperitoneal administration, and greater levels of IgA during intranasal delivery., However, this chimeric VLP was tested to be lethal in mice model when administered intramuscularly. VLPs-based vaccines are the promising candidate for the prevention of the SARS-CoV-2 pandemic. Earlier VLP-based coronavirus vaccines were developed using a variety of antigen combinations or different expression systems. They showed positive response and may pave way for future clinical applications. The strategy used earlier for the hepatitis B virus expressing the S antigen might be followed to express SARS-CoV-2 epitopes on chimeric VLPs.,,,, The above-mentioned approach may give a direction to the production of prospective SARS-CoV-2 VLPs as well. Balke and Zeltins comprehensively discussed the development of CPMV-based vector to produce vaccines which may shade some light on plant-based vaccine production against SARS CoV-2. A flow diagram for the possible production of genetically engineered VLP is represented in [Figure 3]. Medicago Inc, Canada has initiated Phase I clinical trials for its plant-based COVID-19 vaccine candidate on July 17, 2020 testing randomly on 180 healthy human volunteers followed by a combined Phase 2 and 3 trial by October 2020, provided it gets approval to do so based on satisfactory data from its Phase I trial. The active compound consists of coronavirus-like particles (CoVLPs) which will be tested with and without adjuvant.
|Figure 3: A flow diagram showing the use of plants to produce antigenic virus proteins that self-assemble into virus-like particles and may be used as clinical-grade SARS-CoV-2 vaccines after purification|
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This approach is based on the design of a vaccine by choosing multiple epitopes of T- or B-cells that have the potential to evoke protective immune responses. To design the multiepitope vaccine against COVID-19, it should have the ability to elicit cytotoxic T lymphocytic response. This technique needs a carrier to harbor these epitopes to increase the immunogenicity of the vaccine, these epitopes singly can induce adaptive immunity only. Hence, these carriers act as an adjuvant and ensure proper polarization of the epitopes. Apart from VLPs which are the most common carriers for single or multiple epitopes, other possible carriers can be the B subunit of cholera toxin or enterotoxin of Escherichia coli used for the delivery of antigen into the submucosa. These antigens may be captured and presented by the antigen-presenting cells after processing, and subsequently, they induce Th1 responses in the lymph nodes.
| Role of New Plant Breeding Techniques in Plant-based Vaccine Production|| |
The new age plant breeding techniques are applied for the production of the improved variety of crops that are difficult to achieve using traditional breeding techniques or through transgenic modifications. The CRISPR-Cas9 is a potential tool, widely used to improve the efficiency of PMF platforms such as Nicotiana tabacum and N. benthamiana, and for rapid protein expression by the plants. Here, no genetic modification is created or introduced and this way several legal issues of commercial exploitation of GM plants may be bypassed. CRISPR-Cas12b system is an efficient plant genome engineering system that is versatile, customizable, and provides effective gene editing. Abbott et al. 2020 demonstrated a CRISPR-Cas 13-based strategy, prophylactic antiviral CRISPR in human cells (PAC-MAN) which aims to degrade SARS-CoV 2 RNA sequences in human lung epithelial cells. They have designed and screened CRISPR RNAs (cr RNAs) which target the conserved regions of the virus. Using bioinformatics approach, they predicted that only a group of six crRNAs can destroy more than 90% of all coronavirus RNAs. They described that Cas 13d PAC-MAC system can effectively target and cleave SARS-CoV2 RNAs. One major constraint in the vaccine development of SARS-CoV-2 is its rapid mutability. Thus, the knowledge of genomic variability and host-pathogen interaction analysis is very important. The use of techniques such as next-generation deep sequencing and genome-wide association study can address this issue. Computer-aided molecular dynamic analysis (MDA) and reverse vaccinology can help in epitope mapping of the pathogen efficiently followed by experimental validation. MDA can also predict the possible binding targets and binding energy, the dissociation constant of the vaccine candidates. These approaches may prevent the number of failures by designing efficient vaccines and can reduce the time lag for vaccine development during pandemics.
| Social Perception of Plant Molecular Pharming and Its Commercial Approach|| |
There are two separate but complementary Horizon 2020 projects viz “Newcotiana” and “Pharma-Factory” which work extensively on the regulatory and societal issues related to PMF and new plant breeding techniques in the European Union. The project “Pharma-Factory” is led by St. Georges University of London with 14 other participating Institutions aims to consider the social aspects, economic and technical bottlenecks of harnessing the field of PMF., The project “Newcotiana” led by the CSIC (Consejo Superior de Investigaciones Cientı´ficas) of Spain with 19 participating institutes aims to the implementation of new breeding techniques, improvement of the tobacco plant (Nicotiana tabacum, N. benthamiana) as a model crop which could be harnessed as a platform for the development of plant-based high-value recombinant products. Therefore, the joint venture of them could be applied to gain commercial production of antigens by utilizing the tobacco plant as the platform. The various commercial interventions on PMF are mentioned in [Table 2].
|Table 2: List of companies producing plant-based vaccines or antibodies against SARS-CoV-2|
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| Future Scope and Conclusion|| |
Rapid transmissibility of SARS-CoV-2 and its ability to fast mutation has jeopardized the pandemic. The use of VLPs and ELPs can be an attractive approach for the plant-based vaccine development of SARS-CoV-2 complemented by new breeding techniques to enhance the efficiency of PMF. The vaccines developed from transplastomic lines may give long-term effects but manipulating the antigenic spike protein in this route may not be a good idea, as the protein is naturally heavily glycosylated and processed via the ER. The greatest successes in this field have been achieved with Agrobacterium-mediated infiltration and transient expression system. Earlier initiatives to produce plant-based oral vaccines have been superseded by purification and formulation for the large part of injectable vaccines. Plants can also serve as the platform for the production of antibodies and can be successfully delivered to the human system for passive immunotherapy. Mapp Biopharmaceutical (USA) produced ZMapp against ebolavirus where three monoclonal antibodies that were produced in N. benthamiana. The plants harvesting antibodies against SARS-CoV-2 could also be used as an alternative to plasma convalescent therapy and provide an immediate response to critical patients when injected intravenously. Therefore, the plant is the mixed bag platform that can produce antigenic protein, antibodies as well as synthesize antivirals (based on GMP). Earlier, during the preclinical evaluation of vaccine candidates against coronavirus, exacerbating lung disease was reported. Therefore, vaccine candidates developed should undergo rigorous testing using suitable animal models before clinical trials for the safety concern. The use of a suitable vaccine adjuvant will be protective and nullify the chance of immunopathology. The stability of immune response and the requirement of single or multiple doses of the plant vaccine is also a critical issue to be optimized.
The approach of molecular pharming for the development of plant-based vaccines can be the de novo approach for the discovery of a vaccine against SARS-CoV-2 and has great potential to meet the large scale demand of a safe and effective vaccine during the sudden outbreak of the COVID-19. One main constraint is the limited public acceptability of the genetically modified plant-based biopharmaceuticals. All applications and commercial products focus on the cultivation of plants in specially designed containment greenhouses. However, with the advancement of PMF techniques that have the potential to produce very large-scale plant-derived biopharmaceuticals would be able to address the vaccine demand to control COVID-19 and other infectious diseases.
| Pathway of Plant Vaccine|| |
As we are all aware of the fact about global pandemic COVID-19, there is utmost need for its prevention, correct diagnosis, and treatment. In most of the cases, it is misdiagnosed., There has been the development of few vaccines globally but more quantities of vaccine must be produced to meet the population need. To meet the global requirement or demand for vaccines suitable platform must be developed, of which plant-based platform is the key to the solution. Plants have been used as the production platform for diagnostic and pharmaceutical reagents and proteins since several years ago and this technique called molecular farming. The plant vaccine concept has evolved from molecular farming harvesting the idea and techniques of genetic engineering and plant biotechnology. It was the effort during 1990s to produce plant-based edible, subunit vaccines that can be administered orally. The plant-based vaccine had answers to many viral, bacterial, and parasitic diseases. The plant-based vaccine can be produced by incorporating the desired antigenic gene into the plant either directly (inserting the DNA or RNA into the plant cells) or by indirect methods using bacteria or virus (serves as cloning vectors) to infect the cells. The plant vaccine development involves several steps, of which the selection of antigenic epitope or gene is the primary condition. One requisite for the selection of the antigen is to identify the gene which encodes a protein that will elicit proper immune response. The second criteria are the ability of the gene to be assembled in the cloning vectors (bacterial plasmid or on virus). The further requirements of the gene are its viability in the gastrointestinal tract and to arouse oral and mucosal immune responses. Earlier, plant-based vaccine was formulated for SARS (severe acute respiratory syndrome) caused due to SARS CoV. The SARS-CoV-2 has similar virion and genomic organization to other CoVs. Among all the, the spike (S) protein is the most efficient antigen of choice to induce immune response. Therefore, S1 subunit of S protein is targeted due to its diverse structure to elicit neutralizing antibodies by antibody-dependent cell-mediated cytotoxicity to achieve specific cellular immunity by binding to ACE2 receptor. Consequently, a subunit vaccine can be developed unlike conventional (inactivated/attenuated) which will have quicker, safer approach, and have minimal side effects. The subunit vaccine can also be induced with adjuvant or VLP for greater immunogenic reactivity. The second requisite step includes the selection on plant. The plant serving to be the platform should be readily available, grown in local environment, ability to transform and amplify the required protein, and should not harbor any toxic compounds. The third criteria are to insertion of desired gene into the plant platform and then further harvesting and clinical evaluation of vaccines. These plant-based platforms can produce millions of doses within short span. The company Medicago and GlaxoSmithKline (GSK) have announced Phase III testing of plant-based vaccines against COVID-19 developed by Medicago along with the adjuvant developed by GSK. It uses CoVLP technology, the vaccine is composed of recombinant spike (S) glycoprotein which is expressed as VLPs, and is co-administered along with GSK's pandemic adjuvant. This vaccine was granted Fasttrack designation by FDA on February 2021 and it had been directed to administer two doses of 3.75 micrograms of CoVLP within interval of 21 days. This had paved a new opening for further research on plant-derived vaccines.
The authors would like to acknowledge all their respective departments.
List of Abbreviations-
- ACE2 – Angiotensin-converting enzyme 2
- ADCC – Antibody-dependent cell-mediated cytotoxicity
- Covid-19 – Novel coronavirus disease-19
- CoV – Coronavirus
- CT-B – Cholera toxin B-subunit
- DNA – Deoxyribonucleic acid
- dsDNA – Double-stranded DNA
- dsRNA – Double-stranded RNA
- ELP – Elastin-like polypeptides
- ER – Endoplasmic reticulum
- FMDV – Foot and mouth disease virus
- GMP – Good manufacturing practice
- HA – Hemagglutinin
- HBsAg – Hepatitis B surface antigen
- HBV – Hepatitis B virus
- IFN – Interferon
- IgA – Immunoglobulin A
- IgG – Immunoglobulin G
- IL – Interleukin
- M cells-microfold
- mAb-monoclonal antibody
- MERS – Middle-East Respiratory Syndrome
- MHC II – Major histocompatibility complex
- MALT – Mucosal-associated lymphoid tissue
- Osp – Outer surface protein
- PMF – Plant molecular pharming
- RBD – Receptor-binding domain
- RNA – Ribonucleic acid
- S protein – Spike glycoprotein
- SAG1 – Surface antigen 1
- SARS-CoV-2 – Severe Acute Respiratory Syndrome Coronavirus 2
- VLP – Virus-like particles
- VP – Viral protein
- WHO – World Health Organization
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]