Poultry is one of the largest segment of livestock and is a domineering section of agriculture sector in the world as it provides meat, income and employment, however, every year the growth of the poultry industry is severely threatened by a number of infectious viral, bacterial and parasitic diseases. Currently, economic losses due to poultry diseases are 10 to 20% of the gross value of production in developed countries and are likely to be higher in developing countries. Most of these diseases are controlled by preventive vaccine.
With advancement of vaccinology, there are a number of vaccines available to control these diseases including inactivated virus vaccines, attenuated virus vaccines, live virus vaccines, and subunit vaccines. The major constraints of existing vaccines are high production cost, difficulty in maintaining cold chain, vaccine safety, problems associated with mass vaccination, manpower and technical skill needed for vaccine administration, complexity in production and purification etc.
Problems about unavailability of vaccines for the treatment of severe diseases have driven worldwide attention towards production of safer, easier, and more effective vaccines, which initiates the development of plant-based vaccines.
In recent times, plant cell cultures have been widely used to design new biopharmaceutical systems, which facilitate the suitable folding of exogenous proteins and are economically viable. This is also known as “molecular farming,” where marketing-value biomolecules of genetically engineered crops were generated. Numerous on-going clinical trials are underway using purified antigens temporarily generated in injectable vaccine formulations from tobacco plants (Nicotiana benthamiana).
The science of “molecular farming”, or the use of plants and plant cell cultures to produce high-value recombinant proteins, started with the production via transgenic tobacco and sunflower of chimaeric human growth hormone in 1986, then of monoclonal antibodies in transgenic tobacco in 1989 and human serum albumin in transgenic tobacco and cell cultures. This was followed from 1992 onwards with the expression of the first candidate virus vaccine antigens: these were Hepatitis B virus (HBV) surface antigen (HBsAg) in 1992, and a VP1 epitope of foot-and-mouth disease virus (FMDV) expressed on the surface of particles of recombinant Cowpea mosaic virus (CPMV) in 1993 (see Table 1).
Plant cell expression: Although in the past plant geneticists have largely concentrated on crop improvement, some recent studies have shown that plants may provide a useful expression system for mammalian proteins. To express foreign genes in plants, it is necessary to splice a plant promoter, terminator, and, generally, a regulatory sequence onto cloned complementary DNA. Selectable markers may also be incorporated to facilitate identification of recombinants, and the expression hosts can be plant either cell cultures or whole plants. The first licensed vaccine to use this expression system was against Newcastle disease virus (NDV) infection in poultry, and it is being investigated for many other vaccine applications, including infectious bronchitis virus, infectious bursal disease virus and bovine herpes virus(fig.1)
Foreign immunogenic gene is isolated from infected chicken and introduced into plantsthrough plant expression vector. Plant-based vaccines provide a better option to control the disease in low profit margin poultry industry. Still there are some challenges in the field of plant-based, so called ‘green’ vaccines. Injection-based oral priming is a big challenge for commercialisation of green vaccines so, new techniques are needed in the field of plant-based vaccine to pass these barriers for commercialisation.
Plant-based vaccines are a kind of recombinant vaccines that introduce antigens against particular pathogens into the selected plant. By far, scientists have developed over 200 proteins expressed in plants. These encouraging results demonstrate a brighter future for plant-based vaccines. Hiatt and his colleagues firstly made attempt to produce vaccines using plants since 1989. National Institute of Allergic and Infectious Diseases (NIAID) certified that plant-based vaccines could induce sufficient immunogenicity in inoculated individuals in 1998. After 8-year development, world’s first plant-based vaccine against Newcastle disease virus (NDV) was approved by the United States Department of Agriculture (USDA) for poultry.
Production of dozens of viral and bacterial subunit vaccines is attempted in transgenic plants. Recombinant subunit vaccines are safer than traditional vaccines, because they contain no live pathogens. Various plants such as tobacco, rice, maize, potato, alfalfa, lettuce, tomato, carrot, peanut, and soybean are used as hosts for gene introduction, which is achieved in vitro by using protoplast or cell culture, or hairy root culture. Nuclear or chloroplast genome recombination is routinely used to obtain transgenic plants. The choice of the plant species and technology determines the vaccine administration route because some plants can be consumed only when processed, whereas heat or pressure treatments may destroy the antigen. Cereal crops are attractive for subunit vaccine production because vaccines produced in seeds are stable over long storage periods.
Fig 1. Schematic presentation of plant based vaccines production against poultry diseases
(N. SHAHID1 etal. World’s Poultry Science Journal, Vol. 73, September 2017)
Advantages of plant based Vaccines
Vaccines produced from plants are associated with several advantages, some of which include:
- No handling of birds, no stress
- Cost-effective to produce in large quantities.
- Antigens derived from plants such as potatoes and corn are stable and have a high shelf-life.
- Low probability of adverse effects due to contamination caused by a plant virus.
- Do not require sophisticated storage
- Can be handled easily
- Can be administered orally or delivered through the mucosal route, thus eliminating the need for needles.
- Impossibility of reverse virulence
- Easy to expand production scale
- Free from pyrogens and microbial toxins,
- Needle free administration oral vaccines
Other advantages of plant based vaccines are ease of production, scale up and administration, biological encapsulation of candidate antigen, ability to evoke serum and mucosal response, protection against mucosal pathogens, low production cost, room temperature stability, trouble free storage, devoid of human or animal pathogens, free from pyrogens and microbial toxins, needle free administration etc.
Challenges
Despite their advantages, the development of plant-derived vaccines is also associated with several challenges. Some of the major challenges associated with the production of these vaccines include selecting the antigen and plant expression systems, maintaining dosage consistency, and ensuring that all manufacturing processes abide by the Good Manufacturing Practice (GMP) guidelines.
| LIVESTOCK/ANIMALS | POULTRY | ||
| Disease | Reference | Disease | Refrence |
| FMD | Widorovitz et al.1999 | ND/RD | Yoshibo et al2011 Berinsteine et.al.2005 |
| Rabies | Rubio etal.2012 | IBD | Wuetal.2004 |
| Brucellosis | Maritinz et.al 2012 | Chen etal,2012 | |
| Bovine Rota Virus | Widorovitz et al2004 | IB | Zhou etal.2004 |
| Faciola hepatica | Legociki et.al.2005 | Avian Influenza LPAI HPAI | Kanakarajan et.al.2012 Hwangetal2012 Shoji etal.2012 |
| Taenia solium | Hemandez et.al.2007 2008 | Coccidiosis | Sathish et.al.2012 |
| Anthrax | Gorantala et.al 2014 | Adenoviral infection | Wuo et.al. 2009 |
| E .coli | Mason et.al. 1998 | Chicken Infectious anaemia | Lacorte et.al.2007 |
| Canine parvovirus | Dalsgaard et.al.1997 | ||
| Plague | Alvaraj et.al.2006 | ||
| Schistosoma Japanicum | Warang et.al.2011 | ||
| Echinococcus | Yan ju et.al 2010 |
In the poultry vaccine arena, several infectious pathogens of economic importance have been the attention of development of plant-made vaccines. The NDV is one of them, and the virus surface glycoprotein fusion and/or hemagglutinin neuraminidase are the targets of expression.
Immunogenic proteins of avibirnavirus (VP2), avian reo virus (σC), Newcastle disease virus (HN, F), avian coronavirus (S1), avian influenza virus (rHA0), chicken infectious anaemia virus (VP1) and Eimeria tenella (EtMI C2) were expressed in selected plants.
Infectious bursal disease virus : plant-based vaccine candidate for IBD by means of a transient VP2 expression in Nicotiana benthamiana (Gómez et al., 2013) and demonstrated that it was able to protect chicken against infection with IBDV when 7.5 μg of VP2 were administered in a prime/boost scheme through the intramuscular (im) route but not when delivered by the oral or intranasal routes.
Newcastle disease (ND) is a viral disease that causes labored breathing, periorbital oedema, and ataxia in the majority of avian species. The most frequently used method of vaccination against NDV is intranasal. Intranasal vaccination induces mucosal immunity but always poses some potential risks to the birds. Plant-based vaccines are an alternative approach to inducing mucosal immunity, which is the most important factor for providing protection against viral respiratory diseases.
The available vaccines against Newcastle disease virus (NDV) are limited, owing to their low reactivity and multiple dosage requirements. Plant-based machinery provides an attractive and safe system for vaccine production.
Almost 2–7.1-fold higher expression of F gene mRNA in transgenic corn leaves and 8–28-fold higher expression of HN gene mRNA in transgenic corn seeds were observed, when the expression was analyzed by real-time PCR on a relative basis as compared to non-transgenic control plant material (Leaves and seeds).
The IBV, S1 glycoprotein contains virus neutralizing and hemagglutination-inhibiting epitopes have been the component of interest for vaccine development. By stably transforming the S1 glycoprotein gene into the potato plant, tuber extracts from the transgenic potatoes were used for vaccination and protective efficacy studies in chicken [
There are two options for vaccine administration: injection (intramuscular or subcutaneous) and mucosal (oral or nasal) administration. Injection-type vaccines elicit strong protective immunity by preferentially inducing IgG production. They are most suitable against pathogens that infect via a systemic or respiratory route; however, the antigens have to be purified before administration. These vaccines are often produced in tobacco plants using transient expression(Table.2)
Future potential
Plant-based vaccines are a novel class of vaccines that have great potential to treat diseases. Though the development procedure has advanced considerably, there remains a need for more suitable gene delivery methods to achieve efficient and optimum vaccine production.
Several bioethical concerns surrounding the production of plant-based vaccines, such as the risk of transferring allergens from transgenic plants to humans and animals, are also important to consider. Additionally, various technical challenges, such as the development of regulatory requirements, must be addressed before these vaccines can be produced on a large scale.
Plant based edible Vaccines
Edible vaccines are called by several alternative names such as ‘food vaccines’, oral vaccines, subunit vaccines, and green vaccines. They seem to be a viable alternative especially for the poor and developing countries. They have come up as great boon in medicinal science for which biotechnologists should be given all credit. The concept of edible vaccines lies in converting the edible food into potential vaccines to prevent infectious diseases. It involves introduction of selected desired genes into plants and then inducing these altered plants to manufacture the encoded proteins. Oral feeding of transgenic maize expressing the viral fusion protein was shown to be immunogenic and conferred protective immunity to chicken.
Plant based edible vaccines are one of the novel branches of vaccinology. Candidate antigen can be expressed on selected plant species through various biotechnological approaches. Stable integration of selected antigen into plant genome can be achieved through vector mediated or biolistic method of transgenesis.
Another method is transient expression of candidate antigen using agroinfiltration or infection with modified RNA viruses. These types of vaccines have been developed against poultry diseases also. Immunogenic proteins of avibirnavirus (VP2), avian reo virus (σC), Newcastle disease virus (HN, F), avian coronavirus (S1), avian influenza virus (rHA0), chicken infectious anaemia virus (VP1) and Eimeria tenella (EtMIC2) were expressed in selected plants.
Edible vaccines against poultry diseases are cost effective, thermostable and devoid of human or animal pathogens and microbial toxins. Other points in credit for edible vaccines include suitability for mass vaccination, ease of administration, storage stability, least stress, needle free delivery, no muscle damage etc. Commercial preparations of plant based edible vaccines are likely become a reality in near future.
Ideal properties of Edible Vaccines
Application of plant based Vaccines in Aquaculture
Aquatic diseases also threaten human livelihood and health by reducing food security. Unhealthy aquatic production and drug residues from production may have harmful effects on humans and the environment. People increasingly pay attention to the quality and safety of aquatic products and the risk to these from environmental pollution. To control disease occurrence, chemicals and antibiotics are employed. Antibiotics, such as florfenicol, norfloxacin, and flavomycin, can protect farmed fish from bacterial diseases efficiently and cost-effectively. However, these may pose serious risks to the environment, human health, and food security. Drug residues with the potential risk of drug resistance, allergic reactions, and poisoning reactions seriously endanger the farmed species, the environment, and human health, for which the emerging antimicrobial resistance (AMR) has become a global threat. Therefore, health management of aquaculture is of great importance for food security, food safety, the environment, and sustainable development of the aquaculture industry. Moreover, candidate alternatives, e.g., probiotics and plant bioactive compounds to replace antibiotics, could also be promising, contributing to the management of aquaculture fish health.
Plant biotechnology provides a perfect option for production and vaccination for fish production. More research should be carried out to meet this urgent need. Since the rapid advance of aquaculture, the high densities and other artificial conditions (artificial propagation, polyculture, transportation etc.) in which fish are farmed exponentially increase the risks of outbreaks from infectious diseases. There may also be an ecological hazard in terms of pathogen spread to wild fish. It is estimated that approximately 10% of cultured aquatic animal production are lost due to infectious diseases globally, leading to over the loss of 10 billion USD. Outbreaks of diseases in Atlantic salmon (Salmo salar), oyster and marine shrimp have led to partial or sometimes total loss of production in several countries worldwide. Natural disasters, diseases, and pollution have together caused serious production loss globally.
Vaccine types mainly include live vaccines, inactivated vaccines, and genetically engineered vaccines. Recombinant subunit vaccines, one kind of genetically engineered vaccine, represent promising options for high safety, high stability, easy production, easy control, and good immunogenicity. Subunit antigens have been produced in bacteria, yeast, transgenic plants, insects, mammalian cell cultures, and cell-free platforms.
Oral vaccination provides a non-stressful and energy-saving administration for both fish and farmers. Vaccination via feeding appears to be an ideal method to provide protective immunity without extortionate cost of time and effort. Although taking into account the vaccination dosage and the cost of plant treatment including homogenization, drying, and briquetting, the use of edible plants for production of pathogen subunits decreases the need for expensive fermentation, purification, cold storage, transportation, and sterile delivery and oral recombinant plant-produced vaccine has the advantages of simplicity and safety. Thus, oral vaccine produced in edible crops offers unique cost advantages and antigen stability at room temperature.
Conclusion Plant-based vaccines are the emerging type of vaccines that have a higher therapeutic value to treat many human and animal diseases. “Plant molecular farming” is a term used to describe the application of molecular biological techniques to the synthesis of commercial products in plants, which include a variety of carbohydrates, fats, and proteins, as well as secondary products. Research on plant-based vaccines focusses mostly on increasing the amount and purity of antigen in transgenic plants to stimulate adequate immune responses. Targeting the most suitable subcellular compartment in plant cells is also of great importance for optimal quantity and quality of antigen. At present, production and application of plant-made vaccines still face several challenges, yet the promise and potential of better plant-based vaccines are attractive. Plant-based vaccine is an emerging type of vaccine and it is anticipated that regulatory approval will be granted eventually. Plant based vaccines against various poultry diseases may become an alternative to conventional vaccination programmes in coming decades.
Prof. R.N.S.Gowda
Former VC, KVAFSU, Bidar, Former Director. IAH&VB, Bangalore, Former Prof and Head, Veterinary College, UAS, Bangalore.